Here, we conducted a randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of a treatment regimen consisting of 12 weeks of oral intake of MMFS-01, a compound containing L-TAMS, in older adults with cognitive impairment. We used three inclusion criteria, including subjective memory complaints (SMC), sleep disorder, and anxiety, to select subjects who had cognitive impairment. Their cognitive impairment was later confirmed by an object cognitive test (Trail Making Test - Part B).Sleep and anxiety disorder were used as inclusion criteria to increase the chance of recruiting subjects who had cognitive impairment with an underlyingneurodegenerative condition. This was necessary because previous studies show that SMC is not a good sole indicator of mild cognitive impairment (MCI). Many people who think they have memory issues actually have a normal cognitive profile when tested with objective memory tests. There is only a 30% chance that someone with SMC has MCI [ 16 ]. It is common that patients with brain atrophy not only have MCI but also have neuropsychiatric symptoms [ 17 ]. The common core non-cognitive symptoms in MCI patients are anxiety [ 18, 19 ] and sleep disorder [ 20, 21 ]. 47% of MCI patients have anxiety symptoms [ 22 ], and 83% of those with MCI and anxiety develop AD compared to only 41% of those with MCI without anxiety [ 23 ]. Recent studies show sleep disorder is strongly correlated with cognitive impairment [ 20 ], and even the chance of getting AD [ 24 ].

In our pre-clinical studies, we found that the level of brain magnesium is a critical factor controlling synapse density and plasticity. Elevating neuronal intracellular magnesium can increase functional synapse density and plasticity in cultured hippocampal neurons [ 6, 7 ]. Mechanistically, we found that intracellular magnesium in neurons serves as a critical second messenger controlling neuronal energy supply and functional synapse density [ 7 ]. In an intact rodent, treatment with conventional magnesium salts is ineffective at elevating brain magnesium and improving memory function [ 8 ]. This is because active transport systems tightly control the amount of magnesium that crosses first from digestive tract into blood, and then from blood to cerebrospinal fluid (CSF) [ 9 ]. In human, increasing blood magnesium by up to 300% only changes CSF magnesium by less than 19% [ 10 ]. To overcome this problem, we developed L-Threonic acid Magnesium salt (L-TAMS, formerly MgT), a compound that can effectively enhance CSF magnesium concentration via oral intake [ 8 ]. L-TAMS treatment increases synapse density in brain regions critical for executive function and memory, such as the prefrontal cortex and hippocampus [ 8, 11–13 ]. Furthermore, L-TAMS treatment increases the number of NR2B containing NMDA receptors, resulting in an enhancement of synaptic plasticity in aging rats and Alzheimer’s disease (AD) model mice [ 8, 14 ]. At a functional level, L-TAMS treatment reverses cognitive impairment in aging rats and AD model mice [ 8, 14 ]. The increase of synapse density in aging rats is linearly correlated with memory improvement [ 8 ]. L-TAMS treatment also enhances fear memory extinction and prevents fear memory over-generalization, leading to a reduction of anxiety in rats [ 13, 15 ].

Cognitive function declines with aging [ 1 ]. Cognitive impairment in elderly is a major problem that can affect activities of daily living (ADL) and qualityof life [ 2, 3 ]. Although the neuropathological process underlying cognitive impairment remains elusive, the best correlate to cognitive impairment is brain atrophy [ 1, 4 ]. Brain atrophy is associated with neuronal, axonal, and synaptic loss. So far, the best structural predictor of cognitive decline is the degree of synaptic loss [ 5 ]. Since synapses are the elemental units of neural communication, synapse loss and reduction of synaptic plasticity should have a major impact on neural signaling, resulting in impaired cognition. Therapeutic strategies that prevent net synapse loss and increase synapse density may have great potential for cognitive impairment.

MATERIALS AND METHODS

Study design This was a 12-week parallel-designed, randomized, single-site, double-blind, placebo-controlled clinical trial that compared MMFS-01 and placebo. MMFS-01is a compound containing L-TAMS, trademarked under the name ClariMem®.

