N-DOSE AD: A Dose Optimization Trial of Nicotinamide Riboside in Alzheimer's Disease
Phase: PHASE2
Conditions: Alzheimer Disease, Dementia
Sponsor: Haukeland University Hospital

N-DOSE AD Protocol v4.0 
 
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N-DOSE AD: A randomized, double blind, dose optimization 
trial of nicotinamide riboside in Alzheimer’s disease.  
 
 
 
 
 
Protocol Identification Number:   v4.0 18.Jun25 
 
 
SPONSOR: 
Haukeland University Hospital 
Address: Jonas Lies 65  
Tel : 55975000 
 
 
PRINCIPAL INVESTIGATOR (PI): 
Kristoffer Haugarvoll 
Tel : 55975301 
E-mail: 
kristoffer.haugarvoll@helse-
bergen.no 
 
 
 
 
 
 
 
 
 
 
 
PROTOCOL VERSION NO. 4.0 – 18Jun2025 

N-DOSE AD Protocol v4.0 
 
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CONTACT DETAILS  
 
Sponsor: 
Haukeland University Hospital 
Address: Jonas Lies 65  
Tel: 55975000 
 
Study Director 
Kristoffer Haugarvoll 
Tel : 55975301 
E-mail: kristoffer.haugarvoll@helse-bergen.no 
 
 
 
 
Principal investigator (at each 
participating site or if single site study): 
Haukeland University Hospital 
Kristoffer Haugarvoll 
E-mail: kristoffer.haugarvoll@helse-bergen.no 
 
Haraldplass Deaconess Hospital 
Ragnhild Eide Skogseth  
E-mail: ragnhild.eide.skogseth@haraldsplass.no  
 
Participating Departments: 
Haukeland University Hospital, 
Department of Neurology 
 
 
Haraldplass Deaconess Hospital, 
Department of Medicine 
 
  
Monitor: 
Section of Innovation and Research Haukeland University 
Hospital, department of Research and Development 
Tel: +47 92860178 
 
 
 
 

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SIGNATURE PAGE 
 
Title 
N-DOSE AD: A randomized, double blind, dose optimization trial of 
nicotinamide riboside in Alzheimer’s disease.  
 
Protocol ID no: 
428878 
 
 
I hereby declare that I will conduct the study in compliance with the Protocol, ICH GCP and the 
applicable regulatory requirements: 
To be signed by sponsor’s representative, Coordinating Investigator, Principal Investigator, statistician etc. 
This section and table above should be completed as appropriate for the type of study conducted and the 
parties involved.   
Name 
Title 
Role 
Signature 
Date 
Torhild Næss 
Vedeler 
Neurology clinic 
Director 
Sponsor 
representative 
 
 
Kristoffer 
Haugarvoll 
Researcher 
Principal Investigator 
 
 
Ragnhild Eide 
Skogseth 
Postdoc 
Principal Investigator 
 
 
 
 
 
 

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PROTOCOL SYNOPSIS 
 
N-DOSE AD: A randomized, double blind, dose optimization trial of 
nicotinamide riboside in Alzheimer’s disease. 
 
Sponsor 
Haukeland University Hospital 
Phase and study type 
Phase II, Dose-Optimization study 
Investigational 
Product (IP) (including 
active comparator and 
placebo):        
Nicotinamide Riboside  
 
Placebo  
Centers: 
Haukeland University Hospital 
Haraldsplass Deaconess Hospital 
 
Study Period: 
 
Estimated date of first patient enrolled: 20.10.2022 
Anticipated recruitment period: 20.10.2022 – 20.04.2025 
Estimated date of last patient completed: 30.06.2025. 
Treatment Duration: 
12 weeks 
 
Objectives and endpoints 
Objective 
Endpoint 
Primary 
To compare the effect of orally administered 
nicotinamide riboside (NR), escalated to 1500 
mg twice per day (3000 mg/day) in the dose-
escalation group (DE-group) - versus stable 
dosing of 500 mg twice per day (1000 
mg/day) in the dose-stable group (DS-group) 
on cerebral NAD-levels, at week 12. 
Change in cerebral NAD/ATP-α ratio 
measured by 31 Phosphorus magnetic 
resonance spectroscopy (31P-MRS) in the 
posterior brain (encompassing the occipital, 
parietooccipital and posterior parts of the 
temporal cortex). 
Secondary 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in cerebral NAD 
levels from baseline to weeks 4, 8 and 12. 
Change in cerebral NAD/ATP-α ratio 
measured by 31 Phosphorus magnetic 
resonance spectroscopy (31P-MRS) in the 
posterior brain (encompassing the occipital, 
parietooccipital and posterior parts of the 

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temporal cortex). 
To compare the effectiveness of orally 
administered nicotinamide riboside (NR) 
1500 mg twice per day versus 500 mg twice 
per day in augmenting the NAD-metabolome 
in the central nervous system (CNS) at week 
12. 
 
 
Change in the cerebrospinal fluid (CSF) levels 
of NAD or other metabolites of the NAD 
metabolome*, measured by LC-MS. 
Exploratory 
Neuroimaging 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
NR related metabolic pattern (NRRP) 
expression at week 12. 
Change in NRRP expression, measured by 
FDG-PET. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in NRRP expression 
from baseline to weeks 4, 8 and 12. 
Change in NRRP expression, measured by 
FDG-PET. 
To compare the effect of orally administered 
NR DE-group versus DS-group on the AD-
related pattern (ADRP) expression at week 12. 
Change in ADRP expression, measured by 
FDG-PET. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in ADRP expression 
from baseline to weeks 4, 8 and 12. 
Change in ADRP expression, measured by 
FDG-PET. 
Metabolism & molecular markers 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
NAD metabolome* in the blood, urine and 
central nervous system (CNS) at week 12. 
Change in levels of NAD metabolites in blood, 
urine and CSF, measured by HPLC-MS and/or 
the NADMed method. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in the NAD 
metabolome* in blood and urine from baseline 
to weeks 4, 8 and 12. 
Change in levels of NAD metabolites in blood 
and urine, measured by HPLC-MS and/or the 
NADMed method. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on serum 
and CSF inflammatory markers at week 12. 
Change in inflammatory cytokines in serum 
and CSF, measured by ELISA. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in serum 
inflammatory markers from baseline to weeks 
4, 8 and 12. 
Change in inflammatory cytokines in serum, 
measured by ELISA. 
Clinical – cognitive & non motor symptom severity, quality of life 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
clinical severity of AD symptoms at week 12. 
Change in the total ADAS-Cog 13 score, CDR 
sum of boxes, MoCA score, TMT. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and change in clinical severity of 
PD symptoms from baseline to weeks 4, 8 and 
12.                
Change in the total ADAS-Cog 13 score, CDR 
sum of boxes, MoCA score, TMT. 
To compare the effect of orally administered 
Change in IADL and PADL scores. 

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NR in the DE-group versus DS-group on ADL 
severity in AD  at week 12. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and change in ADL scores in  AD 
from baseline to weeks 4, 8 and 12.                
Change in IADL and PADL scores.. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on  
depressive symptoms in AD at week 12. 
Change in MADRS score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and depressive symptoms in AD 
from baseline to weeks 4, 8 and 12.                
Change in MADRS score. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
neuropsychiatric symptoms in AD at week 12. 
Change in the NPI-Q score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and change in neuropsychiatric 
symptoms in AD from baseline to weeks 4, 8 
and 12. 
Change in the NPI-Q score. 
Hypothesis-generating or resource-dependent endpoints (may be reported in follow-up 
or secondary publications). 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on gene 
expression at week 12. 
Change in gene expression, measured by RNA 
sequencing (RNAseq). 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in gene expression 
from baseline to weeks 4, 8 and 12. 
Change in gene expression, measured by RNA 
sequencing (RNAseq). 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
protein expression at week 12. 
 Change in protein levels, measured by LC-MS. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in protein expression 
from baseline to weeks 4, 8 and 12. 
Change in protein levels, measured by LC-MS. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on serum 
and CSF inflammatory markers at week 12. 
Change in inflammatory cytokines in serum 
and CSF, measured by ELISA. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in serum 
inflammatory markers from baseline to weeks 
4, 8 and 12. 
Change in inflammatory cytokines in serum, 
measured by ELISA. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
histone acetylation in AD at week 12. 
Change in histone panacetylation, measured 
by immunoblotting. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in histone 
acetylation in AD from baseline to weeks 4, 8 
Change in histone panacetylation, measured 
by immunoblotting. 

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and 12. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
H3K27 and H4K16 histone acetylation in AD at 
week 12. 
Changes in levels of H3K27 and H4K16 
acetylation, measured by immunoblotting. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in on H3K27 and 
H4K16 histone acetylation in AD from baseline 
to weeks 4, 8 and 12. 
Changes in levels of H3K27 and H4K16 
acetylation, measured by immunoblotting. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
genomic distribution of H3K27 and H4K16 
histone acetylation in AD at week 12. 
 Change in the genomic distribution of H3K27 
and H4K16 acetylation, measured by 
chromatin immunoprecipitation sequencing 
(ChIPseq). 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in the genomic 
distribution of H3K27 and H4K16 histone 
acetylation in AD from baseline to weeks 4, 8 
and 12. 
Change in the genomic distribution of H3K27 
and H4K16 acetylation, measured by 
chromatin immunoprecipitation sequencing 
(ChIPseq). 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on folate 
and one-carbon metabolism in AD at week 12. 
Change in folate and one-carbon metabolites 
in blood and CSF, measured by HPLC-MS. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in folate and one-
carbon metabolism in AD from baseline to 
weeks 4, 8 and 12. 
Change in folate and one-carbon metabolites 
in blood, measured by HPLC-MS. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
methyl donors in AD at week 12. 
Change in methyl-donors (e.g., SAM), 
measured by HPLC-MS, in the blood and/or 
CSF. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in methyl-donors in 
AD from baseline to weeks 4, 8 and 12. 
Change in methyl-donors (e.g., SAM), 
measured by HPLC-MS, in the blood. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on DNA 
methylation at week 12. 
Change in level and genomic distribution of 
DNA methylation, measured by Illumina 
Infinium MethylationEpic kit. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in methyl-donors in 
AD from baseline to weeks 4, 8 and 12. 
Change in level and genomic distribution of 
DNA methylation, measured by Illumina 
Infinium MethylationEpic kit. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
synthesis of neurotransmitters in AD at week 
12. 
Change in neurotransmitters in CSF, measured 
by HPLC-MS. 
Determine whether NR-therapy affects the gut 
microbiome in a dose-responsive manner at 
week 12. 
Change in gut microbiome composition, 
measured by metagenomics in fecal samples. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
gut metabolome at week 12. 
Change in fecal metabolomics, measured by 
LC-MS in fecal samples. 

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Safety 
To determine the safety and tolerability of NR 
at a dose of 1000 mg, 2000 mg, and 3000 mg 
per day in AD. 
Number and severity of adverse events from 
baseline to week 12 across treatment groups 
and NR dose levels. 
*The NAD metabolome is comprised of: Nicotinamide adenine dinucleotide oxidized (NAD+), Nicotinamide adenine 
dinucleotide reduced (NADH), NAD+/NADH ratio, total NAD (sum of NAD+ and NADH), Nicotinamide adenine 
dinucleotide phosphate oxidized (NADP+), Nicotinamide adenine dinucleotide phosphate reduced (NADPH), 
NADP+/NADPH ratio, total NADP (sum of NADP+ and NADPH, 1-methyl nicotinamide (Me-Nam), nicotinic acid-adenine 
dinucleotide (NAAD), N1-methyl-2-pyridone-5-carboxamide (Me-2-PY), Nicotinamide (Nam), Nicotinamide N-oxide 
(Nam N-oxide), ADP-ribose (ADPR), Nicotinic acid riboside (NAR), Nicotinamide riboside (NR), Nicotinamide 
mononucleotide (NMN), Nicotinic acid (NA).  
 
 
 
 
 
 
 
 
 
  
Study Design: 
Multi-center, double-blinded, randomized, placebo controlled, dose-
optimization 
 
Inclusion Criteria:    
● Diagnosis of probable AD according to the core clinical criteria 
updated in the NIA and Alzheimer’s Association guidelines. 
● Biomarker evidence consistent with AD neuropathologic change, 
defined by CSF markers. 
● Diagnosed with AD within two years from enrollment. 
● CDR 0.5-1 (inclusive) at enrolment.  
● Age 50 to 85 years (inclusive) at the time of enrollment. 
● A study partner able to study data and assist the participant in 
the study drug administration, i.e. contact ≥ 3 times weekly. 
● Capacity to provide written informed consent for study 
participation defined as Montreal  
Cognitive Assessment (MoCA)  score ≥ 16 or Mini Mental State 
Evaluation (MMSE)  score ≥ 20. MMSE or MoCA must have 
been performed within 6 months prior to baseline. If there is 
any doubt regarding the participants capacity to give informed 
consent we will ask for an independent evaluation by a 
consultant clinician who is 
not associated with the N-DOSE AD study. 
● Cholinesterase inhibitors and memantine can be used if stable 
for 8 weeks prior to baseline visit. 
● Able to undergo lumbar puncture 
● Able to undergo MRI 
 
 
 

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Exclusion Criteria 
● Diagnosis of dementia other than probable AD. 
● Comorbidity that precludes study participation or data 
interpretation.  
● Any psychiatric disorder that would interfere with compliance in 
the study. 
● Any severe somatic illness that would interfere with compliance 
and participation in the study.  
● Use of high dose vitamin B3 supplementation within 30 days of 
enrollment. 
● Metabolic, neoplastic, or other physically or mentally 
debilitating disorder at baseline visit. 
● Current treatment with Oral Anticoagulation Therapies 
● Implants that preclude MRI examinations, e.g. DBS, pacemaker  
 
Sample Size: 
80 patients (20 in placebo group, 60 in treatment groups) 
Efficacy Assessments: 
Primaryendpoint: Cerebral NAD levels as measured by 31P-MRS (see 
details under Endpoints).  
Safety Assessments: 
Biochemistry: Routine blood analysis (see Lab-manual).  
Vital signs: pulse, blood-pressure. 
Registration of adverse events. 
 
 
 
 

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TABLE OF CONTENTS  
CONTACT DETAILS 
2 
SIGNATURE PAGE 
3 
PROTOCOL SYNOPSIS 
4 
TABLE OF CONTENTS 
10 
List of Abbreviations and Definitions of Terms 
15 
1 INTRODUCTION 
16 
1.1 Background – Disease 
16 
1.1.1. Alzheimer’s disease (AD) is a major societal challenge 
16 
1.1.2. NAD-replenishment therapy shows promise as neuroprotective therapy for AD 
16 
1.1.3. Phase-I evidence for NR-therapy in neurodegenerative diseases 
17 
1.2 Background - Therapeutic Information 
20 
1.3 Pre-Clinical & Clinical Experience with Investigational Product (IP) 
20 
1.4 Rationale for the Study and Purpose 
21 
2 STUDY OBJECTIVES AND RELATED ENDPOINTS 
21 
2.1 Primary Objective 
24 
2.2 Secondary Objectives 
25 
2.3 Experimental Objectives 
25 
2.4 Primary Endpoint 
27 
2.5 Secondary Endpoints 
27 
2.6 Experimental endpoints 
27 
The between visit difference in the 1000mg NR group will be assessed to identify any time effects and 
differentiate those from dose-effects. 
27 
3 OVERALL STUDY DESIGN 
28 
3.1 STUDY DESIGN 
28 
4 STUDY POPULATION 
29 
4.1 Selection of Study Population 
29 
4.2 Number of Patients 
29 

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4.3 Inclusion Criteria 
29 
4.4 Exclusion Criteria 
29 
5 TREATMENT 
30 
5.1 Drug Identity, Supply and Storage 
30 
5.2 Dosage and Drug Administration 
30 
5.3 Duration of Therapy 
30 
5.4 Cognitive Therapy During Screening and IP treatment period 
30 
5.5 Concomitant Medication 
31 
5.6 Subject Compliance 
31 
5.7 Drug Accountability 
31 
5.8 Drug Labeling 
31 
5.9 Subject Numbering 
31 
6 STUDY PROCEDURES 
31 
6.1 Flow Chart 
32 
6.2 By Visit 
33 
6.2.1 Screening Visits/ Before start of Investigational Product (IP) 
33 
6.2.2 Baseline/Week 0 
34 
6.2.3 During treatment (visits 2-4) 
35 
6.2.4 End of study (Visit 4) 
35 
6.3 Criteria for Patient Discontinuation 
35 
6. 4 Procedures for Discontinuation 
36 
6.4.1 Patient Discontinuation 
36 
6.4.2 Trial Discontinuation 
36 
7 ASSESSMENTS 
36 
7.1 Safety and Tolerability Assessments 
36 
7.2 Clinical Assessments 
36 
7.3 Routine Laboratory Tests 
38 
7.4 Imaging studies 
38 

