Benfotiamine in Alzheimer's Disease: A Pilot Study
Phase: PHASE2
Conditions: Alzheimer's Disease
Sponsor: Burke Medical Research Institute

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Benfotiamine in Alzheimer’s Disease: A Pilot Study 
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NCT02292238 
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September 2, 2020 
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ABSTRACT 
 
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Background. In preclinical models, benfotiamine efficiently ameliorates the clinical and 
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biological pathologies that define Alzheimer’s disease (AD) including impaired cognition, beta 
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amyloid plaques, neurofibrillary tangles, diminished glucose metabolism, oxidative stress, 
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increased advanced glycation end products (AGE) and inflammation.   
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Objective. To collect preliminary data on feasibility, safety and efficacy in individuals with 
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amnestic mild cognitive impairment (aMCI) or mild dementia due to AD in a placebo-controlled 
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trial of benfotiamine. 
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Methods. A twelve-month treatment with benfotiamine tested whether clinical decline would be 
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delayed in the benfotiamine group compared to the placebo group. The primary clinical outcome 
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was the AD-Assessment-Scale-Cognitive Subscale (ADAS-Cog). Secondary outcomes were 
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the clinical dementia rating (CDR) score and fluorodeoxyglucose (FDG) uptake, measured with 
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brain positron emission tomography (PET). Blood AGE were examined as an exploratory 
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outcome.  
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Results. Participants were treated with benfotiamine (34) or placebo (36). Benfotiamine 
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treatment was safe. The increase in ADAS-cog was 43% lower in the benfotiamine group than in 
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the placebo group, indicating less cognitive decline, and this effect was nearly statistically 
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significant (p=0.125). Worsening in CDR was 77% lower (p=0.034) in the benfotiamine group 
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compared to the placebo group, and this effect was stronger in the APOE ε4 non-carriers. 
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Benfotiamine significantly reduced increases in AGE (p=0.044), and this effect was stronger in 
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the APOE ε4 non-carriers. Exploratory analysis derivation of an FDGPET pattern score showed 
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a treatment effect at one year (p=0.002). 
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Conclusions Oral benfotiamine is safe and potentially efficacious in improving cognitive 
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outcomes among persons with MCI and mild AD. 
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INTRODUCTION 
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Alzheimer’s disease (AD) therapies targeting brain amyloid-β (Aβ) have in most cases 
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shown a lack of efficacy, suggesting that AD treatment development should consider alternative 
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targets. In addition to plaques, tangles and cognitive decline, multiple changes accompany AD 
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including inflammation, oxidative stress and metabolic dysregulation. Cerebral glucose 
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metabolism as measured by fluorine-18 (18F) fluorodeoxyglucose positron-emission tomography 
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(FDGPET) changes decades before AD is typically diagnosed [1], and in AD patients reductions 
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in glucose utilization correlate highly with cognitive decline [2].  
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Abnormalities in glucose metabolism, vascular changes and inflammation are closely 
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linked and common features of AD [3, 4]. Thiamine diphosphate (ThDP)- dependent enzymes 
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regulate key steps in brain glucose metabolism, and the activities of ThDP-dependent enzymes 
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decline in blood and brain of AD patients. The reduction in the activity of these enzymes provide 
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a plausible underlying mechanism for the metabolic abnormalities [5-7]. In pre-clinical models, 
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thiamine deficiency induces inflammation and change in vasculature [8]. Abnormal metabolism 
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often leads to over production of free radicals that damage other molecules. At autopsy, 
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oxidative stress in the brain is as widespread as plaques and tangles [9]. Increases in Advanced 
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Glycation End products (AGE), toxic protein modifications that are indicative of altered glucose 
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metabolism, and their receptor, RAGE, occur in the brain [10] and periphery [11] of AD patients, 
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in both plaques and tangles [12].  
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Benfotiamine, a synthetic thiamine precursor, has direct actions on multiple metabolic 
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enzymes and pathways, inflammation and oxidative stress [13, 14]. Benfotiamine’s activation of 
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the enzyme transketolase [15] accelerates the shunting of the precursors of AGE towards the 
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pentose phosphate pathway thereby reducing the production of AGE [16, 17]. The reduction in 
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AGE decreases metabolic stress, which reduces vascular complications [18-21]. By being more 
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effective in raising blood thiamine concentrations than direct thiamine administration, 
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benfotiamine may overcome the reduction in activity of ThDP dependent enzymes in AD [18, 
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19]. For example, mice [22] and humans [23] that have genetic defects in the thiamine 
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transporter can be treated with high dose benfotiamine. Benfotiamine is an antioxidant  [24-26], 
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modulates arachidonic acid inflammation pathways, nuclear transcription Factor κB (NF-κβ), 
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protein kinase B, mitogen-activated protein kinases (MAPK) and vascular endothelial growth 
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factor receptor 2 (VEGFR2) signaling pathways [14]. Recent studies suggest that restoring 
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cerebral perfusion by preventing neutrophil adhesion may provide another strategy for improving 
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cognition in AD participants [27]. Benfotiamine prevents lipopolysaccharide (LPS)-induced 
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macrophage death and monocyte adhesion to endothelial cells [28]. Multiple approaches suggest 
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that benfotiamine inhibits inflammatory mediators and enhances anti-inflammatory factor 
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production in activated microglia [28, 29].  
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Benfotiamine diminishes pathology in multiple pre-clinical models of disease including 
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animal models of AD, which have human gene mutations that cause AD [25]. In a transgenic 
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mouse model of tauopathy, benfotiamine treatment diminishes tangles, activates the Nrf2/ARE 
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pathway, neuroprotective and improves behavioral deficits [30]. In animal models of amyloid 
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plaque formation, benfotiamine reduces amyloid plaque numbers and phosphorylated tau levels, 
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elevates the phosphorylation of glycogen synthase kinase-3α and -3β, and improves memory 
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[31]. In other animal models, benfotiamine modulates activation of GSK3-β [32], restores 
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neurogenesis [26, 33], modulates AMPA receptor expression [25] and decreases oxidative stress 
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[26]. Together, these results suggest that benfotiamine may be therapeutically beneficial for AD.  
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Benfotiamine also diminishes AGE. Measures of AGE in the serum assess peripheral 
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abnormalities and may mirror CNS abnormalities in glucose homeostasis. AGE are a biomarker 
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implicated in aging and the development, or worsening of many degenerative diseases, such as 
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diabetes, atherosclerosis, chronic renal disease, and AD. High concentrations of AGE appear 
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predictive of long-term decline in cognition-related daily living performance in patients with AD 
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as measured by Clinical Dementia Rating (CDR) [11] or Mini Mental Status Exam (MMSE)[34]. 
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Thus, AGE may be a promising therapeutic target to prevent or delay the progression of AD 
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[35]. Numerous studies in patients with diabetes show that benfotiamine diminishes AGE [21]. A 
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preliminary study of five patients without placebo control that was published after our trial was 
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initiated showed promise [36]. 
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Benfotiamine is safe compound in AD patients as demonstrated in trials conducted for 
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the treatment of peripheral neuropathy in diabetes [13, 20, 37]. The dosage studied most 
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extensively in diabetics is 300 mg in the morning and night, but dosages as high as 900 mg per 
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day show no significant toxicity [20].  
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 The aim of this study was to conduct a double-blind early phase II randomized placebo-
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controlled trial of benfotiamine with the objective of collecting preliminary data on feasibility, 
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safety, and efficacy. The goal was to test whether benfotiamine treatment could delay clinical 
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decline in amyloid positive patients with amnestic mild cognitive impairment (aMCI) or mild 
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dementia due to AD with MMSE scores of >21. ADAS-cog served as the primary endpoint. 
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Brain glucose utilization, measured using FDG PET imaging, was assessed as a secondary 
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endpoint. Cerebral glucose metabolism declines in temporoparietal regions with the progression 
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of AD, correlates with clinical decline, and is also a sensitive measure of changes in regional 
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neuronal function associated with disease or treatment effect [1, 2]. AGE levels were used as a 
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peripheral marker of efficacy. Measures of thiamine and its esters thiamine diphosphate (ThDP) 
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and thiamine monophosphate (ThMP) provided blood markers of efficacy of drug delivery.  
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101 
MATERIALS AND METHODS 
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This clinical trial was a collaborative study between investigators at the Burke 
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Rehabilitation Center including the Burke Rehabilitation Hospital and the Burke Neurological 
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Institute [an affiliate of Weill Cornell Medicine (WCM)], WCM, and investigators at Columbia 
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University Irving Medical Center (CUMC). The trial was approved by the Institutional Review 
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Boards of the Burke Rehabilitation Hospital, WCM and CUMC. 
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Patient population. 
108 
 
