Efficacy and Safety of N-acetylcysteine (NAC) in Patients With Mild Vascular Cognitive Impairment
Phase: PHASE2
Conditions: Vascular Cognitive Impairment no Dementia
Sponsor: Sunnybrook Health Sciences Centre

Version 1.9 Oct 8, 2021 
1 
Efficacy and safety of N-acetylcysteine (NAC) in 
patients with mild vascular cognitive impairment 
A randomized, double-blind, placebo-controlled parallel group design study 
Version 1.9 
Principal Investigator:
Dr. Krista L. Lanctôt, PHD
Qualified Investigator:
Dr. Damien Gallagher, MD FRCP(C) 
Regulatory Sponsor:
Sunnybrook Research Institute 
Funding Agency:
Canadian Institutes of Health Research 
Investigational Product:
N-acetylcysteine (NAC)
NCT03306979

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2 
SPONSOR STATEMENT OF COMPLIANCE
This study will comply with the International Conference on Harmonization Good Clinical 
Practice E6 (ICH-GCP), World Medical Association Declaration of Helsinki: Ethical Principles for 
Medical Research Involving Human Participants, as well as applicable regulatory and 
institutional requirements.  
 
Name: 
(Print) 
Dr. Krista L. Lanctôt 
Title:  
(Print) 
Principal Investigator
Signature: 
 
Date of Approval: 
(yyyy-mmm-dd) 
Name: 
(Print) 
Dr. Damien Gallagher 
Title:  
(Print) 
Qualified Investigator 
Signature: 
 
Date of Approval:
(yyyy-mmm-dd)

Version 1.9 Oct 8, 2021 
 
3 
PROTOCOL SIGNATURE PAGE
I have read this protocol in its entirety l and its appendices.  I agree to comply with the 
requirements of the study protocol and procedures for data recording/reporting and 
acknowledge my responsibility for the well-being of each research participant, and to ensure 
that all persons involved in study activities are adequately informed about the protocol, the 
investigational product, and their trial-related duties.  The signature below constitutes the 
agreement to conduct this study in accordance with the REB approved protocol, GCP and 
applicable regulatory requirements, including confidentiality, ethical guidelines and regulations 
regarding the conduct of research in humans. 
Qualified Investigator:   
Name & Title: 
(Print) 
Dr. Damien Gallagher, MD FRCP (C) 
Institution:  
(Print) 
Sunnybrook Health Sciences Centre 
Signature: 
 
Date of signature: 
(yyyy-mmm-dd) 

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4 
TABLE OF CONTENTS
PAGE 
SPONSOR STATEMENT OF COMPLIANCE .............................................................................. 2
PROTOCOL SIGNATURE PAGE ................................................................................................. 3
TABLE OF CONTENTS ................................................................................................................ 4
LIST OF ABBREVIATIONS .......................................................................................................... 6
PROTOCOL SUMMARY .............................................................................................................. 7
1
KEY ROLES AND CONTACT INFORMATION....................................................................8
2
INTRODUCTION .................................................................................................................. 9
2.1 
Background and Rationale ....................................................................................... 9
2.1.1
Vascular cognitive impairment-no dementia.................................................9
2.1.2 
Oxidative stress ............................................................................................ 9
2.1.3 
The therapeutic potential of antioxidant supplementation N-
acetylcysteine for cognition ........................................................................ 10
3
STUDY OBJECTIVES ........................................................................................................ 11
3.1 
Primary Objective ................................................................................................... 11
3.2 
Secondary Objective(s) .......................................................................................... 11
3.3 
Exploratory Hypotheses ......................................................................................... 12
4
METHODS ......................................................................................................................... 14
4.1 Study Design ................................................................................................................ 14
4.2 Subjects ....................................................................................................................... 14
5
PARTICIPANT SELECTION AND WITHDRAWAL ............................................................ 15
5.1 
Inclusion Criteria ..................................................................................................... 15
5.2 
Exclusion Criteria ................................................................................................... 15
5.3 
Participant Recruitment .......................................................................................... 16
5.4 
Participant Withdrawal and Discontinuation of IP ................................................... 16
5.4.1 
Reasons for Withdrawal/Discontinuation of IP ........................................... 16
5.4.2 
Data Collection and Follow-up for Withdrawn Participants ......................... 16
6
INTERVENTIONS .............................................................................................................. 17
6.1 
Investigational Product ........................................................................................... 17
6.1.1
Intervention.................................................................................................17
6.1.2
Randomization and Masking ......................................................................17
6.1.3 
Receiving and Storage ............................................................................... 17
6.1.4 
Participant Compliance Monitoring ............................................................. 18
7
STUDY SCHEDULE AND PROCEDURES ....................................................................... 19
7.1 
Assessments .......................................................................................................... 19
7.2 
MRI SCAN .............................................................................................................. 19
7.2.1 
MRS Data Acquisition ................................................................................. 20
7.3 
Blood Samples ....................................................................................................... 20
7.4 
Statistical Analyses ................................................................................................ 20
7.5 
Power Calculation .................................................................................................. 22
7.6 
Protocol Deviations ................................................................................................ 22
8
ASSESSMENT OF SAFETY .............................................................................................. 23
8.1 
Definitions ............................................................................................................... 23

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8.1.1
Adverse Events ..........................................................................................23
8.1.2 
Serious Adverse Events ............................................................................. 23
8.1.3 
Unexpected Adverse Event ........................................................................ 23
8.1.4
Unexpected Adverse Drug Reaction (ADR) ...............................................23
8.2 
Assessment of an Adverse Event .......................................................................... 24
8.2.1 
Relationship (Causality/Relatedness) ......................................................... 24
8.2.2
Expectedness .............................................................................................24
8.2.3 
Seriousness ................................................................................................ 24
8.2.4 
Severity ....................................................................................................... 24
8.3
Adverse Event Recording.......................................................................................24
8.3.1 
Investigator reporting: Notifying the REB ................................................... 24
8.3.2
Investigator reporting: Notifying the Sponsor .............................................24
8.3.3 
Sponsor Reporting of SUADRs: Notifying Health Canada ......................... 25
8.3.4 
Sponsor Reporting of SUADRs: Notifying Sites ......................................... 25
8.4 
Type and Duration of Follow-up for Adverse Events .............................................. 25
8.5
Reporting and Entry Timelines ...............................................................................25
8.6 
Rescue Medication ................................................................................................. 25
9
SITE MONITORING, AUDITING AND INSPECTING ........................................................ 26
9.1 
Site Monitoring Plan ............................................................................................... 26
9.2 
Auditing and Inspecting .......................................................................................... 26
10 
DATA HANDLING AND RECORD KEEPING .................................................................... 27
10.1 
Confidentiality ......................................................................................................... 27
10.2 
Source Documents ................................................................................................. 27
10.3 
Data Management Responsibilities ........................................................................ 27
10.4 
Data Capture .......................................................................................................... 27
10.4.1 Case Report Forms .................................................................................... 27
10.5 
Records Retention .................................................................................................. 27
10.6 
Clinical Trial Registration ........................................................................................ 28
11 
ETHICS CONSIDERATIONS ............................................................................................. 29
11.1 
Ethical Standard ..................................................................................................... 29
11.2 
Research Ethics Board (REB) ................................................................................ 29
11.3 
Consent .................................................................................................................. 29
12 
PANDEMIC CONTINGENCY PLAN .................................................................................. 30
12.1 
Alternative Methods for Consenting ....................................................................... 30
12.1.1 Consent obtained remotely by phone or video conference ........................ 30
12.2 
Remote Assessments ............................................................................................ 30
12.3 
Investigational Product Administration and Monitoring .......................................... 30
12.4 
Blood Samples ....................................................................................................... 30
13 
BIBLIOGRAPHY ................................................................................................................ 31
14 
SUPPLEMENTAL MATERIALS ......................................................................................... 37
Appendix A: Study Assessment Schedule ........................................................ 37
Appendix B: Study Assessment Schedule ........................................................ 38

