Study design and participants. This was a randomized, double-blind, placebo-controlled study of the effects of galantamine treatment in patients diagnosed with MetS. The study was performed at a clinical research center, The Heart Institute (InCor), University of São Paulo, Brazil. Study participants were recruited from outpatient settings. Individuals of both sexes between the ages of 18 and 59 years, and with BMI measures between 25 and 39 kg/m2 were screened for the presence of MetS according to the ATP III criteria (1). These criteria require the presence of at least 3 of the following 5 parameters: increased abdominal circumference (≥102 cm for men and ≥88 cm for women); low plasma HDL cholesterol levels (<40 mg/dl for men and <50 mg/dl for women); increased values for plasma triglycerides (≥150 mg/dl); elevated BP (≥130 mm Hg SBP or ≥85 mm Hg DBP); and increased plasma glucose levels (≥100 mg/dl). All subjects were evaluated to exclude conditions that restricted their inclusion in the study, which included symptoms and signs of cardiovascular disease or previous diagnosis of cardiac arrhythmias; coronary artery disease; valvular disease; heart failure; chronic obstructive pulmonary disease; chronic inflammatory diseases; cancer; positive status for HIV; abuse of alcohol or other illicit substances in the months prior to study entry; chronic use of medications, including drugs that have known or probable interaction with galantamine (serotonin reuptake inhibitors, amitriptyline, fluoxetine, fluvoxamine, ketoconazole, oxybutynin, paroxetine, quinidine); symptoms and signs of neurologic and autonomic diseases; past history of major depression, suicidal ideation, and history of eating disorders; triglyceride levels 400 mg/dl or higher; known history of liver disease or levels of aspartate transaminase (AST) or alanine transaminase (ALT) 200 U/l or higher; office BP 160 mmHg or higher or DBP 110 mmHg or higher; and abnormal renal and thyroid function. All women were tested for pregnancy as an exclusion criterion. CONSORT 2010 guidelines (http://www.consort-statement.org) were followed during the preparation of this manuscript.

Outcomes. The primary outcome measures of this study were plasma levels of cytokines and adipokines with a recognized role in mediating the MetS inflammatory state and linked to insulin resistance and cardio-metabolic derangements, including TNF, IL-1β, IL-6, IL-10, leptin, and adiponectin. Secondary endpoints were fasting plasma glucose and insulin levels and the associated HOMA-IR, HRV, fat tissue depots (measured by computed tomography), metabolic and cardiovascular indices, including HDL cholesterol, LDL cholesterol, total cholesterol, triglycerides, arterial BP, and heart rate. No changes in the trial outcomes were made on the protocol after the trial commenced. We did, however, make some edits to the text initially posted on ClinicalTrials.gov to better reflect and summarize information in the protocol; cytokines and adipokines as primary outcome measures and metabolic and cardiovascular indices as secondary outcome measures were specifically listed.

Randomization, masking, and blinding. In order to reduce sex bias, subjects were stratified by sexual phenotype prior to being randomized to the galantamine or placebo arm. Recruitment of study participants took place until 30 men and 30 women were recruited to allow randomization in equally divided groups by sex. Study participants were randomly assigned to 1 of 2 parallel groups at a 1:1 ratio: 30 subjects (15 males and 15 females) were assigned to galantamine treatment and 30 subjects (15 males and 15 females) to placebo. A computer-generated random sequence method was used to allocate the participants to the corresponding groups. Randomized packages of placebo and active drug were numbered from 1 to 60 (http://graphpad.com/quickcalcs/randomize1/). Members of the staff of the Central Pharmacy that did not participate in the study conducted the randomization and were responsible for the drug delivery and inventory. Specific drug accountability included quantities dispensed/received (capsule counting) every month, serial numbers, expiration date, and drug code number. To achieve masking (blinding), galantamine capsules were reencapsulated at the Central Pharmacy of the Clinical Hospital, Medical School of University of São Paulo. Active drug and placebo capsules were presented in identical medication packaging. The study drug packages were stored in a secure, temperature-controlled medication room according to standard operating procedures. Subjects received packages of study drug for 30 ± 3 days, every 4 weeks for a total of 12 weeks. Study participants, investigators, and outcome assessors were blinded to the group assignment.