 Seventy amyloid positive patients 60 years and older with aMCI (21<MMSE < 26) or 
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mild AD dementia (MMSE≥26) were included. Table 1 shows the inclusion and exclusion 
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criteria for what we define as AD in this trial.  These criteria are especially important because 
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new imaging capabilities will likely redefine AD [38].  
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Study design.  
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Sample size justification. In addition to literature that states a four-point change on the 
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ADAS-Cog is considered clinically significant, several randomized clinical trials have found 
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ADAS-Cog change scores differed by 3-4 points between placebo and treatment groups over a 6-
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month time period. Moreover, other studies report annual changes in the ADAS-Cog among 
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those who are untreated to average 9.6 points (SD=8.2) [39, 40]. Power was calculated based on 
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expected difference in change on the ADAS-COG of 3 points between the treatment and control 
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groups. Estimates based on using a two-sided alpha of 0.05 and a standard deviation of 4, 
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enrolling 29 patients per group, (N=58) suggest 80% power to detect a mean change of 3 
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between treatment and placebo.  
122 
 
Assignment of patients. A randomized, placebo-controlled, double-blinded trial of 
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benfotiamine in persons with aMCI or AD dementia with a duration of 12 months was 
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conducted. Using blocked, stratified randomization design, patients were assigned to the 
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treatment or control group. By the inclusion criteria all subjects had MMSE of >21.  Within this 
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group, a separate randomization schedule was generated using the proc plan function in SAS 
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statistics program for those with an MMSE greater than or less than or equal to 26 to balance 
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their allocation patients to placebo or treatment groups. Using a block size of four for a total of 
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seventy-six patients, 19 blocks were created to help ensure balanced recruitment into treatment 
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and control groups within strata. The schedule was generated in advance by the statistician and 
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provided to the blinded pharmacist in charge of executing the randomization. Two randomization 
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worksheets stratified by MMSE were provided to the pharmacist, who randomized the patients. 
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One sheet had MMSE Scores >=26 (randomized to Active or Placebo). The other sheet had 
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MMSE scores <26. The patients were enrolled by the clinical study team and randomized by the 
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pharmacist. The assignment to the treatment or placebo group was known only to the pharmacist 
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and kept behind a triple lock. The patients received numbered bottles.  
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Study procedures.  
139 
 
The trial was registered in ClinicalTrials.gov (NCT02292238 (Figure 1). Participants 
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were pre-screened from the database of the Memory Evaluation Treatment Service (METS) at 
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Burke Rehabilitation Center or referrals from the Center for the Aging Brain (CAB) at 
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Montefiore/Einstein Medical College, Alzheimer’s Association, primary care physicians, and 
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private neurologists from the lower Hudson Valley region. aMCI or mild AD dementia were 
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diagnosed according to NIA-AA workgroups criteria [41, 42]. Patients who met the inclusion 
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criteria for aMCI or mild dementia due to AD were invited for a screening initial visit at the 
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Memory Evaluation and Therapeutic Service (METS) outpatient department at the Burke 
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Rehabilitation Hospital. After informed consent was obtained from patients and their health care 
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proxies, a physical examination including EKG, laboratory tests (Complete Blood Count, 
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Complete Metabolic Panel, Vitamin B12, folate, Thyroid function tests), a neurological exam 
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and the MMSE were administered. If eligible (Table 1), participants were referred to 
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Westchester Imaging Center for an Amyloid PET/CT scan of the brain. Only participants with a 
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positive amyloid scan were sent to CUIMC for a baseline F-18-FDG PET/CT scan of the brain. 
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At the baseline visit, the cognitive tests were performed and blood drawn for measurement of 
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thiamine, ThDP and ThMP by HPLC [43] and APOE genotyping. Enrolled patients returned to 
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the Burke outpatient clinic at month 3, 6, 9 and 12 for subsequent visits. At month 12 the final 
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FDG/PET scan was performed at CUIMC. 
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The trial duration per participant was twelve months. Participants in the treatment group 
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took one 300 mg capsule of benfotiamine in the morning and one in the evening. The 
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participants in the placebo group took one 300 mg capsule in the morning and evening with 
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microcrystalline cellulose without benfotiamine. At each visit, the patients returned the pill 
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bottles for that period. The number of pills returned was used to assess compliance (the percent 
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of pills consumed). 
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Characterization procedures. 
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Amyloid scans. Amyloid-β was assessed using PET imaging with 18F-Betapir F18 PET 
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[44] to help confirm the presence of AD pathology in study participants. Positivity was 
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determined by a visual read. 
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APOE Genotyping Method. Total nucleic acid was isolated from whole blood samples 
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for APOE genotyping using the MasterPure™ Complete DNA and RNA purification kit 
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(Lucigen) with a starting volume of 150 µl of blood, according to the manufacturer’s 
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instructions. Genotyping of the two human APOE polymorphisms was carried out using the 
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TaqMan® SNP genotyping assays (ThermoFisher Scientific): C_3084793_20 for SNP rs429358 
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and C_904973_10 for SNP rs7412. An initial 5 min step at 95°C was followed by 40 cycles of 
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15 s at 95°C and 30 s at 60°C. Genotyping was performed in duplicate with controls for all six 
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possible APOE genotypes and no DNA controls using a QuantStudio™ 12K Flex real-time PCR 
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system (ThermoFisher Scientific). 
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Treatment.  
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The trial duration per participant was twelve months. Participants in the treatment group 
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took one 300 mg capsule of benfotiamine in the morning and one in the evening. The 
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participants in the placebo group took one 300 mg capsule in the morning and evening with 
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microcrystalline cellulose without benfotiamine. At each visit, the patients returned the pill 
181 
bottles for that period. The number of pills returned was used to assess compliance (the percent 
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of pills consumed).The benfotiamine and placebo were manufactured and provided by the 
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Advanced Orthomolecular Research, Canada. They prepared the benfotiamine according to an 
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FDA-approved IND, which was prepared by the Cornell Translational Science Center, and 
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issued to the Burke Neurological Institute.  
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Cognitive measures.  
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The following cognitive tests were conducted at the intervals indicated in Figure 1:  
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• AD Assessment Scale-Cognitive Subscale (ADAS-Cog) was the primary outcome 
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measure. It indicates the severity of the most important symptoms of Alzheimer's disease. 
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It consists of 11 tasks measuring the disturbances of memory, language, praxis, attention 
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and other cognitive abilities [45] [46]. 
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• Clinical dementia rating (CDR) score. CDR is a 5-point scale used to characterize six 
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domains of cognitive and functional performance applicable to Alzheimer disease and 
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related dementias: Memory, Orientation, Judgment & Problem Solving, Community 
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Affairs, Home & Hobbies, and Personal Care. A higher score indicates greater dementia 
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[47]. 
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• The Buschke Selective Reminding Test (SRT) [48] is a standard diagnostic tool in the 
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assessment of verbal memory. Several studies attest to its predictive value for dementia 
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[49, 50].  
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• Neuropsychiatric Inventory (NPI). The NPI assesses a wide range of behaviors 
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encountered in dementia patients to provide a means of distinguishing frequency and 
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severity of behavioral changes. Ten behavioral and two neuro-vegetative domains are 
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evaluated through an interview with the caregiver [51-53]. 
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• Alzheimer’s Disease Cooperative Study- Activities of Daily Living (ADCS-ADL). 
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The ADCS-ADLSIV is a caregiver-based ADL scale composed of 19 items developed 
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for use in dementia clinical studies [54]. It assesses the patient's performance of both 
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basic and instrumental activities of daily living such as those necessary for personal care, 
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communicating and interacting with other people, maintaining a household, conducting 
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hobbies and interests, as well as making judgments and decisions. Higher numbered 
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scores and answers of "yes" reflect a more self-sufficient individual. Therefore, the 
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higher total score correlates with higher cognitive function. The total score is the sum of 
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all items and sub-questions [55]. 
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Biomarker outcomes.  
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AGE are formed during the Maillard reaction where reducing carbohydrates react with 
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lysine side chains and N-terminal amino groups of various macromolecules, particularly 
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proteins. AGE can adversely affect the function of these macromolecules. One of the most 
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prevalent advanced glycation end products, N-epsilon-(Carboxymethyl) Lysine, has been 
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implicated in oxidative stress and vascular damage. The quantity of AGE adduct in protein 
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samples is determined by comparison with that of a known AGE-BSA standard curve. 
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 AGE levels were measured on plasma sample with a kit from ABCAM (AB238539), 
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Cambridge, MA., USA 
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Fluorodeoxyglucose (FDG) Positron Emission Tomography (PET).  
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Image acquisition, processing, and measurement  
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FDG PET imaging of glucose metabolism was acquired at baseline and after 12 months 
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of treatment. All scans were acquired on a Siemens MCT 64 PET/CT PET-CT scanner at 
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CUIMC. Study participants were maintained in an awake, at rest state with eyes and ears open in 
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dim lighting during tracer uptake. Forty minutes after injection of the tracer, the emission image 
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was acquired in four contiguous 5-minute frames. Frames were aligned with SPM 12, averaged, 
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and then spatially normalized to the MNI template using SPM12 
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https://www.fil.ion.ucl.ac.uk/spm/software/spm12/, resulting in one image per participant for 
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each time point. Average voxel values within 90 regions of interest (ROIs) in the Automated 
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Anatomic Labeling (AAL) Atlas [56] were computed. A subset of 16 pre-specified bilateral 
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regions of interest (ROIs) were chosen for the group analysis due to their relevance to AD 
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including: posterior cingulate, precuneus, frontal, inferior parietal, mid temporal, hippocampus, 
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paracentral lobule, and cerebellum. The paracentral lobule and cerebellum were included as 
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reference regions given their relative preservation during AD progression. 
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Derivation of spatial covariance patterns for Glucose FDGPET.  
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A multivariate machine learning approach was also applied to evaluate the FDG PET 
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data (Figure 10). Pattern-based methods have been increasingly applied to the evaluation of 
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neurodegeneration and therapeutic response as they address the issue of complexity in comparing 
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multiple regions and can increase signal to noise for analysis. Feature reduction was performed 
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through use of the Scaled Subprofile Model (SSM) [57-61], a form of Principal Components 
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Analysis (PCA). The resulting components were used in regression modeling that determined 
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spatial patterns of hypometabolism and hypermetabolism (or preservation relative to other 
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regions) associated with the Clinical Dementia Rating (CDR) score. 
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Specifically, SSM by performing PCA on the PET-data array was run, with a subsequent 
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brain-behavioral regression to derive a best-fitting pattern whose pattern scores correlates with 
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the CDR score in a negative direction (i.e. the higher the pattern score, the lower the CDR). The 
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best-fitting set of Principal Components was obtained via the Akaike criterion [62], and came out 
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as PC1-2. 
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To help with the imputation of the multivariate analysis, a generic multivariate 
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decomposition was written as: Y(s,x) = w(s) v(x) + ε (s,x), where Y denotes the (log-
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transformed) data which depends on a participant and time index s and the voxel location x. The 
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pattern score w(s) is a scalar that solely depends on subject and time, but not voxel location, 
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whereas the derived pattern v(x) depends on voxel location, but shows invariance across 
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participants and time, i.e. does not depend on index s; ε(s,x) denotes residual signal that is 
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dependent on participant, time and voxel location, but which was discarded for our purposes. 
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The pattern score w(s) was chosen to correlate negatively with CDR across the data. The pattern 
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v(x) is normalized to have unity Euclidean norm, i.e. ||v||=1. This means that the pattern score 
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carries all information about the strength of the signal associated with the spatial pattern. Higher 
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values of w(s) imply higher values of pattern-associated FDGPET signal in direct proportion in 
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all regions. 
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To estimate the topographic robustness of any patterns of interest, a bootstrap resampling 
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procedure [63, 64] was performed 10,000 times, for which data were resampled with 
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replacement and the complete analytic recipe was executed on the resampled data, generating 
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distribution for pattern loadings. Regional loadings were considered robust if the 95% coverage 
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interval (= [2.5%, 97.5%]) did not overlap with, and lay to one side of, zero. For the correct 
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interpretation, it is important to keep in mind that positive and negative loadings describe only 
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relative, and not absolute differences, in the signal associated with any covariance pattern. Since 
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the residual signal in ε(s,x) was stripped off, there cannot be assurance that there are absolute 
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differences in the total data for the regions with robust loadings. 
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After deriving and estimating the topographic robustness of the pattern, the pattern score 
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was inspected for an effect of treatment at baseline and follow-up, also broken down by APOE 
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ε4 status. 
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Statistical methods [65, 66]  
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Our primary clinical outcome was ADAS-Cog and secondary outcomes were the CDR 
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score and fluorodeoxyglucose (FDGPET) imaging of the brain. AGE levels were an exploratory 
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outcome. 
281 
  