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6 
LIST OF ABBREVIATIONS
The following abbreviations describe terms, documents and study personnel used in the 
conduct of this study protocol. 
AD 
Alzheimer’s disease  
AE 
Adverse Event/Adverse Experience 
ARWMC 
Age-Related White Matter Change 
CAD 
Coronary Artery Disease 
ccf-mtDNA 
Circulating cell-free mitochondrial DNA 
CR 
Cardiac Rehabilitation 
CRF 
Case Report Form 
CTS 
Clinical Trial Services 
ICH 
International Conference on Harmonization 
IP 
Investigational Product 
pCRF 
Paper Case Report Form 
GCP 
Good Clinical Practice 
GSH 
Glutathione 
ICF 
Informed Consent Form 
MCI 
Mild Cognitive Impairment 
NAC 
N-acetylcysteine 
NINDS-CSN 
National Institute of Neurological Disorders and Stroke-Canadian Stroke Network 
OS 
Oxidative Stress 
PHI 
Personal Health Information 
PI 
Principal Investigator 
PlGF 
Placental Growth Factor 
PM 
Product Monograph 
QI 
Qualified Investigator 
REB 
Research Ethics Board 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
SRI 
Sunnybrook Research Institute 
SUADR 
Serious and Unexpected Adverse Drug Reaction 
VCI 
Vascular Cognitive Impairment 
 
 
 
 
 
 

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PROTOCOL SUMMARY
Protocol Title 
(Short Title) 
Efficacy and Safety of N-acetylcysteine in patients with mild 
vascular cognitive impairment (MOVE-IT) 
Protocol 
Number 
1.9
Phase 
II
Study Design 
Randomized, double-blind, placebo-controlled parallel group  
Study Duration 
5 years
Setting 
Single-centre 
Sample Size 
60 
Main Inclusion 
Criteria 
Males or females aged 55-85 years, with mild vascular cognitive 
impairment (VCI), who are enrolled in a supervised cardiac 
rehabilitation program.  
Primary 
Outcome(s): 
In patients with mild VCI, those randomized to NAC (2,400 mg/day) 
will demonstrate greater improvement in executive function over 24 
weeks as compared to those randomized to placebo. 
Secondary 
Outcome(s): 
In patients with mild VCI, those randomized to NAC (2,400 mg/day) 
will demonstrate greater improvement in processing speed, 
memory and working memory over 24 weeks as compared to those 
randomized to placebo. 
Investigational 
Product and 
Planned Use 
N-acetylcysteine (NAC) Natural Health Products Directorate (NPN 
#: 80004844).  
Patients will be randomized to receive NAC (four 600 mg capsules 
given as 2 capsules in the morning and 2 capsules in the evening) 
or matching placebo capsules. The initial NAC dosage will be 
600mg/day (one 600mg capsule in the morning) for the first week, 
followed by 1,200 mg/day (one 600 mg capsule in the morning, one 
600mg capsule in the evening) for the second week, followed by 
1,800 mg/day (two 600mg capsules in the morning, one 600mg 
capsule in the evening) for third week, followed by 2,400mg/day 
(two 600mg capsules in the morning, two 600mg capsules in the 
evening) for the following 21 weeks.
Statistical 
Analysis: 
Differences in executive function composite z scores between the 
groups at 24 weeks will be compared between groups using a two 
sample two sided t-test. A linear mixed model will be run to 
compare the rate of change in the executive function measure over 
time. 

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1
KEY ROLES AND CONTACT INFORMATION
Regulatory Sponsor: 
Sunnybrook Research Institute (SRI) 
2075 Bayview Avenue 
Toronto, ON Canada M4N 3M5 
Principal Investigator:   
Dr. Krista L. Lanctôt, PHD  
Sunnybrook Health Sciences Centre  
2075 Bayview Avenue  
Toronto, Ontario M4N 3M5 
Tel: (416) 480-6100 x2241 
Academic Research 
Organization: 
Clinical Trial Services, SRI 
2075 Bayview Avenue 
Toronto, ON Canada M4N 3M5 
Qualified Investigator: 
Damien Gallagher, MD FRCP(C) 
Sunnybrook Health Sciences Centre  
2075 Bayview Avenue  
Toronto, Ontario M4N 3M5 
Tel: (416) 480-4098 

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2
INTRODUCTION
This study document is the protocol for research involving human participants. This study is to 
be conducted according to Canadian and international standards, and in compliance with the 
protocol, International Conference on Harmonisation Good Clinical Practice E6 (ICH-GCP), 
World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research 
Involving Human Participants, as well as applicable regulatory and institutional requirements 
and research policies.  
2.1 Background and Rationale 
2.1.1
Vascular cognitive impairment-no dementia
The prodromal stage of major neurocognitive disorders has become the current target for 
interventional research, as this stage may provide an optimal therapeutic window for 
neuroprotection. Mild vascular cognitive impairment is a prodromal stage defined by early 
cognitive deficits due to cerebrovascular disease that are not severe enough to be classified as 
dementia and is the most prevalent form of vascular cognitive impairment (VCI) among those 
aged 64 to 84 years1. Probable mild VCI is characterized by neuroimaging findings confirming 
vascular pathology; clinical symptoms indicating vascular disease in the absence of these 
neuroimaging findings is categorized as possible mild VCI2.  
 
Consistent with the recognition of vascular contribution to cognitive impairment and dementia3, 
those with coronary artery disease (CAD) are particularly at risk for mild VCI. Clinical trials 
evaluating the efficacy of cholinesterase inhibitors and memantine, as well as primary 
prevention trials to control traditional vascular risk factors have demonstrated no significant 
benefit of these interventions in the prodromal stage4-6. Regular exercise is promising7,8, but has 
variable efficacy on cognition9-11, particularly in the preclinical stages11. There is currently no 
effective treatment for those at increased risk for dementia4.  
2.1.2 
Oxidative stress 
Oxidative stress (OS) plays a major role in age-related neurodegeneration12. Cumulative 
reactive oxygen species cause cellular damage, mitochondrial dysfunction and impair the DNA 
repair system, all of which are key factors involved in the propagation of neuronal injury leading 
to apoptosis. Consistent with this, increased oxidative damages have been implicated as early 
events in the evolution of the neurodegenerative processes13,14. Similarly, decreased levels of 
glutathione (GSH), the primary antioxidant in the brain, are observed in patients with 
neuropsychiatric disorders 15. Antioxidant supplementation has been proposed for dementia 
prevention and therapy16-18. Observational and epidemiological studies with dietary or 
supplemented antioxidants showed benefits18,19, although findings from controlled trials have 
been mixed20. Of those studies, therapy with the antioxidant N-acetylcysteine (NAC) showed 
promise, consistent with action as an effective GSH precursor21.  
Importantly, emerging evidence suggests that altered OS might represent an early event in the 
neurodegenerative process. A recent meta-analysis showed that lipid peroxidation was 
significantly elevated while a range of antioxidant levels were depleted in mild cognitive 
impairment (MCI) patients 22-28. OS may be particularly important in mild VCI as cerebral 

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10 
hypoperfusion can cause ischemia- and inflammation- related increases in OS29.  Our pilot data 
showed that high OS predicted less cognitive improvement following a 6-month exercise 
intervention, suggesting that elevated OS levels might interfere with the benefit of exercise on 
cognition.   
2.1.3 
The therapeutic potential of antioxidant supplementation N-acetylcysteine 
for cognition 
GSH depletion plays an important role in OS-mediated neuronal loss13. As such, increasing 
central GSH has been proposed as a promising therapeutic strategy in Alzheimer’s disease 
(AD)21. NAC protects the cells against OS damage by increasing the endogenous intracellular 
GSH level. NAC bioavailability, metabolism and plasma levels have been studied in humans30-
32. NAC has been shown to cross the blood brain barrier and accumulate in the central nervous 
system30,33,34, where it lowers lipid peroxidation35 and beta-amyloid deposition36. NAC also 
protects neuronal function and improves cognitive deficits by decreasing acetylcholinesterase 
activity and increasing acetylcholine levels37. Clinical studies in patients with AD suggest that 
NAC may have pro-cognitive effects. Patients with mild to moderate AD treated with NAC for 24 
weeks showed significant improvement in performance on the Letter Fluency Task and 
Wechsler Memory Scale Immediate Number Recall38. In a 12-month small open label trial, a 
vitamin/nutraceutical formulation containing NAC produced a significant effect on executive 
function in individuals with early stage AD and MCI39-41. However, to date, no studies have 
evaluated the effects of NAC on cognition in patients with mild VCI.   
Those with mild VCI show deficits across all cognitive domains, with greater impairment typically 
being found in executive function. Impairment in cognitive subdomains of memory, processing 
speed and working memory have also been found in mild VCI populations42. Our recent pilot 
data showed CAD patients with possible mild VCI had elevated levels of OS compared to CAD 
controls. In addition, higher baseline ratios of late to early lipid peroxidation markers (8-
isoprostane (8-ISO) to lipid hydroperoxides (LPH) ratio) predicted less improvement in executive 
function following an exercise intervention. Since OS damage occurs early in the pathogenesis 
of the disease and might precede other neuropathological alterations23,43, an antioxidant 
intervention would be most beneficial if started before the onset of symptomatic dementia. We 
propose to combine the synergistic effects of NAC supplementation and exercise to increase 
the efficacy of each individual intervention to reduce OS and improve cognitive performance in 
patients with mild VCI. 44,45. If successful, this study will demonstrate that NAC, a widely 
available and safe supplement, can improve cognitive outcomes in patients at high risk of 
cognitive and functional decline.  