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7.5 Molecular analyses 
38 
8 SAFETY MONITORING AND REPORTING 
39 
8.1 Definitions 
39 
8.1.1 Adverse Event (AE) 
39 
8.2.1 Serious Adverse Event (SAE) 
39 
8.2 Time Period for Reporting AE and SAE 
40 
8.3 Recording of Adverse Events 
40 
8.4 Reporting Procedure 
41 
8.4.1 AEs and SAEs 
41 
8.5 Clinical Study Report 
41 
9 DATA MANAGEMENT AND MONITORING 
41 
9.1 Electronic - Case Report Forms (e-CRFs) 
41 
9.2 Study Monitoring 
42 
9.3 Confidentiality 
42 
9.4 Database management 
43 
10 STATISTICAL METHODS AND DATA ANALYSIS 
43 
10.1 Determination of Sample Size 
43 
10.2 Randomization 
44 
Randomization is done by e-CRF upon enrollment to the study. Participants will be randomized into one 
of three groups (Placebo, NR 1000 mg for the duration of the study, NR dose escalation group). 
44 
10.3 Population for Analysis 
44 
10.4 Planned analyses 
45 
10.5 Statistical Analysis 
46 
11 STUDY MANAGEMENT 
47 
11.1 Investigator Delegation Procedure 
47 
11.2 Protocol Adherence 
47 
11.3 Study Amendments 
47 
11.4 Audit and Inspections 
47 

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12 ETHICAL AND REGULATORY REQUIREMENTS 
47 
12.1 Ethics Committee Approval 
47 
12.2 Other Regulatory Approvals 
47 
12.3 Informed Consent Procedure 
48 
12.4 Subject Identification 
48 
13 TRIAL SPONSORSHIP AND FINANCING 
48 
14 TRIAL INSURANCE 
48 
15 PUBLICATION POLICY 
48 
16 REFERENCES 
48 
17 LIST OF APPENDICES 
52 
Standard Operating Procedure (SOP) for sampling and preparation of biological material (Lab manual) 53 
Project Management Committee for the study 
53 
Collaborators and investigators for sub-studies: 
53 
1 GENERAL INFORMATION 
54 
TABLE OF CONTENTS 
55 
2 HEMATOLOGY, BIOCHEMISTRY, HORMONE, SEROLOGY AND SAFETY LABORATORY PARAMETERS 
56 
2.1.1 Safety laboratory (blood) 
57 
3 SAMPLING FOR BIOBANK AND RESEARCH 
58 
3.1 Mandatory research and biobank samples 
59 
3.1.1 Whole blood EDTA 
59 
3.1.2 Whole blood EDTA – snap-frozen 
59 
3.1.3 RNA PAXgene tubes 
59 
3.1.4 Platelets isolation 
59 
3.1.5 Whole blood for Serum 
59 
3.1.6 Cerebrospinal fluid (CSF) 
59 
3.1.7 Fecal sample biobanking 
59 
3.1.8 Urine sample biobanking 
59 
4 SAMPLE PROCESSING AND STORAGE 
59 

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4.1 Whole blood (EDTA): standard 
60 
4.2 Whole blood (EDTA): snap-frozen 
61 
4.3 RNA PAXgene tubes 
62 
4.4  
63 
4.5 Serum for biobanking 
63 
4.6 Collection, of cerebrospinal fluid (CSF) 
64 
4.6.1              Collection of cerebrospinal fluid 
65 
Absolute contraindications for the procedure: 
65 
4.6.2              Processing and storage of cerebrospinal fluid 
65 
4.7 Fecal sample biobanking 
66 
4.8 Urine for biobanking 
67 
5 CONTACT PERSONS 
68 
5.1 Helse Bergen 
68 
5.1.1 Biobank Haukeland 
68 
5.1.2 Laboratory Clinic 
68 
5.1.3 Haraldsplass (HDS) Laboratory 
68 
5.1.4 Department of Neurology 
68 
APPENDIX B – MRI AND FDG-PET – PROTOCOL 
69 
APPENDIX C - CLINICAL RATING SCALES 
69 
 
 

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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
Abbreviation or special term 
Explanation 
AD 
Alzheimer’s Disease 
ADL 
Activities of Daily Living 
ADAS-Cog 
The Alzheimer's Disease Assessment Scale 
ADRP 
Alzheimer’s disease related pattern 
AE 
Adverse Event  
CDR 
Clinical Dementia Rating 
CRF 
Case Report Form (electronic/paper) 
CSF 
Cerebrospinal fluid 
CSA 
Clinical Study Agreement 
CTC 
Common Toxicity Criteria 
CTCAE 
Common Terminology Criteria for Adverse Event 
DAE 
Discontinuation due to Adverse Event 
DE-group 
Dose-escalation group 
DS-group 
Dose-stable group 
EC 
Ethics Committee, synonymous to Institutional Review Board (IRB) 
and Independent Ethics Committee (IEC) 
GCP 
Good Clinical Practice 
HPLC-MS 
High performance liquid chromatography-mass spectrometry 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
IP 
Investigational Product (includes active comparator and placebo) 
IND 
Investigational New Drug 
LC-MS 
Liquid chromatography-mass spectrometry 
MoCA 
Montreal Cognitive Assessment 

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MRS-responder 
An individual susceptible to NR-induced increase in cerebral NAD 
levels, detectable by 31P-MRS 
MRS-non-responder 
An individual showing no NR-induced increase in cerebral NAD 
levels, detectable by 31P-MRS 
NAD 
Nicotinamide adenine dinucleotide 
NPI-Q 
Neuropsychiatric Inventory Questionnaire 
NR 
Nicotinamide Riboside 
NRRP 
NR-related metabolic pattern 
OBD 
Optimal biological dose 
PD 
Parkinson’s Disease 
PDRP 
Parkinson’s Disease-Related Pattern 
NAD 
Nicotinamide adenine dinucleotide 
SAE 
Serious Adverse Event   
SAM 
S-denosyl methionine 
SOP 
Standard Operating Procedure 
TMT 
Trail Making Test 
 
1 INTRODUCTION 
1.1 
Background – Disease  
1.1.1. Alzheimer’s disease (AD) is a major societal challenge  
Alzheimer’s disease (AD) is the most common neurodegenerative progressive dementia and a leading cause 
of disability, loss of independence and mortality. The pathological hallmarks of Alzheimer’s disease are 
extracellular accumulation of amloid β (Aβ) peptide in the brain (“senile plaque”; SP) and by intraneuronal 
accumulation of hyperphosphorylated tau protein in neurofibrillary tangles (NFT). This is accompanied by 
synaptic and neuronal losses1. The prevalence of Alzheimer’s disease is about one percent at age 65 and 
increases dramatically to between 20-50% in those older than 85 years1. AD will have a dramatic increasing 
emotional and economic impact as the median population age increases, yet available treatments remain 
largely ineffective. 
 
1.1.2. NAD-replenishment therapy shows promise as neuroprotective therapy for AD 
Increasing evidence supports that boosting cellular levels of nicotinamide adenine dinucleotide (NAD) 
confers neuroprotective effects in both healthy aging and neurodegeneration2. NAD, which constantly 
shuttles between its oxidized (NAD+) and reduced (NADH) state, is an essential cofactor for metabolic redox 
reactions, including mitochondrial respiration. Furthermore, NAD+ is substrate to vital signaling reactions 
involved in DNA repair, histone- and other protein deacylation, and second messenger generation3. These 
reactions consume NAD+ at high rates, requiring constant replenishment via NAD biosynthesis. NAD levels 

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have been shown to decline with age and this is believed to contribute to age-related diseases3,4. Increasing 
the NAD replenishment rate (e.g., via supplementation of precursors), and/or enhancing the NAD+/NADH 
ratio (e.g., via caloric restriction) have shown beneficial effects on life- and healthspan in multiple model 
systems, and evidence of neuroprotection in models of neurodegeneration and other age-related diseases3–
5. Enhancing NAD replenishment could potentially help ameliorate several major processes implicated in the 
pathogenesis of AD, including mitochondrial respiratory dysfunction6, neuroinflammation7, epigenomic 
dysregulation8 and increased neuronal DNA damage9. 
NAD can be replenished via supplementation of nicotinamide riboside (NR), a vitamin B3 molecule and 
biosynthetic precursor of NAD3,10. NR has undergone extensive preclinical testing11 and is Generally 
Recognized as Safe (GRAS) for use in food products by the United States Food and Drug Administration12 and 
by the European Food  Safety Authority13. NR is well tolerated by adult humans, showing no evidence of 
toxicity with doses up to at least 2000 mg daily14. Trials in healthy individuals have shown that oral intake of 
1000 mg NR daily substantially elevates total levels of NAD and related metabolites in blood and muscle, 
boosts mitochondrial bioenergetics and decreases circulating inflammatory cytokines11,15–17. Moreover, 
evidence from cell and animal studies suggests that NR supplementation promotes healthspan and has 
neuroprotective effects in models of Cockayne syndrome18, noise-induced injury19,20, amyotrophic lateral 
sclerosis21, Parkinson’s disease22, and AD23,24. 
1.1.3. Phase-I evidence for NR-therapy in neurodegenerative diseases    
Two phase I studies of NR in PD have been completed, the NADPARK25 study and the NR-SAFE study26. These 
are described briefly below. 
I. The NADPARK study (ClinicalTrials.gov: NCT03816020), is a phase I randomized, double blinded trial, 
aiming to assess the tolerability, cerebral bioavailability and molecular effects of NR therapy in Parkinson’s 
disease (PD)25. A total of 30 individuals with newly diagnosed, drug-naïve PD were randomized to NR 500 mg 
x2/day or placebo for 30 days. The study showed promising results, which are briefly summarized below:  
1) NR is well-tolerated: NR 1000 mg per day has excellent compliance, tolerability and no signs of toxicity or 
adverse side effects in neurodegenerative disease.  
2) NR achieves brain penetration: In vivo measurement of cerebral NAD levels using phosphorus magnetic 
resonance spectroscopy (31P-MRS) of the brain showed a highly significant (paired t-test: P = 0.016) increase 
in cerebral NAD levels in the NR group, while no change was observed in the placebo group. Cerebral 
penetration was further validated by detecting the metabolite Me-2-PY in the CSF of participants receiving 
NR, but not placebo. While a significant NR-induced increase in cerebral NAD levels was detected at the group 
level, this effect was not uniform at the individual level. The magnitude of the cerebral NAD-increase showed 
high interindividual variation. Moreover, three out of 13 patients showed no evidence of cerebral NAD 
response, despite a clear peripheral metabolic response, confirming treatment compliance and an impact on 
the NAD metabolome, in CSF, blood, and muscle. Thus, the variable cerebral NAD response observed by 31P-
MRS may reflect interindividual variability in cerebral penetration and/or cerebral NAD metabolism. 3) NR is 
associated with clinical improvement of PD: NR was associated with a significant decrease in the total MDS-
UPDRS (I-III) score between visits (mean decrease: 2.33 ± 2.35; paired t-test: p = 0.017). 4) NR has a major 
impact on cerebral metabolism: 18F-fluorodeoxyglucose positron emission tomography (FDG-PET), 
performed at baseline and 30 days of treatment, revealed that NR altered cerebral metabolic activity. The 
analysis revealed a significant ordinal trend pattern (i.e., metabolic network), which was represented by the 
first principal component (PC1), accounting for 20.6% of the variance in the paired data. This novel NR-
related metabolic pattern (NRRP) was characterized by multiple regional metabolic changes, including 
bilateral metabolic reductions in the caudate and putamen, extending into the adjacent globus pallidus, and 
in the thalamus. Interestingly, the NRRP overlapped spatially with the Parkinson’s Disease-Related Pattern 
(PDRP)27, and  changes in NRRP expression in the NR group resulted in partial normalization of the striatal 
and thalamic hypermetabolism, typically characterizing the PD brain27. Furthermore, changes in NRRP 
expression in the NR group correlated significantly (r=-0.59, p = 0.026) with a decrease of the UPDRS ratings 
recorded at the time of PET (Fig 1F). These results indicate that NR ameliorates the cerebral metabolic pattern 
of PD, and this is associated with significant clinical improvement. 

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5) NR has widespread metabolic and regulatory effects: Metabolomics revealed a highly significant increase 
in NAD-related metabolites in blood, muscle and CSF (Fig 1G), indicating that NR supplementation boosts 
NAD metabolism across tissues. Intriguingly, RNA-sequencing in blood and muscle biopsy showed a highly 
significant (FDR < 10-8) upregulation of the mitochondrial, proteasomal and lysosomal pathways in the NR 
group. These findings indicate that NR supplementation increases both mitochondrial respiration and 
proteostasis – two hallmark pathogenic processes involved in AD9,28. 
 
II. The NR-SAFE study (ClinicalTrials.gov: NCT05344404)21 is a phase I randomized, double blinded trial, 
aiming to assess the safety, tolerability, and bioavailability of NR in PD at an oral dose of 3000 mg daily. A 
total of 20 individuals with PD were randomized, in a 1:1 ratio, to NR 1500 mg x2/day or placebo for 30 days. 
The study was concluded in July 2022 and is currently in preparation. The main results are summarized below: 
1) NR at a dose of 3000 mg daily is safe and well-tolerated: the treatment had excellent compliance, 
tolerability, and no signs of toxicity or clinically significant adverse effects in PD.  
2) NR, 3000 mg daily, augments NAD-metabolism: oral NR intake at a dose of 3000 mg daily induced a potent 
(up to 5-fold) and highly significant augmentation of NAD+ and NADP+ levels, as well as a clear increase of the 
redox ratio (NAD+/NADH) and the NADP+/NADPH ratio (Fig. 2A).  
3) NR, 3000 mg daily, is associated with clinical improvement of PD: the treatment was associated with a 
significant and substantial decrease in the total MDS-UPDRS (I-IV) score between visits (mean decrease 14 ± 
13.7; paired t-test: P = 0.01). No significant change was seen in the placebo group (Fig. 2B). The UPDRS change 
(delta) in the NR-group was significantly higher than that of the placebo (t-test, P =0.02) (Fig. 2C).   
 
 
Fig 1. Results of the NR-SAFE trial. A: Metabolomics were performed in snap-frozen blood from the NR-SAFE 
participants at baseline (V2) and after 30 days of treatment (V9). Oral NR intake at a dose of 3000 mg daily induced a 
potent (up to 5-fold) and highly significant augmentation of NAD+ and NADP+ levels, as well as a clear increase of the 
redox ratio (NAD+/NADH) and the NADP+/NADPH ratio. P-values indicate the results of paired t-tests in the NR-group. 
There were no significant changes in the placebo group.  B: Oral NR intake at a dose of 3000 mg daily was associated 
with a significant and substantial decrease in total MDS-UPDRS in the NR (mean decrease 14 ± 13.7; paired t-test: p = 
0.01), but not in the placebo (mean decrease 1 ± 12.7; paired t-test: p = 0.81). C: At the group level, the change (delta) 
in total MDS-UPDRS in the NR group was significantly (t-test, P = 0.02) larger than that of the placebo group (B).  
Key: V2: baseline; V9: after 30 days of treatment; NR: the group receiving NR 3000 mg daily; PL: the placebo group.  
 
 

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The results from our phase I trials suggest that NR holds promise as a neuroprotective therapy against 
neurodegenerative diseases. These results must now be replicated in AD. Several essential knowledge gaps 
(KG) must now be addressed in order to further develop NR towards an AD-drug, so that we may harness its 
full therapeutic potential and maximize clinical benefit and impact.  
 
KG1 – What is the Optimal Biological Dose (OBD) of NR in AD? We define the OBD of NR as the dose required 
to achieve an optimal neurometabolic response in AD, i.e., maximal cerebral NAD increase or optimal change 
in cerebral metabolic pattern, in the absence of toxicity.  
While our trials show that the treatment responses to NR in neurodegenerative diseases are clearly dose-
dependent, the OBD of NR in AD remains undetermined. Based on the results of NR-SAFE, it is likely that 
improved biological and clinical responses can be achieved by escalating the dose. Moreover, the NR OBD 
may not be universal. NADPARK showed that the NR-mediated increase in cerebral NAD-levels, and 
accompanying metabolic and clinical response,are not universal and vary across individuals25. The fact that 
all NR-recipients showed a robust metabolic response in blood, muscle and CSF, suggests that the variable 
cerebral NAD response may reflect interindividual variability in cerebral NAD metabolism (i.e., variation in 
the rate of NAD-synthesis or consumption). It is likely that such differences can be modulated by varying the 
substrate concentration (i.e., the intake dose of NR). This question is critical to address, so that NR-therapy 
can be correctly dosed and tailored to individual patients to achieve an optimal neurometabolic response. 
  
KG2 – Investigate whether NR has a symptomatic clinical effect in AD and assess its dose-responsiveness. 
The NADPARK and NR-SAFE studies showed that NR was associated with a clinical improvement, and this 
correlated significantly with the increase in cerebral NAD levels and brain metabolic network (NRRP) change. 
Furthermore, the clinical improval was more pronounced with  mg NR in the NR-SAFE study, compared to 
1000 mg in the NADPARK study. These findings suggest that NR may be ameliorating neuronal function in 
neurodegenerative disease resulting in symptom improvement. Exploring the dose-responsiveness of this 
effect will allow us to: 1) confirm the clinical impact of NR, 2) determine the optimal clinical dose of NR and 
3) allow us to account for symptomatic effects in neuroprotection trials.   
 
KG3 Determine the dose-dependence of the metabolic response to NR therapy. The NADPARK study 
showed that a dose of 1000 mg NR daily augmented the NAD metabolome in PBMC, muscle and CSF. The 
NR-SAFE study suggested that a dose of 3000 mg NR daily leads to a more potent augmentation of the NAD 
metabolome in blood. The relationship between NR dose and metabolic response needs to be further 
explored in order to determine optimal dosing regimens. 
 