Our primary analysis followed Intention-to-Treat (ITT) and the secondary analysis was 
282 
per-protocol. The per-protocol analysis omitted one placebo participant who took benfotiamine 
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from a commercial vendor. The ITT and per-protocol analysis are presented for the primary 
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outcome ADAS-cog and the secondary measure CDR. For the other measures only per-protocol 
285 
analysis are presented.  
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Spearman correlation coefficient was used to assess the correlation between continuous 
287 
variables. Student’s t-test was used to compare the continuous variables between placebo and 
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treatment groups; Fisher's exact test was used to compare categorical variables between placebo 
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and treatment groups. Specifically, two-sample Student’s t-test was used to compare the score 
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changes (ADAS score, normalized PET-related scores, etc.) from baseline between Placebo and 
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Treatment groups when normality was satisfied, otherwise Wilcoxon Rank-sum test was used. 
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ANCOVA was used to test the group difference while adjusting for covariates.  
293 
  
The primary analysis was done on the ITT data. The Last-Observation-Carry-Forward 
294 
(LOCF) method was used to impute the missing values of ADAS total score and the secondary 
295 
endpoints such as CDR as well for each time point. The primary analysis was done on ITT data 
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which were imputed with LOCF method. Per-protocol analysis was done as a sensitivity analysis 
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and as observational comparisons [66]. 
298 
 
In the time to event analysis, time to ≥ 3 points of ADAS change was calculated based on 
299 
whether the ADAS score changed from baseline ≥3 (event) at each time point. When no change 
300 
≥3 points was observed at any time point, the observation is censored and the last follow-up time 
301 
(12 month) was used to calculate the duration. Kaplan-Meier estimator was then used to estimate 
302 
probability of time-to-event. The difference between groups was tested by log-rank test for 
303 
statistical significance.  
304 
 
As sensitivity analyses, repeated-measure ANOVA, generalized estimating equation 
305 
(GEE) and Mixed effect model and Wilcoxon Rank-sum test were also performed on primary 
306 
endpoints with and without imputation to compare differences between placebo and treatment 
307 
groups.  
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Subgroup analyses in MMSE, APOE and sex were either in the per-protocol analysis or 
309 
exploratory. A Student’s t-test was used in each of the subgroup comparisons. An ANCOVA was 
310 
also used to analyze the treatment difference while adjusting for each of these covariates. 
311 
Interaction between MMSE and ADAS-cog responses was assessed by ANCOVA with 
312 
interaction term. Multiple comparisons were present in our analyses with secondary endpoints, 
313 
subgroup analyses, or analyses with multiple PET-related scores. Due to exploratory nature of 
314 
those analyses and early trial of this study, we did not apply correction of p-values for multiple 
315 
comparisons. All statistical tests were two-sided with an alpha level of 0.05 as the significance 
316 
cutoff. All analyses were performed in statistical software SAS Version 9.4 (SAS Institute, Cary, 
317 
NC). 
318 
 