Participants Subjects were men or women between 50 and 70 years of age with self-reported complaints of cognition (memory and concentration), and with anxiety and sleep disorder. Subjects had a Mini-Mental State Examination score (MMSE) equal to or greater than 24. Sleep difficulties defined by a score of greater than 5 on the Pittsburgh Sleep Quality Index (PSQI), and the presence of mild-to-moderate anxiety, with scores ≥12 and ≤ 28 on the Hamilton Anxiety Questionnaire sub-score A (HAM-A), were required for inclusion in the study [26]. Exclusion criteria included active heart disease; uncontrolled high blood pressure (≥140/90 mmHg); renal or hepatic impairment/disease; Type I or II diabetes; bipolar disorder; Parkinson’s disease; AD; dementia; unstable thyroid disease; diagnosed major affective disorder; psychiatric disorder (hospitalized in the past year); immune disorder (such as HIV/AIDS); a history of cancer (except localized skin cancer without metastases or in situ cervical cancer) within 5 years prior to screening; current use of calcium channel blockers, SSRIs or anxiolytics other than benzodiazepines as needed, with “as needed” defined as less than 5 times per month; current use of any medications that are known to interact with magnesium including loop, thiazide, or potassium-sparing diuretics, muscle relaxants, penicillamine, corticosteroids, magnesium-containing antacids or other magnesium containing products; use less than 7 days before the randomization visit of calcium channel blockers, any anxiolytics or SSRIs; current use of antibiotics (a washout period of 2 weeks was allowed); presence of an unstable dose of medication (defined as fewer than 90 days at the same dose); presence of an allergy or sensitivity to any ingredient in the test product; hepatic or renal dysfunction as evidenced by ALT, AST, AP being ≥2 times the upper limit of normal or serum creatinine value ≥2.0 mg/; history of drug or alcohol abuse in the past 12 months or had begun/stopped smoking ≤ 6 months ago or had plans to begin/quit smoking; possibility that the subject may become pregnant as shown by lack of birth control use, pre-menopausal status or absence of hysterectomy; status of pregnancy, lactation or plans to become pregnant during the study period; participation in another research study either presently or within 30 days prior to the screening visit; any condition, abnormality, medication usage or clinically significant clinical laboratory findings that, in the opinion of the investigator, would compromise the safety of the subject or the quality of the study data. Subjects were allowed to take medications if the medication was not part of the exclusion criteria and the dose was unchanged at least 90 days before screening and throughout the study. Subjects stopped taking any dietary supplements at least 7 days prior to randomization, and maintained cessation during the study. They refrained from alcohol consumption or exercise for at least 24 hours prior to each test visit. No changes to the methods, including eligibility requirements and dosing, were made after commencement of the trial.

Recruitment and randomization Subject randomization began in November 2012, and recruitment was completed in June 2013. A total of 51 subjects (age 50–70) were recruited by Miami Research Associates (MRA) and enrolled in a randomized double-blind, placebo-controlled trial conducted at MRA (Miami). Data for all subjects at every time point were collected by MRA at their Miami clinical laboratory. Before the study began, the protocol, informed-consent form, and other information provided to subjects and caregivers were reviewed and approved by the Aspire IRB (September 20, 2012). Subjects were randomly assigned to the MMFS-01 or placebo group in a ratio of 1:1, using a block-2 randomization schedule. Subjects received a sequential number corresponding to the order in which they entered the study. Study sponsors, investigators, research coordinators, attending care teams, and subjects were blinded to treatment group. The consulting statistician locked the database of data elements and unblinded it by accessing the table of randomized assignments and merging them into the data tables.

Dosage Dosage was set to correspond to approximately 25 mg/kg/day. To accomplish this, subjects between 50 and 70 kg took 1.5 g/day, and subjects between 70 and 100 kg took 2 g/day of MMFS-01. At conclusion of the study, 8 subjects (35 percent) were taking 1.5 g of MMFS-01 per day, and 15 subjects (65 percent) were taking 2 g of MMFS-01 per day.