KG4 – Determine whether  NR therapy enhance proteostasis in AD and characterize the dose dependence 
of this effect. Impaired proteostasis plays a central role in AD and other neurodegenerative disorders, 
including Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS).  Our transcriptomic analyses in the 
NADPARK study indicated that NR therapy may enhance proteostasis by inducing the expression of both 
proteasomal and lysosomal pathways. If confirmed, this would suggest that NR targets multiple major 
processes implicated in the pathophysiology of AD, including mitochondrial respiratory dysfunction, 
oxidative damage, lysosomal and proteasomal impairment, and neuroinflammation.  
 
KG5 – Does NR therapy influence histone acetylation status? Histone hyperacetylation has been shown in 
the brain of individuals with AD8. Increasing neuronal NAD levels would boost the activity of the NAD-
dependent histone deacetylases of the sirtuin family, potentially ameliorating histone hyperacetylation in 
AD.  
 
KG6 – Does NR decrease neuroinflammation? While it is known that NR has anti-inflammatory properties in 
peripheral tissues21, the results of the NAD-PARK trial suggest it also downregulates multiple inflammatory 
cytokines in the central nervous system. If confirmed this would be of importance for AD and other 

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neurodegenerative and neuroinflammatory disorders.  
 
KG7 – Determine whether NR therapy alters methylation metabolism and characterize any dose 
dependence of such an effect. In theory, NAD replenishment via NR administration could decrease/deplete 
the cellular methylation capacity. NR boosts the NAD-metabolome, leading to increased production of the 
degradation product nicotinamide (NAM), which is eliminated via methylation to MeNAM, Me-2-PY, and Me-
4-PY, and excreted in the urine.  Synthesis of Me-Nam requires the methyl-donor S-adenosylmethionine 
(SAM). This, in turn, could limit SAM availability for other essential methylation reactions, such as DNA and 
histone methylation, and neurotransmitter synthesis, including dopamine29. Thus, in theory, NR would cause 
an increased consumption of SAM, limiting methylation reactions and generating higher levels of 
homocystein.  Such a phenomenon would be a particular concern for the ~60% of the population that carries 
MTHFR variants that reduce the efficiency of methylation. The NADPARK study showed no change in serum 
homocystein levels, or any other evidence of methylation depletion associated with NR 1000 mg daily. It is, 
however, unknown whether such effects may occur with higher NR doses.  
 
KG8 – Determine how orally ingested NR interacts with the microbiome in AD. Current evidence suggests 
that the gut microbiome may be involved in the pathogenesis of AD and that individuals with AD host a 
different microbiome composition compared to neurologically healthy individuals. It is possible that NR 
therapy may beneficially affect the gut microbiome in AD restoring normal patterns. On the other hand, it is 
also possible that variation in the gut microbiome may affect local metabolism of NR and absorption in the 
bloodstream. These effects have not been studied.  
 
KG9 – Determine whether NR therapy influences gastrointestinal (GI) motility in AD. GI dysmotility due to 
dysfunction and degeneration of both central and local neuronal populations controlling GI motility occurs 
in AD. It is possible that NR may improve the function of these neurons and preserve them against the 
neurodegenerative process.  
 
To address these pertinent questions, we will conduct N-DOSE AD, a multi-center randomized double-
blinded placebo-controlled trial to assess the optimal biological dose for NR in AD.  
  
1.2 
Background - Therapeutic Information 
NR (Niagen®, Chromadex) is fully approved for human use and no evidence of toxicity has been found. NR has 
undergone extensive preclinical testing16 and is Generally Recognized as Safe (GRAS) for use in food products 
by the United States Food and Drug Administration31 and by the European Food Safety Authority32. As 
mentioned above multiple animal studies suggest that NR has strong neuroprotective effects as well as our 
NADPARK study showing possible therapeutic effects in neurodegenerative disease. NR is well tolerated with 
no evidence of toxicity in adult humans with doses up to at least 3000 mg daily11,13,14,25,26,30. This evidence 
includes our recently concluded safety and tolerability trial NR-SAFE (clinicaltrials.gov: NCT05344404), which 
revealed no adverse events of clinical relevance with 3000mg NR daily for 30 days (manuscript in 
preparation). We therefore propose that dosages up to 3000 mg is highly unlikely to cause toxicity. Niagen 
and placebo will be provided from Chromadex, https://chromadex.com/. Active study drug capsules will 
contain 250 mg NR. Placebo will contain microcrystalline cellulose, which will be identical in appearance and 
taste.  
 
1.3 
Pre-Clinical & Clinical Experience with Investigational Product (IP)  
NR (Niagen®, Chromadex) is fully approved for human use and no evidence of toxicity has been found. Dose 
selection is based on pharmacokinetic studies of oral bioavailability in humans33. See details under section 
1.1.2 above. 

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1.4 
Rationale for the Study and Purpose 
Our phase I trial (NADPARK) showed that NR holds great promise as a potential neuroprotective, disease-
modifying therapy for neurodegenerative diseases. Encouraged by these findings, this project aims to 
determine the optimal biological dose of NR in AD and to further explore its neuroprotective potential. We 
propose that oral administration of the NAD precursor NR (Niagen®, Chromadex) in increasing dosages up to 
3000 mg will show a dose-responsive improvement in the cerebral metabolism in AD patients and show signs 
of improved mitochondrial function, histone acetylation, neuroinflammation and clinical symptoms.  
The outcomes of this project will take us several steps closer to developing NR into an AD-drug, so that we 
may harness its full therapeutic potential and maximize its clinical benefit and impact. 
 
2 STUDY OBJECTIVES AND RELATED ENDPOINTS 
DS-group refers to the dose-stable group, i.e. participants receiving NR 1000 mg daily for 3 months. DE-group 
refers to the dose-escalation group, i.e. participants receiving NR 1000 mg in an escalating dose with 1000 
mg from baseline to week 4, 2000 mg from week 4 to week 8 and 3000 mg from week 8 to week 12. PL-group 
refers to the placebo group. 
Objective 
Endpoint 
Primary 
To compare the effect of orally administered 
nicotinamide riboside (NR), escalated to 1500 
mg twice per day (3000 mg/day) in the dose-
escalation group (DE-group) - versus stable 
dosing of 500 mg twice per day (1000 
mg/day) in the dose-stable group (DS-group) 
on cerebral NAD-levels, at week 12. 
Change in cerebral NAD/ATP-α ratio 
measured by 31 Phosphorus magnetic 
resonance spectroscopy (31P-MRS) in the 
posterior brain (encompassing the occipital, 
parietooccipital and posterior parts of the 
temporal cortex). 
Secondary 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in cerebral NAD 
levels from baseline to weeks 4, 8 and 12. 
Change in cerebral NAD/ATP-α ratio 
measured by 31 Phosphorus magnetic 
resonance spectroscopy (31P-MRS) in the 
posterior brain (encompassing the occipital, 
parietooccipital and posterior parts of the 
temporal cortex). 
To compare the effectiveness of orally 
administered nicotinamide riboside (NR) 
1500 mg twice per day versus 500 mg twice 
per day in augmenting the NAD-metabolome 
in the central nervous system (CNS) at week 
12. 
 
 
Change in the cerebrospinal fluid (CSF) levels 
of NAD or other metabolites of the NAD 
metabolome*, measured by LC-MS. 
Exploratory 
Neuroimaging 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
NR related metabolic pattern (NRRP) 
expression at week 12. 
Change in NRRP expression, measured by 
FDG-PET. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in NRRP expression 
from baseline to weeks 4, 8 and 12. 
Change in NRRP expression, measured by 
FDG-PET. 
To compare the effect of orally administered 
NR DE-group versus DS-group on the AD-
related pattern (ADRP) expression at week 12. 
Change in ADRP expression, measured by 
FDG-PET. 

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To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in ADRP expression 
from baseline to weeks 4, 8 and 12. 
Change in ADRP expression, measured by 
FDG-PET. 
Metabolism & molecular markers 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
NAD metabolome* in the blood, urine and 
central nervous system (CNS) at week 12. 
Change in levels of NAD metabolites in blood, 
urine and CSF, measured by HPLC-MS and/or 
the NADMed method. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in the NAD 
metabolome* in blood and urine from baseline 
to weeks 4, 8 and 12. 
Change in levels of NAD metabolites in blood 
and urine, measured by HPLC-MS and/or the 
NADMed method. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on serum 
and CSF inflammatory markers at week 12. 
Change in inflammatory cytokines in serum 
and CSF, measured by ELISA. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in serum 
inflammatory markers from baseline to weeks 
4, 8 and 12. 
Change in inflammatory cytokines in serum, 
measured by ELISA. 
Clinical – cognitive & non motor symptom severity, quality of life 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
clinical severity of AD symptoms at week 12. 
Change in the total ADAS-Cog 13 score, CDR 
sum of boxes, MoCA score, TMT. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and change in clinical severity of 
PD symptoms from baseline to weeks 4, 8 and 
12.                
Change in the total ADAS-Cog 13 score, CDR 
sum of boxes, MoCA score, TMT. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on ADL 
severity in AD  at week 12. 
Change in IADL and PADL scores. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and change in ADL scores in  AD 
from baseline to weeks 4, 8 and 12.                
Change in IADL and PADL scores.. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on  
depressive symptoms in AD at week 12. 
Change in MADRS score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and depressive symptoms in AD 
from baseline to weeks 4, 8 and 12.                
Change in MADRS score. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
neuropsychiatric symptoms in AD at week 12. 
Change in the NPI-Q score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and change in neuropsychiatric 
symptoms in AD from baseline to weeks 4, 8 
Change in the NPI-Q score. 

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and 12. 
Hypothesis-generating or resource-dependent endpoints (may be reported in follow-up 
or secondary publications). 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on gene 
expression at week 12. 
Change in gene expression, measured by RNA 
sequencing (RNAseq). 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in gene expression 
from baseline to weeks 4, 8 and 12. 
Change in gene expression, measured by RNA 
sequencing (RNAseq). 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
protein expression at week 12. 
 Change in protein levels, measured by LC-MS. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in protein expression 
from baseline to weeks 4, 8 and 12. 
Change in protein levels, measured by LC-MS. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on serum 
and CSF inflammatory markers at week 12. 
Change in inflammatory cytokines in serum 
and CSF, measured by ELISA. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in serum 
inflammatory markers from baseline to weeks 
4, 8 and 12. 
Change in inflammatory cytokines in serum, 
measured by ELISA. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
histone acetylation in AD at week 12. 
Change in histone panacetylation, measured 
by immunoblotting. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in histone 
acetylation in AD from baseline to weeks 4, 8 
and 12. 
Change in histone panacetylation, measured 
by immunoblotting. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
H3K27 and H4K16 histone acetylation in AD at 
week 12. 
Changes in levels of H3K27 and H4K16 
acetylation, measured by immunoblotting. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in on H3K27 and 
H4K16 histone acetylation in AD from baseline 
to weeks 4, 8 and 12. 
Changes in levels of H3K27 and H4K16 
acetylation, measured by immunoblotting. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
genomic distribution of H3K27 and H4K16 
histone acetylation in AD at week 12. 
 Change in the genomic distribution of H3K27 
and H4K16 acetylation, measured by 
chromatin immunoprecipitation sequencing 
(ChIPseq). 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in the genomic 
distribution of H3K27 and H4K16 histone 
acetylation in AD from baseline to weeks 4, 8 
and 12. 
Change in the genomic distribution of H3K27 
and H4K16 acetylation, measured by 
chromatin immunoprecipitation sequencing 
(ChIPseq). 

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To compare the effect of orally administered 
NR in the DE-group versus DS-group on folate 
and one-carbon metabolism in AD at week 12. 
Change in folate and one-carbon metabolites 
in blood and CSF, measured by HPLC-MS. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in folate and one-
carbon metabolism in AD from baseline to 
weeks 4, 8 and 12. 
Change in folate and one-carbon metabolites 
in blood, measured by HPLC-MS. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
methyl donors in AD at week 12. 
Change in methyl-donors (e.g., SAM), 
measured by HPLC-MS, in the blood and/or 
CSF. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in methyl-donors in 
AD from baseline to weeks 4, 8 and 12. 
Change in methyl-donors (e.g., SAM), 
measured by HPLC-MS, in the blood. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on DNA 
methylation at week 12. 
Change in level and genomic distribution of 
DNA methylation, measured by Illumina 
Infinium MethylationEpic kit. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 
mg per day) and changes in methyl-donors in 
AD from baseline to weeks 4, 8 and 12. 
Change in level and genomic distribution of 
DNA methylation, measured by Illumina 
Infinium MethylationEpic kit. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on 
synthesis of neurotransmitters in AD at week 
12. 
Change in neurotransmitters in CSF, measured 
by HPLC-MS. 
Determine whether NR-therapy affects the gut 
microbiome in a dose-responsive manner at 
week 12. 
Change in gut microbiome composition, 
measured by metagenomics in fecal samples. 
To compare the effect of orally administered 
NR in the DE-group versus DS-group on the 
gut metabolome at week 12. 
Change in fecal metabolomics, measured by 
LC-MS in fecal samples. 
Safety 
To determine the safety and tolerability of NR 
at a dose of 1000 mg, 2000 mg, and 3000 mg 
per day in AD. 
Number and severity of adverse events from 
baseline to week 12 across treatment groups 
and NR dose levels. 
*The NAD metabolome is comprised of: Nicotinamide adenine dinucleotide oxidized (NAD+), Nicotinamide adenine 
dinucleotide reduced (NADH), NAD+/NADH ratio, total NAD (sum of NAD+ and NADH), Nicotinamide adenine 
dinucleotide phosphate oxidized (NADP+), Nicotinamide adenine dinucleotide phosphate reduced (NADPH), 
NADP+/NADPH ratio, total NADP (sum of NADP+ and NADPH, 1-methyl nicotinamide (Me-Nam), nicotinic acid-adenine 
dinucleotide (NAAD), N1-methyl-2-pyridone-5-carboxamide (Me-2-PY), Nicotinamide (Nam), Nicotinamide N-oxide 
(Nam N-oxide), ADP-ribose (ADPR), Nicotinic acid riboside (NAR), Nicotinamide riboside (NR), Nicotinamide 
mononucleotide (NMN), Nicotinic acid (NA).  
 
 
2.1 
Primary Endpoint Measure  
Change in cerebral NAD level, measured by 31P-MRS. 
 
31P-MRS assesses the levels of key-energy metabolites in the brain, including NAD, ATP, free inorganic 
phosphate (Pi), and phosphocreatine, and others, as previously shown25.  Using this method, we will assess 

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total brain NAD levels normalized to ATP-α levels (i.e., the NAD/ATP-α ratio) in the posterior brain - 
encompassing the occipital, parietooccipital and posterior parts of the temporal cortex. 
 
 
 
2.2 
Secondary Endpoint Measure 
Change in the CSF level of NAD or other metabolites of the NAD metabolome in CSF, measured by HPLC-MS. 
 
Using high performance liquid chromatography mass spectrometry (HPLC-MS), we will measure the NAD 
metabolome in CSF, including the following specific measures: Nicotinamide adenine dinucleotide oxidized 
(NAD+), Nicotinamide adenine dinucleotide reduced (NADH), NAD+/NADH ratio, total NAD (sum of NAD+ and 
NADH), Nicotinamide adenine dinucleotide phosphate oxidized (NADP+), Nicotinamide adenine dinucleotide 
phosphate reduced (NADPH), NADP+/NADPH ratio, total NADP (sum of NADP+ and NADPH, 1-methyl 
nicotinamide (Me-Nam), nicotinic acid-adenine dinucleotide (NAAD), N1-methyl-2-pyridone-5-carboxamide 
(Me-2-PY), Nicotinamide (Nam), Nicotinamide N-oxide (Nam N-oxide), ADP-ribose (ADPR), Nicotinic acid 
riboside (NAR), Nicotinamide riboside (NR), Nicotinamide mononucleotide (NMN), Nicotinic acid (NA).  
 
 
 
2.3 Exploratory  Endpoint Measures 
 
Change in the following parameters listed below: 
 
NR related pattern (NRRP), an ordinal trend pattern (i.e., metabolic network), associated with NR treatment 
identified in the NADPARK study25, measured by 18F-fluorodeoxyglucose positron emission tomography (FDG-
PET).  
AD related pattern (ADRP), an ordinal trend pattern (i.e., metabolic network), associated with AD, measured 
by 18F-fluorodeoxyglucose positron emission tomography (FDG-PET). 
The ADAS-Cog 13 (Alzheimer’s Disease Assessment Scale – Cognitive Subscale, 13-item version) is a widely 
used tool for assessing cognitive function in individuals with Alzheimer’s disease and other forms of 
dementia. The total score ranges from 0 to 85, with higher scores indicating greater cognitive impairment. 
Clinical Dementia Rating scale sum of boxes (CDR-SB). The CDR integrates assessments from 3 domains of 
cognition (memory, orientation, judgment/problem-solving) and 3 domains of function (community affairs, 
home/hobbies, personal care). Following caregiver interview and systematic participant examination, the 
rater assigns a score describing the participant's current performance level in each of these domains of life 
functioning. The "Sum of boxes" scoring methodology (CDR-SB) sums the score for each of the 6 domains and 
provides a value ranging from 0 to 18 with higher scores indicating greater impairment. Positive change from 
baseline indicates greater impairment. 
The Montreal Cognitive Assessment (MoCA) scale. The MoCA scale (minimum score = 0, maximum score = 
30) and a dichotomized cut-off score for normality of 26 or over. High scores indicate less cognitive 
impairment than low scores. 