319 
Results 
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Characteristics of the populations at baseline. 
321 
 
The first participant entered the trial on February 12, 2015 and the final participant 
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finished July 9, 2019. This allowed us to exceed our enrollment goal of 58. Pre-screening of 634 
323 
patients at the METS at the Burke Rehabilitation Hospital excluded all but 120 participants 
324 
(Figure 1). Only eighty-three of these patients were amyloid positive. Twelve declined to 
325 
participate. Seventy-one of these participants agreed to be part of the trial, and were randomized 
326 
to receive either placebo or benfotiamine. Eight subjects were prematurely discontinued from the 
327 
trial prior to Month 12.   Three participants were withdrawn due to non-compliance <80%; three 
328 
withdrew consent due to unwillingness to complete study procedures; one participant was lost to 
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17 
 
follow-up and one was withdrawn by PI due to physical limitations.  Patients with uncontrolled 
330 
diabetes were excluded. Eight patients were being successfully managed for diabetes.  Patients 
331 
had to have an HbA1c < 8% trial and/or a fasting glucose < 200 mg/dl to be enrolled in the trial. 
332 
None of the participants randomized to the treatment group withdrew due to adverse reactions or 
333 
adverse effects. Since the ones who withdrew did not have final scores, their dropout did not 
334 
affect 12 month scores. After the trial completion and after the data were locked, one patient in 
335 
the placebo group was determined to be on benfotiamine from another source and was excluded 
336 
from the per-protocol analysis. Thus, 37 (placebo) and 34 (benfotiamine) were included in the 
337 
ITT analysis, and 36 (placebo) and 34 (benfotiamine) were included in the per-protocol analysis.  
338 
 
Whether the patient took the required medication was referred to as compliance. If the 
339 
patients who withdrew are included, the percent compliance in the placebo group was 87.7 
340 
(3.5)% and in the treatment group was 89.8 (3%). If the patients that withdrew are not included, 
341 
the percent compliance in the placebo group was 94.1 (1.3)% and in the treatment groups percent 
342 
compliance was 94.8 (1.4)% . 
343 
 
The demographic characteristics of the patients are described in Table 2. The 
344 
randomization procedure was based on the order of patient entry into the study. There were no 
345 
statistically significant differences in age, race, MMSE, and demographic or clinical 
346 
characteristics. The goal to recruit patients with an average MMSE of 26 was met. The 
347 
percentage of females in the benfotiamine group (67.6%) was higher than in the placebo group 
348 
(50%). Although the distribution by race was similar, only 2.9% of the population was Non-
349 
Hispanic Black. The distribution of APOE ε4 carriers and non-carriers (60% and 40%, 
350 
respectively) in the whole population was reflected in the benfotiamine (64.7 and 35.3%, 
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18 
 
respectively) and placebo (55.6% and 44.4%, respectively) groups. Nearly identical proportions 
352 
were also observed for males (58.6% and 41.4%) and females (61% and 39%). The scores on the 
353 
neuropsychological tests at baseline did not differ between the two groups, with the exception of 
354 
NPI, which differed between groups at baseline (p=0.040) (Table 2B). 
355 
 
Baseline thiamine and ThMP, but not ThDP distributions were similar in the two groups. 
356 
Blood thiamine diphosphate (ThDP) was lower (p=0.038) in the benfotiamine group (Table 2C). 
357 
In agreement with the literature [67], ThDP was lower in females than males (p=0.0003). At 
358 
baseline, ThDP did not correlate with MMSE (p=0.644), CDR (p=0.618), ADAS-cog (p=0.883) 
359 
or whole brain glucose utilization (p=0.644).  
360 
 
Baseline FDGPET measures are presented in Table 2D. In agreement with prior findings 
361 
FDG PET in whole brain at baseline correlated with the MMSE (Spearman correlation, r=0.288, 
362 
p=0.015). Brain glucose utilization was 4.4% higher in females than males (p=0.003). At 
363 
baseline, FDG PET in the mid-temporal region was significantly higher in the benfotiamine 
364 
treatment group than placebo group (p=0.020), and the cingulate was higher in the treatment 
365 
group at trend level (p=0.069) (Table 2D). 
366 
 
 Safety profile. No adverse events related to the 2 X 300 mg benfotiamine per day were 
367 
observed and patients did not complain about the medication (Table 3A).  
368 
 
Benfotiamine and ADAS-Cog changes (Figure 2, Table 3B). A comparison of 
369 
unadjusted changes from baseline to 12 months with ITT analysis revealed a difference between 
370 
the benfotiamine and placebo groups favoring benfotiamine using a mixed effect model 
371 
(p=0.071), GEE (p=0.137), and a non-parametric Wilcoxon rank sum test (p=0.098) (Figure 2, 
372 

 
19 
 
Table 3B). At 12 months, the change in the placebo group was 3.26 whereas in the benfotiamine 
373 
group the change was 1.39. This difference was not apparent at 3, 6 or 9 months. The per-
374 
protocol analysis (Table 3B) suggested that the differences were significant when analyzed by a 
375 
mixed effect model (p=0.035), GEE (p=0.069) or Wilcoxon Rank-Sum (p= 0.049). The sub-
376 
category exploratory analysis of ADAS-cog revealed that the changes from baseline in the 
377 
commands component (p=0.001) and the word finding difficulty (p=0.033) were significant at 12 
378 
months.  
379 
 
An exploratory analysis of effect modification by sex suggests that males might have 
380 
been more responsive to benfotiamine, although none of the differences were statistically 
381 
significant. Furthermore, there was no effect modification by APOE ε4 allele carrier status. 
382 
Finally, no significant correlation occurred between blood thiamine, ThDP or ThMP values and 
383 
ADAS-cog. No significant interaction was found between MMSE score and ADAS-cog 
384 
response (p=0.122), but a post-hoc analysis suggested that benfotiamine had a stronger 
385 
response among those with a higher MMSE at baseline (MMSE >= 26 difference in change 
386 
ADAS-cog was significant (p=0.027) whereas this was not the case for MMSE<26 (p = 0.99).  
387 
 
CDR. Mean change in global CDR from baseline to 12 months was significantly 
388 
different between placebo and benfotiamine groups (p=0.034), favoring the benfotiamine group 
389 
(Figure 3). The difference in the placebo group was 0.22 whereas the change in the benfotiamine 
390 
group was 0.05, corresponding to a reduction of deterioration by 77%. The mean change in 
391 
CDR-SB from baseline to 12 months showed a difference at trend level between placebo and 
392 
benfotiamine groups (p=0.078). In an analysis of individual CDR subscores, the “home and 
393 
hobbies score” differed between groups (p=0.032) whereas other subscores did not differ.  
394 

 
20 
 
 
APOE ε4 status (Figure 3C), but not sex (Figure 3B), was associated with a differential 
395 
response to benfotiamine. The performance of males and females was not significantly different 
396 
(Figure 3B). The change from baseline in females 0.219 was nearly identical to that in males. 
397 
However, the non- APOE ε4 group seemed to respond much more than those with the ε4 allele 
398 
(Figure 3C). Indeed, the change from baseline was significant in the non- APOE ε4 group 
399 
(p=0.013) although only eleven participants were in this category. No significant interaction was 
400 
found by comparing patients that had MMSE values >=26 vs <26 (p=0.878).  
401 
 
The Buschke SRT (Figure 4). No significant change in the SRT (p=0.177) nor the 
402 
change in score (0.315) (Figure 4) occurred. Placebo treated participants showed a downward 
403 
trend while benfotiamine treated participants had stable scores. Trend analysis shows that the 
404 
non-APOE ε4 are the most responsive at 6 months (compared baseline p=0.028) and 12 months 
405 
(compared to baseline p=0.066).  
406 
 
NPI (Figure 5). No differences in change in NPI were observed with benfotiamine 
407 
treatment when the whole population was analyzed (Figure 5A). However, benfotiamine was 
408 
associated with significantly reduced scores in males at month 9 (0.014) and month 12 (p=0.035) 
409 
(Figure 5B). The effects of benfotiamine were not altered by APOE4 status (Figure 5C). 
410 
 
ADCS-ADL (Figure 6).  No significant differences were observed in ADCS-ADL. In the 
411 
sub-analysis of sex and APOE, a trend was observed that was consistent with a beneficial effect 
412 
of benfotiamine (Figure 6). 
413 
 
Response of thiamine, ThDP and ThMP to benfotiamine treatment (Table 3C; 
414 
Figures 7 and 8). 
415 