Power analysis Enrollment for this study was targeted at 50 subjects (25 per group). Prior to this study, this compound was given open-label to a small number of subjects(L-TAMS has self-affirmed/FDA-affirmed GRAS status). These subjects’ subjective feeling was a significant improvement in anxiety, sleep, and mental clarity. They had significant improvement in anxiety based on the HAM-A questionnaire. Therefore, we powered the analysis in this trial by reduction of HAM-A score. We predicted the treatment would lead to a 50% reduction in HAM-A score, with a SD of HAM-A scores of approximately ± 10 score points [27]. Assuming a serial coefficient correlation of about 0.5 for HAM-Ascores at baseline and 12 weeks, the within-group SD of the 12-week changes would also be ± 10 score points. With the use of an unpaired Student t-test with a significance level of 0.05, a total enrollment of 50 subjects (40 completers if 20% attrition) was required for the study to be able to detect differences of about a 45% reduction in HAM-A score. We assumed an attrition rate of 20% in line with previous experience by the contract research organization that ran the study, MRA. Even if the attrition rate had been as high as 32% , there would have been enough analyzable subjects (34 subjects) to provide 87% power in detecting a clinically meaningful 50% HAM-A score reduction.

Efficacy endpoints Efficacy assessments were made at Baseline Visit, Week 6 Visit, and Week 12 Visit. The change in the body’s magnesium status was quantified by assessing blood magnesium concentration (plasma Mg2 +), urine magnesium concentration normalized by the estimated glomerular filtration rate (uMg2 +/GFR), and intracellular magnesium concentration (Red Blood Cell; RBC Mg2 +). The key functional efficacy outcome measures included measurements of cognitive abilities, sleep quality, and affect.

TMT-B test The Trail Making Test – Part B (TMT-B) assesses executive function as well as impulsivity, visual search, visual attention, and motor speed [28]. In the test, subjects were required to connect a series of label circles that constituted a trail. Scores were calculated as the inverse of the time (in milliseconds) it took the subject to complete the task (all 25 circle connections), representing speed. Scores from subjects unable to complete the task in the maximum allotted time (360 seconds), or from those who quit prior to the maximum allotted time, were scaled to the time to complete 25 circle connections before converting to speed. Six out of 44 (13%) subjects did not complete the task at least once with a total of 9 occurrences, 5 at baseline, 3 at Week 6, and 1 at Week 12. Higher speeds reflected better performance.

DigitSpan test The DigitSpan test assesses working memory performance. Scores were based on the length of the longest sequence of digits (consecutive numbers) subjects could remember and thus ranged from 0 without an upper bound, with higher scores reflecting better performance.

Eriksen Flanker Congruent/Incongruent test The Eriksen Flanker Congruent/Incongruent test assesses attention, that is, cognitive processes involved in detection and recognition of targets in the presence of distracting information [29]. A target directional arrow was flanked by either arrows in the same (congruent) or opposite direction (incongruent). The average time to correctly select the target arrow’s direction was recorded. The incongruent task was more difficult than the congruent task because the congruent task did not require response inhibition and was less confounded by training effects. Therefore, the response times in the congruent condition were subtracted from those in the incongruent condition to remove training effects and discern effects on attention. The opposite of this difference was reported so higher scores reflected better performance.

Face-Name association test Finally, the Face-Name Association test assesses hippocampal-dependent episodic memory [30]. Twenty faces with twenty fictional popular first names were shown on screen. Subjects were then asked to remember and later recognize each face and name pair when presented with the same or novel face and name pairs. Using signal detection theory, the hit rate, false alarm rate, and sensitivity index (d’) were calculated, where d’ = z (hit rate) -z (false alarm rate) .d’ showed how well the subject distinguished old from new. Hit rate was defined as a correct identification of an old face and name pair and false alarm as an incorrect identification of a new face and name pair. Higher scores reflected improved performance with scores above three indicative of a near perfect score.