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The Trail Making Test (TMT). The TMT is a timed test and the goal is to complete the test as accurately and 
as quickly as possible. Raw scores are reported in seconds to complete the test. For Part B, an average score 
is 75 seconds and a deficient score is greater than 273 seconds. 
 The Lawton Instrumental Activities of Daily Living (IADL) Scale. The IADL scale consists of 8 items providing 
information about telephone use, preparing food, shopping, doing daily household chores, doing laundry, 
using transport, medication managing, and managing money. Scores range from 0 (dependent) to 8 
(independent). 
 The Physical Self-Maintenance Scale (PSMS). The PSMS includes 6 items, testing the following areas: toilet 
use, eating, dressing, physical appearance, deambulation and bath. The PSMS ranges from 1 to 30, with 
higher scores indicating WORSE functioning. 
The Neuropsychiatric Inventory brief questionnaire form (NPI-Q). The NPI-Q measures the burden of 12 
neuropsychiatric 
symptoms 
of 
dementia: 
delusions, 
hallucinations, 
agitation/aggression, 
depression/dysphoria, anxiety, elation/euphoria, apathy/indifference, disinhibition, irritability/lability, 
motor disturbance, nighttime behaviors, and appetite/eating. Symptom severity is rated on a 3-point scale 
with higher scores indicating worse symptoms. Minimum score would be 0 and maximum score would be 36. 
The Montgomery-Asberg Depression Rating Scale (MADRS). MADRS is a clinician-rated scale designed to 
measure depression severity and detects changes due to antidepressant treatment. The scale consists of 10 
items, each of which is scored from 0 (item not present or normal) to 6 (severe or continuous presence of 
the symptoms), for a total possible score of 60. Higher scores represent a more severe condition. 
NAD metabolome. Using liquid chromatography mass spectrometry (HPLC-MS) and/or the NADMed 
method, we will measure the NAD metabolome in whole blood (and/or PBMCs), CSF and urine including the 
following specific measures: Nicotinamide adenine dinucleotide oxidized (NAD+), Nicotinamide adenine 
dinucleotide reduced (NADH), NAD+/NADH ratio, total NAD (sum of NAD+ and NADH), Nicotinamide adenine 
dinucleotide phosphate oxidized (NADP+), Nicotinamide adenine dinucleotide phosphate reduced (NADPH), 
NADP+/NADPH ratio, total NADP (sum of NADP+ and NADPH, 1-methyl nicotinamide (Me-Nam), nicotinic acid-
adenine dinucleotide (NAAD), N1-methyl-2-pyridone-5-carboxamide (Me-2-PY), Nicotinamide (Nam), 
Nicotinamide N-oxide (Nam N-oxide), ADP-ribose (ADPR), Nicotinic acid riboside (NAR), Nicotinamide 
riboside (NR), Nicotinamide mononucleotide (NMN), Nicotinic acid (NA).  
 
Gene and protein expression levels in PBMC, measured by RNA sequencing (RNAseq) and proteomics (HPLC-
MS), respectively. 
 
Levels of inflammatory cytokines in serum and CSF, measured using enzyme-linked immunosorbent essay 
(ELISA). 
 
Levels of histone panacetylation, and levels and genomic distribution of H3K27 and H4K16 acetylation in 
PBMC, measured by immunoblotting and chromatin immunoprecipitation sequencing (ChIPseq). 
 
Levels of methyl-donors (e.g., SAM) in blood and/or CSF, measured by HPLC-MS.  
 
Levels of neurotransmitters in CSF, measured by HPLC-MS. 
 
Levels of one carbon metabolism metabolites, measured by HPLC-MS metabolomics in PBMC and CSF. 
 
Levels and genomic distribution of DNA methylation, measured by the Illumina Infinium MethylationEPIC 
Kit.  
  
Gut microbiome composition, assessed by metagenomics in fecal samples.  

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Fecal metabolomics, including fatty acid profiling, assessed by HPLC-MS in fecal samples. 
 
● Determine whether NR-therapy affects increase in the NRRP in a dose-responsive manner. 
● Determine whether NR-therapy affects increase in the ADRP in a dose-responsive manner. 
● Determine whether NR-thearpy augments the NAD metabolome in the blood, urine and CSF in a 
dose-responsive manner. 
● Determine whether NR-therapy improves cognitive dysfunction in AD in a dose-responsive 
manner. 
● Determine whether NR-therapy improves ADL in AD in a dose-responsive manner. 
● Determine whether NR-therapy affects neuropsychiatric symptoms in AD in a dose-responsive 
manner. 
 
 
 
 
 
 
 

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3 OVERALL STUDY DESIGN 
This is a multi-center, phase II, double blinded, randomized, placebo controlled dose-optimization study. 
Study Period 
 
Estimated date of first patient enrolled: 01.10.2022 
Anticipated recruitment period: 01.10.2022 – 14.03.2025 
Estimated date of last patient completed: 30.06.2025 
Treatment Duration: 
12 weeks.  
3.1 Study design 
N-DOSE AD is a multi-center, phase II, double blinded, randomized, placebo-controlled dose-optimization 
clinical trial with a dose escalation design. Patients with AD (n = 80) who fulfill participation criteria at 
screening (see section 4) will be randomized (1:3) into one of two three groups and followed for a total of 3 
months: 1) The placebo group (PL-group, n = 20) will receive placebo for the duration of the study. 2) The 
NR 1000 mg group (SD-group, n=20) will receive NR 1000 mg (500 mg x 2) per day for the duration of the 
study. 3) The NR dose escalation group (DE-group, n=40) will receive first 1000 mg (500 mg x 2) NR per day 
for 30 days, then 2000 mg (1000 mg x 2) NR per day for 30 days, and finally 3000 mg (1500 mg x 2) NR per 
day for 30 days (Fig. 3). The selected dose range is within safety limits for healthy humans (see section 1.3). 
After eligibility screening, eligible participants will be randomly assigned to one of two study groups and 
assessed at baseline (Visit-1, V1) and three more visits (Visit-2, Visit-3 and Visit-4; V2, V3, V4) spaced 30 days 
apart (Fig. 3).  Participants and investigators will be blinded to treatment group. Participants will be followed 
for a total of 90 days. All participants will be recruited from the Neuro-SysMed Center, HUS and Haraldsplass 
Deaconess Hospital. 
 
Fig 2. N-DOSE AD study design. A total of 80 patients will be recruited in three arms. Patients will be assessed at baseline (V1) and 
three additional visits, at 4 (V2), 8 (V3) and 12 (V4) weeks. 

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4 STUDY POPULATION 
4.1 Selection of Study Population 
The study setting is at the Geriatrics outpatient clinic at Haraldsplass Deaconess Hospital.   
4.2 Number of Patients 
A total of n=80 patients will be included in this study and randomized to placebo (PL-group, n=20), NR 500 
mg twice per day (DS-group, n=20), or NR escalating dose (DE-group, n=40), starting at 500 mg twice per day, 
increasing to 1000 mg twice per day, and finally increasing to 1500 mg twice per day. 
4.3 Inclusion Criteria 
The following condition must apply to the prospective patient at screening prior to receiving study agent: 
● Diagnosis of probable  AD according to the core clinical criteria updated in the NIA and Alzheimer’s 
Association guidelines. 
● Biomarker evidence consistent with AD neuropathologic change, defined by CSF markers31. 
● Diagnosed with AD within two years from enrolment. 
● CDR32 0.5-1 (inclusive) at enrolment.  
● Age 50 to 85 years (inclusive) at the time of enrollment. 
● A study partner (i.e. a family member or a friend) able to study data and assist the participant in the study 
drug administration, i.e. contact ≥ 3 times weekly. 
● Capacity to provide written informed consent for study participation defined as Montreal 
Cognitive Assessment (MoCA)33 score ≥ 16 or Mini Mental State Evaluation. 
(MMSE) score ≥ 2034. MMSE or MoCA must have been performed within 6 months prior to baseline. If 
there is any doubt regarding the participants capacity to give 
informed consent we will ask for an independent evaluation by a consultant clinician who is 
not associated with the study. 
● Cholinesterase inhibitors and memantine can be used if stable for 8 weeks prior to 
baseline visit. 
● Able to undergo lumbar punction. 
● Able to undergo MRI 
 
4.4 Exclusion Criteria 
Patients will be excluded from the study if they meet any of the following criteria: 
● Diagnosis of dementia other than probable AD. 
● Comorbidity that precludes study participation or data interpretation.  
● Any psychiatric disorder that would interfere with compliance in the study. 
● Any severe somatic illness that would interfere with compliance and participation in the study.  
● Use of high dose vitamin B3 supplementation within 30 days of enrolment. 
● Metabolic, neoplastic, or other physically or mentally debilitating disorder at baseline visit. 
● Current treatment with Oral Anticoagulation Therapies 
● Implants that preclude MRI examinations, e.g. DBS, pacemaker  
 

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5 TREATMENT 
For this study NR (Niagen®, Chromadex) is defined as the Investigational Product(s) (IP). IP includes also 
active comparator and placebo.  
5.1 Drug Identity, Supply and Storage 
NR (Niagen®, Chromadex) and placebo will be manufactured and provided from Chromadex. Both NR and 
Placebo will be prepared as identical capsules. The drug has marketing consent in Norway but is not 
registered by Statens legemiddelverk (SLV)/Direktoratet for medisinske produkter (DMP) as a medical drug, 
but as a supplement. The NR and placebo have a 1-year expiry date. Both the NR and placebo will be stored 
in room temperature with temperature <25 degrees (according to instructions given by Chromadex).  
5.2 Dosage and Drug Administration  
Each NR capsule  contains 250mg.. To keep the study fully blinded, all participants will receive the same 
number of daily capsules irrespective of which treatment group they belong to. To achieve this, NR capsules 
will be combined with placebo capsules as necessary (see below): 
● Patients in the NR 1000mg group will administer orally [2 NR capsules (500mg) +  4 placebo 
capsules] x 2 times daily (1000mg NR daily in total).  
● Patients in the NR dose escalation group will administer orally the following doses: 
o Weeks 0-4: [2 NR capsule (500mg) + 4 placebo capsules] x 2 times daily (1000mg NR daily in 
total).  
o Weeks 5-8: [4 NR capsule (1000mg) + 2 placebo capsule] x 2 times daily (2000mg daily 
total).  
o Weeks 9-12: [6 NR capsules (1500mg) + 0 placebo capsule] x 2 times daily (3000mg daily 
total).  
● The placebo group will administer orally [6 placebo capsules] x 2 times daily. 
There is no specified time of day the dosages should be taken, only that they should be taken with about 12 
hours apart, if possible. If a dose is missed, the patient can take the missed dose as soon as it is 
remembered, provided it is shorter time to the missed dose than the next scheduled dose. There are no 
restrictions with respect to combining the dose with other medication and/or food. The study medication 
is to be taken every day during the treatment period, including prior to study visits.  
5.3 Duration of Therapy 
Therapy duration for the study is 3 months (12 weeks). 
5.4 Cognitive Therapy During Screening and IP treatment period 
Eligible and consenting men and women with AD may use standard treatments with Cholinesterase inhibitors 
and memantine can be used if stable for 8 weeks prior to 
screening and baseline visits. The treatment regime will then be frozen and remain unchanged for the study 
period (3 months). At the end of study visit (month 3), the physician determines (yes/no) whether the patient 
is still adequately treated for his/her AD with their current standard treatment.  
 

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5.5 Concomitant Medication 
There are no restrictions on any other use of medications. All patients should use medications prescribed 
prior to enrollment in the study. There are no restrictions with respect to starting new medications that are 
necessary for the patient. 
The Patient should not take any vitamin B3 supplements for the duration of the study.    
All concomitant medication (incl. vitamins with the exception of vitamin B3, herbal preparation and other 
“over-the-counter” drugs) used by the patient will be recorded in the patient’s file and CRF. 
5.6 Subject Compliance 
Patient compliance will be determined based on self-report at study visits (using information from the study 
participant and study partner). A pill count of remaining medication will be performed when providing new 
study medication and at the end of the study.  
5.7 Drug Accountability 
The responsible site personnel will confirm receipt of study drug and will use the study drug only within the 
framework of this clinical study and in accordance with this protocol. Receipt, distribution, return, and 
destruction (if any) of the study drug must be properly documented according to the sponsor’s agreed and 
specified procedures.  
Study drugs are stored locally at the study site and distributed by the study nurse upon registration in CRF. 
Remaining study drug is returned for pill count.   
5.8 Drug Labeling 
The investigational product will have a label permanently affixed to the outside and will be labeled according 
with ICH GCP and national regulations, stating that the material is for clinical trial / investigational use only 
and should be kept out of reach of children.  
 
Label will include: 
● Patient’s initials 
● Study number (CRF number) 
● Date dispensed 
 
5.9 Subject Numbering 
 
At the screening visit, patients will be sequentially allocated a patient study number. The subject will be 
identified by this patient study number for the remainder of the study. Once a patient number has been 
assigned, no attempt will be made to use that number again.  
The patient study number will be in the following format: XX-YYY where XX is the study site number and 
YYY is the patient id number. If a patient number is allocated incorrectly, no attempt will be made to 
remedy the error once study treatment has been dispensed. Any replacement patients will be given the 
next patient number in the sequence. 
 
6 STUDY PROCEDURES 
 
 

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6.1 Flow Chart 
Table 1.  Trial flow chart  
 
Screening Period 
Treatment Period 
Study Visit: 
End of study 
visit 
Event 
First 
Screening 
Next/Last 
screening1 
Visit-1 
Baseline1 
Visit-2 
Visit-3 
Visit-4 
Time 
 
 
Week 0 
Week 4  
Week 8 
Week 12 
Informed consent 
X 
 
 
 
 
 
Informed consent biobank, 
optional 
X 
 
 
 
 
 
AD Clinical diagnosis Criteria 
X 
 
X 
 
 
 
Inclusion/exclusionEvaluation 
X 
 
X 
 
 
 
Medical history1 
X 
 
X 
 
 
 
Neurologic exam2 
X  
 
X 
X 
X 
X 
Physical Examination3 
X 
 
X 
X 
X 
X 
Body height 
 
 
X 
 
 
 
Body weight and BMI 
 
 
X 
X 
X 
X 
Vital signs4 
 
 
X 
X 
X 
X 
Record of concomitant 
medication 
X 
 
X 
X 
X 
X 
MoCA5 
X 
 
X 
X 
X 
X 
CDR5 
X 
 
X 
X 
X 
X 
ADAS-COG 13 
 
 
X 
X 
X 
X 
Trail Making Test (TMT)5 
X 
 
X 
X 
X 
X 
31P-MRS, 1H-MRSand FDG-
PET imaging 
 
 
X 
X 
X 
X 
MADRS5 
X 
 
X 
X 
X 
X 
NPI-Q, and IADL and PADL5 
X 
 
X 
X 
X 
X 
Dietary registration (3 days 
prior to visit) 
 
 
X 
 
 
X 
Routine blood tests6 
 
 
X 
X 
X 
X 
hCG7 
X 
 
 
 
 
 

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Blood for biobanking8 
 
 
X 
X 
X 
X 
Cerebrospinal fluid collection9 
X 
 
 
 
 
X 
Fecal sample collection 
 
 
X 
 
 
X 
Urine sample collection 
 
 
X 
X 
X 
X 
Treatment (IP) dispensation10 
 
 
X 
X 
X 
 
Cognitive treatment stable11 
 
 
X 
 
 
X 
Adverse event 
 
 
 
X 
X 
X 
Superscripts refer to the specifications below. 
Specifications 
1. Medical history includes: family history of neurological illness (what and who), family history of 
dementia (what and who), smoking history (period, pack-years), first AD symptoms, months since first 
AD symptoms,. 
2. General Neurological examination 
3. Heart and lung auscultation, abdominal palpation, any other examination dictated by patient’s 
condition/symptoms 
4. Blood pressure, pulse, temperature 
5. Not repeated at baseline if conducted at screening within 4 weeks prior to the baseline visit. 
6. CRP, ALAT, ASAT, GT, bilirubin, ALP, creatinine, urea, RBC, Hb, WBC with differential, platelets, CK, FT4, 
TSH, B12, folic acid, homocysteine, methylmalonic acid, sodium, potassium, glucose.  
7. Women of childbearing potential will also have a pregnancy test performed. 
8. EDTA blood, snap-frozen EDTA blood, PAXgene, platelet isolation, serum (see lab manual for details). 
9. CSF, including Amloid-β and Tau protein levels to verify the AD diagnosis. 
10. To ensure correct dosages during dose escalation and if necessary to resupply. 
11. The patient has to be stable if on a standard cognitive treatment for 8 weeks prior to baseline. There 
should not be more than 3 months from last screening to baseline visit.  
 
 
6.2 By Visit 
6.2.1 Screening Visits/ Before start of Investigational Product (IP) 
The first screening visit aims to determine if the patient is eligible to be included in the study. A full physical 
examination, anamnestic medical history, cognitive testing and lumbar puncture to establish AD pathology 
is performed. If the patient fulfills the inclusion/exclusion criteria and gives informed consent, standard 
treatment with cholinesterase inhibitors and memantine must be stable for 8 weeks prior to screening and 
baseline visits. 
If the patient fulfills the inclusion/exclusion criteria, the patient is called in for the baseline study visit (week 
0 study visit). The Baseline study visit should be within 8 weeks from the time the lumbar puncture was 
performed. There should not be more than 3 months from last screening to baseline visit.  

