We aim to assess the effect of 8 mg of oral TA-65MD given twice daily for 12 months on immunosenescence in older patients following ACS.

The secondary objectives of this trial are as follows:

TL will be determined from cryopreserved peripheral blood mononuclear cells (PBMCs) by flow cytometry-fluorescent in situ hybridization (flow-FISH) [9]. TL will be measured in total leukocytes and CD8+ T cells. CD8+ T cell TL is significantly reduced in patients with CHD and post-MI [9]. We expect TL in CD8+ T cells to decrease less in patients treated with TA-65MD. TL will be measured in total leukocytes and in CD8+ T cells at baseline, 6 months, and 12 months.

Microvascular endothelial function will be assessed by measuring FMD at baseline, 6 months, and 12 months. Using plethysmography at the fingertips of both hands, the EndoPAT (Peripheral Arterial Tone) system (Itamar Medical Ltd) will calculate an index of pulse wave amplitude after cuff occlusion to before occlusion of the test arm divided by the same ratio of the control arm, namely the reactive hyperemic index. FMD has been shown to be compromised in patients with CHD and microvascular dysfunction as a predictor of adverse clinical outcome [20].

Systemic inflammation will be assessed using the hsCRP, which is a downstream biomarker of inflammation associated with an increased risk of cardiovascular events [21].

Cardiac function will be assessed by NT-proBNP and transthoracic echocardiography at baseline and 12 months. Together, these measures will determine myocardial function, strain (NT-proBNP), and global longitudinal strain, reflecting the pathophysiological targets of heart failure. Lack of telomerase and presence of shortened telomeres in cardiomyocytes from preclinical models (ie, mice) have been shown to be critical in the development of heart failure in that species [22]. Patients with chronic heart failure have shorter TL compared to age- and gender-matched controls with incremental attrition according to the presence and extent of coronary artery disease (CAD) [23].

TA will be assessed by a modified telomeric repeat amplification protocol (TRAP) assay, using digital droplet polymerase chain reaction (PCR) [24,25]. This provides an indication of drug effect: TA-65MD is expected to increase TA in PBMCs.

Oxidative stress will be measured with the thiobarbituric acid reactive substances (TBARS) colorimetric assay (Oxford Biomedical Research) from freshly collected and cryopreserved plasma. TBARS is an established assay to quantify lipid peroxides [26].

The effect of CMV seropositivity at baseline will be correlated with study outcomes using an exploratory analysis. CMV is a highly prevalent human herpes virus with CMV seropositivity present in a large proportion of elderly patients. CMV-directed cells increase with age, constituting a large proportion of the total CD8+ T cell pool in elderly individuals [27]. T cell responses to CMV are restricted to a limited number of epitopes, resulting in progressive, prolonged oligoclonal expansion of CMV-specific CD8+ T cells in a process known as memory inflation [28]. We have previously shown that individuals who are seropositive have a much higher proportion of CD8+ TEMRA cells and have a greater risk of heart attacks [7,29].

Major clinical events will be measured for 12 months from the start of treatment. All-cause death, MI, and stroke will be presented as a composite outcome—major adverse cardiac and cerebrovascular events (MACCE)—and also reported separately. All AEs, including those considered related and unrelated to the intervention, will be recorded and reported from the time of randomization for 12 months or until a patient has completed all trial activities.

Potential participants will be identified by the clinical team following admission to hospital with ACS and having agreed to undergo coronary angiography or having already had an angiography that confirms CHD. Patients may also be identified following discharge. Potentially eligible participants will be invited to participate by a delegated member of the study team. The trial will be explained by the clinical research team and, after time for questions, written consent will be sought from the patient. Written informed consent will be obtained prior to any trial specific procedures and prior to randomization. There will be additional consent for storage of blood samples for up to 5 years for future use in ancillary studies. Patients have the right to withdraw from the trial at any time and without providing a reason. With the explicit consent of patients, general practitioners will be informed of their participation.

Following consent, the principal investigator or a subinvestigator on the delegation log will confirm the patient’s eligibility. Patients who do not meet trial eligibility criteria prior to randomization will be considered a screen failure and withdrawn from the trial with no further data collected.

Randomization will be performed using the Sealed Envelope Ltd system with a minimization scheme to ensure patients randomized to each group are comparable at baseline. The minimization scheme will account for (1) gender (male or female), (2) type of ACS (ST elevation myocardial infarction [STEMI] or non-ST elevation myocardial infarction [NSTEMI]), and (3) CD8+ TEMRA (high >45% or low ≤45%) at baseline.

Eligible patients will be randomized by delegated and trained members of the research team at each center using the 24-hour, central, secure, web-based randomization system with concealed allocation. Eligible patients will be randomized in a 1:1 ratio to receive TA-65MD (intervention under study) or placebo (control arm).

Assignment to either the TA-65MD or placebo groups will be blinded to the patient, treating clinicians, and the clinical research team, including the pharmacy, research nurses, echocardiogram assessors, laboratory staff, and the chief investigator. Blinding will be maintained in the randomization system, the electronic case report forms (eCRFs), and on investigational medicinal product (IMP) labels. All members of the Newcastle Clinical Trial Unit (NCTU) and the statistics team will be blinded, with the exception of the trial data manager to enable reporting to the independent data monitoring committee as appropriate. The IMP will be labeled using a unique identification code, which will be linked to the study randomization system. TA-65MD and its matched placebo will be identical and presented in the same packaging to ensure blinding of the IMP.