 
21 
 
 
The 161 fold increase in in blood thiamine indicated the administration of the drug was 
416 
successful. In the placebo group, small increases for the levels of thiamine (5.5 to 13.6; p= 
417 
0.044) and ThDP (74.5 to 91.7; p=0.044) occurred, but not ThMP (3.4 to 4.0; p=0.382) (Table 
418 
3C). After completion of the trial, it was discovered that one patient in the placebo group took 
419 
commercial benfotiamine during the trial. Consequentially, data from the patient was excluded 
420 
for all per-protocol analysis. The twelve-month treatment with benfotiamine significantly 
421 
elevated blood thiamine from 6.2 to 999 (161 fold) above baseline, ThDP (two fold) and ThMP 
422 
(five fold) (Table 3C). Although the differences were significant, the scatter grams revealed large 
423 
variations (Figure 7). These changes were apparent even though the timing between the taking 
424 
the last capsule and taking blood were not standardized. The much larger changes than expected 
425 
may be related to the duration of the treatment or the purity of the benfotiamine.  
426 
 
There was a trend for APOE ε4 and sex related differences in thiamine response to 
427 
benfotiamine but the differences were not significant (Figure 8). Thiamine levels after 
428 
benfotiamine were about two times higher in females than males. Thiamine values were 
429 
approximately 50% higher in APOE ε4 carriers than non- APOE ε4 carriers.  
430 
 
The concentrations of blood thiamine, ThDP, ThMP after benfotiamine treatment did not 
431 
correlate with ADAS-cog scores (p=0.736, 0.917, 0.500, respectively) nor CDR (p=0.762, 0.896, 
432 
0.767, respectively).  
433 
 
The response of advanced glycation end products (AGE) to benfotiamine treatment. 
434 
Benfotiamine inhibited the increase in AGE over the course of the disease and the effect was 
435 
more apparent in non- APOE ε4 patients (Figure 9).  
436 

 
22 
 
 
The response of FDGPET to benfotiamine treatment.  
437 
 
The comparison of regions of interest is presented in Table 3D, using the paracentral 
438 
lobule and cerebellum as the reference region. No significant differences were observed between 
439 
the benfotiamine and placebo populations in the pre-specified regions of interest.  
440 
 
The multivariate pattern derived through the regression against CDR correlated 
441 
negatively with CDR (p=0.002) (Figure 10B) across all participants and time points. Robust 
442 
positive loadings, i.e. with more than 97.5% of bootstrap loadings larger than zero, were found in 
443 
the right precuneus, inferior parietal and mid frontal cortex: higher relative signal in these areas 
444 
was associated with a better (=lower) CDR score. Robust negative loadings, i.e. with more than 
445 
97.5% of bootstrap loadings smaller than zero, were found in the bilateral paracentral lobules and 
446 
bilateral cerebellum: higher relative signal at these locations was associated with a higher 
447 
(=worse) CDR score. Pattern scores showed a significantly higher change from baseline to 12 
448 
months in treated than untreated participants (Figure 10A). However, a difference was observed 
449 
between placebo and treatment arm at baseline (T=2.1582, p=0.034) when APOE status was not 
450 
considered. Calculation of differences with sex was complicated by differences in the rates of the 
451 
two groups at baseline.  
452 
 
Stratification by APOE ε4 revealed that that the CDR-derived FDG PET pattern showed 
453 
a treatment effect at 12 months in APOE ε4 negative population (p=0.019) but not in APOE ε4 
454 
positive population (p=0.255) (Figure 10C). In the APOE positive population there was no 
455 
difference between treatment groups at baseline (p=0.164); in the APOE negative population, 
456 
pattern scores were higher at trend level (p=0.086) in the benfotiamine group. 
457 

 
23 
 
 
For 59 participants who completed follow-up, the longitudinal change in pattern score 
458 
(follow-up minus baseline) also correlated negatively with the accompanying change in CDR 
459 
score (R=-0.446, p<0.001). No difference in longitudinal change was observed between treated 
460 
and untreated participants (p=0.638). Additional analyses to adjust for any baseline differences 
461 
and to explore other baseline heterogeneity effects or comparison patterns were deferred for 
462 
subsequent evaluation. 
463 
 
464 
Discussion 
465 
 
The results show that benfotiamine administration in patients with aMCI and dementia 
466 
due to AD is safe and successful in increasing peripheral thiamine levels. The trial provides 
467 
preliminary evidence of efficacy of benfotiamine on cognitive and functional outcomes. In 
468 
aggregate, our results provide proof of principle that justify testing the efficacy of benfotiamine 
469 
in ameliorating cognitive and functional decline among participants with aMCI and dementia due 
470 
to AD in a trial with a larger sample size and study duration. Measures of blood thiamine (a 
471 
pharmacokinetic marker of drug delivery), FDGPET patterning (a CNS biomarker of synaptic 
472 
activity) and serum AGE (a peripheral biomarker of metabolic dysregulation) provided further 
473 
evidence of the effects of benfotiamine that could benefit cognition.  
474 
 
  
475 
 
The results support benfotiamine’s effectiveness which was reported in a preliminary 
476 
study of five patients without placebo control that was published after our trial was initiated [36]. 
477 
That study found that 300 mg daily of commercial benfotiamine over 18 months improved 
478 

 
24 
 
MMSE by three points in with greater severity of dementia (i.e., MMSE of 12-25) than our 
479 
patient population (MMSE>21). Levels of blood thiamine and thiamine esters were not 
480 
reported in the previous study and too few patients were reported to examine sex or APOE 
481 
effects. 
482 
 
The large increases in whole blood thiamine, ThDP and ThMP provided a robust 
483 
indication that oral tablets effectively delivered the treatment. Indeed, the 161-fold increase 
484 
serum thiamine was more robust than predicted, but the variation was large. The large increase in 
485 
thiamine with relatively small increases in ThDP (twofold) and ThMP (fivefold) was also 
486 
reported following benfotiamine in mouse brain [30]. Appreciable differences in thiamine levels 
487 
were observed by sex (two-fold) and APOE-epsilon4 carrier status (three-fold) following 
488 
treatment, but these observations need to be replicated in a larger sample.  
489 
 
The ability of blood thiamine or its esters to predict AD at baseline that was suggested by 
490 
other trials [67-70] was not evident in our patients. Unlike previous studies which included more 
491 
severe patients [68, 70], blood ThDP did not correlate significantly with MMSE (0.664), CDR 
492 
(0.618) or ADAS-cog (0.883) at baseline or following benfotiamine. Thus, the baseline studies 
493 
are not supportive of a critical role of blood ThDP in AD. Our studies do support the finding that 
494 
ThDP is lower in females than males [67, 70]. These results suggest that thiamine, ThDP and 
495 
ThMP should be tested in any subsequent study, and additional aspects of thiamine homeostasis 
496 
such as cellular localization or ThDP effect on transketolase should be tested as well. At 
497 
minimum, the blood measures provide a measure of drug delivery. 
498 
The significant correlation of MMSE and the normalized FDG PET at screening, as well 
499 
as the correlation between CDR and the derived multivariate pattern, are consistent with the 
500 

 
25 
 
well-documented tight relation of glucose metabolism to AD. Several factors may have 
501 
contributed to the lack of FDG PET treatment effect findings despite the large measured changes 
502 
in blood thiamine and observed differences in ADAS-cog changes. These include baseline 
503 
heterogeneity in regional hypometabolism, the number of participants having both initial and 
504 
post-treatment scans, use of CDR as the sole target outcome for the progression pattern, and the 
505 
very small longitudinal changes that occur in FDG PET over 12 months in this mild population. 
506 
Next steps include alternate apriori and data driven pattern-based analyses to further understand 
507 
these relationships. As other potential considerations, the positive effects of 
508 
benfotiamine/thiamine, including improved cognition, in neurodegeneration occur with minimal 
509 
change in ThDP [33] [30, 31]. Thus, benfotiamine/thiamine could be acting at steps of glucose 
510 
metabolism that do not change brain glucose uptake or by one of thiamine/benfotiamine’s 
511 
actions not directly linked to metabolism. Thiamine also regulates activities of enzyme like 
512 
malate dehydrogenase and glutamate dehydrogenase [71]. Thiamine can act as an antioxidant 
513 
[13] [19, 26, 72, 73] and may act directly in cholinergic transmission [74]. Thiamine serves as an 
514 
allosteric regulator of many proteins [73]. Benfotiamine and thiamin may act as Nrf2 activators 
515 
[30], which would help the brain deal with many oxidative insults. Finally, 
516 
benfotiamine/thiamine could be acting on endothelial cells as has been demonstrated in studies 
517 
of diabetes [20, 21, 37].  
518 
 