Study procedures. Galantamine hydrobromide extended-release capsules of 8 mg and 16 mg, commercially available as Reminyl ER (manufactured by Janssen-Cilag Pharmaceuticals, Johnson & Johnson) or placebo were used in the study. Galantamine was administered orally in a dose of 8 mg for 4 weeks, and then titrated to 16 mg for 8 weeks, for a total of 12 weeks. Following informed consent, at the initial screening all subjects underwent complete clinical and neurological evaluations including BP, waist circumference, height, weight, and BMI. Blood tests, including triglycerides, total cholesterol, HDL cholesterol and LDL cholesterol, fasting glucose, serum creatinine, thyroid and liver function tests, renal function panel, pregnancy testing (if applicable), and complete blood count were performed. Electrocardiogram and chest x-ray were also performed. Based on results from the screening visit, eligible subjects (fulfilling the criteria for enrollment in the study) returned 7–10 days later for additional assessments that included determination of intra-abdominal, subcutaneous abdominal, and epicardial fat tissue depots, heart rate, office and 24-hour ABPM, HRV analysis, blood tests for plasma cytokine, adipokine and insulin levels, and HOMA-IR assessment. After completing all evaluations, subjects were randomized and instructed to start taking the study drug. All subjects were assessed at 4 and 8 weeks for safety monitoring with clinical and neurological exams. After 12 weeks, at the end of the treatment period, subjects repeated the specific assays for fat tissue depot determination, heart rate determination, office and 24-hour ABPM, HRV analysis, blood tests and plasma cytokine, adipokine and insulin determination, and HOMA-IR assessment. The principal investigator, coinvestigators, and the principal coordinator monitored all study participants who were also able to communicate with the investigators by phone or email if necessary. Subject self-reporting of medication adherence and capsule counts were used to establish treatment compliance.

Methods and assays. Intra-abdominal (visceral) and subcutaneous abdominal fat were determined by computed tomography scans at the Cardiac Tomography and Resonance Service Center at the same hospital. Briefly, intra-abdominal fat and subcutaneous abdominal fat were measured by computed tomography (Aquilion One, Toshiba Medical Systems) at the level of the 4th and 5th (L4 and L5) lumbar vertebrae. All study participants were examined in the supine position with both arms stretched above the head, and 10-mm slices were measured. Intra-abdominal and subcutaneous abdominal fat areas (cm2) were obtained by delineating and computing the adipose tissue surface using attenuation range of –150 to –50 Hounsfield units (HU) (48). For analysis an automated selection of visceral fat area was taken by using threshold technique in the range of –30 to –190 HU with subsequent volume calculation. For this analysis, we used a workstation with Aquarius Intuition software (Terarecon), which automatically separates visceral abdominal fat from subcutaneous fat, and exports the corresponding volumes of the 2 compartments. To evaluate the total chest fat around the heart, a Vitrea workstation (Vital Images) was used with a volumetric threshold instrument defining fat as voxels with a tissue density between –30 and –190 HU. Epicardial fat was defined as the one internal to the pericardium and in direct contact with the epicardial surface of the heart. This assessment was performed using the Vitrea workstation volumetric tool with manual planimetry, drawing a dividing line at the pericardium. Thus, average density and volume (in ml) of the epicardial compartment was generated.

Twenty-four–hour ABPM was performed using a SpaceLabs healthcare device (model 90207). BP was measured every 10 minutes during the day (8 AM to 11 PM) and every 20 minutes during the night (11 PM to 8 AM) with an appropriate cuff placed on a nondominant arm. Participants were instructed to perform their ordinary daily activities and not to move their arm during the ongoing measurement. Activity, bedtime, and time on awakening from sleep were recorded by participants in their diaries. Therefore, daytime and nighttime BP based on 24-hour ABPM was evaluated using actual sleep and wake times recorded by participants.

For HRV analysis all subjects were asked to abstain from exercise 24 hours before the study and from drinking caffeinated products on the morning of the test. The test was performed in a quiet room. BP waveforms were obtained with a digital photoplethysmograph device (Finometer; Finapres Medical System BV) while subjects were awake in a supine position during a 15-minute rest period. These stored data were subsequently analyzed to calculate HRV values. All of the segments were visually inspected, and the nonstationary data were discarded. A Hanning window was used to attenuate the side effects, and the spectrum of each segment was computed using a direct fast Fourier transform (FFT). Spectral bands were defined according to literature references of evaluation in humans: LF (0.04–0.15 Hz), HF (0.15–0.4 Hz), and total power. Spectral components were expressed in absolute values (ms2) and nu. The nu were obtained by calculating the percentage of LF and HF with respect to the total power after subtracting the power of the very-low-frequency component (frequencies of <0.04 Hz). To assess the sympathovagal balance, the LF/HF ratio of the HRV was calculated (49, 50).

For plasma cytokine, adipokine, and insulin determination venous blood was collected from the participants and plasma samples obtained. Samples were stored at –80°C prior to analysis. Cytokines, adipokines, and insulin were analyzed using multiplex immunoassay (all from Millipore): HCYTMAG-60K-PX41 for TNF, IL-1β, IL-6, and IL-10; HADK2MAG-61K for leptin and insulin; and HADK1MAG-61K for adiponectin. Individual values for leptin and adiponectin were used to calculate the leptin/adiponectin ratio.

