Clinical trial NCT02380573
Conditions: Mild Cognitive Impairment MCI Aging Alzheimer's Disease AD
Keywords: methylene blue fMRI
Sponsor: The University of Texas Health Science Center at San Antonio
Start date: 2015-07-01; Completion date: 2023-07-01
Enrollment: 117 participants; Allocation: RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
A double-blind, placebo-controlled study that aims to investigate the effect of 2-week and 12-week administration of USP methylene blue (MB) on cerebral blood flow, functional connectivity, memory and attention cognitive abilities using fMRI and behavioral measures in healthy aging, mild cognitive impairment (MCI) and mild Alzheimer's disease (AD) subjects.

Detailed description:
Healthy aging and aging human subjects with mild cognitive impairment and mild Alzheimer's disease from the TARCC cohort and South Texas will be studied using a double-blind, placebo-controlled design. After informed consent and familiarity with the tasks and the MRI environment, the subject will enter an MRI scanner and perform the following 6 tasks.

fMRI and behavioral data will be collected simultaneously while inside the scanner.

Delayed match-to-sample task: The subject views a pattern for a few seconds and then is prompted to recall the memorized pattern using a response system (approx. 10 mins).

Face-name task: The subject is shown blocks of stimuli where a novel or familiar face is paired with a name. In a later run, the subjects are asked whether the correct name is matched with the correct face. (approx. 10 mins).

Psychomotor vigilance task: The subject receives a visual cue that alerts them to press a button as fast as possible. (approx. 10 mins).

Cerebral Blood Flow and Resting State fMRI: Subject scanned with eyes closed and told to not think about a particular topic, each lasting about 10 minutes.

fMRI data acquisition: fMRI and neuropsychological battery measurements will be made before the intervention. These measurements will then be repeated after 2 weeks and 12 weeks.

fMRI will image changes in regional brain activity associated with these tasks. The MRI pulse sequences include diffusion tensor imaging, standard and non-invasive anatomical and quantitative MRI for coregistration and blood-oxygen-level dependent (BOLD) fMRI.

CO2 challenge: Cerebral blood flow measurements will be obtained while the subject rests in the scanner after administration of medical-grade 5% CO2 in air for 3-5 minutes. This will be repeated on weeks 2 and 12.

Data analysis: Standard fMRI analysis will be analyzed using established fMRI software. Statistical parametric analysis will be performed to generate activation maps. fMRI data will be corrected for multiple comparisons using a false discovery rate (q \< 0.05) and threshold for cluster values to conservatively control for type I error. Behavioral data will be analyzed with paired t-test and ANOVA calculations used for group comparison with p \< 0.05 (with Bonferroni correction) considered statistically significant.

Expected results: The investigators predict that, compared to placebo, MB will: i) improve working memory retention in a delayed match-to-sample task by memory performance and enhanced fMRI responses in the prefrontal cortex and parietal lobes, ii) improve episodic memory as determined by fMRI activation in the hippocampus, medial temporal lobes and prefrontal cortex iii) reduce reaction time in a psychomotor vigilance test and enhance fMRI responses within a cortical sustained attention network iv) improve CBF and v) improve fMRI connectivity in default mode and visuospatial and memory networks/subnetworks. The fMRI and behavioral performance effects on memory will be greater in the MCI and mild AD groups than in the healthy aging group. The effects will be greater in the MCI and AD groups than in the control groups.

Power analysis: Sample sizes were calculated using an fMRI power tool based on pilot data from the current study for a power of 80%, alpha = 0.05, False Discovery Rate \< 0.05, to detect statistical difference between MB and placebo23. The investigators estimate they will need 20-25 subjects per arm of group (complete studies) and thus will recruit 200-240 subjects to account for potential failed studies.

Arms: arm 1: Methylene Blue (USP grade, 282mg oral, daily, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks) arm 2: Drug: FD\&C Blue # 2 (USP grade, 282 mg oral, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks) arm 3: Methylene Blue (USP grade, 282 mg oral, daily, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks) arm 4: Drug: FD\&C Blue # 2 (USP grade, 282 mg oral, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks) arm 5: Methylene Blue (USP grade, 282 mg oral, daily, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks) arm 6: Drug: FD\&C Blue # 2 (USP grade, 282 mg oral, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks) arm 7: Methylene Blue (USP grade, 282 mg oral, daily, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks) arm 8: Drug: FD\&C Blue # 2 (USP grade, 282 mg oral, 2 weeks, 12 weeks) Phenazopyridine hydrochloride (97.5 mg oral, 2 weeks, 12 weeks)
Interventions: intervention 1: Methylene Blue intervention 2: FD&C Blue # 2 intervention 3: Phenazopyridine hydrochloride
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: None