The CDR, FDGPET data, and AGE response to benfotiamine suggest that AD patients 
519 
without APOE ε4 were more responsive to benfotiamine in this study population. The 
520 
diminished response did not seem to be a difference in drug availability since blood thiamine 
521 
(+46%), and its esters were all higher in patients with APOE ε4 following benfotiamine (not 
522 
statistically significant). Patients with APOE ε4 may have a more severe form of the disease 
523 

 
26 
 
since they have more plaques and they occur earlier [75-77]. APOE ε4 carriers have higher 
524 
levels of the glyoxal, fluorescent AGEs, Nε-carboxymethyllysine, and the receptor for AGE 
525 
(sRAGE) (p= 0.018) when compared to non-carriers [78] .  
526 
 
 The role of AGE in AD as a biomarker and progression of disease is not well developed. 
527 
Recent studies demonstrate that the development of AGE parallels the development of the 
528 
cognitive deficit [11]. The AGE pentosidine is an indicator of Alzheimer’s disease [79]. 
529 
Methylglyoxal and glyoxal levels in serum are higher in MCI patients. Methylglyoxal in serum 
530 
distinguishes MCI from controls but not from AD. Meanwhile, serum glyoxal levels differentiate 
531 
MCI from control and AD groups [35]. The levels of carboxymethylysine in serum correlate 
532 
negatively with the clinical cognitive as measured by MMSE [34]. AGE increase in healthy 
533 
APOE ε4 and this may provide a link between APOE ε4 and AGE and our responses [78]. Both 
534 
sex and APOE status alter the AD serum metabolome [80]. In animals, even mild thiamine 
535 
deficiency leads to increases in AGE [81]. Increased AGE are common in diabetes, which 
536 
predisposes to the development of AD, and there are many intriguing overlaps between diabetes 
537 
and AD [18]. Benfotiamine prevents the micro and macro vascular damage in diabetes related to 
538 
AGE [20, 37, 82]. The mechanisms for the protection have been studied extensively [83].  
539 
 
Our study has several limitations. Our sample size, while appropriate for a pilot study, 
540 
was relatively small and of short duration, which particularly affected our subgroup analyses. 
541 
Some significant findings in the secondary endpoints, subgroup analyses and multiple PET-
542 
related scores could be due to chance in the context of multiple comparisons without p-value 
543 
correction.  However, we believe that this approach is appropriate in the setting of a pilot study 
544 
and inform the proposal of a larger confirmatory clinical trial. It is also important to point out 
545 

 
27 
 
that the observed effects for primary and secondary outcomes were consistently in the direction 
546 
of benefit for benfotiamine. Another potential limitation is our definition of AD. Our study 
547 
participants had aMCI and dementia that met the criterion for the Alzheimer’s continuum in 
548 
the NIA/AA research framework [38], which we ascertained through amyloid positivity on 
549 
PET scans. However, we cannot say with certainty that amyloid was the primary pathology 
550 
causing cognitive impairment, as other pathologies that we did not ascertain could have caused 
551 
the cognitive impairment. Lastly, the lack of ethnic and racial diversity is also of concern, and a 
552 
larger trial must aim to recruit a sample with representation of all ethnic and racial groups.  
553 
In summary, benfotiamine is safe and cost effective, and the results of this pilot study are 
554 
encouraging, providing preliminary evidence of efficacy. Our next step is to propose a larger 
555 
clinical trial appropriately powered to replicate our findings.  We believe that further studies 
556 
would be very valuable to determine whether benfotiamine may be helpful in delaying onset or 
557 
treating AD.  
558 
Acknowledgements. 
559 
We thank the patients and caregivers for their time and commitment. We also thank the 
560 
following:  Alzheimer Drug Discovery Foundation, NIA R01AG043679 (GEG), Burke 
561 
Rehabilitation Hospital, Burke Neurological Institute, NIA R56AG062305 (SAF). NIH 
562 
K24AG045334 (JAL) for funding, Advanced Orthomolecular Medicine for providing the 
563 
benfotiamine (Dr. Traj Nibber), Dawn C. Matthews, M.S., M.B.A., ADM Diagnostics, 
564 
Northbrook, IL, USA for help interpreting the imaging, Dr. Michael Reding, Dr. Mary Beth 
565 
Walsh and Marina Harmon (Burke Rehabilitation Hospital) for their help. Sarah Mink and 
566 
Tiffany Moy of the Burke Neurological Institut for their help with patient care. 
567 

 
28 
 
Research reported in this publication was supported by the National Center For Advancing 
568 
Translational Sciences of the National Institutes of Health under Award Number 
569 
UL1TR000457. The content is solely the responsibility of the authors and does not 
570 
necessarily represent the official views of the National Institutes of Health”. Sonia Sequeir 
571 
from this center was critical in preparing the IND. Lucien Bettendorff is Research Director of 
572 
the Fund for Scientific Research (FNRS, Belgium, 
573 
 
574 
Conflicts of Interests/Disclosure Statement. None of the authors has any conflicts to declare. 
575 
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843 
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845 
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846 
Benfotiamine blocks three major pathways of hyperglycemic damage and prevents 
847 
experimental diabetic retinopathy. Nat Med 9, 294-299. 
848 
 
 
 
849 
 
 
850 

 
34 
 
 
851 
Table 1. Selection criteria for the patients 
852 
Inclusion criteria. Each patient met the following criteria:  
853 
• 
Subjects who are able and willing to provide informed consent. 
854 
• 
Male and non-pregnant, non-lactating, postmenopausal, or surgically sterilized female 
855 
subjects at least 60 years of age or older.  
856 
• 
Clinical diagnosis of amnestic MCI by the Peterson criteria or probable AD dementia 
857 
according to the National Institute of Neurological Disorders and stroke and the 
858 
Alzheimer’s Disease related Disorders Association (NINCDS/ADRDA) 
859 
• 
 MMSE score >21, CDR score >0.5 and <1 Cornell Scale for Depression in Dementia 
860 
(CSDD) score <10.  
861 
• 
Ambulatory or ambulatory with aide 
862 
• 
Has a caregiver willing to accompany the patient to each visit, accept responsibility for 
863 
supervising treatment and provided input to clinical outcome assessments  
864 
• 
Reside at home 
865 
• 
Speak English  
866 
• 
Amyloid positive PET-scan  
867 
• 
Patients taking FDA approved medications for the treatment of Alzheimer’s Disease 
868 
[e.g.,donepezil (Aricept), galantamine (Razadyne), rivastigmine (Exelon), or memantine 
869 
(Namenda)] for three months prior to baseline.  Patients not on these medications did 
870 
not initiate them during the study. 
871 
Exclusion criteria  
872 
• 
Significant neurological disorder other than AD including hypoxia, stroke, traumatic brain 
873 
injury  
874 
• 
A current psychiatric disorder according the DSM-IV diagnosis of major depression 
875 
unless successfully treated on a stable dose of an antidepressant for at least 4 weeks 
876 
and continues on stable dose throughout the study  
877 
• 
Any other DSM-IV Axis l diagnosis including other primary neurodegenerative dementia, 
878 
schizophrenia or bipolar depression  
879 
• 
A current diagnosis of uncontrolled Type I or Type II diabetes mellitus [Hemoglobin A1 C 
880 
(Hb A1C <8]. Patients with uncontrolled diabetes (i.e.,  if glucose values exceed 200 
881 
mg/ml.  
882 
• 
A current diagnosis of active, uncontrolled seizure disorder.  
883 
• 
A current diagnosis of probable or possible vascular dementia according to NINDS-
884 
AIREN.  
885 
• 
An investigational drug during the previous 4 weeks.  
886 
• 
Any previous exposure to Benfotiamine.  
887 
• 
A current diagnosis of severe unstable cardiovascular disease.  
888 
• 
A current diagnosis of acute severe, or unstable asthmatic condition (e.g., severe 
889 
chronic obstructive pulmonary disease (COPD),  
890 
• 
A current diagnosis of cardiac, renal or hepatic disease.  
891 
• 
A current diagnosis of cancer including any active treatment.  
892 
• 
History of alcoholism, current or within past 5 years.  
893 
• 
A disability that may prevent the patient from completing all study requirements (e.g., 
894 
blindness, deafness, severe language difficulty).  
895 

 
35 
 
 Table 2. Baseline comparison between benfotiamine (n = 34) and 
placebo (n = 36) 
 
Table 2A. Baseline demographic characteristics. 
 