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3
STUDY OBJECTIVES
3.1 
Primary Objective 
As highlighted above, NAC preferentially improved executive function. Our primary goal is to 
evaluate the therapeutic potential of direct antioxidant supplementation using NAC to enhance 
the effect of exercise on executive function in patients with mild VCI. We hypothesize that:  
 
 
In patients with mild VCI, those randomized to NAC (2,400 mg/day) will demonstrate greater 
improvement in executive function over 24 weeks as compared to those on placebo.  
Executive function will be based on verbal fluency and trail test A & B found in the 60-minute 
battery recommended by the National Institute of Neurological Disorders and Stroke-Canadian 
Stroke Network (NINDS-CSN) harmonized standards. It is the cognitive domain most frequently 
impacted in mild VCI42,46, and it reflects white matter lesion burden, the key neuroimaging finding 
defining probable mild VCI.  The 24 week treatment duration was chosen based on a previous 
clinical trial of NAC in patients with probable AD38. The daily dose is consistent with the upper 
dosing range in published clinical trials, in which oral NAC supplementation was investigated in a 
variety of populations including AD, where evidence of tolerability and efficacy were 
observed34,38,47-50. 
3.2 
Secondary Objective(s)
Individuals with mild VCI also show deficits in processing speed, memory and working 
memory42. Our secondary goal is to evaluate the therapeutic potential of NAC to enhance the 
effect of exercise on additional cognitive domains in these patients. We hypothesize that: 
 
In patients with mild VCI, those randomized to NAC (2,400 mg/day) will demonstrate greater 
improvement in processing speed over 24 weeks as compared to placebo. 
 
In patients with mild VCI, those randomized to NAC (2,400 mg/day) will demonstrate greater 
improvement in memory over 24 weeks as compared to placebo. 
In patients with mild VCI, those randomized to NAC (2,400 mg/day) will demonstrate greater 
improvement in working memory over 24 weeks as compared to placebo. 
Processing speed will be based on the Symbol Digit Modalities Test and the memory domain, will 
consist of the Rey Complex Figure Test-immediate/delayed recall and recognition and Hopkins 
Verbal Learning Test (HVLT) -immediate/delayed recall and recognition, found in the 60-minute 
battery recommended by the NINDS-CSN. Working Memory will be based on Digit Span test 
(Forward, Backward, and Sequencing subtests). 
NAC crosses the blood brain barrier and acutely increases blood and brain GSH concentrations51. 
To demonstrate target engagement, we aim to evaluate the effect of NAC on the GSH antioxidant 
system and OS. The antioxidant levels will be evaluated by measuring levels of glutathione 
peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferases (GST)15. OS will be 
quantified by measuring concentrations of late markers (e.g., 8-ISO and 4-HNE) and early marker 
(LPH) lipid peroxidation products52. LPH is one of the primary and earliest products of lipid 
peroxidation, which are inherently unstable and susceptible to further free radical-mediated 

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12 
reactions, which results in the generation of late-stage oxidative stress products. Higher OS will 
be indicated by higher ratio between late marker (e.g., 8-ISO) and early marker (LPH) of lipid 
peroxidation as supported by our pilot data.  
In patients with mild VCI, the plasma levels of GPx, GR and GST will be significantly higher
in those receiving NAC supplementation relative to those who received exercise intervention 
alone.
 
In patients with mild VCI, the 8-ISO to LPH ratio will be significantly lower in those receiving 
NAC supplementation relative to those who received exercise intervention alone 
Evidence from neuroimaging studies in VCI patients indicated greater severity and progression 
of white matter lesion burden was associated with greater declines in executive function and 
processing speed53-55. We will investigate whether baseline WMH severity significantly predicts 
the cognitive response to NAC.  
 
In patients with mild VCI, those with lower WMH volume at baseline will demonstrate greater 
cognitive improvement in response to 24 weeks of NAC treatment  
Previous trials have shown that NAC at these doses is well-tolerated in the elderly38,56,57. We will 
investigate tolerability of NAC.  
 
In patients with mild VCI, measures of safety (including incidences of adverse events and 
drop-outs) will be comparable between treatment with NAC and placebo. 
3.3 
Exploratory Hypotheses 
The antioxidant defense molecule GSH serves as the primary antioxidant in the brain by 
scavenging harmful reactive oxygen species generated during cellular metabolism. GSH 
depletion plays an important role in OS-mediated neuronal death53, precedes 
neurodegeneration54, and contributes to the etiology of various neurodegenerative diseases55. 
Advances in magnetic resonance spectroscopy (MRS) now allow in vivo non-invasive 
quantification of GSH in the brain. We propose an innovative methodology to investigate the 
association between brain GSH and cognitive performance in subjects with mild VCI who are 
likely to have higher oxidative stress and GSH depletion.
 
 In these patients, those with higher GSH levels at baseline will demonstrate greater 
improvement in executive function over 24 weeks.  
 In these patients, those with higher GSH levels at baseline will demonstrate greater 
improvement in processing speed over 24 weeks. 
 
In these patients, those with higher GSH levels at baseline will demonstrate greater 
improvement in memory over 24 weeks. 
 
In these patients, those with higher GSH levels at baseline will demonstrate greater 
improvement in working memory over 24 weeks. 
Apathy is a prominent neuro-behavioral symptom of vascular dementia;58 it is defined as an 
impairment in motivation that persists over time. Unfortunately, apathy has rarely been 
assessed in mild VCI. We propose to assess apathy in these patients using the Apathy 
Evaluation Scale (AES) and the Mild Behavioral Impairment Checklist (MBI-C).59,60 We will 
explore the possible associations between apathy, blood biomarkers, and the therapeutic 
potential of NAC in these patients. 

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13 
White matter lesions is the key neuroimaging finding defining probable mild VCI2. We will define 
those with clinical symptoms indicating vascular disease, and negative neuroimaging findings as 
possible mild VCI. We hypothesize that patients with probable mild VCI will have higher OS than 
patients with possible mild VCI.  We will explore baseline brain GSH levels in patients with 
probable mild VCI compared to patients with possible mild VCI. Where possible, we will also 
test our primary and secondary outcomes in the probable mild VCI subgroup.  
Oxylipins are lipid species formed from the oxygenation of polyunsaturated fatty acids; they play 
a role in inflammation and vascular function61,62. Oxylipins will be explored in association with 
cognitive and mood symptoms. Mitochondrial dysfunction has been linked to inflammation and 
several diseases including Alzheimer’s disease and cardiovascular disease. Mitochondrial 
dysfunction will be measured by circulating cell-free mitochondrial DNA (ccf-mtDNA). We will 
explore its relationship with blood biomarkers, cognition, and neuroimaging biomarkers. 
Placental growth factor (PlGF) is a protein belonging to the vascular endothelial growth factor 
family and higher levels are associated with CAD63. It is proposed to stimulate angiogenesis as 
a response to pathological events, such as cardiovascular disease and ischemia64, and can be 
enhanced through exercise 65. We will explore the clinical relevance of PlGF, its relationship 
with neuroimaging biomarkers, and its relationship with cognition. Astrocyte performs a crucial 
role in central GSH synthesis and homeostasis. Astrocyte depletion may be a biomarker of OS 
mediated astrocyte dysfunction that precipitates change in brain GSH. Astrocytic damage and 
dysfunction will be quantified by measuring the S100 calcium-binding protein B (S100B). S100B 
is a biomarker secreted by astrocytes that enters the bloodstream66. Evidence shows that 
S100B was consistently elevated in the brain, cerebrospinal fluid and serum among 
mild/moderate AD67-69. We will explore possible associations between S100B and brain GSH 
levels. 
 