Unblinding should not occur except in the case of medical emergencies, where the appropriate management of the patient requires the knowledge of the randomization allocation. To avoid unnecessary unblinding, it will be assumed that the patient is on active treatment within the trial. The Sealed Envelope Ltd online web-based randomization service will be used for emergency unblinding. The primary trial analysis will be performed prior to unblinding. All patients will be informed of which arm they were assigned to, once analysis of the trial data is complete.

TA-65MD is marketed as a dietary supplement. The active ingredient of TA-65MD is 1.8% CAG, isolated from roots of the Astragalus species. The active ingredient has been identified in an empirical screen of traditional Chinese medicine plant extracts and compounds. TA-65MD is an activator of telomerase, an enzyme whose actions protect the ends of chromosomes from shortening associated with repeated cellular replication. Patients allocated to the intervention will be given 8 mg of TA-65MD twice daily.

The matched placebo has been manufactured to ensure that it is consistent with TA-65MD in appearance, taste and smell, labeling, packaging, and batch number. Patients allocated to the placebo group will be given this twice daily.

An independent expert panel has determined TA-65MD to be Generally Recognized As Safe (GRAS) for use in a medical food under the provisions of the Federal Food, Drug, and Cosmetic Act, administered by the US Food and Drug Administration. T.A. Sciences, Inc, provided extensive animal and human clinical data to support the status of GRAS. The safety of TA-65MD has been assessed in an observational study of 114 adults over the course of 1 year [30]. At doses up to 50 mg per day there were no AEs reported for TA-65MD. Safety was also assessed in a randomized, placebo-controlled study involving 117 adult volunteers over the course of 1 year, with no toxicities detected in the liver, kidneys, and metabolic functions as assessed by biochemical markers [29]. An anticipated risk for the use of TA-65MD relates to its ability to activate telomerase. This is the subject of scientific conjecture in consideration of its relationship to cancer [31,32]. An in vitro assay testing the telomerase-activating potency of TA-65 (ie, CAG not in capsules) in Medical Research Council cell strain-5 (MRC-5) fibroblasts suggested there was dose-related TA (Sierra Sciences Labs). While telomerase activation has been observed, TA-65MD does not increase the lifespan of cells. In fact, the preliminary observation of a controlled in vivo cancer study using 5 mL/kg of TA-65 (ie, CAG not in capsules) or sham drug administered by oral gavage for up to 40 days in mice xenografted with four different human tumors—lung (H460), colon (HT29), breast (MDA-MB-435), and prostate (PC3)—suggests a trend toward tumor growth retardation in two cancer types; as well, there was no statistically significant adverse effects on body weight or tumor size for any of the cell lines nor on the growth rate of the tumors (internal T.A. Sciences document, 2008). Given that the population under study in the TACTIC trial are elderly with ACS, and that there were no data on the impact of the drug on the outcomes or population, we chose to conduct the trial using a conservative dose of 8 mg twice daily.

TA-65MD may interfere with medications that suppress the immune system and may also affect blood sugar and blood pressure. In an observational study, two subjects self-reported an “anxious” feeling shortly after voluntarily increasing their daily consumption of TA-65MD to 100 mg/day—a self-imposed choice without physician approval at a consumption level two times above the intended dose for this observational study; the feelings resolved in both subjects when daily consumption was returned to 50 mg/day.

A pragmatic decision was taken to recruit and analyze data on 90 patients: 45 in each group. This sample size is the minimum conventional threshold for making parameter estimates in pilot studies [33]. The parameter estimates in this trial will be used to inform a large, multicenter clinical trial.

Study data are recorded in each patient’s medical notes before being recorded onto eCRFs, which are developed and managed by the NCTU. The eCRF has been built using the Red Pill system supplied by Sealed Envelope Ltd. Data entered onto the eCRF must be consistent with the information in the medical notes. Patients are identified using a unique study ID; all data passed to the NCTU will have patient identifiers removed, with the exception of date of birth, gender, ethnicity, and study ID. Data cleaning is provided by staff within the NCTU.

Primary analysis will follow intention-to-treat principles with patient data analyzed according to randomization and irrespective of intervention received; other analysis groups, such as per-protocol groups, may be considered subsequently. Every effort will be made to retain and include all patients who are part of the trial.

Data will be summarized by study group. Mean or median will summarize continuous variables, whereas number and percentage will be used to summarize categorical variables. A general linear model will be used to analyze the primary outcome considering all covariates used in the randomization scheme. Similar methods will be used to analyze all continuous secondary outcomes. Data of other types will be analyzed using generalized linear models with appropriate distributions.

The numbers of MACCE after 12 months will be compared between arms by the use of standardized rates (eg, by consideration of the number of events per patient month in each arm). Impact of missing data will be explored by tabulating the proportion of missing data in each arm. A full statistical analysis plan will be developed for the outcome measures and agreed upon with the IDMEC and the chief investigator prior to any analysis being undertaken.