The REVAMP trial is an investigator-initiated, randomized, double-blind, placebo-controlled study that aims to investigate the safety and efficacy of the long-term intake of resveratrol 150 mg/day in patients with asymptomatic CASO and will be performed at the NCVC. One hundred participants will be randomly assigned to either the resveratrol or placebo control group in a 1:1 ratio (Figures 1, 2). During the REVAMP trial, participants will be given either resveratrol or a placebo for 35 weeks (Figure 3).

The physicians involved in the study will obtain written informed consent for the trial and the NCVC biobank from all trial patients before participating in the study. In case of any revision in the written informed consent form, the physicians involved in the study will explain the revised procedure again to the participants, revise the written informed consent form, and obtain the subject’s voluntary consent for continuation of participation.

The primary objective will be change of ADAS-Cog total score from baseline to assess the effectiveness of resveratrol in preserving or improving cognitive function in patients with asymptomatic CASO. The principal secondary objectives will be to evaluate the impact of resveratrol on the following: (i) the preservation or enhancement of cognitive function, as evaluated by a change in the total MoCA score and the trail-making test score from baseline; (ii) the preservation or improvement of cerebral hemodynamic parameters, including CBF, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO₂) as measured by ¹⁵O-gas PET (Table 1).

The REVAMP trial will be conducted by investigators at the NCVC. We will include the following categories of participants: (i) patients aged ≥20 years and < 90 years at the time of consent; and (ii) individuals with asymptomatic carotid artery stenosis of ≥50%, or occlusion. A peak systolic velocity of ≥130 cm/s at a stenotic lesion, as measured by carotid Doppler ultrasonography, indicated a stenotic diameter of approximately ≥50% (22). The asymptomatic status will be confirmed based on the absence of stroke or transient ischemic attack caused by the development of a carotid lesion within 6 months (3). Patients with hypertension, cardiovascular diseases, or diabetes mellitus will not be excluded. Details of our eligibility criteria are presented in Table 2.

Before enrollment, all potential participants will undergo an eligibility screening process by reviewing their medical charts to check the results of carotid ultrasonography. Personal information of potential and enrolled patients will be shared in a database accessible only within the project group to personnel responsible for patient inclusion, to protect confidentiality before, during and after the trial. At the first visit, the physicians involved in the study will provide detailed information about the REVAMP trial to the patients and/or their families and obtain written informed consent. Participants will be informed in the consent forms that they have the right to withdraw from the trial without any negative consequences. The attending physician will conduct a physical examination of all participants, and the clinical research coordinators (CRCs) will collect necessary clinical data such as height, body weight, vital signs, medical history, supplements, and demographics.

After the initial screening, all participants will undergo neuropsychological assessments such as ADAS-Cog, MoCA, and the trail-making test. They will also undergo ¹⁵O-gas PET and brain magnetic resonance imaging, randomized at visit 2 (Figure 2). Blood samples for analyses will be collected before taking either resveratrol or placebo tablets and stored in the NCVC biobank. The tablets will be then distributed to participants after randomization (See the Randomization, allocation, and blinding section) by each physician involved in the study. To ensure proper medication adherence, all participants will be instructed to fill out a schedule book specific to the REVAMP trial every day and return the remaining tablets at each visit.

All participants will be required to visit the NCVC at weeks 12 and 35, which will be the final visit. During the week 35 visit, carotid ultrasonography, neuropsychological assessments, ¹⁵O-gas PET, and brain magnetic resonance imaging will be conducted, and blood samples will be collected and stored in the NCVC biobank. At weeks 12 and 35 (Figure 2), each physician involved in the study will conduct a physical examination, and the CRCs will conduct interviews to inquire about changes in prescription, over-the-counter medications and supplements, and occurrence of adverse events. To promote participant retention and complete follow-up, the CRCs will call the participants at least once a month to check whether the participants are continuing taking the resveratrol or placebo tablets.

The physicians involved in the study will generate the allocation sequence, enroll patients, and assign patients to interventions. Registered patients will be assigned randomly to either the resveratrol group or the control group. The assignment method will be minimization, and the stratification factors considered will be sex, the presence of left CASO, and a total score of ADAS-Cog ≥15. The randomization process will be executed using computer-generated random allocation treatment codes. Both patients and physicians will remain blinded to the assigned therapy throughout the study. The randomization list will be maintained by an independent investigator who is not involved in patient care, assessment, data collection, or analysis. Emergency unblinding will only occur if the principal investigator deems it necessary to reveal the assigned intervention to manage any potential adverse events among participants.