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Screening checklist:  
1. Patient signed informed consent for N-DOSE AD 
2. Patient signs informed consent for consent for storage and analysis of biological material 
(“Samtykke for lagring av biologisk material i biobank for demens og aldring”) 
3. Physical examination (general neurological examination) at first screening 
4. MoCA 
5. Clinical AD diagnosis Criteria 
6. Record current use of medication. Advise to stop any use of vit B3 supplements 
7. Plan/conduct Lumbar puncture to verify AD diagnosis 
8. If the patient is ready for enrollment, they are referred to baseline study visit (including MRI and 
PET).   
9. The study partner is given the 3-day dietary registration form along with instructions on how to fill 
it out and deliver to the study nurse at baseline. 
10. Give instructions on how to collect urine and fecal sample before the baseline visit. 
  
6.2.2 Baseline/Week 0 
1. At the first study visit the investigator needs to verify the informed consent for the study and offer 
the subject to sign the informed consent for storage and analysis of biological material (Samtykke for 
lagring av biologisk materiale i FORSKNINGSBIOBANKEN FOR ALDRING, DEMENS OG NEVROLOGI).  
2. Investigator verifies anamnestic information gathered at screening, current use of medication and 
medical history. 
3. Investigator verifies fulfillment of inclusion and exclusion criteria.  
4. The study identification number is assigned (see section 5.9).  
5. Study medication is dispensed to the subject by the study nurse and the patient is reminded to take 
the study medication every day including at the morning of each visit. 
6. The following clinical examination are performed by the study nurse or investigator (see flowchart 
in section 6.1):  
a. Vital signs  
b. Hight, body weight and BMI 
c. MoCA 
d. ADAS-COG  
e. CDR 
f. Trail Making Test (TMT) 
g. MADRS  
h. NPI-Q 
i. 
IADL and PADL  
7. Routine blood tests are done (see flowchart in section 6.1) 
8. Samples for biobanking are taken (see flowchart in section 6.1): 
a. EDTA whole blood 
b. Snap-frozen whole blood 
c. PAXgene blood for RNA 
d. Platelets  

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e. Serum 
f. Fecal sample 
g. Urine sample 
9. Imaging is conducted 
a. 31P-MRS & 1H-MRS 
b. FDG-PET 
10. All data gathered during study visits is either written into the electronic journal or filled out in paper 
format with appropriate date and signatures. Each patient will have their own folder with gathered 
clinical data. The clinical tests are recorded in paper format. 
11. The patient's study partner is given the 3-day dietary registration form along with instructions on 
how to fill it out and deliver to the study nurse at the next visit. 
12. The patient and study partner is instructed to take the study medication every day for the remainder 
of the study. The study medication or placebo is to be taken as outlined in section 5.2. The capsules 
are taken in the morning and evening. There is no specified time of day the dosages should be taken, 
only that they should be taken with about 12 hours apart if possible. If a dose is missed, the patient 
can take the missed dose as soon as it is remembered, provided it is shorter time to the missed dose 
than the next scheduled dose. There are no restrictions with respect to combining the dose with 
other medication and/or food.  
 
 
6.2.3 During treatment (visits 2-4) 
See flowchart (section 6.1) for which clinical examinations are performed at each study visit. Some particular 
considerations follow: 
● Should the patient need replenishment of study medication, it will be logistically handled by the 
study nurse.  
● Points 6-12 from section 6.2.2 are repeated. 
6.2.4 End of study (Visit 4) 
● End of study (Week 12): Patients bring with them remaining study medication which is gathered by 
the study nurse for a pill count. Clinical examinations are performed as listed in section 6.1.  
● Points 6-10 from section 6.2.2. are repeated. 
● At the end of the study visit, consider changes in standard treatment with cholinesterase inhibitors 
or memantine. 
6.3 Criteria for Patient Discontinuation  
Patients may be discontinued from study treatment and assessments at any time. Discontinuation and the 
reason for discontinuation (withdrawn from the study) will be registered. Specific reasons for discontinuing 
a patient for this study are: 
● Voluntary discontinuation by the patient who is at any time free to discontinue his/her participation 
in the study, without prejudice to further treatment. 
● Any form of expression, both verbal and non-verbal, by a participant opposing participation, either 
before or during the study, will be respected. 
● Safety reason as judged by the Principal Investigator. 

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● Incorrect enrollment, i.e. the patient does not meet the required inclusion/exclusion criteria for the 
study. 
● Deterioration in the patient's condition which in the opinion of the Principal Investigator warrants 
study medication discontinuation (to be recorded as an AE or under Investigator Discretion). 
6. 4 Procedures for Discontinuation 
6.4.1 Patient Discontinuation 
Patients who are withdrawn from the study before start of treatment, will be replaced. Withdrawn patients 
are not followed up.   
6.4.2 Trial Discontinuation 
The whole trial may be discontinued at the discretion of the PI or the sponsor in the event of any of the 
following: 
● Occurrence of AEs unknown to date in respect of their nature, severity and duration. 
● Medical or ethical reasons affecting the continued performance of the trial. 
● Difficulties in the recruitment of patients. 
 
The sponsor and principal investigator will inform all investigators and the Ethics Committees of the 
termination of the trial along with the reasons for such action. If the study is terminated early on grounds 
of safety and Ethics Committees will be informed within 15 days. 
 
 
 
 
7 ASSESSMENTS  
The schedule of assessments is indicated in Section 6 (see section 6.1, Flow Chart). 
7.1 Safety and Tolerability Assessments  
Safety will be monitored by the assessments described below as well as the collection of AEs at every visit. 
Significant findings that are present prior to the signing of informed consent must be included in the relevant 
medical history/ current medical condition page of the e-CRF. For details on AE collection and reporting, refer 
to Section 8.  
For the assessment schedule refer to study flow chart in Section 6.1.  
7.2 Clinical Assessments 
1. Medical history: 
Performed by: investigator  
a. Family history of parkinsonism:  
i. Who   
ii. Which illness. 

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b. Family history of Dementia: 
i. Who 
ii. Was the diagnosis certain, probable or possible 
c. Family history of cardiovascular disease 
d. Smoking history: 
i. Active smoker, previous smoker, or never smoker? 
ii. Years since smoking cessation (if applicable)  
iii.  
iv.  
e. Alcohol use 
i. Average alcohol units per week (if applicable)  
f. First AD symptoms: 
i. Which symptoms: memory, orientation, language, behavior, other (if other = free text 
comment). 
 
2. Vital signs: 
Performed by: study nurse  
a. Blood pressure. 
b. Pulse. 
c. Temperature. 
3. Body metrics: 
Performed by: study nurse 
a. Body weight. 
b. Height. 
c. BMI. 
4. Physical examination: 
Performed by: investigator 
a. Heart and lung auscultation. 
i. Heart: normal / findings (free field) 
ii. Lungs: normal / findings (free field) 
b. Abdominal palpation: normal / findings (free field) 
c. Any other examination dictated by patient’s condition/symptoms: free field. 
5. General neurological examination  
Performed by: investigator 
a. Register any findings not related to the subject’s parkinsonism: free field. 
6. Clinical scales & registrations:  
Performed by: see individual tests below 
a. MoCA:  
Performed by: investigator or study nurse 
b. ADAS-COG 
Performed by: investigator or study nurse 
c. CDR: 
Performed by: Investigator or study nurse 
d. Trail Making Test (TMT)  
Performed by: Investigator or study nurse 
e. MADRS:  

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Performed by: investigator study nurse 
 
f. NPI-Q: 
Performed by: study nurse 
g. IADL and PADL 
Performed by: study nurser 
h. 3-day dietary record:  
Performed by: self-filled by the patient and patient’s study partner 
7.3 Routine Laboratory Tests 
These will include: CRP, ALAT, ASAT, GT, bilirubin, ALP, creatinine, urea, RBC, Hb, WBC with differential, 
platelets, CK, FT4, TSH, B12, folic acid, homocysteine, methylmalonic acid, sodium, potassium, glucose. 
Women of childbearing potential will also have a pregnancy test performed. 
Laboratory tests and biosampling are listed and described in detail in Appendix A. 
7.4 Imaging studies 
1. 31P-MRS (CSI, multinuclear coil – 15 min) will be conducted on a 3T Biograph mMR MR-PET scanner 
(Siemens Healthcare, Germany) to assess the intracerebral concentration of NAD, as we have done 
previously25. 
2. FDG-PET imaging will be performed on the same MR-PET scanner and in the same session, to assess 
the metabolic response to NR treatment, as we have previously shown25. Following standard 
preprocessing protocols and spatial normalization, the NRRP will be assessed using using ordinal 
trends/canonical variates analysis (OrT/CVA), a supervised form of principal component analysis 
(PCA). This multivariate approach is designed to detect and quantify regional covariance patterns 
(i.e., metabolic networks) for which expression values (i.e., subject scores) increase or decrease with 
treatment in all or most of the subjects. 
 
The MRI protocol is also summarized in Appendix B. 
 
7.5 Molecular analyses  
1) Metabolomics analyses will be performed in snap-frozen blood, PBMC, and CSF, using liquid 
chromatography-mass spectrometry (LC-MS) as described37. Absolute metabolite concentrations will be 
determined using in house standards. We will assess the entire NAD-metabolome, and key-metabolites 
involved in the Krebs’ cycle, fatty acid beta-oxidation, and methylation reactions (e.g., SAM, homocystein, 
folate).  
2) Gene and protein expression. The transcriptome will be mapped in PBMC and/or PAXgene samples by 
RNA-sequencing, using ribosomal depletion and sequencing at 125 bp paired-end and 100 million read pairs 
per sample, as we have previously described38. Quantitative proteomics will be performed in PBMC, muscle 
and CSF, using TMT (Tandem Mass Tags) labeling and mass spectrometry (LC-MS/MS Q-Exactive HF).  
3) Histone acetylation profiling. As in our previous work12, we will first assess quantitative changes in global 
histone acetylation status in PBMC and muscle, by immunoblotting with a pan-acetyl-lysin antibody. Next, 
acetylation levels of specific lysine residues (e.g., H3K27 and H4K16) will be assessed with targeted 
immunoblotting. Finally, genome-wide changes in the acetylation status of histone lysine residues found to 
be quantitatively altered by the treatment, will be assessed by chromatin-immunoprecipitation sequencing 
(ChIP-Seq)12.  

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4) Inflammatory cytokine concentration will be determined in CSF using ELISA as in the NADPARK trial18.  
5) DNA methylation will be mapped in PBMC and/or snap-frozen blood using the Illumina Infinium Epic Chip. 
6) Neurotransmitter levels. Monoamine levels will be determined in the CSF using HPLC-MS. 
7) Gut microbiome. Using fecal samples, we will assess the microbiota profile (i.e., estimates of composition 
and abundance) by metagenomics, and function by microbial metabolomics. Metagenomics analyses will 
comprise 16S-rRNA sequencing-based count of operational taxonomic units (OTUs), Illumina NovaSeq short-
read sequencing, and long-read sequencing by MinION technology. For microbial metabolomics, feces will 
be analysed for short-chained fatty acids (SCFA) and, if feasible, NAD-related metabolites.  
8 SAFETY MONITORING AND REPORTING 
The investigator is responsible for the detection and documentation of events meeting the criteria and 
definition of an adverse event (AE) or serious adverse event (SAE). Each patient will be instructed to contact 
the investigator immediately should they manifest any signs or symptoms they perceive as serious. 
The methods for collection of safety data are described below. 
8.1 Definitions 
8.1.1 Adverse Event (AE) 
An AE is any untoward medical occurrence in a patient administered a pharmaceutical product and which 
does not necessarily have a causal relationship with this treatment.  
An adverse event (AE) can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of an investigational product, 
whether or not related to the investigational product.  
The term AE is used to include both serious and non-serious AEs. 
If an abnormal laboratory value/vital sign is associated with clinical signs and symptoms, the sign/symptom 
should be reported as an AE and the associated laboratory result/vital sign should be considered as additional 
information that must be collected on the relevant CRF. 
Only intensity 2 and 3 is registered as AE in eCRF, see section 8.3. An AE has to interfere with everyday life 
to be of intensity 2 or 3.  
8.2.1 Serious Adverse Event (SAE) 
Any untoward medical occurrence that at any dose:  
● Results in death 
● Is immediately life-threatening 
● Requires in-patient hospitalization or prolongation of existing hospitalization  
● Results in persistent or significant disability or incapacity 
● Is a congenital abnormality or birth defect 
● Is an important medical event that may jeopardize the subject or may require medical intervention 
to prevent one of the outcomes listed above. 
Medical and scientific judgment is to be exercised in deciding on the seriousness of a case. Important medical 
events may not be immediately life-threatening or result in death or hospitalization, but may jeopardize the 
subject or may require intervention to prevent one of the listed outcomes in the definitions above. In such 
situations, or in doubtful cases, the case should be considered as serious. Hospitalization for administrative 

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reasons (for observation or social reasons) is allowed at the investigator’s discretion and will not qualify as 
serious unless there is an associated adverse event warranting hospitalization. 
8.2 Time Period for Reporting AE and SAE  
Recording AE and SAEs will begin after baseline (week 0) and continue to be monitored and registered 
throughout the duration of the study up until 7 days after the last study visit.   
During the course of the study all AEs and SAEs will be proactively followed up for each patient; events should 
be followed up to resolution, unless the event is considered by the investigator to be unlikely to resolve due 
to the underlying disease. Every effort should be made to obtain a resolution for all events, even if the events 
continue after discontinuation/study completion. 
8.3 Recording of Adverse Events 
If the patient has experienced adverse event(s), the investigator will record the following information in the 
e-CRF: 
● The nature of the event(s) will be described by the investigator in precise standard medical terminology 
(i.e. not necessarily the exact words used by the patient). 
● The duration of the event will be described in terms of event onset date and event ended data. 
● The intensity of the adverse event: Only intensity 2 and 3 is registered as AE in eCRF.  
Assessment of Intensity 
The investigator will make an assessment of intensity for each AE and SAE reported during the study 
and assign it to 1 of the following categories:  
● 1 Mild: An event that is easily tolerated by the participant, causing minimal 
discomfort and not interfering with everyday activities. 
● 2 Moderate: An event that causes sufficient discomfort to interfere with normal 
everyday activities. 
● 3 Severe: An event that prevents normal everyday activities. An AE that is assessed as 
severe should not be confused with an SAE. Severe is a category utilized for rating the 
intensity of an event; and both AEs and SAEs can be assessed as severe. 
 
 
The Causal relationship of the event to the study medication will be assessed as one of the following: 
Unrelated: 
There is not a temporal relationship to investigational product administration (too early, or late, or 
investigational product not taken), or there is a reasonable causal relationship between non-
investigational product, concurrent disease, or circumstance and the AE. 
 
Unlikely:  
There is a temporal relationship to investigational product administration, but there is not a 
reasonable causal relationship between the investigational product and the AE. 
 

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Possible:  
There is a reasonable causal relationship between the investigational product and the AE. 
Dechallenge information is lacking or unclear. 
 
Probable:  
There is a reasonable causal relationship between the investigational product and the AE. The event 
responds to dechallenge. Rechallenge is not required. 
 
Definite:  
There is a reasonable causal relationship between the investigational product and the AE.  
 
● It will be recorded in the eCRF the outcome of the adverse event, the action taken and whether the event 
is resolved or still ongoing. 
● It is important to distinguish between serious and severe AEs. Severity is a measure of intensity 
whereas seriousness is defined by the criteria in Section 8.1. An AE of severe intensity need not 
necessarily be considered serious. For example, nausea that persists for several hours may be 
considered severe nausea, but is not an SAE. On the other hand, a stroke that results in only a limited 
degree of disability may be considered a mild stroke, but would be an SAE. 
8.4 Reporting Procedure 
8.4.1 AEs and SAEs 
All adverse events and serious adverse events that should be reported as defined in section 8.1.1 will be 
recorded in the patient's CRF. 
SAEs must be reported by the investigator to the sponsor, (PI Kristoffer Haugarvoll, see contact information) 
within 24 hours after the site has gained knowledge of the SAE. Every SAE must be documented by the 
investigator on the SAE pages to be found in e-CRF. The initial report shall promptly be followed by detailed, 
written reports if necessary. The initial and follow-up reports shall identify the trial subjects by unique code 
numbers assigned to the latter.  
The sponsor keeps detailed records of all SAEs reported by the investigators and performs an evaluation with 
respect to seriousness, causality and expectedness. 
 
8.5 Clinical Study Report 
The adverse events and serious adverse events occurring during the study will be discussed in both the main 
results and the safety evaluation part of the Clinical Study Report.  
9 DATA MANAGEMENT AND MONITORING   
9.1 Electronic - Case Report Forms (e-CRFs) 
The study nurse/investigator will enter the data required by the protocol into the electronic Case report 
forms (e-CRF) online. The electronical CRF that will be used is Viedoc. The Investigator is responsible for 
assuring that data entered into the e-CRF for his/her patient is complete, accurate, and that entry is 
performed in a timely manner. The signature of the investigator will attest the accuracy of the data on each 
CRF. If any assessments are omitted, the reason for such omissions will be noted on the e-CRFs.  