Total 
Placebo        Benfotiamine 
P value 
 
 
 
  
Age 
 
 
 
 
T 
   Mean(SD) 
75.77 (7.01) 
75.81 (7.19) 
75.74 (6.91) 
0.967  
 
Gender 
 
 
 
 
F 
   Female 
41 (58.6) 
18 (50.0) 
23 (67.6) 
0.153  
   Male 
29 (41.4) 
18 (50.0) 
11 (32.4) 
  
 
Race 
 
 
 
 
F 
   Black 
2 (2.9) 
1 (2.8) 
1 (2.9) 
1.000  
   White 
68 (97.1) 
35 (97.2) 
33 (97.1) 
  
 
Ethnicity 
 
 
 
 
F 
   Hispanic/Latino 
4 (5.7) 
4 (11.1) 
0 (0.0) 
0.115  
   Not Hispanic/Latino 
66 (94.3) 
32 (88.9) 
34 (100) 
  
 
MMSE total 
 
 
 
 
T 
   Mean(SD) 
25.33 (2.63) 
25.33 (2.52) 
25.32 (2.78) 
0.988  
 
Dichotomized MMSE 
 
 
 
 
F 
   < 26 
34 (48.6) 
18 (50.0) 
16 (47.1) 
0.816  
   >= 26 
36 (51.4) 
18 (50.0) 
18 (52.9) 
  
 
APOE genotype 
 
 
 
 
F 
   '2, 3' 
4 (5.7) 
2 (5.6) 
2 (5.9) 
0.883  
   '2, 4' 
1 (1.4) 
0 (0.0) 
1 (2.9) 
  
   '3, 3' 
24 (34.3) 
14 (38.9) 
10 (29.4) 
  
   '3, 4' 
34 (48.6) 
17 (47.2) 
17 (50.0) 
  
   '4, 4' 
7 (10.0) 
3 (8.3) 
4 (11.8) 
  
T: t-test (with equal variances); “Fisher’s extact t” test 
 
 
 
896 
 
 
897 

 
36 
 
Table 2B. Baseline neuropsychological outcome measures  
 
 
 
 
 
Total 
Placebo               Benfotiamine 
P value 
 
 
 
 
 
ADAS total score 
15.34 (6.36) 
15.50 (6.61) 
15.19 (6.16) 
0.835 t 
  (ITT) 
 
 
 
 
 
 
 
 
 
ADAS total score 
15.34 (6.40) 
15.48 (6.70) 
15.19 (6.16) 
0.849 t 
  (Per protocol) 
 
 
 
 
 
 
 
 
 
CDR score    
0.50 (0.50 - 1.00) 0.50 (0.50 - 1.00) 0.50 (0.50 - 1.00) 0.334 w 
Median(range) 
 
 
 
 
 
 
 
 
 
ADCS-ADL total score 
47.44 (4.29) 
47.42 (4.65) 
47.47 (3.95) 
0.959 t 
 
 
 
 
 
NPI 
13.50 (10.44) 
11.03 (10.15) 
16.12 (10.23) 
0.040 t 
 
 
 
 
 
Buschke score 
27.09 (9.74) 
26.03 (9.01) 
28.21 (10.49) 
0.354t  
 
 
 
 
 
Values are Mean (SD). T denotes t-test (with equal variances).  W Wilcoxon rank sum test 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
898 
 
 
899 

 
37 
 
Table 2C.   Baseline Thiamine, ThDP and ThMP . 
900 
 
Total 
Placebo      Benfotiamine 
P Value 
 
 
 
 
 
 
Thiamine 
 
 
 
 
 
   Mean(SD) 
5.72 (11.31) 
5.26 (4.50) 
6.20 (15.56) 
0.735 
 
 
Thiamine diphosphate 
 
 
 
 
 
   Mean(SD) 
69.71 (19.40) 
74.46 (20.21) 
64.82 (17.50) 0.038  
 
Thiamine monophosphate 
 
 
 
 
 
   Mean(SD) 
3.21 (1.72) 
3.46 (1.83) 
2.97 (1.59) 0.250  
 
 
 
 
  
Comparisons were by t-test (with equal variances);   
901 
 
902 
 
 
903 

 
38 
 
All values were normalized to the cerebellum as described in methods. All values are mean 
904 
(SD). All comparison were by the t-test (equal variances). 
905 
 
906 
 
 
907 
Table 2D. Baseline comparison of FDGPET  
 
 
 
 
  
 
 
 
 
  
 
Total 
Placebo 
Benfotiamine 
P value 
Posterior cingulate 
 
 
 
 
 
   Left      
0.85 (0.09) 
0.83 (0.10) 
0.87 (0.08) 0.087  
  Right 
0.81 (0.07) 
0.79 (0.07) 
0.82 (0.06) 0.069  
 
Percuneus 
 
 
 
 
 
   Left  
1.09 (0.09) 
1.08 (0.10) 
1.10 (0.08) 0.332  
   Right  
1.09 (0.10) 
1.08 (0.11) 
1.11 (0.09) 0.287  
 
Medial temporal 
 
 
 
 
 
   Left   
0.97 (0.11) 
0.94 (0.11) 
1.00 (0.10) 0.022  
   Right 
1.01 (0.12) 
0.98 (0.12) 
1.04 (0.10) 0.020  
 
Frontal cortex 
 
 
 
 
 
   Left  
0.99 (0.09) 
0.98 (0.09) 
0.99 (0.09) 0.480  
   Right 
1.01 (0.09) 
1.00 (0.09) 
1.03 (0.08) 0.268  
 
 Hippocampus 
 
 
 
 
 
   Left   
0.74 (0.08) 
0.74 (0.08) 
0.75 (0.08) 0.938  
   Right  
0.76 (0.09) 
0.75 (0.10) 
0.76 (0.08) 0.764  
 
Entorhinal_cortex 
 
 
 
 
 
   Left  
0.88 (0.13) 
0.88 (0.14) 
0.87 (0.13) 0.693  
   Right  
0.89 (0.18) 
0.87 (0.21) 
0.90 (0.14) 0.477  
 
Prefrontal_cortex 
 
 
 
 
 
   Left  
0.82 (0.09) 
0.81 (0.09) 
0.83 (0.08) 0.417  
   Right  
0.87 (0.09) 
0.86 (0.10) 
0.88 (0.08) 0.293  
 
Whole brain 
0.88 (0.05) 
0.87 (0.06) 
0.89 (0.05) 0.122  
    
 
 
 
  

 
39 
 
Table 3.  Consequences a 12 month treatment with benfotiamine. 
908 
Table 3A. Benfotiamine did not cause any adverse events. 
909 
SYMPTOM 
PLACEBO (n = 36) 
TREATMENT (n = 34) 
Anxiety 
4 (11%) 
5 (14%) 
Bruise 
5 (14%) 
2 (6%) 
Cold symptoms 
3  (8%) 
3 (8%) 
Depression 
                2 (6%) 
1 (3%) 
Dizziness 
3 (8%) 
3 (8%) 
Fall 
12 (34%) 
6 (17%) 
Head injury 
2 (6%) 
0 (0%) 
Heart arrhythmia 
2 (6%) 
1 (3%) 
Pain 
4 (11%) 
5 (14%) 
Pneumonia 
3 (8%) 
0 (0%) 
Sprain 
       2 (6%) 
        0 (0%) 
Surgery 
       3 (8%) 
       1 (3%) 
Allergy 
      2 (2%) 
        0 (0%) 
Gastrointestinal problem 
             12 (34%) 
         9 (26%) 
Stroke 
     0 (0%) 
       2 (6%) 
Total 
59 
38 
 
910 
 
 
911 

 
40 
 
Table 3B.  Changes in ADAS-cog following twelve month treatment 
912 
with placebo or benfotiamine (ITT and per-protocol analysis) 
913 
  
914 
[Unadjusted comparison of the changes form baseline to month_12 in ADAS  
score between intervention and control (ITT data after LOCF imputation)] 
 
Variable 
Total 
Placebo  Benfotiamine 
P 1 
 
ADAS score 
change 
 
 
 
 
 
   Mean(SD) 
2.37 (5.61) 3.26 (5.52) 
1.39 (5.63) 0.162 [T] 
 