Recent studies suggest a role of innate immune activity, including the effects of neutrophils, in 
neurodegeneration and cognitive decline70,71. Myeloperoxidase (MPO), an enzyme primarily 
found in the auzophilic granules of neutrophils, catalyses the formation of reactive oxidant 
species (ROS)72. Previous research has reported higher blood concentrations of MPO in CAD 
patients73 and serum MPO concentration has been associated with future risk of developing 
CAD74. High concentration of peripheral MPO have also been reported in individuals with 
vascular dysfunction, such as high WMH burden75 and AD76. We will explore possible 
associations between MPO and specific cognitive domains. Examining this relationship will 
enable us to identify upstream contributors of inflammation and OS associated with symptoms 
of mVCI. 
 

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14 
4
METHODS
4.1 Study Design 
This is a 24-week randomized, double-blind, placebo-controlled parallel group design study 
involving mild VCI patients to evaluate the efficacy and safety of oral NAC supplementation 
(2,400 mg daily) as an add-on therapy to improve cognitive function in patients undergoing 
cardiac rehabilitation (CR). The CR program consists of a harmonized aerobic and resistance 
training in a supervised group setting. Eligible patients will be randomized to receive NAC (four 
600 mg capsules given as 2 capsules in the morning and 2 capsules in the evening) or 
matching placebo capsules. The initial NAC dosage will be 600mg/day (one 600mg capsule in 
the morning) for the first week, followed by 1,200 mg/day (one 600 mg capsule in the morning, 
one 600mg capsule in the evening) for the second week, followed by 1,800 mg/day (two 600mg 
capsules in the morning, one 600mg capsule in the evening) for third week, followed by 
2,400mg/day (two 600mg capsules in the morning, two 600mg capsules in the evening) for the 
following 21 weeks. 
4.2 Subjects 
This study will be approved by the Research Ethics Board of Sunnybrook Health Sciences 
Centre (Sunnybrook) and the University Health Network (UHN). All patients will provide written, 
informed consent.  
Mild VCI will be diagnosed by a clinical history of vascular disease, the presence of mild 
cognitive impairment as defined by modest deficits (1 SD below population norm) in executive 
function, memory, processing speed or working memory. Subjects will be categorized as 
“possible mild VCI”, those that meet neuroimaging criteria (below) will be categorized as 
“probable mild VCI”.  
Probable mild VCI will be diagnosed using the core diagnostic criteria for Subcortical Ischemic 
Mild Cognitive Impairment3,77, which include (A) Presence of mild cognitive impairment, as 
defined by modest deficits (1 SD below population norm) in executive function, memory, 
processing speed or working memory; (B) Absence of prior symptomatic stroke (silent brain 
infarcts are allowed); (C) Presence of “diffuse, subcortical cerebrovascular disease”, defined as 
1) the presence of two or more silent brain infarcts in supratentorial locations or 2) extensive 
white matter disease (Age-Related White Matter Change [ARWMC] scale score78 of 2 or higher, 
indicating “beginning confluence of lesions” or greater) in any brain region.  

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15 
5
PARTICIPANT SELECTION AND WITHDRAWAL 
A total of 60 mild VCI patients will be recruited from the CR program at the University Health 
Network Toronto Rehabilitation Institute.  
5.1 
Inclusion Criteria 
Each mild VCI participant must meet all of the following inclusion criteria at baseline: 
 
Males or females aged 55-85 years. 
 
MoCA score indicating cognitive impairment (less than 28 out of 30; Telephone MoCA: 
less than 20 out of 22)
 
Modest deficits (1 SD below population norm) in executive function, memory, processing 
speed or working memory based on the 60-minute battery recommended by the NINDS-
CSN. 
 
Speaks and understands English. 
 
Enrollment in the Cardiac Rehabilitation program at the University Health Network 
Toronto Rehabilitation Institute.  
 
In addition, to meet criteria for probable mild VCI 
 
 
Presence of “diffuse, subcortical cerebrovascular disease”, defined as 1) the presence of 
two or more silent brain infarcts in supratentorial locations or 2) extensive white matter 
disease (Age-Related White Matter Change [ARWMC] scale score78 of 2 or higher, 
indicating “beginning confluence of lesions” or greater) in any brain region. 
 
Participants who exhibit modest deficits (1 SD below population norm) in executive function, 
memory, processing speed or working memory based on the 60-minute battery recommended 
by the NINDS-CSN but do not have MRI findings of “diffuse, subcortical cerebrovascular 
disease” will be categorized as possible mild VCI.  
 
5.2
Exclusion Criteria
All participants meeting any of the following exclusion criteria at baseline will be excluded from 
participation in this study:  
 
A history of stroke 
 
A history of epilepsy 
 
Uncontrolled asthma (requiring hospitalization or ER visit in the last 3 months by patient 
report) 
 
Uncontrolled diabetes (clinical determination) 
 
Severe hypo/hypertension (clinical determination) 
 
Uncontrolled hypercholesterolemia (clinical determination) 
 
Presence of significant medical illnesses: 
 Severely disturbed liver function 
 Severely disturbed kidney function 
 Severely disturbed lung function 
 HIV, HBV and/or HCV infection 
 Malignant tumors 

Version 1.9 Oct 8, 2021 
 
16 
 
A current neurological condition 
 Parkinson’s disease 
 Multiple sclerosis 
Significant traumatic brain injury
 
Major psychiatric condition 
 Current major depressive disorder 
 Schizophrenia 
 Bipolar disorder 
Substance use disorder (alcohol abuse, heavy smoking (20 cigarettes or 
more/day)) 
 
Contraindication to MRI or MRS (e.g. metal in body, pacemaker). 
 
Contraindication to NAC (documented allergy) or allergy to lactose.  
 
Daily nitroglycerin use.  
 
Bleeding disorders (e.g. hemophilia, thrombotic thrombocytopenic purpura) and/or 
elective surgery within 30 days.  
 
Volunteers who currently participate in another pharmacological study.  
5.3 
Participant Recruitment  
Participants will be recruited for the study from the cardiac rehabilitation program at Toronto 
Rehabilitation Institute. Study personnel will make initial contact with potential subjects and will 
explain all the details of the study. Participants will be given as much time as they need to 
review the information sheet and consent form in order to make an informed decision to consent 
to participate in the study.  
5.4 
Participant Withdrawal and Discontinuation of IP
5.4.1 
Reasons for Withdrawal/Discontinuation of IP
At their own discretion, participants may withdraw from the study at any time and for any 
reason. Study participants will be withdrawn from the study if consent is withdrawn, and will be 
considered for study withdrawal for non-compliance, significant protocol violation, NAC 
intolerance and/or severe adverse events, the development of new medical conditions which 
prevent the continuation of participation, being uncooperative during the study, or if continuing in 
the study is in conflict with the best interest of the patient. Any patients with emergent AEs will 
be followed until the Aes are resolved. 
5.4.2 
Data Collection and Follow-up for Withdrawn Participants 
Participants who withdraw from the study will be contacted by the study research team 
to request a final visit and to follow up any unresolved adverse events. After the early 
termination visit, no further study procedures or evaluations will be performed, or 
additional study data collected. Any data collected prior to the withdrawal of consent 
may be retained and used by the sponsor.
 