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At study visit the following source date should be registered either on paper or hospital records. See section 
6.1 and 6.2 for when the relevant information is gathered. This data will then be transferred to eCRF by the 
study nurse/investigator.  
● That the patient is participating in the study, e.g. by including the enrollment number and the study code 
or other such study identification 
● Date when Informed Consent was obtained from the patient and statement that patient received a copy 
of the signed and dated Informed Consent 
● Results of all assessments confirming a patient’s eligibility for the study 
● Diseases (relevant past and current with date; both the disease studied and others, as relevant) 
● Medical history 
● Laboratory data 
● Results of clinical assessments performed during the study 
● Treatment given  
● Non-Serious Adverse Events and Serious Adverse Events (if any) including causality assessments 
● Date of, and reason for, discontinuation from study treatment 
● 
Date of, and reason for, withdrawal from study 
9.2 Study Monitoring  
The investigator will be visited on a regular basis by the Clinical Study Monitor, who will check the following: 
● Informed consent process 
● Reporting of adverse events and all other safety data 
● Adherence to protocol 
● Maintenance of required regulatory documents 
● Study Supply accountability log 
● Data completion on the e-CRFs including source data verification (SDV). 
The monitor will review the relevant e-CRFs for accuracy and completeness, based on a risk assessment, and 
will ask the site staff to adjust any discrepancies as required.  
 
 
9.3 Confidentiality 
The investigator shall arrange for the secure retention of the patient identification and the code list. Patient 
files shall be kept for the maximum period of time permitted by each hospital. The study documentation (e-

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CRFs, Site File etc) shall be retained and stored during the study and for 5 years after study closure. All 
information concerning the study will be stored in a safe place inaccessible to unauthorized personnel. 
9.4 Database management 
● Access to study after the study completion is granted by Kristoffer Haugarvoll.  
● Data for each patient will be recorded on the eCRF. Data collection must be completed for each 
patient who signs an informed consent form and receives at least one dose of study treatment.  
● eCRFs will be designed and produced by the Investigator and should be completed in accordance 
with instructions. The Investigator is responsible for maintaining adequate and accurate medical 
records from which accurate information will be transcribed directly into the eCRFs using a secure 
internet connection. The eCRFs should be filled out completely by the Investigator or designee as 
stated on the delegation of responsibilities form.  
● The eCRFs must be reviewed, signed and dated by the Investigator.  
● Data entered into the eCRF will be validated as defined in the data validation plan. Validation 
includes, but is not limited to, validity checks (e.g. range checks), consistency checks and 
customised checks (logical checks between variables to ensure that study data are accurately 
reported) for eCRF data and external data (e.g. laboratory data). A majority of edit checks will be 
triggered during data entry and will therefore facilitate efficient ‘point of entry’ data cleaning.  
● Data management personnel will perform both manual eCRF review and review of additional 
electronic edit checks to ensure that the data are complete, consistent and reasonable. The 
electronic edit checks will run continually throughout the course of the study and the issues will be 
reviewed manually online to determine what action needs to be taken.  
● Manual queries may be added to the system by clinical data management or study monitor. Clinical 
data managers and study monitors are able to remotely and proactively monitor the patient eCRFs 
to improve data quality.  
● Pharmacokinetic data will be transferred electronically into the study database. Discrepancies will 
be queried to the site and/or the laboratory until the electronic data and the database are 
reconciled.  
● All updates to queried data will be made by authorised study centre personnel only and all 
modifications to the database will be recorded in an audit trail. Once all the queries have been 
resolved, eCRFs will be locked by password protection. Any changes to locked eCRFs will be 
approved by the Investigator.  
● Once the full set of eCRFs have been completed and locked, the Sponsor will authorise database 
lock and all electronic data will be sent to the designated statistician for analysis. Subsequent 
changes to the database will then be made only by written agreement.  
● Adverse events and medical history will be coded from the verbatim description (Investigator 
term). Prior and concomitant medications and therapies will be coded according to the World 
Health Organization drug code. 
10 STATISTICAL METHODS AND DATA ANALYSIS 
10.1 Determination of Sample Size 
Our primary null hypothesis (H0) is that the NR-induced increases in cerebral NAD levels (measured by 
31P-MRS) are not dose-responsive. The alternative hypothesis (HA) is that this measure is dose 
responsive. In the NADPARK study, all three measures showed a highly significant increase in the group 

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receiving 1000 mg NR (n = 15) compared to the placebo group (n = 15). In the NADPARK study25, treatment 
with 1000mg of NR led to an increase in cerebral NAD-levels by a factor of 1.27 from baseline in the 
treatment group, whereas the change in the placebo group was negligible at -0.43%. Under the HA, we 
assume that cerebral NAD levels will increase in a linear fashion in the 2000 mg NR and 3000 mg NR 
groups, respectively. Based on these assumptions and given a type I error rate of 5% (α = 0.05) and a type 
II error rate of 10% (β = 0.1, power = 90%), we estimated that a sample size of 30 individuals will be 
required in the dose escalation group. Accounting for drop-out and statistical safety margin, we estimate 
that the study requires 40 subjects in this group. 
 
 
For the secondary and exploratory outcomes, we assume that the metabolomic, transcriptomic and 
inflammatory cytokine analyses will have sufficient power, since they produced very large effect sizes and 
highly significant results in the NADPARK study with 15 individuals per group. Since no pilot data exist for the 
proteomic and epigenomic analyses, these will be exploratory in nature. However, in our previous 
experience, a sample size of 20 per group should be sufficient to detect treatment-induced differences of 
biological relevance.  
10.2 Randomization 
Randomization is done by e-CRF upon enrollment to the study. Participants will be randomized into one of 
three groups (Placebo, NR 1000 mg for the duration of the study, NR dose escalation group). 
10.3 Population for Analysis 
Intention to treat (ITT)/Full analysis set (FAS)  population: All participants, regardless of protocol 
adherence.  
The Safety Analysis Set (SAS) will include all patients having received at least one study treatment infusion 
after randomisation. 
 
The Per Protocol Analysis Set (PPS) will include all randomized patients meeting the study eligibility criteria 
and with no major protocol deviations affecting the treatment efficacy. 
 
The following are pre-defined major protocol deviations regarded to affect the efficacy of the intervention: 
• 
Entering the trial when the eligibility criteria should have prevented trial entry.  
• 
Discontinuation of intervention prior to 84 ± 7 days.  
• 
Received or used other intervention than allocated.  
• 
Adherence to allocated treatment below 80%.  
• 
Visit date interval larger than 28 ± 7 days between individual visits (i.e.: V1 and V2, V2 and V3 and V3 
and V4)*. 
• 
Visit date interval larger than 84 ± 7 days from V1 to V4*. 
• 
Not fasting before neuroimaging. 
*Due to updated findings from a pharmacokinetic study (ClinicalTrials.gov, NCT: NCT05698771, in 
revision) that NAD levels increase and reach a plateau within 1-2 weeks of NR treatment both in healthy 

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individuals and persons with Parkinson’s disease. A 3 week period should therefore be sufficient to assess 
the effects of increasing NR dosage. 
 
10.4 Planned analyses 
All statistical analysis is planned after the completion of the study.  
 
All randomized patients will be included in the primary analyses. All randomized patients will be included in 
the primary analyses and sensitivity analyses will be carried out comparing results from the ITT, SAS and PPS 
data sets. In the case of missing assessments, the subject will be included if possible. 
 
The primary analysis of the primary endpoint will be performed using analysis of covariance (ANCOVA) 
between the dose stable (DS-group) and dose escalation group (DS-group), i.e. the regression of NAD levels 
measured by 31P-MRS at week 12 depending on randomization group and adjusted for 31P-MRS at baseline. 
No additional adjustments/covariates will be used. As this is the single primary outcome, alpha will be set at 
0.05. No correction for multiple testing will be performed. 
 
 
The secondary analysis of the primary endpoint will be the comparison of change over time from baseline 
to week 4, week 8 and week 12, between the DE-group and the DS-group. This will be assessed using a linear 
mixed model (LME), i.e. NAD (31P-MRS) at weeks 4,8 and 12 depending on randomization group, time and 
their interaction adjusted for random individual intercept. 
 
The primary analysis of the secondary endpoint will be performed using ANCOVA between the dose stable 
(DS-group) and dose escalation group (DS-group), i.e. the regression of NAD or NAD metabolite levels 
measured by HPLC-MS at week 12 depending on randomization group and adjusted for NAD or NAD 
metabolite at baseline. No additional adjustments/covariates will be used. 
 
The analysis of exploratory endpoints will be performed in a similar manner as for the primary and secondary 
endpoints. ANCOVA will be used to compare endpoints at week 12, and LME will be used to compare change 
from baseline to week 4, week 8 and week 12. 
 
In addition, for PET analyses, changes in network scores with treatment will be evaluated for each group 
separately using permutation tests. Relationships between network values, brain NAD levels and cognitive 
ratings or between treatment-related changes in these variables will be evaluated using Pearson’s product-
moment correlations, whereas Spearman rank-order correlation coefficients will be computed for non-
normally distributed variables.  
 
Omics data will undergo rigorous quality control and filtering according to established best practice 
procedures. The between visit change in this data will be assessed by a pairwise comparison between each 
NR group and the placebo group, using linear models with appropriate covariates. Comparison of adverse 
events and abnormal laboratory test results between the treatment and placebo groups will be analyzed 
descriptively. 
 
Further details for the analysis are outlined in the statistical analysis plan (SAP) for the trial, and we refer the 

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reader to this document. 
 
10.5 Statistical Analysis  
Dependent variable 
Primary analysis: 
- 
Cerebral NAD levels (measured by 31P-MRS) 
Secondary, exploratory and safety analysis:  
- 
Adverse effects, categorized as either moderate or severe. 
- 
Levels of CSF NAD and NAD-metabolites (measured by LS-MS) 
- 
Level of ADRP expression (measured by FDG-PET) 
- 
 
- 
Routine blood tests.  
- 
Total ADAS-COG. Assessed by the ADAS-COG 13 scale. 
- 
CDR sum of boxes. Assessed by the CDR questionnaire. 
- 
ADL score. Assessed by the IADL and PADL questionnaires. 
- 
MoCA total score. Assessed by the MoCA questionnaire. 
- 
MADRS score. Assessed by the MADRS scale. 
- 
NPI-Q score. Assessed by the NPI-Q questionnaire. 
 
- 
Levels of NAD-related and other metabolites in, frozen blood, urine and CSF 
- 
Expression of RNA and protein of genes and pathways involved in proteasomal and lysosomal 
biogenesis and function. 
- 
Histone panacetylation levels 
- 
Levels of specific lysine residues H3K27 and H4K16 acetylation 
- 
Genome-wide distribution of histone lysine residues found to be quantitatively altered by the 
treatment (measured by ChIP-Seq). 
- 
Level of inflammatory cytokines in patient serum and CSF. 
- 
Gene and protein expression levels in PBMC and/or PAXgene blood. 
- 
Levels of one carbon metabolism metabolites, measured by HPLC-MS metabolomics in PBMC and 
CSF. 
- 
Levels of monoamine neurotransmitters in CSF. 
- 
Levels and genomic distribution of DNA methylation. 
- 
Gut microbiome composition.  
- 
Fecal metabolomics, including fatty acid levels. 
 
Statistical hypothesis 
Primary analysis:  
 
- 
Our primary null hypothesis (H0) is that the NR-induced increases in cerebral NAD levels (measured 
by 31P-MRS) is not dose-responsive. The alternative hypothesis (HA) is that this increase is dose 
responsive. 
Secondary analysis:  

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- 
Mean change between theNR dose escalation arm, NR 1000 mg arm and placebo arm for the 
dependent variables mentioned above.  
- 
Mean change between and within the NR dose escalation arm, NR 1000 mg arm and placebo arm 
for the dependent variables mentioned above.   
11 STUDY MANAGEMENT 
11.1 Investigator Delegation Procedure 
The principal investigator is responsible for making and updating a “delegation of tasks” listing all the involved 
co-workers and their role in the project. He will ensure that appropriate training relevant to the study is given 
to all of these staff, and that any new information of relevance to the performance of this study is forwarded 
to the staff involved. 
11.2 Protocol Adherence 
Investigators ascertain they will apply due diligence to avoid protocol deviations.  
All significant protocol deviations will be recorded and reported in the Case Report Form (CRF). 
11.3 Study Amendments 
If it is necessary for the study protocol to be amended, the amendment and/or a new version of the study 
protocol (Amended Protocol) must be notified to the Ethics Committee according to EU and national 
regulations. 
11.4 Audit and Inspections 
Authorized representatives of the Ethics Committee may visit the centre to perform inspections, including 
source data verification. Likewise the representatives from sponsor may visit the center to perform an audit. 
The purpose of an audit or inspection is to systematically and independently examine all study-related 
activities and documents to determine whether these activities were conducted, and data were recorded, 
analyzed, and accurately reported according to the protocol, Good Clinical Practice (ICH GCP), and any 
applicable regulatory requirements. The principal investigator will ensure that the inspectors and auditors 
will be provided with access to source data/documents. 
12 ETHICAL AND REGULATORY REQUIREMENTS 
The study will be conducted in accordance with ethical principles that have their origin in the Declaration of 
Helsinki and are consistent with ICH/Good Clinical Practice and applicable regulatory requirements. 
Registration of patient data will be carried out in accordance with national personal data laws. 
12.1 Ethics Committee Approval 
REK has approved the study (REK 428878). The investigator is responsible for informing the ethics committee 
of any serious and unexpected adverse events and/or major amendments to the protocol as per national 
requirements. 
12.2 Other Regulatory Approvals 
Statens legemiddelverket has deemed that the project is not a clinical trial due to the fact that NR is not 
classified as a drug, but as a nutritional supplement.  

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12.3 Informed Consent Procedure 
All subjects will be presented verbally and with written informed consent to be signed prior to enrollment to 
the study. The informed consent will be presented at the first screening. Informed consent will be handled 
according to GCP principles. A copy of the informed consent will be given to the subject.  
12.4 Subject Identification 
Upon entry in screening, each subject is given a patient study number, this study number is used for the 
remaineder of the study. The Patient study number is in the format XX-YYY where XX is the study site number 
and YYY is the patient ID number.  
The investigator is responsible for keeping a list of all patients (who have received study treatment or 
undergone any study specific procedure) including patient’s date of birth and personal number, full names 
and last known addresses.  
The patients will be identified in the CRFs by the patient study number and initials.   
13 TRIAL SPONSORSHIP AND FINANCING 
Research Council of Norway (RCN), Neuro-SysMed, (ES633272) 2020-2028 
14 TRIAL INSURANCE 
The Patients are insured by the government through the “Norsk Pasientskadeerstatning” (NPE).  
15 PUBLICATION POLICY 
Upon study completion and finalization of the study report the results of this study will either be submitted 
for publication and/or posted in a publicly accessible database of clinical study results.  
The results of this study will also be submitted to the Ethics Committee according to EU and national 
regulations. 
All personnel who have contributed significantly with the planning and performance of the study (Vancouver 
convention 1988) may be included in the list of authors. 
16 REFERENCES 
1. Dickson, D. W. Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders. (Wiley-
Blackwell, 2012). 
2. Lautrup, S., Sinclair, D. A., Mattson, M. P. & Fang, E. F. NAD+ in Brain Aging and Neurodegenerative Disorders. 
Cell Metab. 30, 630–655 (2019). 
3. Katsyuba, E., Romani, M., Hofer, D. & Auwerx, J. NAD+ homeostasis in health and disease. Nat. Metab. 2, 9–31 
(2020). 
4. Johnson, S. & Imai, S. NAD + biosynthesis, aging, and disease. F1000Research 7, (2018). 
5. Braidy, N. & Liu, Y. NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis. Exp. Gerontol. 132, 

N-DOSE AD Protocol v4.0 
 
18.06.2025 
 
49 
110831 (2020). 
6. Misrani, A., Tabassum, S. & Yang, L. Mitochondrial Dysfunction and Oxidative Stress in Alzheimer’s Disease. Front. 
Aging Neurosci. 13, 617588 (2021). 
7. Thanan, R. et al. Oxidative stress and its significant roles in neurodegenerative diseases and cancer. Int. J. Mol. Sci. 
16, 193–217 (2014). 
8. Marzi, S. J. et al. A histone acetylome-wide association study of Alzheimer’s disease identifies disease-associated 
H3K27ac differences in the entorhinal cortex. Nat. Neurosci. 21, 1618 (2018). 
9. Hou, Y. et al. Ageing as a risk factor for neurodegenerative disease. Nat. Rev. Neurol. 15, 565–581 (2019). 
10. Bieganowski, P. & Brenner, C. Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes 
establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell 117, 495–502 (2004). 
11. Conze, D. B., Crespo-Barreto, J. & Kruger, C. L. Safety assessment of nicotinamide riboside, a form of vitamin B3. 
Hum. Exp. Toxicol. 35, 1149–1160 (2016). 
12. Claire, K. FDA. GRAS Notification for Nicotinamide riboside chloride. 
https://www.fda.gov/files/food/published/GRAS-Notice-000635--Nicotinamide-riboside-chloride.pdf (2016). 
13. EFSA Panel on Nutrition. European Food Safety Authority Safety of nicotinamide riboside chloride. 
http://www.efsa.europa.eu/en/efsajournal/pub/5775 (2019). 
14. Dollerup, O. L. et al. A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, 
insulin-sensitivity, and lipid-mobilizing effects. Am. J. Clin. Nutr. 108, 343–353 (2018). 
15. Trammell, S. A. et al. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Nat Commun 7, 
12948 (2016). 
16. Martens, C. R. et al. Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy 
middle-aged and older adults. Nat. Commun. 9, 1286 (2018). 
17. Elhassan, Y. S. et al. Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD+ Metabolome and 
Induces Transcriptomic and Anti-inflammatory Signatures. Cell Rep. 28, 1717-1728.e6 (2019). 
18. Okur, M. N. et al. Short-term NAD+ supplementation prevents hearing loss in mouse models of Cockayne 
syndrome. NPJ Aging Mech. Dis. 6, 1 (2020). 
19. Brown, K. D. et al. Activation of SIRT3 by the NAD(+) precursor nicotinamide riboside protects from noise-induced 
hearing loss. Cell Metab 20, 1059–68 (2014). 