Protocol documents:
Prot_SAP_000.pdf: 
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1 
COVER PAGE  
 
 
 
 
 
Official Study Title: Cognitive and Functional Connectivity Effects of Methylene 
Blue in Healthy Aging, Mild Cognitive Impairment and Alzheimer’s Disease 
 
NCT number: NCT02380573 
 
IRB Approval Date: 11-06-2015 
 
 
Unique Protocol ID: HSC20150410H 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Protocol Template Form 
 
Item 1 UTHSCSA 
Tracking Number 
Cognitive and Functional Connectivity Effects of Methylene Blue in Healthy 
Aging, Mild Cognitive Impairment and Alzheimer’s Disease  
 
Item 2 Abstract / Project Summary   
 
Provide a succinct and accurate description of the proposed 
research. State the purpose/aims. Describe concisely the research 
design and methods for achieving the stated goals. This section 
should be understandable to all members of the IRB, scientific and 
non-scientific.   
DO NOT EXCEED THE SPACE PROVIDED. 
Purpose/Objectives: The purpose of the current proposal is to test the hypothesis whether daily oral MB for two week period (and up 
to 12 weeks if subject decides to continue participation) will improve memory, attention, cognition and functional connectivity in 
healthy aging volunteers, mild cognitive impairment (MCI) and Alzheimer’s disease (AD) subjects by using objective MRI readouts 
and standard neuropsychological tests.    
Research Design/Plan: Adult volunteers will be studied using a randomized, double-blinded, placebo-controlled design.  MB and 
Placebo arms will be included for healthy aging, MCI and AD groups.  MB and placebo pills will be compounded into blue immediate 
release capsules.  Each arm will also receive a pill of Azo (phenazopyridine hydrochloride) with each daily dose as urinary tract 
analgesic and to maintain coloration of the urine in both groups. Subjects will have MRI scanning on two different days, fourteen 
days apart. After visit #2 is completed, the subject will be given the option to continue the study for another 10 weeks. 
Methods: This study will use a low-dose oral USP MB (282mg) daily over a two-week period and subjects will take drug daily for two 
weeks combined with an additional daily dose of 97.5mg of Azo.   Subjects will take 3 pills of MB/placebo each day (1 with each meal 
(94mg of MB per pill) and 1 pill of Azo for 14 days. 
Daily dose intake may be extended to 12 weeks after the initial follow up visit. 
Clinical Relevance: Middle-aged and elderly people with MCI are at high risk for developing Alzheimer’s disease, a condition that 
slowly destroys memory and thinking skills.  Approximately 5.2 million Americans are living with AD today and this number is 
predicted to triple by 2050.  There is a critical need for therapies that can arrest the progression from MCI to AD and other 
dementias, and for objective and sensitive measures of efficacy for interventions.   
 
 
Item 3 
Background 
 
Describe past experimental 
and/or clinical findings 
leading to the formulation 
of your study.   
For research involving 
unapproved drugs, describe 
animal and human studies.   
For research that involves 
approved drugs or devices, 
describe the FDA approved 
uses of this drug/device in 
relation to your protocol. 
Middle-aged and elderly people with mild cognitive impairment (MCI) are at high risk for 
developing Alzheimer’s disease (AD), a condition that slowly destroys memory and thinking skills. 
Approximately 5.2 million Americans are living with AD today and this number is predicted to triple 
by 20501.  There is a critical need for therapies that can arrest the progression from MCI to AD and 
other dementias, and for objective and sensitive measures of efficacy for interventions.  
USP 
methylene 
blue 
(MB) 
is 
a 
FDA-grandfathered 
drug 
safely 
used 
to 
treat 
methemoglobinemia, carbon dioxide and cyanide poisoning in humans2. Daily 4 mg/kg oral MB has been 
used safely for one year in clinical trials. Oral MB readily enters the brain and has unique energy-
enhancing and antioxidant properties and acts in the mitochondria to sustain or enhance ATP energy 
production, thereby promoting neuronal cell survival and cognitive enhancement 3. MB has recently 
been shown to reduce behavioral impairments in animal models of Parkinson's disease 4 and Alzheimer’s 
disease 5 6. Our laboratory has shown with magnetic resonance imaging (MRI) that low-dose MB 
(intravenous 1 mg/kg) increases glucose uptake, oxygen consumption, blood flow 7 and evoked 
responses 8 in the rat brain in vivo. We also found that MB treatment reduces MRI-defined lesion and 
behavioral deficits in animal models of traumatic brain injury 9 10 and stroke 11 12.  
As the first step to translate these experimental findings to humans, we are carrying out a 