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6
INTERVENTIONS 
6.1 
Investigational Product  
N-acetylcysteine (NAC), Natural Health Products Directorate (NPN #: 80004844), 
supplementation will be used as an add-on therapy to improve cognitive function in patients 
undergoing cardiac rehabilitation.
6.1.1 
Intervention 
Patients will be randomized to receive NAC (four 600 mg capsules given as 2 capsules in the 
morning and 2 capsules in the evening) or matching placebo capsules. The initial NAC dosage 
will be 600mg/day (one 600mg capsule in the morning) for the first week, followed by 1,200 
mg/day (one 600 mg capsule in the morning, one 600mg capsule in the evening) for the second 
week, followed by 1,800 mg/day (two 600mg capsules in the morning, one 600mg capsule in 
the evening) for third week, followed by 2,400mg/day (two 600mg capsules in the morning, two 
600mg capsules in the evening) for the following 21 weeks. The dose will be escalated (to 
2,400mg), unless there are tolerability issues (assessed by QI), in which case the QI will decide 
which dose is appropriate for the patient.  
The Pharmacy Department at Sunnybrook Health Sciences Centre will prepare, label and 
dispense the experimental and placebo (lactose-filled) capsules. The Pharmacy Department at 
Sunnybrook Health Sciences Centre will prepare the placebo capsules by using empty capsule 
shells supplied by SISU, a Canadian manufacturer, and filling them with a lactose-based filler. 
To make experimental and placebo capsules indistinguishable with regard to taste and 
appearance, a process will be implemented to mimic the smell of NAC, as done previously47. 
The investigational product and placebo capsules will be stored in a secure location at 
Sunnybrook in a temperature monitored area. Placebo and intervention capsules will be 
provided by SISU, and licensed in accordance with Natural Health Products Regulations. 
Details regarding this preparation will be filed in the Natural Health Products Directorate (NPN #: 
80004844).  The Pharmacy Department at Sunnybrook Health Sciences Centre will destroy the 
experimental and placebo capsules.  
6.1.2 
Randomization and Masking 
A block randomization code will be independently computer-generated by the Clinical Trial 
Services at SHSC and remain locked in a secure location in that department. Kits with study 
medication will be consecutively pre-packaged as per randomization sequence prior to 
commencement of the trial. All study personnel will remain blind to treatment allocation and 
block size until the final patient has completed follow-up and the database is locked. Unblinding 
will not be allowed unless there exists exceptional clinical circumstances that justify it, such as if 
acute medical management of an SAE is required. Individuals performing study related 
measures (e.g., plasma analyses, imaging analyses) will also remain blind to treatment 
allocation and all clinical data throughout the entire duration of the study.  
6.1.3 
Receiving and Storage 
6.1.3.1 
Receipt of Investigational Product 
SISU will ship the investigational product to the Pharmacy Department at SHSC and will provide 
the qualified investigator with chain of custody documentation as well as certificates of analysis. 

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Upon receipt of the investigational product, the Pharmacy will count and verify that the shipment 
contains all the items noted in the shipment inventory.  Any damaged or unusable product in a 
given shipment will be documented in the study files and study staff will be informed. 
 
SISU will ship the empty capsule shells to the Pharmacy Department at SHSC and will provide 
the qualified investigator with chain of custody documentation as well as certificates of analysis. 
Upon receipt of the empty capsule shells, the Pharmacy will count and verify that the shipment 
contains all the items noted in the shipment inventory.  Any damaged or unusable capsules will 
be documented in the study files and study staff will be informed. 
6.1.3.2 
Storage and Stability 
The investigational product will be stored in a secure location in a temperature monitored area 
at Sunnybrook Health Sciences Centre. There are no special handling requirements for storing 
NAC.  
6.1.4
Participant Compliance Monitoring
Compliance will be monitored using capsule counts (non-compliance is defined as taking < 80% 
of their capsules). Study participants will not be withdrawn from the study for non-compliance 
unless the PI/QI have safety concerns. 

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7
STUDY SCHEDULE AND PROCEDURES
7.1 
Assessments 
A schedule of assessments is provided in Appendix A. Demographic data, including age, 
gender, BMI, level of education, vascular risk factors (hypertension, smoking, 
hypercholesterolemia, obesity, and diabetes), concomitant medications, and language and
medical history will be collected during the first screening visit. The MRI scan will be done at the 
second screening visit. Individuals that cannot have an MRI can still be enrolled in the study; 
they will be classified as possible mVCI. The effect of exercise will be operationalized using the 
VO2 peak. GST, S100B and lipid peroxidation markers, and other blood-based biomarkers will 
be assessed as stated in section 7.3. APOE4 non-carriers might be more susceptible to 
oxidative stress and thus more likely to benefit from antioxidant treatment17,79. Blood samples 
will be collected, to determine APOE4 genotype. Dietary intake (including antioxidant 
supplementation), vital signs, and safety outcomes will be evaluated as shown on Appendix A.  
Cognitive performance will be assessed using the 60-minute battery recommended by the 
NINDS-CSN harmonized standards80. The NINDS-CSN was chosen based on its ability to 
characterize individuals with VCI, particularly in the early stages80.  In a recent study, the 60-min 
protocol was shown to effectively discriminate between cognitively intact individuals and those 
with cognitive impairment of varying degrees, particularly in patients with cognitive impairment- 
no dementia (AUC range: 0.80- 0.90)46. Additionally, the Digit Span Test (Forward, Backward 
and Sequence) will be administered as part of the cognitive battery. General cognitive function 
will be assessed using the MoCA, which has superior sensitivity to detect cognitive deficits for 
mild VCI81. Apathy will be assessed using the AES and MBI-C.59,60 For each cognitive task, a Z-
score will be determined based on published age- and sex- matched norms. The Z distribution 
maps the population mean of the test variable to 0, with better performance being more positive 
and poorer performance being more negative based on a normal distribution. The composite z- 
scores for each cognitive domain will be calculated by summing the Z-scores of individual test 
scores within the particular domain. 
7.2 
 MRI SCAN 
The MRI will be performed on a 3 Tesla scanner at screening 2 visit to determine baseline WMH 
severity. We will use the standardized Canadian Dementia Imaging Protocol, which allows for 
semiautomatic and simultaneous quantification of regional small vessel disease, including 
WMH, lacunes, and perivascular spaces, as well as cerebrospinal, grey and white matter 
segmentation82-87. Sequences include high resolution 3D T1, Proton Density (PD)/T2 weighted 
sequence, fluid attenuated inversion recovery (FLAIR), Susceptibility Weighted Imaging (for 
microbleed counts). The PD/T2 images will be used for image co-registration and to generate 
intracranial volume mask88. Following tissue segmentation, regional parcellation of 26 bilateral 
brain regions will be generated using Semiautomatic Brain Region Extraction (SABRE; 
Sunnybrook Health Sciences Centre)83. FLAIR or PD/T2 images will be used to identify WMH 
using one of two semiautomatic protocols (Fuzzy Lesion Extractor [FLEX]85 or Lesion Explorer82 
with manual selection editing to obtain reliable quantification of the number, size, location, and 
volume of WMH lesions82,89. All image acquisition, processing, and scoring will be blinded to 
clinical information using our standardized protocol.  
 

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7.2.1
MRS Data Acquisition
MRS data will be acquired to assess GSH concentrations in vivo. The regions of interest in the 
brain will be chosen based on a high-quality signal and relevance to the cognitive assessments 
outlined in section 7.1. The MEGA-PRESS pulse sequence will be used, enabling expression of 
GSH levels as an absolute concentration in mM.    
 