N-DOSE AD Protocol v4.0 
 
18.06.2025 
 
50 
20. Han, S., Du, Z., Liu, K. & Gong, S. Nicotinamide riboside protects noise-induced hearing loss by recovering the hair 
cell ribbon synapses. Neurosci. Lett. 725, 134910 (2020). 
21. Harlan, B. A. et al. Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ 
levels in hSOD1-linked ALS mouse models. Exp. Neurol. 327, 113219 (2020). 
22. Schöndorf, D. C. et al. The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal 
Loss in iPSC and Fly Models of Parkinson’s Disease. Cell Rep. 23, 2976–2988 (2018). 
23. Sorrentino, V. et al. Enhancing mitochondrial proteostasis reduces amyloid-β proteotoxicity. Nature 552, 187–193 
(2017). 
24. Xie, X. et al. Nicotinamide ribose ameliorates cognitive impairment of aged and Alzheimer’s disease model mice. 
Metab. Brain Dis. 34, 353–366 (2019). 
25. Brakedal, B. et al. The NADPARK study: A randomized phase I trial of nicotinamide riboside supplementation in 
Parkinson’s disease. Cell Metab. 34, 396-407.e6 (2022). 
26. Haukeland University Hospital. NR-SAFE: a Safety Study Investigating Treatment With High-dose Nicotinamide 
Riboside (NR) in Parkinson’s Disease. https://clinicaltrials.gov/ct2/show/NCT05344404 (2022). 
27. Hirano, S., Eckert, T., Flanagan, T. & Eidelberg, D. Metabolic Networks for Assessment of Therapy and Diagnosis in 
Parkinson’s Disease. Mov. Disord. Off. J. Mov. Disord. Soc. 24, S725–S731 (2009). 
28. Nowicka, U. et al. Cytosolic aggregation of mitochondrial proteins disrupts cellular homeostasis by stimulating the 
aggregation of other proteins. eLife 10, e65484 (2021). 
29. Jadavji, N. M., Wieske, F., Dirnagl, U. & Winter, C. Methylenetetrahydrofolate reductase deficiency alters levels of 
glutamate and γ-aminobutyric acid in brain tissue. Mol. Genet. Metab. Rep. 3, 1–4 (2015). 
30. Conze, D., Brenner, C. & Kruger, C. L. Safety and Metabolism of Long-term Administration of NIAGEN 
(Nicotinamide Riboside Chloride) in a Randomized, Double-Blind, Placebo-controlled Clinical Trial of Healthy 
Overweight Adults. Sci. Rep. 9, 9772 (2019). 
31. Jack, C. R. et al. NIA-AA Research Framework: Toward a biological definition of Alzheimer’s disease. Alzheimers 
Dement. J. Alzheimers Assoc. 14, 535–562 (2018). 
32. Morris, J. C. The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology 43, 2412–2414 
(1993). 
33. Nasreddine, Z. S. et al. The Montreal Cognitive Assessment, MoCA: a brief screening tool for mild cognitive 

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impairment. J. Am. Geriatr. Soc. 53, 695–699 (2005). 
34. Trzepacz, P. T. et al. Relationship between the Montreal Cognitive Assessment and Mini-mental State Examination 
for assessment of mild cognitive impairment in older adults. BMC Geriatr. 15, 107 (2015). 
 
 
 

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17 LIST OF APPENDICES 
 
A.  Lab Manual 
B.  Imaging Manual 
 
 

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APPENDIX A   
 
LABORATORY MANUAL FOR N-DOSE AD 
 
Helse Bergen, Haukeland University Hospital 
Protocol:   
N-DOSE AD 
 
REK Number: 428878 
 
N-DOSE SAFE: A randomized, double blind, dose optimization trial of nicotinamide 
riboside in Alzheimer’s disease. 
STANDARD OPERATING PROCEDURE (SOP) FOR SAMPLING AND PREPARATION OF BIOLOGICAL MATERIAL (LAB 
MANUAL) 
Project Management Committee for the study  
Kristoffer Haugarvoll 
Ragnhild Eide Skogseth 
Charalampos Tzoulis 
 
 

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1 GENERAL INFORMATION 
The purpose of this document is to give an overview of the biological material collected for biobanking in the 
N-DOSE AD clinical trial. At different time points during the clinical trial the following biological samples are 
collected, processed and stored based on standard operating procedures: whole blood, serum, plasma, 
PBMCs, blood cells. The samples are stored at Biobank Haukeland and at the Neurology Department. Some 
of the biological material is collected for specific analysis. Samples in the general biobank may be distributed 
after approval of the application to the Project Management.    
In the following chapters you will find a detailed description on how to collect, prepare, and store samples at 
HDS and HUS. It is critically important that the samples are prepared correctly and standardized, and that all 
deviations from the protocol are documented. This information will be registered in LabVantage when the 
Biobank Information system is in operation and at the Geriatrics and Neurology Department.   
 
 

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2 HEMATOLOGY, BIOCHEMISTRY, HORMONE, SEROLOGY AND SAFETY 
LABORATORY PARAMETERS 
This chapter describes the biological material that should be collected for hematology, biochemistry, 
hormone and serology analyses and for serum hCG pregnancy test. The collection should be done as 
specified in the Flow Chart. The local study site laboratory will be used for the analyses of these 
components, as indicated in the Study Protocol. Protocols for collection of biological material for 
research and biobanking are described in Chapter 3 and 4. 
 
Flow chart 
 
TUBES 
 
 
 
 
 
Visits 
V0 / 
Baseline 
V2 
V3 
V4 
  
Week 
0 
4 
8 
12 
 
Routine: 
 
 
 
 
 
hCG1 
X 
 
 
 
 
CRP 
X 
X 
X 
X 
 
ALAT 
X 
X 
X 
X 
 
ASAT 
X 
X 
X 
X 
 
GT 
X 
X 
X 
X 
 
Bilirubin 
X 
X 
X 
X 
 
ALP 
X 
X 
X 
X 
 
Creatinine 
X 
X 
X 
X 
 
Urea 
X 
X 
X 
X 
 
RBC 
X 
X 
X 
X 
 
Hb 
X 
X 
X 
X 
 
WBC with differential 
X 
X 
X 
X 
 
Platelets 
X 
X 
X 
X 
 
CK 
X 
X 
X 
X 
 
FT4 
X 
X 
X 
X 
 
TSH 
X 
X 
X 
X 
 
B12 
X 
X 
X 
X 
 
Folic acid 
X 
X 
X 
X 
 
homocystein 
X 
X 
X 
X 
 
Methylmalonic acid 
X 
X 
X 
X 
 
Cobalamin 
X 
X 
X 
X 
 
Sodium 
X 
X 
X 
X 
 
Potassium 
X 
X 
X 
X 
 

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Flow chart 
 
TUBES 
 
 
 
 
 
Visits 
V0 / 
Baseline 
V2 
V3 
V4 
  
Week 
0 
4 
8 
12 
 
Glucose 
X 
X 
X 
X 
 
Biobank: 
 
 
 
 
 
EDTA whole blood 
X 
 
 
 
FluidX, 0,7ml, 8 aliquotes 
Snap-frozen whole blood 
X 
X 
X 
X 
 
PAXgene blood for RNA 
X 
X 
X 
X 
PAXgene tubes 
Platelet isolation and 
cryopreservation 
X 
X 
X 
X 
VACUETTE® TUBE 9 ml ACD-A 
Serum 
X 
X 
X 
X 
FluidX, 0,7ml, 8 aliquotes  
Fecal sample 
X 
 
 
X 
 
Urine sample 
X 
X 
X 
X 
 
Cerebrospinal fluid 
X 
 
 
X 
 
 
 
 
 
2.1.1 
Safety laboratory (blood)  
All safety laboratory parameters will be collected at the timepoints as indicated in the Flow Chart 
(page 4), and include hematology, liver enzymes/parameters, clinical chemistry, thyroid status. All 
safety parameters evaluated during the study are listed below. The samples will be analysed at the 
local laboratory at each study site. The respective reference ranges must be provided to the central 
study administration for uploading into the eCRF.  
 
Hematology 
 
● Hemoglobin 
● WBC/leukocytes 
 
● Platelet count / thrombocytes  
● Differentials: neutrophils, eosinophils, 
basophils, monocytes, lymphocytes 
Liver enzymes/parameters 
 
● ALAT (alanine transaminase, SGPT) 
● ASAT (aspartate transaminase, SGOT) 
 
● Alkaline phosphatase  
● GT (glutamyl transferase) 
● Bilirubin total, fractionated if increased 
Clinical chemistry 
 
● Creatinine  
● CRP  
● Potassium 
● Sodium 
 
Thyroid status 
 
● TSH (thyroid stimulating hormone) 
● Free T4 
 

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3 SAMPLING FOR BIOBANK AND RESEARCH 

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3.1 Mandatory research and biobank samples 
Chapter 3.2 gives an overview of all mandatory samples that should be collected for research and 
biobanking.The sample processing protocols are in Chapter 4.  
 
 
3.1.1 
 
Whole blood EDTA 
EDTA blood will be collected at HDS 
3.1.2 
Whole blood EDTA – snap-frozen  
Snap-frozen EDTA blood will be collected at HDS 
3.1.3 
 
RNA PAXgene tubes 
Blood in PAXgene tubes for RNA extraction will be collected at HDS 
3.1.4 
Platelet isolation and cryopreservation 
Platelet isolation and cryopreservation will be conducted at HUS 
3.1.5 
 
Whole blood for Serum 
Serum will be collected at HDS  
3.1.6 
Cerebrospinal fluid (CSF) 
3.1.7 
Fecal sample biobanking 
3.1.8 
Urine sample biobanking 
 
 
 
 

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4 SAMPLE PROCESSING AND STORAGE 
 
4.1 
Whole blood (EDTA): standard 
Performed at: ALL CENTERS 
 
Material and instrumentation: 
Item 
How 
many 
Supplier 
Cat no 
Comment 
VACUETTE® TUBE 2 ml K2E K2EDTA 
2 
Greiner Bio-One 
International 
454024 
 
0.7 ml FluidX tubes 
8 
Pedro Consulting 
68-0702-
11N 
 
Pipettes & tips 
 
 
 
 
Barcode reader 
 
 
 
 
-80°C freezer 
 
Study center 
 
 
 
Collection, preparation and storage: 
1. Collect whole blood into Vacuette tubes for EDTA blood 
2. Aliquot 500 µL blood into 8 x 0.7 ml FluidX tubes. 
3. Freeze the aliquots at -80°C within 60 min after sample collection. 
4. Use the barcode reader for registration of the sample and log all deviation on the same registration 
form.  
5. Sample aliquotes are stored at -80C and shipped on dry ice in the original FluidX boxes (x 48 tubes). 
6. Samples must never be allowed to warm or thaw except from when to be used in analyses. Any 
deviation from this must be registered. Number and date of thawing an aliquote for analyses must 
be registered.   
 
Contact person for biosampling and laboratory preparation:  
Cesilie Dahll, HDS / Siri Hinteregger, MBF  
 
Contact person for Biobank Haukeland: 
Hilde Kristin Garberg 
 
 
 
 
 
 
 
 
 
 

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4.2 
Whole blood (EDTA): snap-frozen 
Performed at: ALL CENTERS 
 
Snap-frozen whole blood will be used for the analysis of NAD+. NAD degrades very rapidly after sample 
collection and the concern is that measurements in any sample that requires extensive processing may not 
prove reliable.    
 
Material and instrumentation: 
Item 
How 
many 
Supplier 
Cat no 
Comment 
VACUETTE® TUBE 2 ml K2E K2EDTA 
1 
Greiner Bio-One 
International 
454024 
 
Screw cap micro tubes, 0.5 ml, sterile 
8 
Sarstedt 
72.730.006 
 
Multi dispenser pipette & tips 
 
 
 
 
Liquid nitrogen in a thermos 
 
 
 
 
-80°C freezer 
 
Study center 
 
 
Timer 
 
 
 
 
 
NB! The time from blood drawing (i.e. moment the blood starts flowing into the tube) and freezing MUST 
be 2 min! 
Collection, preparation and storage: 
1. Print labels. For one patient: 8x  
2. Mark 8 x 0.5 ml micro tubes with labels 
3. Laboratory technician must be next to the patient at the blood drawing with all equipment ready. 
4. Laboratory technician must be wearing gloves and lab coat. 
5. Collect whole blood into the Vacuette tube.  
6. START THE TIMER when blood starts flowing into the tube. 
7. Gently invert the EDTA tube at least 10 times. DO NOT SHAKE 
8. Aliquot 8 x 200 µl of blood from the 2 ml EDTA tube into 8 x 0.5 ml micro tubes (easiest using a 
multi dispenser pipette). 
9. Close micro tubes 
10. When the timer shows 2min: immerse all the micro tubes simultaneously in liquid nitrogen. 
11. Transfer the frozen EDTA-aliquots -80⁰C freezer for storage. 
12. Sample aliquots are stored at -80C in freezer-compatible cardboard or plastic storage boxes.  
13. Samples are shipped on dry ice. 
14. Samples must never be allowed to warm or thaw except from when to be used in analyses. Any 
deviation from this must be registered. Number and date of thawing an aliquot for analyses must 
be registered.   
 
If the 2 min interval is not respected – still collect the samples but register the time interval. 
 
Responsible: 
Hanne Linda Nakkestad 

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4.3 
 RNA PAXgene tubes 
Performed at: ALL CENTERS  
 
Rationale:  
The Paxgene Blood RNA collection system is intended for the purification of intracellular RNA from whole 
blood and is optimized for the stabilization of 4.8 x10e6 – 1.1 x 10e7 leukocytes/ml. This protocol describes 
the collection of whole blood in Paxgene RNA tubes from Qiagen for longterm storage at -80° C. 
Important: Paxgene collection tubes must be at room temperature prior to collection. Follow 
standard procedure for venipuncture for tubes with stabilizing agents e.g. butterfly collection 
Material and instrumentation:   
● PAXgene blood RNA tubes (PreAnalytix, Cat. No. 762165) 
● BD Vacutainer SafetyLok Blood collection set (BD, Cat. No. 367281) or similar Butterfly 
and safety lock or similar  
● Sample labels capable of storage at -80 °C 
● Phlebotomy materials: Tourniquet, alcohol swabs, gauze 
● Barcode reader 
● -80°C freezer 
Collection, preparation,  storage and shippment: 
Before starting, ensure PAXgene tubes are at ambient temperature and labeled 
appropriately with temperature resistant labels. Draw PAXgene tubes last, after 
other blood tubes. If the PAXgene tubes are the only tubes, draw a small 
amount of blood into a discard tube.  
1. Using the Blood Collection set, collect blood into the PAXgene tube using 
standard venipuncture techniques. Ensure that the donor’s arm is in a 
downward position, and that the PAXgene tube is held vertically below the donor’s arm.  
2. Collect 2.5 ml of blood into each PAXgene tube. so that the tube gets filled with exactly 2.5 ml blood. 
This is essential so that the final concentration of the reagents will be correct. If PAXgene is the first 
sample to be taken, collect some blood in another tube first, to get rid of the air in the collection 
system, so that the PAXgene tube gets exactly 2.5 ml blood. 
3. Allow at least 10 s for the blood draw to occur and ensure that blood has stopped flowing into the tube 
before removing the needle from the tube. 
4.  Immediately after blood collection mix the tube by gentle inversion (180°) 10 times  
5. Temporartily store the PAXgene upright at RT for between 2-24 hrs. 
6. Temporarily store the PAXGene tube for at least 24 hrs at -20°C  
7. After at least 24 hrs at -20°C, move the PAXgene tube for long-term storage at -80 °C  
8. Sample aliquotes are stored at -80C in freezer-compatible cardboard or plastic storage boxes.  
9. Samples are shipped on dry ice. 
10. Samples must never be allowed to warm or thaw except from when to be used in analyses. Any 
deviation from this must be registered.  
 