Nature Holdings Co., Ltd. in Japan will supply standardized resveratrol and placebo tablets. The resveratrol tablets will be manufactured using 99% pure synthetic trans-resveratrol (resVida™, DSM Nutritional Products Ltd., Kaiseraugst, Switzerland), comply with Good Manufacturing Practice. The visual appearance of the placebo tablets was identical to that of the resveratrol tablets. Participants in the resveratrol group will be administered one 75-mg tablet of resveratrol twice daily (in the morning and evening) for 35 weeks. Conversely, those in the control group will receive one placebo tablet twice daily (also in the morning and evening) during the same period. Participants will consume one tablet with water. The physicians involved in the study or CRCs will maintain a comprehensive tablet inventory record, which will include information on the supply, receipt, disposal, and return of the trial tablets.

Discontinuance criteria will be as follows: (i) severe renal dysfunction (estimated glomerular filtration rate of <15 mL/min/1.73m²); (ii) severe gastrointestinal symptoms requiring emergent treatments; (iii) initiation of taking resveratrol or nicotinamide mononucleotide; (iv) initiation of taking antidementia drugs; and (v) administration of carotid endoarterectomy (CEA) or carotid artery stenting (CAS). Relevant concomitant care and interventions that are prohibited during the trial will be (i) resveratrol or nicotinamide mononucleotide; (ii) antidementia drugs; and (iii) CEA or CAS.

A restricted medication will be allowed if the doses are unchanged during the trial. Statins may improve anti-inflammatory, antithrombotic, and endothelial functions (23), and phosphodiesterase inhibitors may contribute to improvements in cerebrovascular regulation (24), potentially increasing CBF. Therefore, statins and phosphodiesterase inhibitors are included in the restricted medication list. Other medications or interventions may be used without restriction. Nonsteroidal anti-inflammatory drugs (NSAIDs) will not be included in the restricted medication list. NSAIDs used for <24 months do not affect cognitive function (25).

Only psychologists who have substantial experience and are blinded to the intervention groups will be authorized to perform cognitive assessments, including ADAS-Cog, MoCA, and the trail-making test.

All patients will undergo a series of ¹⁵O-gas PET examinations to assess CBF, CMRO₂, and OEF. Radioactive ¹⁵O will be produced by accelerating a deuteron (d) beam via the 14 N (d,n) ¹⁵O nuclear reaction using a cyclotron (CYPRIS HM-12, Sumitomo Heavy Industry, Tokyo, Japan). Further, 0.3% oxygen (O₂) in the nitrogen (N₂) target will be used to produce the ¹⁵O–O₂ and ¹⁵O–carbon monoxide (CO) gasses and 1.0% carbon dioxide (CO₂) in the N₂ target gas to produce the ¹⁵O–CO₂ gas.

A PET scanner (Biograph mCT, Siemens Healthinier, Erlangen, Germany) will be used in the study. A PET scan was initiated 3 min after the 2-min inhalation of ¹⁵O–CO for 4 min. An additional dynamic PET scan was performed for 8 min during the sequential inhalation of ¹⁵O-O₂ and ¹⁵O–CO₂ gasses for 1 min each at a 4.5-min interval. The radiochemical purity was confirmed to be >99% before each radio gas inhalation in every patient via two-channel rapid gas chromatography (Micro 990, Agilent Technologies, Inc., Santa Clara, USA), as described in a previous study (26).

PET images will be reconstructed using vendor software, with an adequately selected methodology that considers the presence of gaseous ¹⁵O–radioactivity surrounding the face during images will require following the 2-min inhalation of the ¹⁵O–carbon monoxide gas (27). CBF and OEF images will be calculated using the previously validated dual-table autoradiography technique (28). Then, the functional images of CMRO₂ will be calculated as follows:

according to a previously validated technique (i.e., dual-table autoradiography technique) (28). The arterial input function will be obtained from the radioactivity concentration in the arterial blood that was continuously withdrawn from the brachial artery (28). The metabolized ¹⁵O-water in the arterial blood generated from ¹⁵O–O₂ will be estimated by modeling physiological oxygen metabolism (29).

Referring to aforementioned our preliminary study performed at the NCVC between July 2020 and March 2022, we observed an intergroup difference in the total ADAS-Cog scores of −2.35 with a standard deviation calculated from the pooled variance estimates of 3.86. In line with previous studies (30–32), the effect size, calculated as | − 2.35/3.86|, was deemed sufficient to detect a clinically meaningful difference. Consequently, the sample size calculation for testing the hypothesis of the superiority of resveratrol treatment over nontreatment, with a certain degree of power, was conducted.