7.3 
Blood Samples 
Blood samples will be collected (schedule shown in Appendix A) to measure plasma levels of 
GSH antioxidant system and serum levels of lipid peroxidation as previously described 15,90. 
Briefly, approximately four teaspoons of blood will be collected by antecubital venipuncture from 
each study participant following at least 12 hours of fasting. Blood draws will be scheduled for 
0900 h + 3 h to minimize the effect of diurnal or dietary variation on marker concentrations. Blood 
will be drawn into SST and EDTA-containing vacutainer tubes and centrifuged (The Drucker 
Company; Model 614B) at 1000 x g for 10 minutes. Plasma and serum will be isolated and frozen 
at -80 C.  Stored samples will be batched for analysis. GSH antioxidant system: We will measure 
levels of GPx, GR and GST using standard western blotting technique with appropriate primary 
and secondary antibodies15. S100B Assay: We will measure levels of S100B (pg/mL) using a 
standard sandwich immunosorbent assay (ELISA). LPH Assay: Early-stage oxidative damage to 
l
(Cayman Chemical; Item No. 705002) according to manufacturer’s instructions.  4-HNE Assay: 
Late-stage lipid peroxidation will be assessed by measuring 4-HNE (f
STA-338), which will be quantified using a standard sandwich ELISA designed to detect protein 
adducts formed via 4-HNE Michael Addition to lysine, histidine, or cysteine residues. 8-ISO 
Assay: Late-stage lipid peroxidation will be evaluated by measuring 8-ISO (pg/mL) (Cayman 
Chemical; Item No. 516351), which will be quantified with a standard competitive sandwich ELISA 
using an 8-ISO-acetylcholinesterase conjugate as a tracer and 8-ISO-specific rabbit antiserum. 
The sensitivity and intra- and inter- assay coefficient of variation (CV) of these assays have been 
validated and published previously91. Oxylipins: Oxylipin species will be assayed by ultra-high-
pressure liquid chromatography tandem mass spectrometry92,93. MPO assay: MPO 
concentrations in blood (pg/mL) will be measured using ELISA. Ccf-mtDNA: Mitochondrial 
dysfunction will be assessed by ccf-mtDNA concentrations, which will be measured using 
quantitative polymerase chain reaction from circulating cell free DNA purified by commercially 
available spin columns. PlGF: We will measure levels of PlGF using a standard sandwich ELISA. 
Blood will also be collected at baseline and analyzed for the APOE genotype.  Blood cells will be 
frozen separately for determination of APOE genotype. ApoE genotype will be determined by a 
polymerase chain reaction based assay94 at Sunnybrook.
7.4 
Statistical Analyses 
All analyses will be performed using current versions of SPSS and SAS (SAS Institute, Cary, 
NC, USA). The intention-to-treat analyses will be based on data from all patients who undergo 
randomization, meet the inclusion criteria for the study, receive the trial medication at least 
once, and undergo at least baseline observation. Descriptive statistics will be calculated for 
composite z-scores for executive function, memory, and speed of processing. Categorical 
variables will be summarized using counts and percentages. Continuous variables will be 
compared between groups using two sample two sided t-tests (or Wilcoxon rank sum tests for 
non-normal data), whereas categorical variables will be compared between groups using chi-
square analyses or Fisher’s exact tests. 
 

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21 
Primary Hypothesis: Differences in executive function composite z scores between the groups 
at 24 weeks will be compared between treatment groups using a two sample two sided t-test. A 
linear mixed model will be run to compare the rate of change in the executive function measure 
over time (baseline, 12- and 24-weeks). The linear mixed model is selected as it can 
accommodate missing data in subjects. The effects of age, sex, BMI, total years of education, 
VO2 peak, physical activity levels, serum homocysteine, will be included as possible covariates. 
The same analyses will be repeated with the composite z-scores of the other cognitive domains 
as repeated variables to test for the effect of NAC treatment on the secondary outcome 
measures.  
Secondary Hypotheses: A linear mixed model will also be used to test the hypothesis that 
patients with mild VCI treated with NAC will show a greater reduction in 8-ISO/LPH ratio over 24 
weeks of treatment. The early to late stage lipid peroxidation marker ratio will be inputted as the 
repeated variable, and treatment group will be added as a fixed factor.  The effects of group by 
time interaction will be evaluated to indicate significant effects of NAC in reducing oxidative 
stress markers as compared to placebo. APOE4 genotype status, BMI, VO2 peak, and sex will 
be added as covariates.  Similar analyses will be conducted separately with GPx, GR and GST 
inputted as the repeated variable. Logistic regression analyses will be performed to examine 
whether baseline severity of WMH is a significant predictor of cognitive response to NAC 
treatment at 24 weeks, adjusting for the same covariates.  
Exploratory Hypothesis: 
A linear mixed model will be used to assess baseline brain GSH as a predictor of change in 
executive function, processing speed, memory and working memory composite z scores over 
24 weeks in patients with mild VCI. A GSH x time interaction will indicate a significant 
relationship between brain GSH and change in executive function, processing speed, memory 
and working memory performance over time. 
 
Post-hoc analyses will be used to assess change in 8-ISO/LPH ratio over 24 weeks of treatment 
between possible and probable mild VCI subgroups using linear mixed models. Between group 
differences in baseline brain GSH levels will also be assessed using analyses of covariance. 
Where possible, the primary and secondary hypotheses will also be tested in the probable and 
possible mild VCI subgroups and compared. 
 
Linear regression will be used to investigate baseline serum S100B as a predictor of baseline 
brain GSH levels, and if serum S100B at baseline will predict change in brain GSH over a 6-
month period. Body fat percentage will be included as a potential confounder in this analysis.  
Linear regression will be used to assess association between mitochondrial dysfunction as 
measured by ccf-mtDNA and oxidative stress markers at baseline and over time. A linear 
regression model will be used to investigate the association between PlGF, oxidative stress 
markers at baseline and termination. A linear regression model will be used to investigate 
peripheral MPO as a predictor of executive function or verbal memory at baseline. Age, use of 
anti-inflammatory medications and inflammatory and vascular comorbidities will be included as 
covariates. Measures of mood, cognitive function, and metabolic markers (oxylipins) will be 
compared between the baseline and follow-up visits, and changes in these measures will be 
compared between individuals relative to each other in a repeated measures linear regression 
analyses. 

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22 
Safety Outcomes: Frequency of adverse events between treatment and placebo groups will be 
compared using chi-square/ Fisher exact tests. In addition, exploratory analyses using logistic 
regression will be performed to determine predictors of cognitive response to NAC, including 
demographics and compliance.  
7.5 
Power Calculation 
Based on a published randomized placebo-controlled trial in patients with probable AD, oral 
NAC treatment resulted in a significant improvement in executive function in the active 
treatment group (p= 0.008) but not in placebo38. Our preliminary analysis of MOVE-IT blinded 
data (n=30) was used to provide an updated estimate of the standard deviation (SD=0.55) of 
executive function Z-scores in our population, controlling for education. Based on this, a sample 
size of 42 (21 in each group) will provide 80% power, at alpha of 0.05, to detect a medium effect 
size in a linear mixed model with a hypothesized intra-cluster correlation of 0.1. The sample size 
calculation was carried out using PASS Version 12 (Kaysville, Utah.). Due to the uncertainties of 
COVID-19 pandemic, a generous drop-out rate was used. To account for a drop-out rate of 
42%, we will inflate the sample size to 60.  
 
Protocol Deviations 
Planned deviations from the protocol will not be implemented without prior agreement from the 
sponsor and approval from Sunnybrook Health Sciences Centre REB, unless to eliminate an 
immediate hazard to a participant. Protocol deviations will be documented and reported as 
required and assessed where necessary during statistical analysis. 

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8
ASSESSMENT OF SAFETY
The safety of research participants is foremost and should always be considered throughout the 
conduct of research.  
8.1
Definitions
8.1.1 
Adverse Events 
An adverse event (AE) means any untoward medical occurrence in a patient or clinical 
investigation participant administered a pharmaceutical product and which does not necessarily 
have to have a causal relationship with this treatment, and includes an adverse drug reaction 
(ADR). 
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding, for example), symptom, or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product. 
8.1.2 
Serious Adverse Events 
A serious adverse event (SAE) or reaction is any untoward occurrence that at any dose: 
 
Results in death, 
 
Is life-threatening, 
 
Requires inpatient hospitalization or prolongation of existing hospitalization, 
 
Results in persistent or in significant disability/incapacity, 
 
Is a congenital abnormality or a birth defect. 
The term "life-threatening" in the definition of "serious" refers to an event in which the participant 
was at risk of death at the time of the event; it does not refer to an event which hypothetically 
might have caused death if it were more severe.  
8.1.3 
Unexpected Adverse Event 
An unexpected adverse event is any AE that is not identified in nature, severity or frequency in 
the current Product Monograph. 
8.1.4 
Unexpected Adverse Drug Reaction (ADR) 
An ADR is an adverse reaction, the severity of which is not consistent with the applicable 
Product Monograph.   All noxious and unintended responses to a medicinal product related to 
any dose should be considered an ADR.  
The phrase "responses to a medicinal product" means that a causal relationship between a 
medicinal product and an adverse event is at least a reasonable possibility, i.e., the relationship 
cannot be ruled out.  
The expression “causal relationship” is meant to convey that in general there are facts, evidence 
or arguments to suggest a reasonable causal relationship.  All serious and unexpected ADRs 
will have expedited reporting to regulatory agencies following ICH-GCP and local regulatory 
requirements. 