Repeated measures ANOVA P-value: 0.5626 
Mixed effect model P value: 0.0708 
GEE P value: 0.1373 
Wilcoxon Rank sum P-value: 0.0980 
1 P values obtained from the statistical tests: [T] t-test (equal variances) 
 
Table 3B. Unadjusted comparison of the baseline to month_12  in ADAS  
score between benfotiamine and control (Per-protocol) 
Variable 
Total 
Placebo  Benfotiamine 
P 1 
 
ADAS score 
change 
 
 
 
 
 
   Mean(SD) 
2.10 (5.59) 
3.2 (5.66) 
0.96 (5.41) 0.125 [T] 
 
Repeated measures ANOVA P-value: 0.355 
Mixed effect model P-value: 0.056 
GEE P-value: 0.107 
Wilcoxon Rank sum P-value :0.069 
  1 P values obtained from the statistical tests:  [T] - T-test (equal variances); 
 
 
915 
 
 
916 

 
41 
 
Table 3C.   Changes in Thiamine, ThDP and ThMP after 12 months of 
917 
placebo (n=36) or benfotiamine (n=34). 
918 
 
919 
 Changes in thiamine and its esters after 12 months of placebo 
920 
  
Baseline  
12 months  
P value 
Thiamine 
5.48±0.77 
13.64±4.06 
0.044 
Thiamine diphosphate 
74.46±3.42 
91.70±7.94 
0.044 
Thiamine monophosphate 
3.38±0.31 
4.05±0.73 
0.382 
 
921 
Changes in thiamine and its esters after 12 months of benfotiamine 
922 
  
Baseline  
12 Months 
P value 
Thiamine 
6.20±2.67 
999.51 ± 147.4 
< 0.001 
Thiamine diphosphate 
64.82±3.00 
197.39  ± 17.75 
< 0.001 
Thiamine monophosphate 
2.97±0.27 
20.73 ± 2.16 
< 0.001 
Comparisons were by t-test (with equal variances);   
923 
 
924 
 
 
925 

 
42 
 
 
926 
 
927 
Table 3D.  Comparison of  the Month_12 - Baseline change in 
FDGPET between benfotiamine (n = 34) and placebo (n = 36) 
 
 
 
 
  
 
Total 
Placebo 
Benfotiamine P value 
Posterior cingulate 
 
 
 
 
 
   Left      
-0.03 (0.03) 
-0.03 (0.04) 
-0.02 (0.03) 0.629  
  Right 
-0.02 (0.03) 
-0.02 (0.03) 
-0.02 (0.03) 0.742  
 
Parietal 
 
 
 
 
 
   Left  
-0.02 (0.04) 
-0.03 (0.04) 
-0.02 (0.05) 0.448  
   Right  
-0.03 (0.04) 
-0.03 (0.04) 
-0.02 (0.04) 0.323  
 
Percuneus 
 
 
 
 
 
   Left  
-0.03 (0.04) 
-0.02 (0.04) 
-0.03 (0.04) 0.719  
   Right  
-0.03 (0.04) 
-0.03 (0.04) 
-0.03 (0.04) 0.722  
 
Medial temporal 
 
 
 
 
 
   Left   
-0.03 (0.04) 
-0.03 (0.04) 
-0.03 (0.04) 0.956  
   Right 
-0.03 (0.05) 
-0.03 (0.05) 
-0.03 (0.05) 0.748  
 
Frontal cortex 
 
 
 
 
 
   Left  
-0.02 (0.04) 
-0.02 (0.04) 
-0.03 (0.04) 0.646  
   Right 
-0.02 (0.04) 
-0.02 (0.04) 
-0.03 (0.04) 0.616  
 
 Hippocampus 
 
 
 
 
 
   Left   
-0.02 (0.04) 
-0.02 (0.05) 
-0.01 (0.04) 0.451  
   Right  
-0.02 (0.04) 
-0.02 (0.05) 
-0.02 (0.04) 0.503  
 
Entorhinal_cortex 
 
 
 
 
 
   Left  
-0.02 (0.11) 
-0.01 (0.10) 
-0.02 (0.11) 0.774  
   Right  
-0.02 (0.09) 
-0.01 (0.08) 
-0.02 (0.10) 0.502  
 
Prefrontal_cortex 
 
 
 
 
 
   Left  
-0.02 (0.04) 
-0.02 (0.04) 
-0.02 (0.04) 0.742  
   Right  
-0.02 (0.04) 
-0.02 (0.04) 
-0.03 (0.04) 0.559  
 
 
 
 
  
Whole_brain 
-0.01 (0.02) 
-0.02 (0.02) 
-0.01 (0.02) 0.753  
    
 
 
 
  

 
43 
 

 
44 
 

 
45 
 
930 

 
46 
 
931 

 
47 
 
932 

 
48 
 
933 

 
49 
 
934 

 
50 
 
935 

 
51 
 
936 

 
52 
 
937 

 
53 
 
938 

 
54 
 
 
939 
 
 
940 

 
55 
 
Figure Legends 
941 
Figure 1. Summary of the treatment protocol for the one year trial. 
942 
Figure 2. Changes in ADAS cog with benfotiamine treatment compared to controls.  
943 
See Table 7 for statistical comparisons.  
944 
Figure 3. Benfotiamine treatment and the CDR.  CDR Placebo = 34, benfotiamine = 29. On the 
945 
figure *** indicates significantly different (p= 0.034) (A). When the groups are also separated by 
946 
sex, large but non-significant differences occur (B). When the groups are separated by APOE4 
947 
only the non-APOE ε4 allele group differs. In the non-APOE4 group the *** indicates values 
948 
significantly different (p=0.013) (C). The APOE4 denotes at least one e4 allele. P values here are 
949 
when there are subgroups are all obtained from subgroup analysis, not interaction from ANOVA 
950 
(C).  
951 
Figure 4. Benfotiamine and the Buschke Selective Reminding Test (SRT) 
952 
Figure 5. Benfotiamine and the Neuropsychiatric Inventory (NPI). No differences were seen in 
953 
the overall scores (Figure 5A). However, separation of the groups by sex revealed a highly 
954 
significant benefit in males but not females. *** indicates p=0.035 (Figure 5B). No significant 
955 
difference was seen with APO ε4 alleles (Figure 5C). 
956 
Figure 6. Alzheimer’s Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) 
957 
Figure 7. Blood thiamine, ThMP, and ThDP concentrations at baseline and month 12.  Each dot 
958 
represents a different patient.  The bar represents the mean value. All values are per protocol 
959 

 
56 
 
after omitting a patient designated as placebo who was taking benfotiamine from another source.  
960 
Figure 8. Relation of sex and APOE ε4 genotype to thiamine, ThDP and ThMP.  Values are 
961 
means ± SEM.  *** Denotes significantly different (p<0.0001) by t-test.  
962 
Figure 9. Advanced Glycation End Products (AGE) after benfotiamine treatment. These were 
963 
done as an exploratory analysis. They were measured on serum and several samples were 
964 
contaminated with RBC. In the left panel, the n’s are 12 placebo and 13 benfotiamine patients. 
965 
The asterisk indicates p=0.043. In the right panel, in the APO ε4 group the n=6. In the non-
966 
APOE4 group n=7.  The APO ε4 denotes at least one e4 allele. 
967 
Figure 10A. Pattern score as function of the 12 month treatment period.  The pattern is a linear 
968 
combination of the first 2 Principal Components whose pattern score is slightly but significantly 
969 
higher for treatment than untreated participants at time point 12 months.  
970 
Figure 10B. The left panel shows the pattern score plotted against CDR status (p-level obtained 
971 
from whole-model F-test.)  A higher pattern score implies lower CDR status. The right panel 
972 
shows loading distributions from a bootstrap test with 90% coverage intervals. We stress that 
973 
these loadings sizes and signs are relative since we removed the whole-brain mean from the 
974 
analysis prior to the pattern derivation. Thus, high positive loadings are found in the right Mid 
975 
temporal and inferior parietal cortex, implying relatively higher signal in participants with lower 
976 
CDR.  Bilateral cerebellum and paracentral lobule on the other hand, had relatively lower signal 
977 
in participants with lower CDR.  
978 
Figure 10C.  Stratification of the pattern score by APOE status reveals that APOE4 negative 
979 
patients show the greatest response. APOE ε4=0 patients show a treatment effect (left panel), 
980 

 
57 
 
APOE ε4=1 do not (right panel). 
981 
 
982