Responsible: 
Contact person for biosampling and laboratory preparation: Cesilie Dahll, HDS / Siri Hinteregger, MBF 
Contact person for Biobank: Hilde Kristin Garberg 

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4.4 Platelet isolation and cryopreservation  
Performed at: ALL CENTERS 
Material and instrumentation 
Item 
How many 
Supplier 
Cat no 
Comment 
VACUETTE® TUBE 9 ml ACD-A 
1 
Greiner Bio-One 
456055 
  
10 ml Falcon centrifugation tubes 
  
  
  
  
DMSO 
  
  
  
  
Pipettes & tips 
  
  
  
  
-80°C freezer 
  
Study center 
  
  
 
Collection, preparation and storage: 
1.       Collect whole blood in vacutainer ACD tubes (yellow cap) 9ml? 
2.       Mix gently by slowly inverting the tube 
3.    Spin at room temperature at 200g for 20 min, no brake 
4.       After the spin, three distinct layers can be observed: 
a.       the top: straw-colored layer contains platelets 
5.       Transfer about two thirds of the top layer into a new 10 ml Falcon tube 
6.       Freeze the cells in their own plasma adding DMSO at a final concentration of 5-6% 
7.    Store at -80 
 
Responsible: 
Contact person for biosampling and laboratory preparation: Cesilie Dahll, HDS / Siri Hinteregger, MBF 
Contact person for Biobank: Hilde Kristin Garberg 
 
4.5 
Serum for biobanking 
Performed at: ALL CENTERS  
 
It is important to let the blood coagulate in an upright position at RT for 60 minutes. Centrifuge the sample 
and immediately aliquot and freeze the aliquots within 90 minutes after sample collection. Register the 
samples according to the local protocol and log all deviation on the same registration form. 
Material and instrumentation: 
● 3 x 5 ml BD VACUTAINER serum glass tube with no additive (367614) 
● 8 x 0.7 ml FluidX (68-0702-11) 
● Centrifuge for vacutainer tubes 
● Barcode reader 
● -80°C freezer 

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Collection, preparation and storage: 
7. Collect whole blood into BD VACUTAINER glass tubes containing no additive.  
8. Incubate in an upright position at room temperature for 60 min (no longer than 60 min) to allow 
clotting. 
9. Centrifuge for 12 min at 1400 x g at room temperature.  
10. Inspect serum for turbidity. Turbid samples should be centrifuged again to remove remaining 
insoluble matter. A new centrifugation must be indicated as a deviation.  
11. Aliquot 500 µL serum into 8 x 0.7 ml FluidX tubes. 
12. Freeze the aliquots at -80°C within 90 min after sample collection. 
13. Use the barcode reader for registration of the sample. Register the samples according to the local 
protocol and log all deviation on the same registration form.  
14. Sample aliquotes are stored at -80C and shipped on dry ice in the original FluidX boxes (x 48 tubes). 
15. Samples must never be allowed to warm or thaw except from when to be used in analyses. Any 
deviation from this must be registered. Number and date of thawing an aliquot for analyses must 
be registered.   
 
 
 
Responsible: 
Contact person for biosampling and laboratory preparation: Cesilie Dahll, HDS / Siri Hinteregger, MBF 
Contact person for Biobank: Hilde Kristin Garberg 
4.6 Collection, of cerebrospinal fluid (CSF) 
Performed at: ALL CENTERS 
  
Material and instrumentation: 
Item 
How many 
Supplier 
Cat no 
Comment 
Standard lumbar puncture equipment   
 Study center 
  
  

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5 ml PolyPropylene (PP) cryo-s tube 
with red PP cap 
2 
Greiner 
Bio-One 
via VWR 
479-4154 
  
0.7 ml FluidX tubes (PP) 
15 
  
68-0702-11N 
  
5 ml PP tube for abeta-42, t-tau and 
p-tau181 
1 
Sarstedt 
63-504-027 
  
Barcode reader 
  
  
  
  
Centrifuge for centrifugation tubes 
  
Study center 
  
  
PP PIPETTE TIPS (ART 1000ΜL 
REACH) 
  
VWR 
732-2215 
  
Pipettes 
  
Study center 
  
  
-80°C freezer 
  
Study center 
  
  
4.6.1              Collection of cerebrospinal fluid  
Absolute contraindications for the procedure: 
●        Thrombocytes less than 40 x 109/L 
●        Use of anticoagulation or antiplatelet drugs (except ASA) 
●        INR >1.7 (warfarin users) 
●        Local infection at the biopsy site 
●        Any other center-specific clinical routines and/or guidelines MUST be followed  
During lumbar puncture the following procedures should take place: 
1.       If noticeably bloody tap, discard the first 1-2 ml until CSF is clear 
2.       10 drops spinal fluid collected in sterile tube for cell count (erythrocytes and leukocytes) 
3.       A total of 10 ml is collected, e.g 5 ml x 2 is collected in 5 ml sterile PolyPropylene tubes (479-4154) for biobanking 
 
 
(see materials and preparation below) 
4.       2 ml is collected in 5 ml PP tube (63-504-027) for clinical analysis of abeta-42, t-tau and p-tau181. 
5.       10 drops spinal fluid collected in sterile tube for cell count (erythrocytes and leukocytes) 
 
NB! Do not use pressure measurements tubing – collect CSF DIRECTLY into the tubes 
 
NB! Only polypropylene (PP) pipette tips should be used to collect and handle the CSF! 
4.6.2              Processing and storage of cerebrospinal fluid 
NB! The sample has to be centrifuged within 1 hour from collection! 
1.    The collected 5ml x 2 CSF in “5 ml PolyPropylene (PP) cryo-s tube with red PP cap” 
a.    Centrifuge within 1 hour at 2,000 g, at room temperature, for 10 min 
b.    Transfer the supernatant to 2 x new “5 ml PolyPropylene (PP) cryo-s tube with red PP cap”  
 
– use  PP pipette tips 

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c.        Mix carefully by inverting the tube x 7-10 times (or mild vortexing) 
d.    Aliquot 500 µl CSF per tube into 0.7 ml FluidX tubes - use PP pipette tips 
e.    Freeze the aliquots at -80°C. 
f.         Register the samples according to the local protocol and log all deviation on the same registration form 
g.       The collected 2 ml CSF in 5 ml PP tube for abeta-42, t-tau and p-tau181 will be sent to Akershus  
 
University Hospital according to their specifications. 
  
Responsible:  
Cesilie Dahll, HDS / Hanne Linda Nakkestad, HUS 
4.7 Fecal sample biobanking 
Performed at: ALL CENTERS  
Fecal samples from the last 24h will be collected according to standard clinical routines and stored at -80C.  
 
 
 

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4.8 
Urine for biobanking 
Performed at: ALL CENTERS  
 
Morning urine will be collected according to standard clinical routines, aliquoted to 2.0 ml FluidX (65-9001) 
(total 8 tubes), frozen and stored at -80C. 

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5 CONTACT PERSONS 
The following chapter summarizes the contact persons and responsible investigators for the sub-
studies: 
5.1 Helse Bergen 
5.1.1 
Biobank Haukeland 
Contact persons regarding biobanking, sample shipment, sample registration 
Ann Cathrine Kroksveen 
55971970 / 92031413, ann.cathrine.kroksveen@helse-
bergen.no   
Hilde Garberg  
55975854 / 55973149, hilde.kristin.garberg@helse-bergen.no  
5.1.2 
Laboratory Clinic 
Contact person regarding safety laboratory, serology and hormone analyses  
Siri Hinteregger 
55973124, siri.hinteregger@helse-bergen.no   
5.1.3 
Haraldsplass (HDS) Laboratory 
Cesilie Dahll 
 
 
55979291/947 83 350, cesilie.dahll@haraldsplass.no  
5.1.4 
Department of Neurology 
Contact person for cells, CSF collection 
Kristoffer Haugarvoll 
55975045 / 98266741, kristoffer.haugarvoll@helse-bergen.no 
Charalampos Tzoulis 
55975045 / 94392305, chtzoulis@gmail.com  
Hanne Linda Nakkestad 
97571149, hanne.linda.nakkestad@helse-bergen.no  
 
5.2 HDS  
Ragnhild Eide Skogseth 
  
99298171, ragnhild.eide.skogseth@haraldsplass.no 
  
 
 
  
 

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APPENDIX B  
MRI and FDG-PET – PROTOCOL,  N-DOSE AD 
Revised May 2025 
Contact persons:  
● Frank Riemer (frank.riemer@helse-bergen.no) 
● Njål Brekke (njal.brekke@helse-bergen.no) 
● Vivian Skjeie (vivian.skjeie@helse-bergen.no) 
● Cecilie Brekke Rygh (cecilie.brekke.rygh@helse-bergen.no) 
 
Equipment 
: 
Scanner:  
Siemens Biograph mMR 3T (PET/MR) 
Software: 
E11P 
Coils:   
RAPID Biomedical 31P – H_Head Coil 3T Human head V-XQ-HQ-030-01921 V01 for 
 
 
 
SIEMENS Biograph mMR 3T 
 
To PACS: 
Clinical protocol 
To fPACS: 
All sequences (entire examination) 
 
Protocol: 
Participant preparation:  
The participant should fast a minimum of 6 hours before injection of 18F-FDG. A peripheral venous 
catheter (PVC) is then placed in the medial cubital vein. Blood glucose is measured. The required 
blood glucose level is below 8 mmol/L. 30 minutes before the scan 250 MBq of 18F-FDG is injected 
into the PVC. The participant is required to lie in a quiet, dark environment during the interval after 
injection and before scanning to minimize brain activity. The participants should not use mobile 
phones or headphones during this period. Eyes must be closed for fMRI sequences. Imaging is to be 
taken up to 72 hours before or after physical examination of participants. 
Positioning:  
Localizer Auto align if possible 
Scan parameters: Scan labelled by study ID. 
MRI sequences:  
 
- 
Localizer for planning of MR and PET – 1.0 min. 
- 
3D T1 (sagital, 1x1x1 mm, 3D-BRAVO eller MPRAGE) – 5.5 min. 
- 
Attenuation correction map, generated using DeepMRAC1 – 2.0 min. 
- 
31P-MRS calibration: FID, multinuclear coil, generated using xnuccalc2 from NMproc-Dockers 
- 2 min.  
- 
31P-MRS sequence: CSI, multinuclear coil – 15 min. 
- 
 
 

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Total imaging time:  
25,5 min imaging time - 35 min including changing the coil for CSI. 
 
Specific procedures for 31P-MRS image analysis: 
Spectra from the occipital region will be aligned using an adaption of the Spectral Registration 
implementation from Gannet 3.0222,3, subject to thresholding on SNR (>=3) to eliminate the majority 
of out-of-brain voxels.  
Voxels will then be averaged before being processed in Matlab 9.5 (the MathWorks, Natick, MA) using 
the OXSA toolbox4 utilizing first order phase correction and fitting with AMARES. 
Custom prior information was created based on literature values for membrane phospholipids (MP), 
glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE), inorganic phosphate (Pi), 
phosphocoline (PC), phosphoethanolamine (PE) as well as alpha-, beta- and gamma resonances of 
adenosine triphosphate (ATP-α, -β, and –γ, respectively) in reference to the phosphocreatine (PCr) 
peak5–7. 
Additional information for the properties of nicotinamide adenine dinucleotide (NAD) was added 
based on the framework developed by Lu and colleagues8 by calculating field-strength dependent 
chemical shift differences, relative amplitudes and frequency separations for oxidized and reduced 
NAD (NAD+ and NADH, respectively).  
Linewidths will be fixed to be equal for NAD+, NADH and ATP-α. At 3T, and to comply with normal-
mode specific absorption rate (SAR) restrictions, peak separation for NAD+ and NADH is likely to be 
limited and therefore only combined values of total NAD (NAD+ and NADH together) will most likely 
be reported. 
Fitted peak amplitudes and areas are used to calculate total NAD in proportion to a normalisation 
constant such as ATP-α or PCr. This normalised NAD-value will be used for the planned statistical 
analysis defined in the trial SAP. 
 
Specific procedures for NRRP and PDRP image analysis of FDG-PET images 
FDG-PET scans will be transferred electronically to the Center for Neurosciences at The Feinstein 
Institutes for Medical Research (Manhasset, NY, USA) and analyzed using automated computing 
pipelines implemented in MATLAB R2023b (MathWorks, Natick, MA). Images will first be pre-
processed using Statistical Parametric Mapping (SPM12) software (http://fil.ion.ucl.ac.uk/spm; 
Wellcome Centre for Human Neuroimaging, London, UK). FDG-PET scans acquired at visits V1, V 2, V3 
and V4 will be aligned to produce a mean image, which will be spatially normalized in standard 
Montreal Neurological Institute (MNI) anatomic space along with the individual scans from each time 
point. The normalized images will then be smoothed with a 10-mm Gaussian filter in three dimensions 
to enhance the signal to noise ratio. 
In the previous NADPARK phase-I study9, we identified a specific NR-related metabolic pattern (NRRP) 
from paired metabolic scan data from participants in the NR 1000 mg group analyzed using ordinal 
trends/canonical variates analysis (OrT/CVA)—a supervised form of principal component 
analysis (PCA)10. This multivariate approach was designed to detect and quantify regional covariance 
patterns (i.e., metabolic networks) for which expression values (i.e., subject scores) increase or 
decrease with treatment in all or most of the subjects11–15. The significance of the resulting OrT/CVA 
topographies, i.e., the NRRP9, was assessed using nonparametric tests, i.e., permutation testing of the 

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subject scores to show that the observed ordinal trend does not occur by chance. Likewise, the 
reliability of the voxel loadings (i.e., region weights) on the resulting NRRP network topography was 
assessed using bootstrap resampling procedures12,16. 
For this study protocol, pre-processed FDG-PET scans of individual subjects will be used to compute 
the expression values (subject scores) of NRRP at baseline and the follow-up timepoints, using the 
GCVA PCA software (available at https://www.nitrc.org/projects/gcva_pca) for Ordinal Trend 
(OrT/CVA) Analysis10. NRRP subject scores will be standardized (z-scored) to computed expression 
values for this pattern in an age-matched group of healthy volunteers scanned at the Feinstein 
Institutes.  
FDG-PET scans will also be used to compute the expression values of the AD-related metabolic pattern 
(termed ADRP) which was identified and validated previously in several independent patient 
populations. Subject scores for PDRP and ADRP of individual subjects will be computed automatically 
on a single-scan basis at each study visit and in a blinded fashion, using in-house Scan Analysis and 
Visualization (ScAnVP) software (available at http://feinsteinneuroscience.org). The subject scores of 
each pattern will be standardized (z-scored) in reference to corresponding scores of age-matched 
healthy controls.  
Outcome measures of the NRRP and ADRP z-scores for individual subjects at each study visit will be 
reported in tabular format and transferred electronically to the Norway group for the planned 
statistical analysis defined in the trial SAP.  
 
References 
1.  
Ladefoged C.N., Hansen A.E., Henriksen O.M., et al. AI-driven attenuation correction for brain 
PET/MRI: Clinical evaluation of a dementia cohort and importance of the training group size. 
Neuroimage 222, 117221 (2020). 
2.  
Edden, R. A., Puts, N. A., Harris, A. D., Barker, P. B. & Evans, C. J. Gannet: A batch-processing 
tool for the quantitative analysis of gamma-aminobutyric acid–edited MR spectroscopy 
spectra. Journal of Magnetic Resonance Imaging 40, 1445–1452 (2014). 
3.  
Near, J. et al. Frequency and phase drift correction of magnetic resonance spectroscopy data 
by spectral registration in the time domain. Magnetic Resonance in Medicine 73, 44–50 (2015). 
4.  
Purvis, L. A. et al. OXSA: An open-source magnetic resonance spectroscopy analysis toolbox in 
MATLAB. PloS one 12, e0185356 (2017). 
5.  
Deelchand, D. K., Nguyen, T.-M., Zhu, X.-H., Mochel, F. & Henry, P.-G. Quantification of in vivo 
31P NMR brain spectra using LCModel. NMR in Biomedicine 28, 633–641 (2015). 
6.  
Peeters, T. H., van Uden, M. J., Rijpma, A., Scheenen, T. W. & Heerschap, A. 3D 31P MR 
spectroscopic imaging of the human brain at 3 T with a 31P receive array: An assessment of 
1H decoupling, T1 relaxation times, 1H-31P nuclear Overhauser effects and NAD+. NMR in 
Biomedicine e4169 (2019). 
7.  
Ren, J., Shang, T., Sherry, A. D. & Malloy, C. R. Unveiling a hidden 31P signal coresonating with 
extracellular inorganic phosphate by outer-volume-suppression and localized 31P MRS in the 
human brain at 7T. Magnetic resonance in medicine 80, 1289–1297 (2018). 

N-DOSE AD Protocol v4.0 
 
18.06.2025 
 
72 
8.  
Lu, M., Zhu, X.-H., Zhang, Y. & Chen, W. Intracellular redox state revealed by in vivo 31P MRS 
measurement of NAD+ and NADH contents in brains. Magnetic resonance in medicine 71, 
1959–1972 (2014). 
9.  
Brakedal, B. et al. The NADPARK study: A randomized phase I trial of nicotinamide riboside 
supplementation in Parkinson’s disease. Cell Metabolism 34, 396-407.e6 (2022). 
10.  
 Habeck, C. et al. A new approach to spatial covariance modeling of functional brain imaging 
data: ordinal trend analysis. Neural computation 17, 1602–1645 (2005). 
11. Ko, J. H. et al. Network modulation following sham surgery in Parkinson’s disease. The Journal of 
clinical investigation 124, 3656–3666 (2014). 
12. Mure, H. et al. Parkinson’s disease tremor-related metabolic network: characterization, 
progression, and treatment effects. Neuroimage 54, 1244–1253 (2011). 
13. Mure, H. et al. Improved sequence learning with subthalamic nucleus deep brain stimulation: 
evidence for treatment-specific network modulation. Journal of Neuroscience 32, 2804–2813 
(2012). 
14. Niethammer, M. et al. Gene therapy reduces Parkinson’s disease symptoms by reorganizing 
functional brain connectivity. Science translational medicine 10, (2018). 
15. Tang, C. C. et al. Metabolic network as a progression biomarker of premanifest Huntington’s 
disease. The Journal of clinical investigation 123, 4076–4088 (2013). 
16. Habeck, C. & Stern, Y. Multivariate data analysis for neuroimaging data: overview and application 
to Alzheimer’s disease. Cell biochemistry and biophysics 58, 53–67 (2010). 
17. Schindlbeck, K. A. & Eidelberg, D. Network imaging biomarkers: insights and clinical applications 
in Parkinson’s disease. The Lancet Neurology 17, 629–640 (2018).