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8.2
Assessment of an Adverse Event
8.2.1
Relationship (Causality/Relatedness)
The causality assessment is the determination, according to the investigator’s clinical judgment, 
of the existence of a reasonable possibility that the study drug (IP) caused or contributed to an 
adverse event.  
If the qualified investigator or delegated sub-investigator is unsure about whether or not the 
study drug caused or is related to the event, then the event will be handled as “related” to the 
study drug for reporting purposes of the trial.  If the causality assessment is "unknown but not 
related” to the study drug, this will be clearly documented in the source documents.  
8.2.2 
Expectedness  
Events will be classified as unforeseen or unexpected if the nature, severity or frequency is not 
consistent with the risk information set out in the Product Monograph or label.     
8.2.3 
Seriousness 
Events will be classified as serious if associated with effects threatening the life or physiological 
functions of a participant.  Refer to the definition for “Serious Adverse Events” in section 8.1.2. 
8.2.4 
Severity  
The term "severe" is often used to describe the intensity (severity) of a specific event (e.g. mild, 
moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor 
medical significance (such as severe headache). This is not the same as "serious," which is 
based on participant/event outcome or action criteria usually associated with events that pose a 
threat to a participant's life or functioning. The terms “serious” and “severe” are not 
synonymous.  Seriousness (not severity) serves as a guide for defining regulatory reporting 
obligations. 
8.3 
Adverse Event Recording 
Investigations into potential adverse events will be conducted throughout the study (in person 
during the baseline visit, and at 4, 8, 12, 16, 20 and 24 weeks; and over the phone as shown on 
Appendix A) using a symptom checklist and will be recorded promptly in the source document. 
This checklist will include all potential side effects listed on the current product monograph for 
NAC. All emerging AEs will be noted and followed-up until resolution in a timely manner. 
8.3.1 
Investigator reporting: Notifying the REB 
Serious adverse events and unanticipated events will be recorded and reported to the REB in 
accordance with local reporting requirements and timelines.    
8.3.2 
Investigator reporting: Notifying the Sponsor 
 

Version 1.9 Oct 8, 2021 
 
25 
The investigator will report serious adverse events and serious and unexpected adverse drug 
reactions (SUADRs) to the sponsor in accordance with applicable regulations and reporting 
requirements and timelines.    
8.3.3 
Sponsor Reporting of SUADRs: Notifying Health Canada 
The regulatory sponsor will report SUADRs to regulatory authorities in accordance with local 
expedited reporting requirements and timelines.  In addition, the Sponsor will complete the ADR 
Expedited Reporting Summary Form and submit this form in conjunction with the completed 
CIOMS Form to the appropriate Health Canada directorate..    
 
8.3.4 
Sponsor Reporting of SUADRs: Notifying Sites 
The regulatory sponsor is responsible for distributing blinded expedited reports of SUADRs to 
each investigator for submission to local Ethics Committees within 15 days of sponsor 
awareness. 
8.4 
Type and Duration of Follow-up for Adverse Events  
 
AEs occurring as of the first administered dose of the investigational product and to a follow up 
period of at least 32 hours (half life of NAC is 6.25hrs)31 after the last administered dose, will be 
collected. AEs recorded during this period will be followed through to resolution, or until the 
event is assessed as chronic or stable. 
8.5 
Reporting and Entry Timelines 
The principal investigator will report SAEs to the sponsor within the following timelines:   
 
All deaths and immediately life-threatening events, whether related or unrelated, will 
be recorded and reported to the sponsor within 24 hours of site awareness.  
 
Serious adverse events other than death and immediately life-threatening events, 
regardless of relationship, will be reported to the sponsor within 72 hours of site 
awareness.  
Adverse event information will be entered into the CRF in a timely manner and no later than 15 
days from the time the investigator becomes aware of the event. 
 
Serious adverse event information will be entered into the CRF in a timely manner/within 72 
hours from the time the investigator becomes aware of the event. 
8.6 
Rescue Medication 
In case of overdose, the managements should be focused on the treatment of symptoms. These 
symptoms should be managed according to current standards of care with general supportive 
measures. Also discontinuation of study drug should be considered. 

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9
SITE MONITORING, AUDITING AND INSPECTING
9.1 
Site Monitoring Plan 
The extent and nature of monitoring is outlined in the Study Monitoring Plan. Monitoring 
activities will be performed both in person and remotely.  Reports of findings identified during 
monitoring activities will be provided to sites detailing any required actions.  Documentation of 
monitoring activities and findings will be provided to the site study team and the study QI. The 
institution and/or local REB reserves the right to conduct independent audits as necessary. 
9.2 
Auditing and Inspecting 
The investigator will provide direct access to source data/documents for the purposes of study-
related monitoring, audits, and inspections by the REB, the sponsor, and applicable regulatory 
bodies.  The investigator will permit the review of all study related documents (e.g. source 
documents, regulatory documents, data collection instruments, study data etc.) and will ensure 
access to applicable study-related facilities (e.g. pharmacy, diagnostic laboratory, etc.). 
 

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10 DATA HANDLING AND RECORD KEEPING
10.1 Confidentiality 
Information about study participants will be kept confidential and managed according to the 
requirements of the Personal Health Information Protection Act of 2004 (PHIPA) and the 
Research Ethics Board.  PHIPA outlines the rules for the collection, use and disclosure of 
personal health information.    
 
10.2 Source Documents
The investigator(s) and research team members listed on the Task Delegation Log will have 
access to participant medical records and will collect only the information needed for the study.  
Sponsor delegated monitors, representatives of institutional committees and regulatory authority 
representatives will also have access to examine records for the purposes of quality assurance 
reviews, audits and evaluation of study safety and progress. 
 
Data will be collected on printed source documents/case report forms (CRFs). Source 
documents/CRFs include physical, cognitive, and clinician assessments.  
 
10.3 Data Management Responsibilities 
As part of the safety plan for this study, the investigator will review individual study participant 
records to ensure that appropriate mechanisms to protect the safety of study participants are 
being followed, that protocol requirements are being adhered to, and that data is accurate, 
complete, and secure. Participant records include, but are not limited to: consent forms, case 
report forms, data forms, laboratory records, inclusion/exclusion forms, and medical charts. All 
study data will be collected by a member of the study research team and recorded in 
accordance with applicable procedures. 
10.4 Data Capture 
10.4.1 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study.  Paper 
case report forms (pCRFs) will be used to collect data for this study. CRFs will be completed by 
data capture personnel and signed off by the investigator in a timely manner. Data from pCRFs 
will be electronically entered into Research Electronic Data Capture (REDCap). Good 
documentation practices will be implemented according to standard operating procedures.  All 
data reported on the CRF will be verifiable by source document.   
10.5 Records Retention 
Study essential documents will be maintained in a secure and confidential manner for a period 
of 25 years.  For the purposes of this study, the start date of the retention period will be the date 
of the final report of the trial.  All study records will be destroyed according to local and national 
policy and requirements. The sponsor will inform the investigator/institution when these 
documents may be destroyed.  

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10.6 Clinical Trial Registration
In accordance with Health Canada’s Notice “Registration and Disclosure of Clinical Trial 
Information, November 30, 2007”, the study will be registered on Clinicaltrials.gov 
(www.clinicaltrials.gov).  