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randomized, double-blinded, placebo-controlled, phase II clinical trial (NCT01836094) to evaluate the 
effects of oral USP MB (280 mg) on memory and cognition by using cerebral blood flow (CBF), evoked 
functional MRI (fMRI) and resting-state functional connectivity fMRI (fcMRI) on young (30 yo) healthy 
volunteers. Our findings suggest the MB has favorable cognitive and memory effects. The subjects, 28 
subjects to date, did not experience any negative adverse event.  
There are also a few MB clinical trials in mild to moderate AD and dementia (NCT01689246, 
NCT01689233, NCT01626378). An abstract of a phase II clinical trial reported that daily oral doses of 300 
mg non-reduced form of MB (methylthioninium chloride), under trade name “Rember” (TauRx 
Therapeutics, Inc.), slowed the progression of AD compared to placebo 13. Ongoing phase III clinical trials 
use a proprietary reduced form of methylene blue (leukomethylene blue, LMTXTM, TauRx) in mild and 
moderate AD and variant frontotemporal dementia. Our clinical trial differs from these clinical trials in 
several aspects: (1) TauRx phase II and phase III trials enrolled subjects with mild to severe Alzheimer’s 
disease and frontotemporal dementia (no MCI). Our study will be the first phase II clinical trial using MB 
in MCI, where early intervention is likely more effective. (2) TauRx phase II clinical trial used a large 
number of subjects with mild to moderate Alzheimer’s dementia and a larger dose up to 300 mg daily for 
one year with RemberTM (non-reduced form of MB). Our phase II trial will offer a markedly lower sample 
size, lower dose, and shorter duration, enabled by objective and powerful fMRI readouts. (3) The TauRx 
phase III clinical trial uses LMTXTM, a new reduced form of MB that is less well studied. Our phase II trial 
will use the non-proprietary non-reduced form of MB (methylthioninium chloride), which has been used 
in most preclinical studies and clinical trials. (4) Subjects will be recruited from among well-characterized 
participants and a large cohort in the Texas Alzheimer's Research and Care Consortium (TARCC). These 
subjects all have longitudinal psychometrics, and serum protein biomarkers on file, and frozen serum 
samples and DNA stored for future analyses. (4) The TauRx trials target to inhibit tau aggregation in 
neurofibrillary tangles of AD and demented patients, whereas we target to support healthy cognitive 
function by increasing mitochondrial oxidative energy metabolism and reducing oxidative stress in aging 
individuals before onset of AD neuropathology and dementia. 
Our proposed phase II trial is very similar to our current phase II trial (NCT01836094) except it 
now includes middle aged, MCI and Alzheimer’s disease subjects and daily MB dose for 2 to 12 weeks (as 
opposed to healthy subjects and single dose) as well as additional neuropsychological tests.  
Item 4 
Purpose and rationale 
Insert purpose, objectives 
and research 
questions/hypotheses here. 
 If you cut and paste from 
another document, make 
sure the excerpted material 
answers the question 
The purpose of the current IND proposal is to test the hypothesis whether daily oral MB for 2 to 
12 weeks will improve memory, attention, cognition and functional connectivity in healthy middle aged, 
and healthy elderly as well as MCI and AD subjects by using objective MRI readouts and standard 
neuropsychological tests. subjects by using objective MRI readouts and standard neuropsychological 
tests. MRI readouts include evoked functional MRI (fMRI), cerebral blood flow (CBF), and resting-state 
functional connectivity fMRI (fcMRI). We will utilize a double-blinded, placebo-controlled phase II clinical 
trial.  
MRI and neuropsychological tests will be administered over about 1.5 hours before oral MB, 
and again about 2 weeks after oral MB. The subject will have the option to continue for another 10 
weeks after the completion of the first two weeks. The primary outcome measures will include fMRI 
readouts to assess episodic memory, working memory and sustained attention, respectively, before and 
two weeks after MB. Secondary outcome measures will include standard neuropsychological tests. 
 