Since the total ADAS-Cog score at baseline is expected to correlate with the change in total score, between-group comparisons will be made via the analysis of covariance using the total ADAS-Cog score at baseline as the covariate when analyzing the primary endpoint. Under this assumption, the minimum number is 45 subjects per group based on the number of subjects required for a two-sided significance level of 5% and 80% power. However, since point estimates of correlation coefficients assessed by the limited available data are difficult to refer to, we conservatively assumed that there was no correlation.

Considering the basis for the above calculation and the difference between the dose in the resveratrol group in the REVAMP trial and the dose in the resveratrol group in the preliminary study, the actual power in the REVAMP trial is expected to be sufficiently higher than assumed. Therefore, we will recruit 100 patients (50 patients per group), considering a dropout rate of 10%. A press release notifying about start of the REVAMP trial at NCVC was issued on the website of NCVC to reach the target sample size.

Web-based electronic data capture system with secure and restricted access will be used to collect clinical data obtained from patient medical records. Geographic data, medical history, medication information, laboratory data, and findings of neuropsychological tests and carotid ultrasonography will be stored in the system. Data will only be de-identified for analysis after this study. Shido Co., Ltd., a contract research organization, assigned to data management will perform quality control at each step of data handling to ensure the reliability of all data related to the trial, and maintain the web-based electronic data capture system.

The individuals assigned to data monitoring will be responsible for safeguarding the interests of trial participants, evaluating the safety and efficacy of the interventions throughout the trial, and monitoring the overall conduct of the trial. A formal data monitoring committee will not be set up because (a) it is known that long-term resveratrol administration has minimal risks (19, 20); (b) asymptomatic CASO is not a life-threatening disease; (c) interim analysis will not be planned; and (d) vulnerable participants such as pediatric patients, patients with severe mental retardation and severely demented patients who fail to undergo neuropsychological examinations will not be included.

We will analyze a full analysis set (FAS), which is defined as data obtained from registered patients who will receive at least some of the assigned treatment and complete at least one assessment at the end of the 35 weeks. However, patients found to be ineligible after the registration will be excluded from the full analysis set. For the primary objective, the changes in total ADAS-Cog scores from the baseline to 35 weeks after starting protocol treatment will be tested using the analysis of covariance with covariates including the baseline ADAS-Cog total score, the existence of left CASO, and sex to assess the improvement in cognitive function brought about by the treatments In addition, the same analysis will be also performed on the per protocol set (PPS) as a supplementary analysis to confirm the robustness of the main analysis using the FAS. The PPS will not include the following categories of participants: (a) patients who will receive the prohibited concomitant care and interventions as outlined in the section 2.8 Intervention; (b) patients who will not attend visits at 35 weeks within the specified allowance; and (c) less than 75% of medication adherence. The REVAMP trial will judge the efficacy of resveratrol using these analyses. Changes in CBF, CMRO₂, and OEF assessed by ¹⁵O–gas PET, white matter volume on brain magnetic resonance imaging and blood pressure from the baseline to 35 weeks will be also analyzed with the same procedure as the primary objective.

Changes from the baseline within a group will be analyzed using paired t-test or Wilcoxon signed-rank test, taking into account the data distribution, which Shapiro–Wilk test will be used to assess. Regarding other evaluation items, for continuous variables, the changes from baseline at each follow-up point will be evaluated using either paired t-test or Wilcoxon signed-rank test based on the data distribution. Comparisons do not take multiplicity into account. Student’s t-test or Wilcoxon rank-sum test will be used to evaluate the difference between the two groups based on the data distribution. For categorical variables, Fisher’s exact or χ² test will be used. All reported p-values will be two-tailed, and p < 0.05 will be considered statistically significant.

An exploratory analysis will be conducted to assess the following issues: (a) temporal changes in plasma NAD⁺ or the NAD/NADH ratio; (b) temporal changes in blood SIRT1 protein concentration or activity; (c) temporal changes in blood eNOS protein concentration; (d) associations between plasma resveratrol and its metabolite concentrations and cognitive function.

If an adverse event occurs, the physicians involved in the study will treat the patient appropriately in terms of safety and report the details of the event. If a severe adverse event occurs, the principal investigator must report it to the Director General of the NCVC and the NCVC Clinical Research Review Board. If the adverse event is caused by protocol treatment, follow-up and best medical treatment will be performed. The patient will be compensated for any study-related injuries by insurance.