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11 ETHICS CONSIDERATIONS
11.1 
Ethical Standard 
This study will be conducted in accordance with the principles set forth in The Belmont Report: 
Ethical Principles and Guidelines for the Protection of Human Participants of Research, and 
codified in the Tri-Council Policy Statement and/or the ICH E6.  
11.2 
Research Ethics Board (REB)  
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the REB for review and approval.  Approval of both the protocol and the consent 
form will be obtained before any participant is enrolled.  Any amendment to the protocol will be 
approved by the REB before the changes are implemented in the study, unless to eliminate an 
immediate hazard.   
11.3 
Consent  
A consent form describing in detail the study procedures and risks will be reviewed with and 
given to each participant.  Consent forms will be REB-approved, and the participant will be 
required to read and review the document or have the document read to him or her.  The 
investigator or designee will explain the research study to the participant and answer any 
questions that may arise. The participant will sign the informed consent form prior to any study-
related assessments or procedures.  Participants will be given the opportunity to discuss the 
study with their surrogates or think about it prior to agreeing to participate.  They may withdraw 
consent at any time throughout the course of the study.  A copy of the signed informed consent 
document will be given to participants for their records.   
 

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12
PANDEMIC CONTINGENCY PLAN
12.1 
Alternative Methods for Consenting 
In order to reduce risk to study participants and staff in pandemic situation (e.g COVID-19), 
alternative consenting methods such as videoconference or telephone consents is available to 
participants. 
12.1.1 Consent obtained remotely by phone or video conference 
The research team will arrange a three-way call or video conference with the participant, an 
impartial witness, and if desired and feasible, additional participants requested by the participant 
(e.g., next of kin). Research staff will mail or email the participant a form ahead of time, explain 
the consent forms and answer any question during the call or video conference. The consent 
forms will be signed by the participant and a witness, and sent back to the research team via 
mail or email. Research team will have PI review and sign the consent form, provide the 
participant with a copy for their records. The process will be documented. 
12.2 
Remote Assessments 
A schedule to reduce in-person contact and risk is provided in Appendix B. Participants will 
complete Baseline visit, Week 4, Week 8, Week 16 and Week 20 visits remotely by phone or 
video conference. 
 
12.3 
Investigational Product Administration and Monitoring 
Participants will receive and return IP via tracked shipment. The research team will give phone 
calls regularly to follow up with the participants and monitor any possible adverse event. 
Compliance will be monitored using capsule counts.
12.4
Blood Samples
Blood samples will be collected at Screening 1 and Screening 2 visits (shown in Appendix B) 
instead of Screening 2 and Baseline visit to reduce the time that the participants have to come 
on-site. 

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13 BIBLIOGRAPHY
1.
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outcomes of vascular cognitive impairment. Vascular Cognitive Impairment Investigators of the 
Canadian Study of Health and Aging. Neurology. Jan 25 2000;54(2):447-51.  
2. 
Skrobot OA, Black SE, Chen C, et al. Progress toward standardized diagnosis of vascular cognitive 
impairment: Guidelines from the Vascular Impairment of Cognition Classification Consensus Study. 
Alzheimer's & Dementia. 2018/03/01/ 2018;14(3):280-292. 
doi:https://doi.org/10.1016/j.jalz.2017.09.007 
3. 
Gorelick PB, Scuteri A, Black SE, et al. Vascular contributions to cognitive impairment and 
dementia: a statement for healthcare professionals from the american heart association/american 
stroke association. Stroke. Sep 2011;42(9):2672-713. doi:10.1161/STR.0b013e3182299496 
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Version 1.9 Oct 8, 2021 
 
37 
14 SUPPLEMENTAL MATERIALS 
Appendix A: Study Assessment Schedule
 
Table 1. Schedule of Assessment 
Visit/phone calls (Weeks) 
-5 
-1
0* 
1*
2* 
3* 
4* 
8* 
12 
16* 
20* 
24 
 
 
± 14 
days 
± 14 
days 
± 2 
days 
± 2 
days 
± 2 
days 
± 7 
days
± 7 
days 
± 7 
days 
± 7 
days
± 7 
days 
± 7 
days 
Informed Consent 
X 
 
Inclusion/Exclusion Criteria 
X 
X 
X 
 
Demographics
Age, gender, education, 
language 
X 
 
Concomitant medications 
X 
X 
 
X
X 
X 
X
X 
X 
Vascular risk factors 
X 
 
Anthropomorphic 
parameters 
 
 
X1 
 
 
 
X1 
 
 
X1 
Medical, Surgical, and 
Psychiatric History
X 
 
 
 
 
Cognitive Outcomes 
NINDCS-60 minute 
cognitive assessment. 
X 
 
X 
X 
MoCA, AES, MBI-C 
X 
 
X 
X 
 
 
 
Dietary Intake 
Food Diary 
 
 
X 
 
X
X 
X 
X
X 
X 
 
 
 
Fasting Blood Draw
GSH markers: GPx, GR, 
GST  
X 
 
X 
X 
Ccf-mtDNA & OS markers: 
LPH, 4-HNE, 8-ISO, MPO, 
oxylipins
X 
 
X 
X 
APOE4 genotype 
X 
 
 
S100B, PlGF
 
 
X 
 
 
 
X 
 
 
X 
Exploratory Neuroimaging Outcome
MRI and MRS
X 
 
Exercise Intervention Outcome
VO2 peak 
X1 
 
X1 
X1 
 
 
 
IP Administration 
Drug Dispensing 
 
 
X 
 
X
X 
X 
X
X 
 
 
 
 
Compliance Measures 
Capsule Count 
 
X
X 
X 
X
X 
X 
 
 
 
Safety Outcomes 
Heart rate, blood pressure 
X 
 
X
X 
X 
X
X 
X 
Plasma electrolyte panel 
and homocysteine level 
X 
 
X 
Phone call
 
 
X 
X 
X 
 
 
 
 
 
X2 
Adverse events review
X 
X 
X 
X 
X
X 
X 
X
X 
X 
1. Collected from TRI on the visit date, as available. 2. At least 32 hours after the final dose.  
 

Version 1.9 Oct 8, 2021 
 
38 
Appendix B: Study Assessment Schedule
Table 1. Schedule of Assessment 
Visit/phone calls (Weeks)
-5
-1
0*
1*
2*
3*
4*
8*
12
16*
20*
24
 
± 14 
days
± 14 
days
± 2 
days
± 2 
days
± 2 
days
± 7 
days
± 7 
days
± 7 
days
± 7 
days
± 7 
days
± 7 
days
Informed Consent 
X
 
 
 
Inclusion/Exclusion Criteria
X
X
X
Demographics
Age, gender, education, 
language 
X
 
 
 
Concomitant medications 
X
X
 
 
 
X 
X 
X 
X 
X
X 
Vascular risk factors 
X
 
 
 
Anthropomorphic parameters
 
 
X1
 
 
 
 
 
X1
 
X1
Medical, Surgical, and 
Psychiatric History
X
 
 
 
 
Cognitive Outcomes
NINDCS-60 minute cognitive 
assessment. 
X
 
 
 
X 
X 
MoCA, AES, MBI-C 
X
 
 
 
X 
X 
 
Dietary Intake 
Food Diary 
 
 
X
 
 
 
X 
X 
X 
X 
X
X 
 
Fasting Blood Draw 
GSH markers: GPx, GR, 
GST  
X 
 
 
 
 
X 
X 
Ccf-mtDNA & OS markers: 
LPH, 4-HNE, 8-ISO, MPO, 
oxylipins
X 
 
 
 
 
X 
X 
APOE4 genotype 
X 
 
 
 
 
S100B, PlGF
 
X 
 
 
 
 
 
 
X 
 
X 
 
Exploratory Neuroimaging Outcome 
MRI and MRS
X 
 
 
 
 
Exercise Intervention Outcome 
VO2 peak 
X1
 
 
 
X1 
X1 
 
IP Administration
Drug Dispensing 
 
 
X3
 
 
 
X3 
X3
X 
X3 
X3 
 
Compliance Measures 
Capsule Count 
 
 
 
X 
X 
X 
X 
X
X 
 
Safety Outcomes
Heart rate, blood pressure 
X
 
 
 
X 
X 
X 
X 
X
X 
Plasma electrolyte panel and 
homocysteine level 
X
 
 
 
X 
Phone call
 
 
 
X 
X 
X 
 
 
 
 
X2 
Adverse events review
X
X 
X 
X 
X 
X 
X 
X 
X
X 
*Visit will be conducted remotely through videoconferencing or telephone. 1. Collected from TRI on the visit date, as available. 2. At least 
32 hours after the final dose. 3. Pill bottle will be shipped and returned via tracked shipment.