 
Item 5 
Study Population(s) Being Recruited 
 
In your recruitment plan, how many 
different populations of prospective 
subjects do you plan to target? 
Provide number: 4  
 
Identify the criteria for inclusion: 
 
 
 
Identify the criteria for exclusion: 


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4 
e.g., a population can be individuals 
with type 2 diabetes controlled with diet 
and/or a population of healthy controls.  
Or a population can be individuals 
attending an education program, etc. 
 
List each different population on a 
separate row and provide a short 
descriptive label:   
(e.g., normal-healthy, diabetics, 
parents, children, etc.) 
 
To add rows use copy & paste 
healthy middle aged 
45 – 64 years old 
All genders 
All minorities 
English, Spanish, or multilingual speakers 
 
Pregnancy or breastfeeding, Contraindication for 
MRI, such as Claustrophobia and magnetic metal 
implants, 
Glucose-6-phosphate 
deficiency, 
methemoglobinemia, Anemia, Allergy to MB, 
Color-blindness, 
Craniotomy, 
craniectomy 
or 
endovascular neurosurgery, A current diagnosis of 
stroke, transient ischemic attack (TIA), any 
primary neurodegenerative disorder, or any other 
causes of neuropsychologic disturbances or 
secondary dementia (MCI or AD does not exclude 
subject), A serious intercurrent illness likely to 
cause death within the next 5 years, such as 
terminal cancer, Alcohol and/or drug abuse, Any 
detection of an unknown disease process (eg. new 
tumor) on the study's neuroimaging at the 
discretion of the investigators, A systolic blood 
pressure ≥180 mmHg and/or a diastolic blood 
pressure ≥105 mmHg, Severe difficulty or an 
inability to perform any one of the 6 Katz 
Activities of Daily Living, Patients who are unlikely 
to comply with trial visit schedule or with trial 
medication, On any psychiatric serotonergic 
antidepressant 
medication 
or 
psychotropic 
medication within the last 5 weeks, Diagnosis of 
epilepsy, traumatic brain injury with loss of 
consciousness, psychosis, panic attacks, Chronic 
kidney disease or Cirrhosis, Liver or renal 
transplants. Known hypersensitivity to thiazide 
diuretics and phenothiazines, Any other condition, 
which in the opinion of the investigator, would put 
the participant at risk and warrant exclusion from 
the study 
 
healthy elderly 
65 – 89 years old 
All genders 
All minorities 
English, Spanish, or multilingual speakers 
 
Pregnancy or breastfeeding, Contraindication for 
MRI, such as Claustrophobia and magnetic metal 
implants, Glucose-6-phosphate deficiency, 
methemoglobinemia, Anemia, Allergy to MB, 
Color-blindness, Craniotomy, craniectomy or 
endovascular neurosurgery, A current diagnosis of 
stroke, transient ischemic attack (TIA), any 
primary neurodegenerative disorder, or any other 
causes of neuropsychologic disturbances or 
secondary dementia (MCI or AD does not exclude 
subject), A serious intercurrent illness likely to 
cause death within the next 5 years, such as 
terminal cancer, Alcohol and/or drug abuse, Any 
detection of an unknown disease process (eg. new 
tumor) on the study's neuroimaging at the 
discretion of the investigators, A systolic blood 


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pressure ≥180 mmHg and/or a diastolic blood 
pressure ≥105 mmHg, Severe difficulty or an 
inability to perform any one of the 6 Katz 
Activities of Daily Living, Patients who are unlikely 
to comply with trial visit schedule or with trial 
medication, On any psychiatric serotonergic 
antidepressant medication or psychotropic 
medication within the last 5 weeks, Diagnosis of 
epilepsy, traumatic brain injury with loss of 
consciousness, psychosis, panic attacks, Chronic 
kidney disease or Cirrhosis, Liver or renal 
transplants. Known hypersensitivity to thiazide 
diuretics and phenothiazines, Any other condition, 
which in the opinion of the investigator, would put 
the participant at risk and warrant exclusion from 
the study 
MCI 
45 – 89 years old 
All genders 
All minorities 
English, Spanish, or multilingual speakers 
 
Participants will meet the criteria for amnestic 
and non-amnestic (single or multiple domain) and 
mild AD such as those currently used by Texas 
Alzheimer's Research and Care Consortium 
(TARCC) consensus diagnosis. The AD subjects will 
only include early stage, late-onset, sporadic-type 
MB. 
 