Composite score Scores from several cognitive tests, evaluating four domains of cognition—executive function, working memory, attention, and episodic memory—were combined to produce a composite score to assess overall cognitive ability [25]. The cognitive tests included TMT-B for executive function [31], DigitSpan for working memory capacity [32], Face-Name Association for episodic memory [33], and Eriksen Congruent/Incongruent Flanker [29] for attention. The composite score was calculated as the average of the four individual z scores ( z ¯ ). z scores were calculated for each subject on each test using the formula z = x - μ b σ b , where μ b is the mean of all subjects (MMFS-01 and placebo combined) at baseline and σ b is the standard deviation (SD) of all subjects at baseline. Baseline means and SDs were used to convert the raw scores of Week 6 and Week 12 to z scores in order to determine the treatment effects (change frombaseline) of MMFS-01 versus placebo for each subject for each test. Effect size (Cohen’s d) was determined for each of the cognitive endpoints at Week 6 and Week 12 using the formula Cohen’s d = ( X ¯ n , Δ MMFS - 01 ) - ( X ¯ n , Δ Placebo ) σ pooled , where X ¯ n was the mean of the change from baseline values in the MMFS-01 or placebo group at either Week 6 or Week 12 and σ pooled was the pooled SD of the change from baseline of the MMFS-01 and placebo groups at either Week 6 or Week 12. Pooled SD was calculated using the formula σ pooled = [ ( n MMFS - 01 - 1 ) ( σ Δ MMFS - 01 ) 2 + ( n Placebo - 1 ) ( σ Δ Placebo ) 2 ] [ ( n MMFS - 01 + n Placebo ) - 2 ] .

Sleep Sleep quality was measured with PSQI [26]. PSQI is a self-rated questionnaire which assesses sleep quality and disturbances over a 1-month time interval. Higher scores indicated worse sleep quality. Based on previous research, a global PSQI score greater than 5 yields a diagnostic sensitivity of 89.6% and specificity of 86.5% (kappa = 0.75, p less than 0.001) in distinguishing good and poor sleepers [26].

Emotion Affective personality was assessed with the HAM-Aand the Positive and Negative Affect Schedule (PANAS). The HAM-A is a rating scale used in both clinical and research settings to measure the severity of psychic and somatic anxiety symptoms [34]. It did not provide any standardized probe questions and was administered by a clinician (subject did not complete the questionnaire by his/herself). Scores ranged from 0 to 56 where ≤ 17 indicated mild severity, 18 to 24 mild to moderate severity, 25 to 30 moderate to severe severity, and >30 severe severity. The PANAS is a self-rated tool used to measure positive and negative affect over a 1-week time interval, and consists of two 10-item scales, one for Positive Affect and the other for Negative Affect [35]. Subjects were asked to rate different feelings and emotions using the following Likert scale: 1 = very slightly or not at all, 2 = a little, 3 = moderately, 4 = quite a bit and 5 = extremely. Scores for each scale ranged from 10 to 50. Higher positive affect scores represented more positive affect, and thus, better outcomes. Higher negative affect scores represented more negative affect, and thus, worse outcomes.

Cognitive ability fluctuation analysis The fluctuation of cognitive ability over time was evaluated by calculating variance of the change in composite score from Week 6 to Week 12 of individual subjects, with the formula σ 2 = ∑ ( X Week 12 - X Week 6 ) 2 n - 1 . The fluctuations of cognition of the placebo group and the MMFS-01 group were calculated separately.

Tolerability and safety Safety evaluations included recording all adverse events, results of laboratory tests (comprehensive metabolic panel, uric acid, and complete blood count with differential), vital signs, body weight, and subjective remarks. Adverse events were listed, MedDRA encoded, grouped by general type of event (gastrointestinal, neurologic, cardiac, etc.), and cross-tabulated by event type and product group. The principal investigator catalogued adverse events as mild, moderate, or severe according to the following definitions: Mild (causing no limitation in normal activities), Moderate (causing some limitation in normal activities), and Severe (causing significant limitation in or the inability to perform normal activities). A central laboratory conducted all laboratory evaluations. Of the 47 adverse events, 13 events, occurring in 10 subjects, were judged by the principal investigator to be probably or possibly related to the study product. Probably and possibly-related adverse events were considerably more prevalent in the placebo group than in the MMFS-01 group (9 and 4 events, in 6 and 4 subjects, respectively). The predominant adverse events were related to gastrointestinal function (affecting 5 of 25 subjects (20.0%) in the MMFS-01 group and 4 of 26 subjects (15.4%) in placebo group, p = 0.726) or infections/infestations (affecting 4 of 25 subjects (16%) in the MMFS-01 group and 6 of 26 subjects (23%) in placebo group, p = 0.726).