Statistics. The proposed sample size for this trial was 60 subjects (n = 30 per group), based on resources available and in consistency with previous clinical studies (51, 52). It should be noted that a sample size of 30 in each group will have 86% power to detect an effect size of 0.8 using a 2-group t test with a 0.05 2-sided significance level. This is consistent with the power analysis previously used in studying the effects of a drug (rosiglitazone) or placebo once daily for 12 weeks on inflammatory and metabolic indices in 60 patients with the metabolic syndrome (51).

Descriptive statistics for all subjects at baseline are reported as means and SD separately by group (Table 1). The Mann-Whitney test was used to determine whether the difference for each of the baseline measures between the placebo and the galantamine groups was significant. As cited below, some variables were analyzed with a log transformation; however, these variables are summarized in their original units of measurement for the columns labeled “Baseline” and “Follow-up” in Tables 1, 2, and 4. For the columns labeled “Treatment × Time Interaction Effect”, they are summarized in the log scale.

Repeated measures analysis of variance (RMANOVA) with a mixed models approach was used to determine if the 2 groups behaved differently over time (pre- vs. post-) for most primary and secondary outcomes/endpoints (i.e., the group × time interaction). For all analyses, the standard assumptions of Gaussian residuals and equality of variance were tested. As the normality assumption was not met for insulin, HOMA-IR, high frequency (ms2) of HRV, LF (ms2) of HRV, and LF/HF ratio, the logarithmic transformation was used for the analysis of these variables. The repeated-within-subjects factor was time and the within-subjects factor was treatment group (galantamine or placebo).

RMANOVA analyses were restricted to those subjects who had measurements at both baseline and follow-up visits. Means and SDs were reported at each time point for each of the 2 randomized treatment arms (Tables 2 and 4). The treatment effect sizes are presented as the arithmetic differences in least-squares means, with corresponding 95% CIs.

IL-1β, IL-6, and IL-10 were subject to lower detectable limits (0.8 pg/ml, 0.9 pg/ml, and 1.1 pg/ml, respectively). Therefore, any values that were below the lower detectable limit were set to 0.79 pg/ml, 0.89 pg/ml, and 1.09 pg/ml, respectively, for analysis (Table 3). Since this approach might render the required assumptions of the mixed-model analyses to be invalid, the bootstrap method using percentiles was used to estimate 95% CIs for the mean treatment × time interaction effect. The Mann-Whitney test was used to determine whether the difference in differences (Post-Pre) for each of these measures between the galantamine and placebo groups was significant.

A result was considered statistically significant at P less than 0.05. All analyses were performed using SAS version 9.4 (SAS Institute Inc.).

Study approval. The study was performed at The Heart Institute (InCor), University of São Paulo, Brazil. The study protocol was reviewed and approved by the Institutional Review Committee and the Human Subject Protection Committee of the Heart Institute (InCor) and the Clinic Hospital (number 11672/555738), University of São Paulo. Informed consent was obtained from all study participants prior to inclusion in the study. The study was conducted in accordance with World Medical Association International Code of Medical Ethics (Declaration of Helsinki, 1964; revised in 2008). The study is registered at www.clinicaltrials.gov with the number NCT02283242 and the following name: “Galantamine effects in patients with metabolic syndrome (GALANTA-MS)”: https://clinicaltrials.gov/ct2/show/NCT02283242?term=galantamine+metabolic+syndrome&rank=1

Adverse effects monitoring and assessment. Prior placebo-controlled clinical trials with galantamine doses of 24 mg/day and 32 mg/day in patients with Alzheimer’s disease have indicated that the most common adverse effects of these galantamine doses are gastrointestinal, particularly nausea (22). However, using a lower galantamine dose or slowly escalating the drug dose has been shown to substantially reduce the frequency of gastrointestinal adverse effects, thus demonstrating a favorable tolerability profile of galantamine (53). Therefore, in this study we implemented a regimen of low-dose galantamine treatment in which the initial drug dose (8 mg/day) for 4 weeks was subsequently increased to 16 mg/day for the remaining period of the study. The patients were informed about possible adverse effects and were encouraged to write down (document) and report any complaints and adverse effects. In addition, the patients were allowed and encouraged to report any adverse effect through unrestricted telephone contact with the investigator. Hospital support was available for the patients. To additionally ensure safety, clinical consultations were performed monthly and the participants in the study were asked about the occurrence of adverse effects, including nausea, vomiting, dizziness, diarrhea, abdominal pain, and tremor.