 
Pregnancy or breastfeeding, Contraindication for 
MRI, such as Claustrophobia and magnetic metal 
implants, Glucose-6-phosphate deficiency, 
methemoglobinemia, Anemia, Allergy to MB, 
Color-blindness, Craniotomy, craniectomy or 
endovascular neurosurgery, A current diagnosis of 
stroke, transient ischemic attack (TIA), any 
primary neurodegenerative disorder, or any other 
causes of neuropsychologic disturbances or 
secondary dementia (MCI or AD does not exclude 
subject), A serious intercurrent illness likely to 
cause death within the next 5 years, such as 
terminal cancer, Alcohol and/or drug abuse, Any 
detection of an unknown disease process (eg. new 
tumor) on the study's neuroimaging at the 
discretion of the investigators, A systolic blood 
pressure ≥180 mmHg and/or a diastolic blood 
pressure ≥105 mmHg, Severe difficulty or an 
inability to perform any one of the 6 Katz 
Activities of Daily Living, Patients who are unlikely 
to comply with trial visit schedule or with trial 
medication, On any psychiatric serotonergic 
antidepressant medication or psychotropic 
medication within the last 5 weeks, Diagnosis of 
epilepsy, traumatic brain injury with loss of 
consciousness, psychosis, panic attacks, Chronic 
kidney disease or Cirrhosis, Liver or renal 
transplants. Known hypersensitivity to thiazide 
diuretics and phenothiazines, Any other condition, 
which in the opinion of the investigator, would put 
the participant at risk and warrant exclusion from 
the study 
AD (Alzheimer’s Early-stage, late-
onset, sporadic-type) 
65 – 89 years old 
All genders 
All minorities 
English, Spanish, or multilingual speakers 
 
Participants will meet the criteria for amnestic 
and non-amnestic (single or multiple domain) and 
mild AD such as those currently used by Texas 
Alzheimer's Research and Care Consortium 
(TARCC) consensus diagnosis. The AD subjects will 
only include early stage, late-onset, sporadic-type 
MB. 
 
 
Pregnancy or breastfeeding, Contraindication for 
MRI, such as Claustrophobia and magnetic metal 
implants, Glucose-6-phosphate deficiency, 
methemoglobinemia, Anemia, Allergy to MB, 
Color-blindness, Craniotomy, craniectomy or 
endovascular neurosurgery, A current diagnosis of 
stroke, transient ischemic attack (TIA), any 
primary neurodegenerative disorder, or any other 
causes of neuropsychologic disturbances or 
secondary dementia (MCI or AD does not exclude 
subject), A serious intercurrent illness likely to 
cause death within the next 5 years, such as 
terminal cancer, Alcohol and/or drug abuse, Any 
detection of an unknown disease process (eg. new 
tumor) on the study's neuroimaging at the 


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discretion of the investigators, A systolic blood 
pressure ≥180 mmHg and/or a diastolic blood 
pressure ≥105 mmHg, Severe difficulty or an 
inability to perform any one of the 6 Katz 
Activities of Daily Living, Patients who are unlikely 
to comply with trial visit schedule or with trial 
medication, On any psychiatric serotonergic 
antidepressant medication or psychotropic 
medication within the last 5 weeks, Diagnosis of 
epilepsy, traumatic brain injury with loss of 
consciousness, psychosis, panic attacks, Chronic 
kidney disease or Cirrhosis, Liver or renal 
transplants. Known hypersensitivity to thiazide 
diuretics and phenothiazines, Any other condition, 
which in the opinion of the investigator, would put 
the participant at risk and warrant exclusion from 
the study 
 
 
 
 
 
Item 6 
Research Plan / Description of the Research Methods a.  Provide a comprehensive narrative describing the research methods.   
Provide the plan for data analysis (include as applicable the sample size calculation). 
 