Statistical analysis The safety population consisted of subjects who received at least one dose of any study product, and who had any subsequent encounter with the study site. The efficacy population included all subjects who completed all scheduled visits, had no protocol deviations that in the judgment of the principal investigator would have invalidated their efficacy data (see product compliance section below). Only data from subjects that completed all visits were included in the statistical analysis; therefore, there were no missing data values in the dataset, and imputation was notrequired. Statistical analyses for cognitive tests and body magnesium status variables were performed with SPSS and R. For categorical variables, difference in the distribution of categories between the different treatment groups was tested for nominal significance by the Chi-Square test, in SPSS or GraphPad Prism. Formal statistical tests were performed for cognitive endpoints and magnesium status using a univariate analysis of covariance (ANCOVA) model at Week 6 and Week 12 with baseline values as a covariate. For safety endpoints, changes were tested for significance by the paired Student t-test, or the non-parametric Wilcoxon signed-ranks test if necessary. Differences in adverse event patterns between product groups were tested by the Fisher’s Exact test. Longitudinal repeated measures ANCOVA analyses using observed data without any data imputation were used to determine the overall effect from baseline of MMFS-01 compared to placebo. The model included the categorical fixed effects of treatment (MMFS-01 versus placebo), week (6 and 12), and treatment-by-week interaction, as well as the continuous fixed covariate of baseline measurement. Normality of distribution and equality of variance were determined using the Shapiro-Wilk’s test and Levene’s test, respectively. For endpoint values that violated either test, additional bootstrapping was employed, using resampling methods. In the ANCOVA analyses, in order to simulate the F-distribution under the null hypothesis, resampling techniques were used to permute the treatment labels, time point labels, and baseline values. For each of the 10,000 random permutations, F-statistics for the ANCOVA model were computed, and used to compute a percentile p-value for the dataset. Bootstrapping was used for TMT-B, Mg2 + Urine, and Mg2 + Plasma. In one exception, to determine treatment differences at Week 6 and Week 12 between the MMFS-01 and placebo groups for percent change in RBC magnesium concentration, an analysis of variance (ANOVA) model was used instead of ANCOVA. As this was not a pivotal Phase-III clinical trial, it was not required to control the study-wise Type-1 error rate to a specified alpha level. Each efficacy endpoint was considered an independent question of interest, with a hypothesized difference, and was tested independently using a two-tailed 0.05 alpha level (p ≤ 0.05 required for a conclusion of statistical significance). No interim analysis was performed for this study. To determine outliers, individual data for each test was analyzed. If a baseline score was greater than 2 SDs away from the mean then that data point was considered to be an outlier, and therefore excluded. Of the four cognitive tests, outliers were only found on the Flanker test. Out of 44 baseline data points, 3 subjects were removed (1 MMFS-01, 2 placebo) from the analysis of the Flanker test. Additionally, we found some ceiling effects in the Face-Name test, in which some subjects had a near perfect baseline score (>3). Therefore, we set 3 as the threshold for the ceiling baseline Face-Name score. Out of 44 data points, 3 subjects were removed (2 MMFS-01, 1 Placebo) from the analysis of the Face-Name test. We removed the contribution of any excluded subject to the composite score so the excluded data points did not erroneously skew the composite score. Except for outliers and scores at the ceiling, all data were included for all subjects for all outcome measurement analyses.

Product compliance Compliance was measured via the pill counting method, by documenting the number of calendar days between visits and the number of pills that should have been taken. Subject compliance was recorded as a percent of the prescribed amount for each visit and then averaged to produce an overall compliance figure. Per the original protocol, 80–120% compliance was considered acceptable. Of 44 subjects in the per protocol population, 41 returned their unused pills and were in the acceptable range. The remaining 3 did not return their pills, but were determined to be within the acceptable range of compliance based on the estimation of the PI, using MRA staff’s familiarity with the subject and/or subject’s compliance during other testing phase(s) of the study to make this decision. Therefore, all 44 subjects were considered compliant.