Step-by-Step Methods:   
Adult volunteers will be studied using a randomized, double-blinded, placebo-controlled 
design. Placebo arms will be included for healthy middle aged, healthy elderly, MCI and AD groups as 
controls. USP Methylene blue (MB) and placebo pills will be compounded into blue immediate release 
capsules. The subject will take 3 pills of MB or placebo (94mg per pill) three times a day with each 
meal. Placebo will be FD&C Blue # 2 which is a powder similar to the powder form of methylene blue. 
Each arm will also receive a pill of phenazopyridine hydrochloride (97.5 mg, marketed as Azo 
Standard) with each daily dosage. Phenazopyridine is a safe OTC product used for urinary discomfort 
that turns urine brownish. This drug will be given to both MB and placebo groups so that both show 
similar urine discoloration. This choice of administration is justified not only to maintain the blind 
procedure but because it would prevent any mild discomfort experienced by MB urinary excretion 
during chronic treatment. The subject, person administering the drug, and data analysts will not know 
whether the intervention is placebo or MB. The design will only be unblinded at the last steps when 
necessary. All subjects will be told that the pill they take may or may not cause urine/feces 
discoloration depending on individual’s metabolism.  
 
Step by Step: 
The healthy elderly, MCI and AD subjects will be recruited from the Texas Alzheimer’s Research and 
Care Consortium (TARCC). A neuropsychological battery of tests (eg. logical memory I, logical 
memory II, verbal fluency/COWA, Instrumental Activities of Daily Living (IADL), Global Depressive 
Scale (GDS), etc.) will be administered for all subjects before MRI study (2 month window).  
 
Baseline MRI scan will be performed during visit 1. 
 
Subject will be given labeled pill organizers with pills to take on days 1 through 14. Subject will be 
contacted via telephone on days 3, 7 and 11 (± 2 days) to document any adverse event or problem. 
After 2 weeks ± 3 days of MB (282 mg, oral x 14 days) or placebo, another neuropsychological tests 
and MRI scan will be obtained. On visit 2, the subject will be given the option to continue for another 
10 weeks. During visit 3, after 10 weeks ± 3 days, the subject will complete the subset of 
neuropsychological tests completed in visit 1, and some of the MRI scans completed in visit 1.  
 
For MRI, task-evoked BOLD fMRI, cerebral blood flow (CBF), resting-state fMRI, and CBF will be 
performed. For the task-evoked BOLD fMRI, subjects will perform the following scans while in the 


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scanner: 
Delayed match-to-sample memory task: The DMS task measures reaction time in terms of 
memory retrieval latency. The subject views a 6x6 pattern for 4 seconds. After six seconds, the 
subject is presented with two patterns and the subject presses the left or right button based on 
whether the picture on the left or the right is the same as the previous picture. The correct and 
incorrect answers are recorded. This exercise is repeated with 22 random patterns for 
approximately 10 mins18 19. 
 
Face-Name Task: The subject is shown blocks of stimuli where a novel or familiar face is 
paired with a name. In a later run, the subjects are asked whether the correct name is matched 
with the correct face. The correct and incorrect answers are recorded.  The acquisition of data 
is divided into encoding and recognition tasks. This exercise is repeated many times lasting 
about 10 minutes 20 21. 
 
Psychomotor vigilance task: The PVT measures attention and time-on-task effects22. The 
subject receives a visual cue that alerts them to press a button as fast as possible. The 
reaction time is recorded. A rest period follows for 4-8 seconds. This exercise is repeated 30 
times, lasting about 7-10 minutes. 
 
Subject behavioral data (ie. Correct responses, reaction time) will be recorded simultaneously while 
the MRI data is acquired by a computer. 
 
For CBF, the subject is instructed to close his or her eyes and relax in the scanner without falling 
asleep for 5-10 minutes.  
 
For resting-state fMRI, the subject is told to close eyes and not to think about a particular topic, lasting 
about 10 minutes. 
 
In addition, CBF will be measured during inhalation of medical-grade 5% CO2 in air for 3-5 minutes for 
calibration on all three visits. Standard anatomical MRI will also be acquired for co-registration. 
Diffusion tensor imaging (DTI) (5-7minutes) will also be acquired at one of the three visits. 
 
Intervention Groups: 
Arms 
Assigned Interventions 
Experimental: 
Healthy Middle 
Aged 
 
Drug: Methylene Blue (USP grade, 282 mg oral, up to 12 weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 12 weeks) 
Placebo: Healthy 
Middle Aged 
Drug: FD&C Blue # 2 (USP grade, 282 mg oral, up to 12 weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 2 weeks) 
Experimental: 
Healthy Elderly 
MB 
Drug: Methylene Blue (USP grade, 282 mg oral, once daily, up to 12 
weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 12 weeks) 
Placebo: Healthy 
Elderly MB 
Drug: FD&C Blue # 2 (USP grade, 282 mg oral, up to 12 weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 12 weeks) 
Experimental: 
MCI MB 
Drug: Methylene Blue (USP grade, 282 mg oral, once daily, up to 12 
weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 12 weeks) 
Placebo:  
MCI MB 
Drug: FD&C Blue # 2 (USP grade, 282 mg oral, up to 12 weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 12 weeks) 
Experimental: 
AD MB 
Drug: Methylene Blue (USP grade, 282 mg oral, up to 12 weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 12 weeks) 


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Placebo:  
AD MB 
Drug: FD&C Blue # 2 (USP grade, 282 mg oral, up to 12 weeks)  
Phenazopyridine hydrochloride (97.5 mg oral, up to 12 weeks) 
 
The daily 282mg of MB or place will be divided into three doses of 94mg each to minimize the 
risk for urinary irritation. A second pill of Azo will be taken during one of these doses. 
 
OUTCOMES: 
Primary Outcome Measures: 
Working memory task fMRI measures 
[Time Frame: baseline, 2 weeks ± 3 days, 12 weeks ± 3 days] 
Delayed Match to Sample Task behavioral (ie. correct number of responses) and fMRI  
measures will be acquired simultaneously 
 
Episodic memory task fMRI measures 
[Time Frame: baseline, 2 weeks ± 3 days, 12 weeks ± 3 days] 
Face-Name Task behavioral (ie. correct recalls) and fMRI measures will be acquired  
simultaneously 
 
Sustained attention task fMRI measures 
[Time Frame: baseline, 2 weeks ± 3 days, 12 weeks ± 3 days] 
Psychomotor vigilance task behavioral (ie. reaction time) and fMRI measures will be acquired  
simultaneously 
 
Neuropsychological Battery 
[Time Frame: baseline, 2 weeks ± 3 days, 12 weeks ± 3 days] 
TARCC designed psychometric tests 
 
Secondary Outcome Measures: 
Cerebral blood flow MRI measures 
[Time Frame: baseline, 2 weeks ± 3 days, 12 weeks ± 3 days] 
Resting measurements will be used to assess response and CBF 
 
Tertiary Outcome Measures: 
Functional Connectivity (resting state) fMRI measures 
[Time Frame: baseline, 2 weeks ± 3 days, 12 weeks ± 15 days] 
fMRI measurements will be obtained while the subject rests in the scanner 
 
CO2 challenge: Cerebral blood flow measurements will be acquired during a brief 
(3-5 minutes) inhalation of medical-grade 5% CO2 in air for fMRI signal  
calibration purposes. 
[Time Frame: baseline, 2 weeks ± 3 days, 12 weeks ± 15 days ] 
 
Data Analysis Plan: Statistical parametric analysis will be performed to generate activation maps using 
established fMRI software. Task-evoked changes in brain activities will be analyzed and contrasted 
between placebo and MB conditions. ANOVAs, paired t and 2-sample t-tests will be used in analyzing 
the reaction times and success/failure rates in the DMS, FNT and PVT, with blocks (pre-MB vs. post-
MB) as a within-subject variable and treatment (MB vs. Placebo) as a between-subject variable.  
Paired t-test will be used for group comparison with p < 0.05 (False Discovery Rate q < 0.05) 
considered statistically significant.  
 
 
References 


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1. Alzheimer's A. 2014 Alzheimer's disease facts and figures. Alzheimer's & dementia : the journal of the 
Alzheimer's Association 2014;10(2):e47-92. 
2. Bruchey AK, Gonzalez-Lima F. Behavioral, Physiological and Biochemical Hormetic Responses to the 
Autoxidizable Dye Methylene Blue. American journal of pharmacology and toxicology 2008;3(1):72-79. 
3. Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and 
neuroprotection of methylene blue. Progress in neurobiology 2012;96(1):32-45. 
4. Telch MJ, Bruchey AK, Rosenfield D, et al. Effects of post-session administration of methylene blue on fear 
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15-410H, Fox, Form BC, 11-06-15, AMD.docx 
vSept3-2014 
10 
Item 7 Risks Section: 
Complete the following table to describe the risks of all research procedures listed in Step 2, Institutional Form (items 28-34).  Do not list risks of Routine care 
procedures here.  
 ☒ N/A, Risks are described in the informed consent document – do not complete this table. 
Research procedures 
 
example: 
• 
History and physical 
• 
Questionnaire 
• 
Laboratory tests 
 
Add or delete rows as needed 
Risks 
 
List the reasonably expected risks 
under the following categories as appropriate: