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

N-DOSE Protocol v3.1 
 
N-DOSE Protocol 
 
N-DOSE: A randomized, double blind, dose optimization trial of 
nicotinamide riboside in Parkinson’s disease. 
 
 
Protocol Identification Number:   v3.1, 5th June 2025 
 
 
SPONSOR: 
Haukeland University Hospital 
Address: Jonas Lies 65  
Tel: 55975000 
 
 
PRINCIPAL INVESTIGATOR (PI): 
Charalampos Tzoulis 
Tel: 55975061 
E-mail: charalampos.tzoulis@helse-bergen.no  
 
 
 
 
 
 
 
 
 
PROTOCOL VERSION NO. 3.1, -June 2025 

N-DOSE Protocol v3.1 
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CONTACT DETAILS  
Sponsor: 
Haukeland University Hospital 
Address: Jonas Lies 65  
Tel: 55975000 
 
Study Director 
Charalampos Tzoulis 
E-mail: charalampos.tzoulis@helse-bergen.no 
Coordinating Investigator  
Haakon Berven cand.med 
Haukeland university hospital 
Tel: 48495197 
E-mail: haakonberven@outlook.com  
Principal investigator (at each 
participating site or if single site study): 
Haukeland university hospital 
Charalampos Tzoulis 
E-mail: charalampos.tzoulis@helse-bergen.no  
 
 
Participating Departments: 
Haukeland university hospital, 
Department of Neurology 
Department of Clinical Medicine,  
University of Bergen  
Monitor: 
Section of Innovation and Research Haukeland University 
Hospital, department of Research and Development 
Tel: +47 92860178 
 
 

N-DOSE Protocol v3.1 
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SIGNATURE PAGE 
Title 
N-DOSE: A randomized, double blind, dose optimization trial of 
nicotinamide riboside in Parkinson’s disease.  
 
Protocol ID no: 
 
 
 
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 
 
 
Charalampos 
Tzoulis 
Professor of 
Neurology 
Principal Investigator 
/ Study Director 
 
 
Haakon Berven 
Neurology 
resident 
Coordinating 
Investigator 
 
 
 
 

N-DOSE Protocol v3.1 
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PROTOCOL SYNOPSIS 
 
N-DOSE: A randomized, double blind, dose optimization trial of 
nicotinamide riboside in Parkinson’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 
 
Study Period: 
 
Estimated date of first patient enrolled: 28.11.2022 
Anticipated recruitment period: 01.10.2022 – 20.01.2025. 
Estimated date of last patient completed: 22.04.2025. 
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 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. 

N-DOSE Protocol v3.1 
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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 PD-related 
pattern (PDRP) expression at week 12. 
Change in PDRP 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 PDRP expression from 
baseline to weeks 4, 8 and 12. 
Change in PDRP 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 – motor & 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 PD symptoms at week 12. 
Change in the total MDS-UPDRS score in the ON-
medication state. 
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 total MDS-UPDRS score in the ON-
medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on severity of 
non-motor symptoms of daily living in PD at week 
12. 
Change in the MDS-UPDRS part I score in the ON-
medication state. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and change in severity of non-motor 
symptoms of daily living in PD from baseline to 
weeks 4, 8 and 12. 
Change in the MDS-UPDRS part I score in the ON-
medication state. 
To compare the effect of orally administered NR 
Change in the MDS-UPDRS part II score in the ON-

N-DOSE Protocol v3.1 
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in the DE-group versus DS-group on severity of 
motor aspects of experiences of daily living in PD 
at week 12. 
medication state. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in severity of motor aspects 
of experiences of daily living in PD from baseline 
to weeks 4, 8 and 12. 
Change in the MDS-UPDRS part II score in the ON-
medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on severity of PD 
motor symptoms at week 12. 
Change in the MDS-UPDRS part III score in the ON-
medication state. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in severity of PD motor 
symptoms from baseline to weeks 4, 8 and 12. 
Change in the MDS-UPDRS part III score in the ON-
medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on severity PD 
motor complications at week 12. 
Change in the MDS-UPDRS part IV score in the 
ON-medication state. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in severity of PD motor 
complications from baseline to weeks 4, 8 and 12. 
Change in the MDS-UPDRS part IV score in the 
ON-medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on clinical 
severity of PD non-motor symptoms at week 12. 
Change in the total MDS-NMS score. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in clinical severity of PD 
non-motor symptoms from baseline to weeks 4, 8 
and 12. 
Change in the total MDS-NMS score. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on clinical 
severity 
of 
gastrointestinal 
non-motor 
dysfunction in PD at week 12. 
Change in the modified GIDS-PD score. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in clinical severity of 
gastrointestinal non-motor dysfunction in PD 
from baseline to weeks 4, 8 and 12. 
Change in the modified GIDS-PD score. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on cognition at 
week 12. 
Change in the MoCA score. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in cognition from baseline 
to weeks 4, 8 and 12. 
Change in the MoCA score. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on quality of life 
in PD at week 12. 
Change in the EQ-5D-5L score. 
To 
assess 
the 
dose-response 
relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
Change in the EQ-5D-5L score. 

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per day) and changes in quality of life in PD from 
baseline to weeks 4, 8 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 PD 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 PD 
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 PD 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 PD 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 PD 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 PD 
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 
Change in folate and one-carbon metabolites in 
blood and CSF, measured by HPLC-MS. 

N-DOSE Protocol v3.1 
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one-carbon metabolism in PD at week 12. 
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 PD 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 PD 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 PD 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 PD 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 PD 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. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on the sense of 
smell at week 12. 
Change in sense of smell, measured by B-SIT 
score. 
Safety 
To determine the safety and tolerability of NR at 
a dose of 1000 mg, 2000 mg, and 3000 mg per day 
in PD. 
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: 
Single-center, double-blinded, randomized, placebo controlled, dose-optimization 
 

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Inclusion Criteria:    
• 
Clinically established diagnosis of idiopathic PD according to the MDS criteria. 
• 
123I-Ioflupane dopamine transporter imaging (DAT-scan) or 18-F-FDOPA 
positron emission tomography imaging (18F-FDOPA PET) confirming 
nigrostriatal degeneration. 
• 
Hoehn and Yahr score < 4 at enrollment. 
• 
Age ≥ 40 years at the time of enrollment. 
• 
Able to undergo lumbar punction. 
• 
Able to undergo MRI 
  
Exclusion Criteria: 
• 
Dementia or other neurodegenerative disorder at baseline visit. 
• 
Diagnosed with atypical parkinsonism (PSP, MSA, CBD) or vascular 
parkinsonism. 
• 
Any psychiatric disorder that would interfere with compliance in the study. 
• 
Metabolic, neoplastic, or other physically or mentally debilitating disorder at 
baseline visit.  
• 
Use of high dose vitamin B3 supplementation within 30 days of enrollment 
Sample Size: 
80 patients (20 in placebo group, 60 in treatment groups) 
Efficacy 
Assessments: 
Primary endpoint: 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. 

N-DOSE Protocol v3.1 
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TABLE OF CONTENTS  
CONTACT DETAILS .......................................................................................................................................................... 2 
SIGNATURE PAGE ............................................................................................................................................................ 3 
PROTOCOL SYNOPSIS .................................................................................................................................................... 4 
TABLE OF CONTENTS ................................................................................................................................................... 10 
1 
INTRODUCTION ...................................................................................................................................................... 15 
1.1 
Background – Disease ....................................................................................................................................... 15 
1.1.1 Parkinson’s disease (PD) is a major societal challenge ................................................ 15 
1.2 
Background - Therapeutic Information ............................................................................................................... 15 
1.2.1 NAD-replenishment therapy shows promise as neuroprotective therapy for PD ........... 15 
1.2.2 Investigational Product (IP) .......................................................................................... 15 
1.3 
Pre-Clinical & Clinical Experience with Investigational Product (IP) ................................................................... 16 
1.3.1 Preclinical evidence for NR-therapy ............................................................................. 16 
1.3.2 Phase I evidence for NR-therapy in PD ........................................................................ 16 
1.4 
Rationale for the Study and Purpose .................................................................................................................. 19 
2 
STUDY OBJECTIVES AND RELATED ENDPOINTS ............................................................................................. 21 
2.1 
Primary Endpoint Measure ................................................................................................................................. 25 
2.2 
Secondary Endpoint Measure ............................................................................................................................ 25 
2.3 
Exploratory Endpoint Measures ......................................................................................................................... 26 
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 
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 
Dopaminergic therapy during Screening and Treatment period ......................................................................... 31 
5.5 
Concomitant Medication ..................................................................................................................................... 31 
5.6 
Subject Compliance ............................................................................................................................................ 31 
5.7 
Drug Accountability ............................................................................................................................................. 32 
5.8 
Drug Labeling ..................................................................................................................................................... 32 
5.9 
Subject Numbering ............................................................................................................................................. 32 
6 
STUDY PROCEDURES ........................................................................................................................................... 32 
6.1 
Flow Chart .......................................................................................................................................................... 32 

N-DOSE Protocol v3.1 
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6.2 
By Visit ............................................................................................................................................................... 34 
6.2.1 Screening Visits/ Before start of Investigational Product (IP) ........................................ 34 
6.2.2 Baseline/Week 0 .......................................................................................................... 34 
6.2.3 During treatment (visits 2-4) ......................................................................................... 36 
6.2.4 End of study (Visit 4) .................................................................................................... 36 
6.3 
Criteria for Patient Discontinuation ..................................................................................................................... 36 
6.4 
Procedures for Discontinuation .......................................................................................................................... 36 
6.4.1 Patient Discontinuation ................................................................................................ 36 
6.4.2 Trial Discontinuation ..................................................................................................... 36 
7 
ASSESSMENTS....................................................................................................................................................... 37 
7.1 
Safety and Tolerability Assessments .................................................................................................................. 37 
7.2 
Clinical Assessments .......................................................................................................................................... 37 
7.3 
Routine Laboratory Tests ................................................................................................................................... 38 
7.4 
Imaging studies .................................................................................................................................................. 38 
7.5 
Molecular analyses ............................................................................................................................................. 39 
8 
SAFETY MONITORING AND REPORTING ............................................................................................................ 39 
8.1 
Definitions ........................................................................................................................................................... 39 
8.1.1 Adverse Event (AE) ...................................................................................................... 39 
8.1.2 Serious Adverse Event (SAE) ...................................................................................... 40 
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 .......................................................................................................................................... 42 
9 
DATA MANAGEMENT AND MONITORING ............................................................................................................ 42 
9.1 
Electronic - Case Report Forms (e-CRFs) .......................................................................................................... 42 
9.2 
Study Monitoring ................................................................................................................................................. 42 
9.3 
Confidentiality ..................................................................................................................................................... 43 
9.4 
Database management ...................................................................................................................................... 43 
10 STATISTICAL METHODS AND DATA ANALYSIS ................................................................................................. 44 
10.1 Determination of Sample Size ............................................................................................................................ 44 
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 

N-DOSE Protocol v3.1 
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11.3 Study Amendments ............................................................................................................................................ 47 
11.4 Audit and Inspections ......................................................................................................................................... 47 
12 ETHICAL AND REGULATORY REQUIREMENTS ................................................................................................. 48 
12.1 Ethics Committee Approval ................................................................................................................................ 48 
12.2 Other Regulatory Approvals ............................................................................................................................... 48 
12.3 Informed Consent Procedure ............................................................................................................................. 48 
12.4 Subject Identification .......................................................................................................................................... 48 
13 TRIAL SPONSORSHIP AND FINANCING .............................................................................................................. 48 
14 TRIAL INSURANCE ................................................................................................................................................. 48 
15 PUBLICATION POLICY ........................................................................................................................................... 49 
16 REFERENCES ......................................................................................................................................................... 49 
17 LIST OF APPENDICES ............................................................................................................................................ 53 
STANDARD OPERATING PROCEDURE (SOP) FOR SAMPLING AND PREPARATION OF BIOLOGICAL MATERIAL 
(LAB MANUAL) ................................................................................................................................................................ 54 
1 
GENERAL INFORMATION ...................................................................................................................................... 55 
2 
HEMATOLOGY, BIOCHEMISTRY, HORMONE, SEROLOGY AND SAFETY LABORATORY PARAMETERS ... 56 
2.1.1 Safety laboratory (blood) .............................................................................................. 58 
3 
SAMPLING FOR BIOBANK AND RESEARCH ....................................................................................................... 58 
3.1 
Mandatory research and biobank samples ......................................................................................................... 58 
3.1.1 Whole blood EDTA ....................................................................................................... 58 
3.1.2 Whole blood EDTA – snap-frozen ................................................................................ 58 
3.1.3 RNA PAXgene tubes .................................................................................................... 58 
3.1.4 Whole blood for Serum ................................................................................................ 58 
3.1.5 Platelets ....................................................................................................................... 58 
3.1.6 Cerebrospinal fluid (CSF) ............................................................................................. 58 
3.1.7 Feces ........................................................................................................................... 58 
3.1.8 Urine ............................................................................................................................ 58 
4 
SAMPLE PROCESSING AND STORAGE .............................................................................................................. 59 
4.1 
Whole blood (EDTA): standard ........................................................................................................................... 59 
4.2 
Whole blood (EDTA): snap-frozen ...................................................................................................................... 60 
4.3 
RNA PAXgene tubes .......................................................................................................................................... 61 
4.4 
Serum for biobanking ......................................................................................................................................... 62 
4.5 
Platelet isolation and cryopreservation for mitochondrial research ..................................................................... 63 
4.6 
Collection, of cerebrospinal fluid ......................................................................................................................... 63 
4.6.1 Collection of cerebrospinal fluid .................................................................................... 64 
4.6.2 Processing and storage of cerebrospinal fluid .............................................................. 64 
4.7 
Fecal sample for biobanking ............................................................................................................................... 66 
4.8 
Urine for biobanking ........................................................................................................................................... 67 
5 
CONTACT PERSONS .............................................................................................................................................. 68 
5.1 
Helse Bergen .................................................................................................................................................... 68 
5.1.1 
Biobank Haukeland ......................................................................................................... 68 

N-DOSE Protocol v3.1 
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5.1.2 
Laboratory Clinic ............................................................................................................ 68 
5.1.3 
Department of Neurology ................................................................................................ 68 
APPENDIX B - .................................................................................................................................................................. 69 
MRI AND FDG-PET – PROTOCOL, N-DOSE .................................................................................................................. 69 
APPENDIX C - .................................................................................................................................................................. 73 
CLINICAL RATING SCALES ........................................................................................................................................... 73 
1 
GENERAL INFORMATION ...................................................................................................................................... 73 
2 
MRS-UPDRS ............................................................................................................................................................ 74 
3 
MDS-NMS .............................................................................................................................................................. 107 
4 
MODIFIED GIDS-PD .............................................................................................................................................. 119 
5 
MOCA ..................................................................................................................................................................... 132 
6 
EQ-5D-EL ............................................................................................................................................................... 136 
List of Abbreviations and Definitions of Terms 
Abbreviation or special term 
Explanation 
AE 
Adverse Event  
ChIPseq 
Chromatin immunoprecipitation sequencing 
CNS 
Central Nervous System 
CRF 
Case Report Form (electronic/paper) 
CSF 
Cerebrospinal fluid 
SAM 
S-denosyl methionine 
DS-group 
Dose-stable group 
DE-group 
Dose-escalation group 
CSA 
Clinical Study Agreement 
CTC 
Common Toxicity Criteria 
CTCAE 
Common Terminology Criteria for Adverse Event 
DAE 
Discontinuation due to Adverse Event 
EC 
Ethics Committee, synonymous to Institutional Review Board (IRB) and 
Independent Ethics Committee (IEC) 
EQ-5D-5L 
A standardized measure of health-related quality of life 
GCP 
Good Clinical Practice 
GIDS-PD 
Gastrointestinal Dysfunction Scale for Parkinson’s Disease  
HPLC-MS 
High pressure 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 
HPLC-MS 
High performance liquid chromatography-mass spectrometry 
MDS-CDC 
Movement Disorders Society – Clinical Diagnosis Criteria for PD 
MDS-NMS 
Movement Disorders Society – Non-Motor rating Scale 
MDS-UPDRS 
Movement Disorders Society – Unified Parkinson's Disease Rating Scale 
MDS-NMS 
Movement Disorders Society – Non-Motor rating Scale 

N-DOSE Protocol v3.1 
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MoCA 
Montreal Cognitive Assessment 
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 
NR 
Nicotinamide Riboside 
NRRP 
NR-related metabolic pattern 
PDRD 
Parkinson’s disease related pattern 
OBD 
Optimal biological dose 
PBMC 
Peripheral Blood Mononuclear Cells 
PD 
Parkinson’s Disease 
PDRP 
Parkinson’s Disease-Related Pattern 
SAE 
Serious Adverse Event   
SOP 
Standard Operating Procedure 
 

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1 
INTRODUCTION 
1.1 
Background – Disease 
1.1.1 Parkinson’s disease (PD) is a major societal challenge  
PD affects 1-2% of the population above the age of 65, and is a major cause of death and disability with a 
devastating global socioeconomic impact1,2. In Europe alone, PD affects an estimated 1.2 million people 
and has a cost of €14 billion per year3. Current treatments for PD are purely symptomatic and have no 
impact on disease progression. As a result, patients confront a future of progressive disability, early 
institutionalization, and premature death. Since demographic studies show that patient numbers will 
continue to grow, effectively doubling by 2040, our failure to make any significant impact to halt or delay 
disease progression means that PD is now a major challenge to health care and society.   
1.2 
Background - Therapeutic Information 
1.2.1 NAD-replenishment therapy shows promise as neuroprotective therapy for PD  
Increasing evidence supports that boosting cellular levels of nicotinamide adenine dinucleotide (NAD) 
confers neuroprotective effects in both healthy aging and neurodegeneration4. 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 
generation5. These reactions consume NAD+ at high rates, requiring constant replenishment via NAD 
biosynthesis. NAD levels have been shown to decline with age and this is believed to contribute to age-
related diseases5,6. 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 diseases5–7. Enhancing NAD replenishment could potentially 
help ameliorate several major processes implicated in the pathogenesis of PD, including mitochondrial 
respiratory dysfunction8–10, neuroinflammation11, epigenomic dysregulation12,13 and increased neuronal 
DNA damage14. 
NAD can be replenished via supplementation of nicotinamide riboside (NR), a vitamin B3 molecule and 
biosynthetic precursor of NAD5,15. NR has undergone extensive preclinical testing16 and is well tolerated 
by adult humans, showing no evidence of toxicity with doses up to at least 2000 mg daily17.  
 
1.2.2 Investigational Product (IP) 
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. NR is well tolerated with no evidence of toxicity in adult humans with doses up to at least 
3,000 mg daily16–21. 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 evidence of toxicity. Niagen and placebo will be provided from Chromadex, 
https://chromadex.com/. Active study drug capsules will contain 250 mg NR. Placebo will contain 

N-DOSE Protocol v3.1 
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microcrystalline cellulose, which will be identical in appearance and taste. 
1.3 
Pre-Clinical & Clinical Experience with Investigational Product (IP) 
1.3.1 Preclinical evidence for NR-therapy 
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 cytokines19,22–24. Moreover, evidence from cell and animal studies 
suggests that NR supplementation promotes healthspan and has neuroprotective effects in models of 
Cockayne syndrome25, noise-induced injury26,27, amyotrophic lateral sclerosis28, Alzheimer's disease 29,30 
and PD31.  
 
1.3.2 Phase I evidence for NR-therapy in PD 
Two phase I studies of NR in PD have been completed, the NADPARK18,32 study and the NR-SAFE21 study. 
These are described briefly below.   
I. The NADPARK study (ClinicalTrials.gov: NCT03816020)32 is a phase I randomized, double blinded trial, 
aiming to assess the tolerability, cerebral bioavailability and molecular effects of NR therapy in PD. 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 were published in 202218 and 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 effects in PD.  
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 (Fig 1A-C). Cerebral penetration was further validated by detecting the metabolite Me-2-PY in the 
CSF of participants receiving NR, but not placebo (Fig 1G). 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 (Fig 1C). Moreover, 
three participants 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 (Fig 1D-E). Interestingly, the NRRP overlapped spatially with 
the Parkinson’s Disease-Related Pattern (PDRP)33, and  changes in NRRP expression in the NR group 
resulted in partial normalization of the striatal and thalamic hypermetabolism (Fig 1D), typically 

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characterizing the PD brain33. 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. 
5) NR has widespread metabolic and regulatory effects: Metabolomics revealed 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 PD34 
  
Fig 1. Results of the NADPARK trial. A-C: 31P-MRS was performed at baseline (v1) and after 30 days of treatment 
(v2). A: exemplary data from one subject showing voxel position. Spectra were acquired for each grid position. B 
(top): average processed spectra from multiple voxels (black) and the model fit (red). B (bottom): the model fit is 
composed of the convolution of all spectral contributions of a simulated dataset fitted to the experimental data. 
Arrow shows the NAD+/NADH spectral peaks. PCr: phosphocreatine. C: comparison of cerebral NAD levels in the 
placebo (PL) and NR groups at baseline (v1) and visit-2 (v2). The Y-axis shows measured levels of NAD normalized to 
α-ATP (which remain unchanged and stable by the intervention). Individual subjects are indicated by dots. 
Connecting lines show the change between v1 and v2. The black points and lines show the mean of each group. The 
treatment group shows a highly significant increase in cerebral NAD levels (*paired t-test p = 0.016), whereas no 
difference is observed in the placebo group (paired t-test p = 0.75). D: FDG-PET data from all subjects in the NR-
group, showing the mean NR-Related Pattern (NRRP, top panel), PD-related pattern (PRDP, middle panel), and the 
overlap of the two (bottom panel). NR partially ameliorates the striatal and thalamic hypermetabolism of PD. E: 
Between visit changes showing increased NRRP expression in the NR versus the placebo group. The red line indicates 
mean values before and after the treatment, black lines indicate individuals with a positive NAD response in the 

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MRS analysis, grey lines indicate individuals without an NAD response in the MRS (*p =0.027, permutation test, 1000 
iterations).  F: the NR-induced change in metabolic pattern (delta NRRP) shows a strong negative correlation 
(P<0.01) with the decrease in the UPDRS score (delta UPDRS). G: Metabolomics in PBMCs, muscle, and CSF. NR-
induced metabolic changes include highly significant (p<<0.001 for all tests) increases in the acid form of NAD 
(NAAD), and nicotinamide (Nam) degradation products: methyl-Nam (Me-Nam) and the methyl pyridone (Me-2-PY).     
 
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 2. 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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1.4 
Rationale for the Study and Purpose 
Our phase I trials (see section 1.3) showed that NR holds promise as a potential neuroprotective, disease-
modifying therapy for PD, and that its effects are likely to be dose-dependent. Encouraged by these 
findings, this project aims to determine the optimal biological dose of NR in PD and to further explore its 
neuroprotective potential. The outcomes of this project will take us several steps closer to developing NR 
into a PD-drug, so that we may harness its full therapeutic potential and maximize its clinical benefit and 
impact. Specifically, the following knowledge gaps (KG) will be addressed by the N-DOSE study: 
 
KG1 – Determine the Optimal Biological Dose (OBD) of NR in PD. We define the OBD of NR as the dose 
required to achieve maximal cerebral NAD increase (measured by 31P-MRS or CSF metabolomics), or 
maximal expression increase in the NRRP (measured by FDG-PET), or maximal proportion of MRS-
responders, in the absence of unacceptable toxicity.  
While our trials show that the treatment responses to NR in PD are clearly dose-dependent, the OBD of 
NR in PD 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 individuals. 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 – Confirm whether NR has a symptomatic clinical effect and assess its dose responsiveness. The 
NADPARK and NR-SAFE studies showed that NR was associated with a clinical improvement, in the form 
of UPDRS decrease, and this correlated significantly with the increase in cerebral NAD levels and change 
in the brain metabolic network (NRRP). Furthermore, the clinical improvement was more pronounced 
with 3000 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 PD, resulting in symptom improvement. 
Confirming this effect and assessing its dose-dependence will allow us to: 1) determine the optimal clinical 
dose of NR in PD, and 2) 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 PD and characterize the dose 
dependence of this effect. Impaired proteostasis plays a central role in PD and other neurodegenerative 
disorders, including Alzheimer’s disease (AD) 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 

N-DOSE Protocol v3.1 
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multiple major processes implicated in the pathophysiology of PD, including mitochondrial respiratory 
dysfunction, oxidative damage, lysosomal and proteasomal impairment, and neuroinflammation. 
Moreover, it is not known whether higher NR doses can induce stronger induction of proteostasis in PD. 
 
KG5 – Determine whether NR therapy influences histone acetylation status and characterize the dose 
dependence of this effect. We have recently shown that genome-wide histone hyperacetylation and 
altered transcriptional regulation occur in the brain of individuals with PD12. Increasing neuronal NAD 
levels would boost the activity of the NAD-dependent histone deacetylases of the sirtuin family, 
potentially, ameliorating histone hyperacetylation in PD. It is currently unknown whether NR-therapy 
influences histone acetylation status in PD, and whether this effect is dose-dependent. 
 
KG6 – Confirm that NR therapy decrease neuroinflammation and characterize the dose dependence of 
this effect. While it is known that NR has anti-inflammatory properties in peripheral tissues24, the results 
of the NADPARK trial suggest it also downregulates multiple inflammatory cytokines in the central 
nervous system. If confirmed this would be of importance for PD and other neurodegenerative and 
neuroinflammatory disorders. Moreover, it is not known whether higher NR doses can induce stronger 
anti-inflammatory effects in PD. 
 
KG7 – Determine whether NR therapy alter 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 dopamine35. 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 PD. Current evidence 
suggests that the gut microbiome is involved in the pathogenesis of PD and that individuals with PD host 
a different microbiome composition compared to neurologically healthy individuals36. It is possible that 
NR therapy may beneficially affect the gut microbiome in PD 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.  
 
  
 
To address these pertinent questions, we will conduct N-DOSE, a single-center randomized double-
blinded placebo-controlled trial to assess the optimal biological dose for NR.  
 
 

N-DOSE Protocol v3.1 
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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 PD-related 
pattern (PDRP) expression at week 12. 
Change in PDRP 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 PDRP expression from 
baseline to weeks 4, 8 and 12. 
Change in PDRP 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 
Change in levels of NAD metabolites in blood, 
urine and CSF, measured by HPLC-MS and/or 
the NADMed method. 

N-DOSE Protocol v3.1 
22 
 
nervous system (CNS) at week 12. 
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 – motor & 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 PD symptoms at week 12. 
Change in the total MDS-UPDRS score in the 
ON-medication state. 
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 total MDS-UPDRS score in the ON-
medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on severity of 
non-motor symptoms of daily living in PD at 
week 12. 
Change in the MDS-UPDRS part I score in the 
ON-medication state. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and change in severity of non-motor 
symptoms of daily living in PD from baseline to 
weeks 4, 8 and 12. 
Change in the MDS-UPDRS part I score in the 
ON-medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on severity of 
motor aspects of experiences of daily living in 
PD at week 12. 
Change in the MDS-UPDRS part II score in the 
ON-medication state. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in severity of motor 
aspects of experiences of daily living in PD from 
baseline to weeks 4, 8 and 12. 
Change in the MDS-UPDRS part II score in the 
ON-medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on severity of 
PD motor symptoms at week 12. 
Change in the MDS-UPDRS part III score in the 
ON-medication state. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in severity of PD motor 
symptoms from baseline to weeks 4, 8 and 12. 
Change in the MDS-UPDRS part III score in the 
ON-medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on severity PD 
motor complications at week 12. 
Change in the MDS-UPDRS part IV score in the 
ON-medication state. 

N-DOSE Protocol v3.1 
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To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in severity of PD motor 
complications from baseline to weeks 4, 8 and 
12. 
Change in the MDS-UPDRS part IV score in the 
ON-medication state. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on clinical 
severity of PD non-motor symptoms at week 
12. 
Change in the total MDS-NMS score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in clinical severity of PD 
non-motor symptoms from baseline to weeks 4, 
8 and 12. 
Change in the total MDS-NMS score. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on clinical 
severity 
of 
gastrointestinal 
non-motor 
dysfunction in PD at week 12. 
Change in the modified GIDS-PD score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in clinical severity of 
gastrointestinal non-motor dysfunction in PD 
from baseline to weeks 4, 8 and 12. 
Change in the modified GIDS-PD score. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on cognition at 
week 12. 
Change in the MoCA score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in cognition from baseline 
to weeks 4, 8 and 12. 
Change in the MoCA score. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on quality of 
life in PD at week 12. 
Change in the EQ-5D-5L score. 
To assess the dose-response relationship 
between NR dose (1000 mg, 2000 mg, 3000 mg 
per day) and changes in quality of life in PD from 
baseline to weeks 4, 8 and 12. 
Change in the EQ-5D-5L 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. 

N-DOSE Protocol v3.1 
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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 PD 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 
PD 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 PD 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 PD 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 PD 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 PD 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 PD 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 PD 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 PD 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 
Change in methyl-donors (e.g., SAM), measured 
by HPLC-MS, in the blood. 

N-DOSE Protocol v3.1 
25 
 
per day) and changes in methyl-donors in PD 
from baseline to weeks 4, 8 and 12. 
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 PD 
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 PD 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. 
To compare the effect of orally administered NR 
in the DE-group versus DS-group on the sense 
of smell at week 12. 
Change in sense of smell, measured by B-SIT 
score. 
Safety 
To determine the safety and tolerability of NR 
at a dose of 1000 mg, 2000 mg, and 3000 mg 
per day in PD. 
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 shown18,37. Using this method, we will 
assess 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 

N-DOSE Protocol v3.1 
26 
 
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 study18, measured by 18F-fluorodeoxyglucose positron emission 
tomography (FDG-PET).  
PD related pattern (PDRP), an ordinal trend pattern (i.e., metabolic network), associated with PD33, 
measured by 18F-fluorodeoxyglucose positron emission tomography (FDG-PET). 
The International Parkinson and Movement Disorder Society – Non-Motor Rating Scale (MDS-NMS)38, 
a 52 item scale that assesses the frequency and severity of non-motor symptoms in PD across 13 non-
motor domains. The MDS-NMS consists of two parts: the Total MDSS-NMS (range 0-832) and the MDS-
NMS Non-Motor Fluctuations (NMF) Subscale to assess changes in non-motor symptoms in relation to 
the timing of anti-parkinsonian medication (range 0-128). Each item in each scale is scored according to 
frequency from 0 to 4 and severity from 0 to 4. Each item’s frequency is then multiplied by its severity to 
calcultate the item scorer. A low score indicates a good outcome, higher scores indicate a poorer 
outcome. 
The International Parkinson’s disease and movement disorders society unified Parkinson’s disease 
rating scale (MDS-UPDRS)39 is a scale assessing clinical impairment and disability in PD. It consists of 4 
parts. Part I assesses non-motor experiences of daily living and has 2 components IA and IB (Range 0-52). 
Part IA comprises 6 questions assessed by the examiner (Range 0-24). Part IB comprises 7 questions 
completed by the participants (Range 0-28). Part II assesses motor experiences of daily living (Range 0-
52). It comprises 13 questions completed by the participant. Part III evaluates motor severity and contains 
33 scores based on 18 items, which are assessed by the rater during clinical examination (Range 0-132). 
Part IV assesses motor complications, which comprises 6 questions assessed by the examiner (Range 0-
24). For each question a numeric score is assigned between 0-4, where 0 = Normal, 1 = Slight, 2 = Mild, 3 
= Moderate, 4 = Severe. MDS-UPDRS Total Score equals the sum of parts I, II, III and IV (Range 0-260). A 
higher score indicates more severe symptoms of PD.  
 
MDS-UPDRS part I is a scale assessing non-motor experiences of daily living and has 2 components (Range 
0-52). Part IA comprises 6 questions assessed by the examiner (Range 0-24). Part IB comprises 7 questions 
completed by the participants (Range 0-28). A higher score indicated more severe symptoms. This will be 
assessed in the ON medication state. 
 
MDS-UPDRS part II assesses motor experiences of daily living (Range 0-52). It comprises 13 questions 
completed by the participant. A higher score indicated more severe symptoms. This will be assessed in 
the ON medication state.  
 
MDS-UPDRS part III evaluates motor severity and contains 33 scores based on 18 items, which are 
assessed by the rater during clinical examination (Range 0-132). For each question a numeric score is 
assigned between 0-4, where 0 = Normal, 1 = Slight, 2 = Mild, 3 = Moderate, 4 = Severe. A higher score 

N-DOSE Protocol v3.1 
27 
 
indicated more severe symptoms. This will be assessed in the ON medication state. 
 
MDS-UPDRS part IV evaluates motor complications and contains 6 questions assessed by the examiner 
(Range 0-24). For each question a numeric score is assigned between 0-4, where 0 = Normal, 1 = Slight, 2 
= Mild, 3 = Moderate, 4 = Severe. A higher score indicated more severe symptoms. This will be assessed 
in the ON medication state. 
 
The International Parkinson and Movement Disorder Society Gastrointestinal Dysfunction Scale in 
Parkinson’s Disease (mGIDS-PD)40 modified*. The following version of GIDS-PD has been modified by 
permission and license from MDS for use in the N-DOSE trial. The modifications in this version are that 
the timeframe for all questions has been reduced from six months to one month. Part 1b has also been 
omitted from the scale.  A low score indicates a good outcome, higher scores indicate a poorer outcome. 
The range is 0 to 108 points. The scale consists of two main parts: Section 1 -12, which are counted as a 
scale. Section 1a, and sections A – F, which are anamnestic and do not count towards the scale score. 
 
MoCA41 is a validated global measure of cognitive ability. This will be assessed in the ON medication state.  
 
EQ-5D-5L42 is a 5 item questionnaire and visual analogue scale that allows calculation of quality adjusted 
life years (QALY) to enable health economic analyses to be performed. The questionnaire covers 5 
domains with scores 0 to 4. A low score indicates a good outcome, higher scores indicate a poorer 
outcome. The EQ VAS score in mm is a general assessment from the participants perspective of how good 
their health is, from 0 to 100. A low score indicates a poor outcome, a high value indicates a better 
outcome. 
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 

N-DOSE Protocol v3.1 
28 
 
MethylationEPIC Kit.  
  
Gut microbiome composition, assessed by metagenomics in fecal samples.  
 
Fecal metabolomics, including fatty acid profiling, assessed by HPLC-MS in fecal samples. 
 
Brief Smell Identification Test (B-SIT) assesses olfaction. It consists of 12 items with 4 alternatives, from 
which the participant attempts to identify the correct smell. It is performed by the participant under 
supervision of the investigator. Not available for all participants as this was added as an exploratory 
outcome during the trial. A low score indicates a poor outcome, higher scores indicate a better outcome. 
The range for the total score is 0 to 12 points. 
 
 
 
3 
OVERALL STUDY DESIGN 
This is a single-center, phase II, double blinded, randomized, placebo-controlled dose-optimization study. 
Study Period 
 
Estimated date of first patient enrolled: 28.11.2022 
Anticipated recruitment period: 28.11.2022 – 20.01.2025B 
Estimated date of last patient completed: 22.04.2025 
Treatment Duration: 
12 weeks.  
 
3.1 
Study design  
N-DOSE is a single-center, phase II, double blinded, randomized, placebo-controlled dose-optimization 
clinical trial with a dose escalation design. Patients with PD (n = 80) who fulfil participation criteria (see 
section 4) will be randomized (1:1:2) into one of three groups: 1) The placebo group (PL-group, n = 20) 
will receive placebo for the duration of the study. 2) The NR 1000 mg group (DS-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 three 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, at the department of Neurology, 
Haukeland University Hospital (HUS).  

N-DOSE Protocol v3.1 
29 
 
 
Figure 3. N-DOSE 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. 
 
4 
STUDY POPULATION 
4.1 
Selection of Study Population 
The study will include individuals ≥ 40 years with a clinically established diagnosis of PD according to the 
MDS criteria43, and Hoehn and Yahr score < 4 (see full list of eligibility criteria below). Participants from 
all of Norway will be recruited at the department of Neurology, Haukeland University 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: 
• 
Clinically established diagnosis of PD according to the MDS criteria. 
• 
123I-Ioflupane dopamine transporter imaging (DAT-scan) or 18-F-FDOPA positron emission 
tomography imaging (18F-FDOPA PET) confirming nigrostriatal degeneration. 
• 
Hoehn and Yahr score < 4 at enrolment.  
• 
Age ≥ 40 years at the time of enrollment. 
• 
Able to undergo lumbar punction. 
• 
Able to undergo MRI 
 
4.4 
Exclusion Criteria 

N-DOSE Protocol v3.1 
30 
 
Patients will be excluded from the study if they meet any of the following criteria: 
• 
Dementia or other neurodegenerative disorder at baseline visit. 
• 
Diagnosed with atypical parkinsonism (PSP, MSA, CBD) or vascular parkinsonism. 
• 
Any psychiatric disorder that would interfere with compliance in the study. 
• 
Metabolic, neoplastic, or other physically or mentally debilitating disorder at baseline visit.  
• 
Use of high dose vitamin B3 supplementation within 30 days of enrollment. 
 
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 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 250 mg. 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 (250mg) + 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 capsules (500mg) + 4 placebo capsules] x 2 times daily (1000mg NR 
daily in total).  
o Weeks 5-8: [4 NR capsules (1000mg) + 2 placebo capsules] x 2 times daily (2000mg daily 
total).  
o Weeks 9-12: [6 NR capsules (1500mg) + 0 placebo capsules] 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, and must be taken prior to 
study visits.  
5.3 
Duration of Therapy 

N-DOSE Protocol v3.1 
31 
 
Therapy duration for the study is 3 months (12 weeks). 
5.4 
Dopaminergic therapy during Screening and Treatment period 
Eligible and consenting men and women with PD will be given optimal dopaminergic therapy, as indicated 
by their clinical disease.  All and any form of dopaminergic therapy formally approved in Norway may be 
used. Patients who do not use dopaminergic therapy are eligible, provided that they have a clinically 
established diagnosis of PD according to the MDS criteria. The treatment regimen will be optimized, if 
necessary, and will then be frozen and remain unchanged for the study period (3 months): 
• 
For patients who are already on dopaminergic therapy, the regimen may be adjusted, if 
necessary, to optimal clinical effect. Adjustment may involve change in dose, formulation, or 
drug. When adjustment of the dopaminergic regimen is undertaken, treatment efficacy will be 
assessed upon reexamination by physical or telephone consultation, until optimal effect or 
exclusion. Once the treatment regimen is deemed optimal, it will be frozen and remain 
unchanged for the study period (3 months).  
• 
Newly diagnosed and/or treatment naïve patients will be given dopaminergic treatment if- and 
as appropriate based on clinical indication. The treatment will be titrated to optimal effect. 
When adjustment of the dopaminergic regimen is undertaken, treatment efficacy will be 
assessed upon reexamination by physical or telephone consultation, until optimal effect or 
exclusion. Once the treatment regimen is deemed optimal, it will be frozen and remain 
unchanged for the study period (3 months).  
• 
Patients who do not use dopaminergic therapy are eligible, provided that they have a clinically 
established diagnosis of PD according to the MDS criteria. 
If adverse effects occur due to the dopaminergic therapy after enrollment, the treatment will be adjusted 
according to good clinical practice. All patients will be enrolled to the main study within 3 months after 
the last screening if inclusion/exclusion criteria are fulfilled.  
At the end of study visit (month 3), the physician determines whether the patient is still adequately 
treated for his/her parkinsonism with their current dopaminergic treatment.  
 
5.5 
Concomitant Medication 
Dopaminergic therapy will be given as described in section 5.4. above.  
In addition, the patient should not take any vitamin B3 supplements for the duration of the study.    
There are no other 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. 
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. A pill count of remaining 
medication will be performed when providing new study medication and at the end of the study.  

N-DOSE Protocol v3.1 
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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: 
• 
Kit number 
• 
Study number (CRF number) 
• 
Expiration date 
5.9 
Subject Numbering 
At the screening visit, each patient will be sequentially allocated a study identification number by the CRF 
(i.e., the registration code of the patient in the CRF). The study identification should be as follows: YYYY, 
where  YYYY is the chronological enrolled subject number (e.g., 0001, 0002, etc.). For example: 002 
(patient number 002). The next enrolled patient is registered as 003.  
The subjects will be identified by this study identification number for the remainder of the study. Once a 
patient number has been assigned, no attempt will be made to use that number again. 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 
6.1 
Flow Chart 
Table 1.  Trial flow chart  
 
Screening Period 
Treatment Period 
Study Visit 
Event 
First 
Screening 
Subsequent/ 
last screening 
Visit-1 
Baseline 
Visit-2 
Visit-3 
Visit-4 
Time 
 
 
Week 1 
Week 4  
Week 8 
Week 12 
Informed consent 
X 
 
 
 
 
 
Informed consent 
biobank 
X 
 
 
 
 
 
MDS Clinical diagnosis 
Criteria (MDS CDC) 
X 
 
 
 
 
 
Inclusion/exclusion 
Evaluation 
X 
 
X 
 
 
 
Medical history1 
X 
 
X 
 
 
 
Physical Examination2 
X 
 
X 
X 
X 
X 

N-DOSE Protocol v3.1 
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Screening Period 
Treatment Period 
Study Visit 
Event 
First 
Screening 
Subsequent/ 
last screening 
Visit-1 
Baseline 
Visit-2 
Visit-3 
Visit-4 
General neurological 
examination 
X 
 
 
 
 
 
Body height 
 
 
X 
 
 
 
Vital signs3 
 
 
X 
X 
X 
X 
Body weight and BMI 
 
 
X 
X 
X 
X 
Record of concomitant 
medication9 
X 
 
X 
X 
X 
X 
Hoehn and Yahr score  
X 
 
X 
X 
X 
X 
DAT-Scan or 18F-FDOPA 
PET4 
X 
 
 
 
 
 
31P-MRS & 1H-MRS of 
the brain 
 
 
X 
X 
X 
X 
FDG-PET of the brain 
 
 
X 
X 
X 
X 
MDS-UPDRS (I-IV) 
 
 
X 
X 
X 
X 
MDS-NMS 
 
 
X 
X 
X 
X 
MoCA 
 
 
X 
X 
X 
X 
EQ-5L 
 
 
X 
X 
X 
X 
GIDS-PD 
 
 
X 
X 
X 
X 
B-SIT 
 
 
X 
 
 
X 
Dietary registration (3 
days) 
 
 
X 
 
 
X 
Routine blood tests5 
 
 
X 
X 
X 
X 
Blood collection for 
biobanking6 
 
 
X 
X 
X 
X 
Cerebrospinal fluid 
collection 
 
 
X 
 
 
X 
Fecal sample collection 
 
 
X 
 
 
X 
Urine sample collection 
 
 
X 
X 
X 
X 
Treatment dispensation7 
 
 
X 
X 
X 
 
Dopaminergic treatment 
stable8 
X 
X 
 
 
 
X 
Adverse event 
 
 
 
X 
X 
X 
Superscripts refer to the specifications below. 
 
Specifications (details in section  
1. Medical history includes: family history of neurological illness (what and who), family history of PD 
(what and who), smoking history (period, pack-years), first PD symptoms, months since first PD 
symptoms, occurrence and duration of REM sleep disorder symptoms, occurrence and duration of 
loss of smell. 
2. Heart and lung auscultation, abdominal palpation, any other examination dictated by patient’s 
condition/symptoms 
3. Blood pressure, pulse, temperature 
4. DAT-Scan or 18F-FDOPA PET must have been performed prior to the Baseline Study Visit.  
5. 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. 
6. EDTA blood, snap-frozen EDTA blood, platelets, serum (see lab manual for details). 
7. To ensure correct dosages during dose escalation and if necessary to resupply.  

N-DOSE Protocol v3.1 
34 
 
8. The patient must be on a stable dopaminergic treatment regimen. When the patient is on a stable 
dopaminergic treatment, the screening is over, and the patient can be included in the study. The 
dopaminergic treatments are listed in section 5.4. There should not be more than 3 months from the 
last screening to the baseline visit. 
9. Record of concomitant medication: in addition to all drugs used by the patient, the type of 
dopaminergic medication used and the time points in the day at which each dose is taken must be 
registered. 
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 and anamnestic medical history is performed. If the patient fulfills the 
inclusion/exclusion criteria and gives informed consent, dopaminergic treatment is initiated/adjusted as 
described in the treatment flowchart in section 5.4.  
If the patient is optimally treated with dopaminergic treatment and fulfills the inclusion/exclusion criteria, 
then the patient is deemed ready for enrolment and may be referred to MRI and PET. If changes are made 
to the dopaminergic treatment, then the subject is contacted by phone after 2-6 weeks to assess if the 
treatment is optimal. If the patient is optimally treated, this dopaminergic treatment is frozen for the 
remainder of the study and the patient is referred to MRI and PET and called in for the baseline study visit 
(week 0 study visit). The patient should at screening be advised to stop using any Vit B3 supplement to 
fulfill inclusion criteria. There should not be more than 3 months from the last screening to baseline visit.  
Screening checklist:  
1. Patient signs informed consent for N-DOSE 
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. MDS clinical diagnosis Criteria 
5. Record current use of medication. Advise to stop any use of vit B3 supplements 
6. Introduce/adjust dopaminergic treatment as described in section 5.4.  
7. If the patient is ready for enrollment, they are referred to baseline study visit (including MRI and 
PET).   
8. The patient 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. 
9. Register when the patient takes their dopaminergic medication (dose and time of the day). 
10. Patient is asked to note the time of dopaminergic therapy intake on the morning of each visit 
11. Visits should be planned so that the patient is at the Department at a time permitting the MDS-
UPDRS to be conducted within no less than 15min and no more than 2 hours after the last 
intake of dopaminergic medication.  
12. MDS-UPDRS must be conducted at the same interval from the last dopaminergic therapy dose 
on every visit (+/- 30min) 
6.2.2 Baseline/Week 0 
1. Investigator verifies the informed consent for the study.  

N-DOSE Protocol v3.1 
35 
 
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 the morning of each visit.  
6. Register the time the patient took dopaminergic medication on the morning of the visit as well 
as the type and dose of the medication. 
7. The following clinical examination are performed by the study nurse or investigator (see 
flowchart in section 6.1):  
o Vital signs 
o Body weight and BMI 
o MDS CDC 
o MDS-UPDRS: section 1&2 by study nurse/investigator, section 3&4 by investigator 
o MDS-NMS 
o MoCA 
o EQ-5L  
o GIDS-PD 
8. Routine blood tests are done (see flowchart in section 6.1) 
9. Samples for biobanking are taken (see flowchart in section 6.1): 
o EDTA whole blood 
o Snap-frozen whole blood 
o PAXgene blood for RNA 
o Serum 
o Fecal sample 
o Urine sample 
o Cerebrospinal fluid 
10. Imaging is conducted 
o 31P-MRS & 1H-MRS 
o FDG-PET 
11. 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 (MDS-UPDRS, MDS-NMS, MoCA, MDS CDC, EQ-5L, GIDS-
PD) are recorded in paper format.  
12. The patient is given the 3-day dietary registration form along with instructions on how to fill it 
out and deliver it to the study nurse at the next visit. 
13. Patient is reminded to note the time of dopaminergic therapy intake on the morning of the next 
visit 
14. Next visit should be planned so that the patient is at the Department at a time permitting the 
MDS-UPDRS to be conducted within no less than 15min and no more than 2 hours after the last 
intake of dopaminergic medication.  
15. MDS-UPDRS must be conducted at the same interval from the last dopaminergic therapy dose 
on every visit (+/- 30min) 
16. B-SIT is performed by either the study nurse or investigator (see flowchart in section 6.1). 

N-DOSE Protocol v3.1 
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6.2.3 
During treatment (visits 2-4) 
• 
Should the patient need replenishing of study medication, it will be logistically handled by the 
study nurse.  
• 
On visit 2 points 5-11 and 13-15 from section 6.2.2 are repeated. 
• 
 
• 
On visit 3, points 5-15 from section 6.2.2 are repeated. 
6.2.4 
End of study (Visit 4) 
• 
The patient brings with them the 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 6-11 from section 6.2.2 are repeated.  
• 
B-SIT is performed by either the study nurse or investigator (see flowchart in section 6.1). 
• 
At the end of the study visit, consider changes in the dopaminergic treatment. 
 
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. 
• 
Safety reasons as judged by the Principal Investigator. 
• 
Incorrect enrolment, 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 records 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 the 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. 
 

N-DOSE Protocol v3.1 
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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 AE registration 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.  
7.2 
Clinical Assessments 
1. Medical history: 
Performed by: investigator  
a. Family history of neurological illness:  
i. Who   
ii. Which illness. 
b. Family history of PD: 
i. Who 
ii. Was the diagnosis certain, probable or possible 
c. Smoking history: 
i. Active smoker, previous smoker, or never smoker? 
ii.  Cigarettes smoked daily (on average) 
iii. Duration of smoking (years) 
iv. Pack-years (pack-years = cigarettes smoked daily / 20 x years of smoking) 
d. First PD symptoms: 
i. Which symptoms: tremor, bradykinesia, gait abnormality, rigidity, loss of smell, other 
(if other = free text comment). 
ii. History of REM sleep behavior disorder (RBD) 
1. RBD: yes/no 
2. If yes – duration (i.e., for how long has the subject had it) 
e. Presence and duration of olfactory changes: 
i. Complete loss of smell: yes/no? 
ii. Decreased sense of smell?  
iii. If yes – duration (i.e., for how long has the subject had it) 
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: 

N-DOSE Protocol v3.1 
38 
 
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. MDS CDC:  
Performed by: investigator 
b. MDS-UPDRS I-IV 
i. NB! The exact time MDS-UPDRS is assessed must be registered in the eCRF.  
Performed by: section 1&2 by study nurse/investigator, section 3&4 by investigator 
c. MDS-NMS: 
Performed by: Investigator or study nurse 
d. MoCA:  
Performed by: study nurse 
e. EQ-5L: 
Performed by: study nurse 
f. GIDS-PD: 
Performed by: study nurse 
g. 3-day dietary record:  
Performed by: self-filled by the patient 
 
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. DAT-scan, performed according to standard clinical routine, will confirm the presence of 
nigrostriatal degeneration.  
2. 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 
before (Fig 1A-C).  
3. 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 shown (Fig 1D-E). Following 
standard preprocessing protocols and spatial normalization, the NRRP will be assessed using ordinal 
trends/canonical variates analysis (OrT/CVA), a supervised form of principal component analysis 
(PCA)33. This multivariate approach is designed to detect and quantify regional covariance patterns 

N-DOSE Protocol v3.1 
39 
 
(i.e., metabolic networks) for which expression values (i.e., subject scores) increase or decrease with 
treatment in all or most of the subjects34–38.  
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 described44. 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, 
homocysteine, 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 described45. 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.  
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 analyzed 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) 

N-DOSE Protocol v3.1 
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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. 
8.1.2 
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 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 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 grade 2 and 3 is registered as AE in eCRF . 

N-DOSE Protocol v3.1 
41 
 
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, and or requires clinical or diagnostic observations. 
2. Moderate: An event that causes sufficient discomfort to interfere with normal everyday 
activities, and or requires medical intervention. 
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: 
o 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. 
o 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. 
o Possible: There is reasonable causal relationship between the investigational product and the 
AE. Dechallenge information is lacking or unclear. 
o Probable: There is a reasonable causal relationship between the investigational product and 
the AE. The event responds to dechallenge. Rechallenge is not required. 
o 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, (coordinating investigator: Haakon Berven or 
PI Charalampos Tzoulis, 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.  

N-DOSE Protocol v3.1 
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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 to 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.  
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 

N-DOSE Protocol v3.1 
43 
 
• 
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 and will ask the site staff to 
adjust any discrepancies as required.  
When the responsible study monitor has checked and verified the e-CRFs, the data will be entered into a 
computer database at the Haukeland University hospital scientific server for further handling and 
statistical evaluation. 
Sponsor’s representatives (e.g., monitors, auditors) and/or competent authorities will be allowed access 
to source data for source data verification in which case a review of those parts of the hospital records 
relevant to the study may be 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 the hospital. The study 
documentation (e-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 Charalampos Tzoulis.  
• 
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 
customized 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 

N-DOSE Protocol v3.1 
44 
 
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 authorized study center 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 authorize 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 
receiving 1000 mg NR (n = 15) compared to the placebo group (n = 15). In the NADPARK study46, 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 exists 
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.  

N-DOSE Protocol v3.1 
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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.  
• 
Major change in concomitant anti-Parkinson medication.  
• 
Received or used other intervention than allocated.  
• 
Adherence to allocated treatment below 85%.  
• 
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 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 analyses will take place after the completion of the study. 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. 
 

N-DOSE Protocol v3.1 
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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 MDS-
UPDRS motor 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, as we have previously described12,45,47,48. 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 as we have previously described12,45,47,48.  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 reader to this document. 
10.5 
Statistical Analysis  
Dependent variable 
Primary analysis: 
- 
Cerebral NAD levels (measured by 31P-MRS) 
Secondary, exploratory and safety analyses:  
- 
Adverse events, categorized as either moderate or severe. 
- 
Levels of CSF NAD and NAD-metabolites (measured by LC-MS). 
- 
Levels of NRRP expression (measured by FDG-PET) 
- 
Levels of PDRP expression (measured by FDG-PET) 
- 
Routine blood tests  
- 
Total MDS-UPDRS 
- 
MDS-UPDRS Part I 
- 
MDS-UPDRS Part II 
- 
MDS-UPDRS Part III 
- 
MDS-UPDRS Part IV 
- 
MDS-NMS 
- 
MoCA 
- 
GIDS-PD 
- 
EQ-5D-5L 
- 
Levels of NAD-related and other metabolites in PBMC, 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 

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- 
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 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. 
- 
Sense of smell, measured by B-SIT. 
 
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:  
- 
Mean change between the NR dose escalation arm and NR 1000 mg 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 and the Ethics Committee according to EU and national 
regulations. 
11.4 
Audit and Inspections 
Authorized representatives of the Ethics Committee may visit the center to perform inspections, including 
source data verification. Likewise, the representatives from the sponsor may visit the center to perform 
an audit. The purpose of an audit or inspection is to systematically and independently examine all study-

N-DOSE Protocol v3.1 
48 
 
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 
Application to REK has been submitted. 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 (SLV)/Direktoratet for medisinske produkter (DMP) 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.  
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 
remainder of the study. The Patient study number is in the format XXYYY 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 Pasientskade Erstatning” (NPE).  

N-DOSE Protocol v3.1 
49 
 
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 
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2.  
Gooch CL, Pracht E, Borenstein AR. The burden of neurological disease in the United States: A 
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Katsyuba E, Romani M, Hofer D, Auwerx J. NAD+ homeostasis in health and disease. Nat Metab 
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Braidy N, Liu Y. NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis. Exp. 
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Schapira AH, Cooper JM, Dexter D, et al. Mitochondrial complex I deficiency in Parkinson’s disease. 
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IV of mitochondria in Parkinson’s disease. Ann. Neurol. 1991;30(4):563–571. 
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the Parkinson’s disease brain, but is not associated with neurodegeneration or mitochondrial DNA 
damage. Acta Neuropathol 2018;135(3):409–425. 
11.  Hirsch EC, Hunot S. Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet 
Neurol 2009;8(4):382–397. 
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13.  Park G, Tan J, Garcia G, et al. Regulation of Histone Acetylation by Autophagy in Parkinson Disease. 
The Journal of biological chemistry 2016;291(7):3531–40. 
14.  Gonzalez-Hunt CP, Sanders LH. DNA damage and repair in Parkinson’s disease: Recent advances and 
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genes establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell 
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16.  Conze DB, Crespo-Barreto J, Kruger CL. Safety assessment of nicotinamide riboside, a form of 
vitamin B3. Hum Exp Toxicol 2016;35(11):1149–1160. 
17.  Dollerup OL, Christensen B, Svart M, et al. A randomized placebo-controlled clinical trial of 
nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects. Am. J. 
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riboside supplementation in Parkinson’s disease. Cell Metabolism 2022;34(3):396-407.e6. 
19.  Conze D, Brenner C, Kruger CL. 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 2019;9(1):9772. 
20.  EFSA Panel on Nutrition. European Food Safety Authority Safety of nicotinamide riboside chloride 
[Internet]. 2019.Available from: http://www.efsa.europa.eu/en/efsajournal/pub/5775 
21.  Haukeland University Hospital. NR-SAFE: a Safety Study Investigating Treatment With High-dose 
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28 ] Available from: https://clinicaltrials.gov/ct2/show/NCT05344404 
22.  Trammell SA, Schmidt MS, Weidemann BJ, et al. Nicotinamide riboside is uniquely and orally 
bioavailable in mice and humans. Nat Commun 2016;7:12948. 
23.  Martens CR, Denman BA, Mazzo MR, et al. Chronic nicotinamide riboside supplementation is well-
tolerated and elevates NAD+ in healthy middle-aged and older adults. Nat Commun 2018;9(1):1286. 
24.  Elhassan YS, Kluckova K, Fletcher RS, et al. Nicotinamide Riboside Augments the Aged Human 
Skeletal Muscle NAD+ Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures. 
Cell Rep 2019;28(7):1717-1728.e6. 
25.  Okur MN, Mao B, Kimura R, et al. Short-term NAD+ supplementation prevents hearing loss in mouse 
models of Cockayne syndrome. NPJ Aging Mech Dis 2020;6:1. 
26.  Brown KD, Maqsood S, Huang JY, et al. Activation of SIRT3 by the NAD(+) precursor nicotinamide 
riboside protects from noise-induced hearing loss. Cell Metab 2014;20(6):1059–68. 
27.  Han S, Du Z, Liu K, Gong S. Nicotinamide riboside protects noise-induced hearing loss by recovering 
the hair cell ribbon synapses. Neurosci. Lett. 2020;725:134910. 
28.  Harlan BA, Killoy KM, Pehar M, 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. 
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29.  Sorrentino V, Romani M, Mouchiroud L, et al. Enhancing mitochondrial proteostasis reduces 
amyloid-β proteotoxicity. Nature 2017;552(7684):187–193. 
30.  Xie X, Gao Y, Zeng M, et al. Nicotinamide ribose ameliorates cognitive impairment of aged and 
Alzheimer’s disease model mice. Metab Brain Dis 2019;34(1):353–366. 
31.  Schöndorf DC, Ivanyuk D, Baden P, et al. The NAD+ Precursor Nicotinamide Riboside Rescues 
Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson’s Disease. Cell Rep 
2018;23(10):2976–2988. 
32.  Haukeland University Hospital. NAD-PARK: A Double-blinded Randomized Pilot Trial of NAD-
supplementation in Drug naïve Parkinson’s Disease [Internet]. clinicaltrials.gov; 2020.[cited 2021 
Mar 18 ] Available from: https://clinicaltrials.gov/ct2/show/NCT03816020 
33.  Hirano S, Eckert T, Flanagan T, Eidelberg D. Metabolic Networks for Assessment of Therapy and 
Diagnosis in Parkinson’s Disease. Mov Disord 2009;24(0 2):S725–S731. 
34.  Bloem BR, Okun MS, Klein C. Parkinson’s disease [Internet]. The Lancet 2021;0(0)[cited 2021 Apr 17 
] Available from: https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(21)00218-
X/abstract 
35.  Jadavji NM, Wieske F, Dirnagl U, Winter C. Methylenetetrahydrofolate reductase deficiency alters 
levels of glutamate and γ-aminobutyric acid in brain tissue. Mol Genet Metab Rep 2015;3:1–4. 
36.  Romano S, Savva GM, Bedarf JR, et al. Meta-analysis of the Parkinson’s disease gut microbiome 
suggests alterations linked to intestinal inflammation. npj Parkinsons Dis. 2021;7(1):1–13. 
37.  Payne T, Appleby M, Buckley E, et al. A Double-Blind, Randomized, Placebo-Controlled Trial of 
Ursodeoxycholic Acid (UDCA) in Parkinson’s Disease. Movement Disorders 2023;38(8):1493–1502. 
38.  Chaudhuri KR, Schrag A, Weintraub D, et al. The Movement Disorder Society Nonmotor Rating Scale: 
Initial Validation Study. Mov Disord 2020;35(1):116–133. 
39.  Goetz CG, Tilley BC, Shaftman SR, et al. Movement Disorder Society-sponsored revision of the 
Unified Parkinson’s Disease Rating Scale (MDS-UPDRS): Scale presentation and clinimetric testing 
results: MDS-UPDRS: Clinimetric Assessment. Mov. Disord. 2008;23(15):2129–2170. 
40.  Camacho M, Greenland JC, Williams-Gray CH. The Gastrointestinal Dysfunction Scale for Parkinson’s 
Disease. Mov Disord 2021;36(10):2358–2366. 
41.  Nasreddine ZS, Phillips NA, Bédirian V, et al. The Montreal Cognitive Assessment, MoCA: a brief 
screening tool for mild cognitive impairment. J Am Geriatr Soc 2005;53(4):695–699. 
42.  Janssen MF, Bonsel GJ, Luo N. Is EQ-5D-5L Better Than EQ-5D-3L? A Head-to-Head Comparison of 
Descriptive Systems and Value Sets from Seven Countries. Pharmacoeconomics 2018;36(6):675–
697. 
43.  Postuma RB, Berg D, Stern M, et al. MDS clinical diagnostic criteria for Parkinson’s disease. 
Movement disorders : official journal of the Movement Disorder Society 2015;30(12):1591–601. 
44.  Trammell SA, Brenner C. Targeted, LCMS-based Metabolomics for Quantitative Measurement of 
NAD(+) Metabolites. Computational and structural biotechnology journal 2013;4:e201301012. 

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45.  Nido GS, Dick F, Toker L, et al. Common gene expression signatures in Parkinson’s disease are driven 
by changes in cell composition. Acta Neuropathol Commun 2020;8(1):55. 
46.  Brakedal B, Dölle C, Riemer F, et al. The NADPARK study: A randomized phase I trial of nicotinamide 
riboside supplementation in Parkinson’s disease. Cell Metab 2022;34(3):396-407.e6. 
47.  Dick F, Nido GS, Alves GW, et al. Differential transcript usage in the Parkinson’s disease brain 
[Internet]. 
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48.  Dick F, Tysnes O-B, Alves GW, et al. Altered transcriptome-proteome coupling indicates aberrant 
proteostasis in Parkinson’s disease. medRxiv 2021;2021.03.18.21253875. 
49.  Ladefoged CN, Hansen AE, Henriksen OM, et al. AI-driven attenuation correction for brain PET/MRI: 
Clinical evaluation of a dementia cohort and importance of the training group size. Neuroimage 
2020;222:117221. 
 
 
 
 
 
 

N-DOSE Protocol v3.1 
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17 
LIST OF APPENDICES 
A. Lab Manual 
B. Imaging Manual 
C. Clinical Rating Scales 
 
 
 

N-DOSE Protocol v3.1 
54 
 
APPENDIX A   
 
LABORATORY MANUAL FOR N-DOSE 
 
Helse Bergen, Haukeland University Hospital 
Protocol:   
N-DOSE 
 
REK Number:  
 
N-DOSE SAFE: A randomized, double blind, dose optimization trial of 
nicotinamide riboside in Parkinson’s disease. 
STANDARD OPERATING PROCEDURE (SOP) FOR SAMPLING AND PREPARATION OF 
BIOLOGICAL MATERIAL (LAB MANUAL) 
Project Management Committee for the study  
Charalampos Tzoulis 
Haakon Berven 
 
 

N-DOSE Protocol v3.1 
55 
 
1 GENERAL INFORMATION 
The purpose of this document is to give an overview of the biological material collected for biobanking 
in the N-DOSE 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 Neuro-
SysMed Center.  
In the following chapters you will find a detailed description of how to collect, prepare, and store 
samples at 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 Neuro-SysMed Center.  
 
 

N-DOSE Protocol v3.1 
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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. 
 

N-DOSE Protocol v3.1 
57 
 
Flow chart 
 
TUBES 
 
 
 
 
 
Visits 
V1 / 
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 
 
homocysteine 
X 
X 
X 
X 
 
Methylmalonic acid 
X 
X 
X 
X 
 
Sodium 
X 
X 
X 
X 
 
Potassium 
X 
X 
X 
X 
 
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 
Platelets 
X 
X 
X 
X 
 
Serum 
X 
X 
X 
X 
FluidX, 0,7ml, 8 aliquotes  
Fecal sample 
X 
 
 
X 
 
Urine sample 
X 
X 
X 
X 
 
Cerebrospinal fluid 
X 
 
 
X 
 

N-DOSE Protocol v3.1 
58 
 
 
2.1.1 
Safety laboratory (blood)  
All safety laboratory parameters will be collected at the timepoints as indicated in the Flow Chart 
(page 47), and include hematology, liver enzymes/parameters, clinical chemistry, thyroid status. The 
samples will be analyzed at the local laboratory at the study site. The respective reference ranges must 
be provided to the central study administration for uploading into the eCRF.  
3 SAMPLING FOR BIOBANK AND RESEARCH 
3.1 Mandatory research and biobank samples 
Chapter 3.1 gives an overview of all mandatory samples 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 HUS 
3.1.2 
Whole blood EDTA – snap-frozen  
Snap-frozen EDTA blood will be collected at HUS 
3.1.3 
RNA PAXgene tubes 
Blood in PAXgene tubes for RNA extraction will be collected at HUS 
3.1.4 
Whole blood for Serum 
Serum will be collected at HUS  
3.1.5 
Platelets 
Platelets will be collected at HUS 
3.1.6 
Cerebrospinal fluid (CSF) 
CSF will be collected at HUS 
3.1.7 
Feces 
Feces will be collected at HUS 
3.1.8 
Urine 
Urine will be collected at HUS 
 
 
 
 
 
 
 
 
 
 
 
 

N-DOSE Protocol v3.1 
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4 SAMPLE PROCESSING AND STORAGE 
4.1 
Whole blood (EDTA): standard 
Performed at: HUS 
 
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 deviations on the same 
registration form.  
5. Sample aliquots 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. The number and date of thawing an aliquot for 
analyses must be registered.   
 
Contact person for biosampling and laboratory preparation:  
Siri Hinteregger, MBF 
 
Contact person for Biobank: 
Hilde Kristin Garberg 
 
 
 
 
 
 
 
 
 
 
FluidX tubes 
FluidX tubes box (x48) 

N-DOSE Protocol v3.1 
60 
 
4.2 
Whole blood (EDTA): snap-frozen 
Performed at: HUS 
 
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 technicians must wear gloves and a 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. The 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 

N-DOSE Protocol v3.1 
61 
 
4.3 
RNA PAXgene tubes 
Performed at: HUS  
 
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 long-term 
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 shipment: 
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. Temporarily 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 aliquots 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: 

N-DOSE Protocol v3.1 
62 
 
Contact person for biosampling and laboratory preparation: Siri Hinteregger, MBF 
Contact person for Biobank: Hilde Kristin Garberg 
 
4.4 
Serum for biobanking 
Performed at: HUS  
 
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 deviations 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 
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 aliquots 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.   
 
 

N-DOSE Protocol v3.1 
63 
 
 
Responsible: 
Contact person for biosampling and laboratory preparation: Siri Hinteregger, MBF 
Contact person for Biobank: Hilde Kristin Garberg 
 
4.5 
Platelet isolation and cryopreservation for mitochondrial research  
Performed at: Bergen  
 
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 20min, 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 10ml Falcon 
tube  
6. Freeze the cells in their own plasma adding DMSO at a final 
concentration of 5-6% 
7. Store at -80°C  
 
Responsible: 
Contact person for biosampling and laboratory preparation: Siri Hinteregger, MBF 
Contact person for Biobank: Hilde Kristin Garberg 
4.6 
Collection, of cerebrospinal fluid 
Material and instrumentation: 
Item 
How many 
Supplier 
Cat no 
Comment 
Standard lumbar puncture equipment 
 
 
 
 
Standard CSF cell count tubes 
2 
 
 
 
5 ml PolyPropylene (PP) cryo-s tube with red 
PP cap  
2 
Greiner Bio-One via 
VWR 
479-4154 
2 uker 
0.7 ml FluidX tubes (PP) 
15 
Pedro Consulting 
68-0702-11N 
 
5 ml PP tube for dementia biomarkers 
1 
Sarstedt 
63-504-027 
 
Barcode reader 
 
 
 
 
Centrifuge for centrifugation tubes 
 
Study center 
 
 

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PP PIPETTE TIPS (ART 1000µL REACH) 
 
VWR 
732-2215 
(2079-HR) 
Alternative tip will 
be used 
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 (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 punction 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 standard tube for cell count (erythrocytes and leucocytes) 
3. 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 for dementia biomarkers (63-504-027) for clinical analysis of 
abeta42, t-tau and p-tau181. 
5. 10 drops spinal fluid collected in standard tube for cell count (erythrocytes and leucocytes) 
 
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 
1. The collected 5ml x 2 CSF in “5 ml PolyPropylene (PP) cryo-s tube with red PP cap”  
a. NB! The sample has to be centrifuged within 30 min from collection! 
b. Centrifuge within 30 min at 2,000 g, at 4 °C, for 10 min  
c. Transfer the supernatant to 2 x new “5 ml PolyPropylene (PP) cryo-s tube with red 
PP cap” – use PP pipette tips 
d. Mix carefully by inverting the tube x 7-10 times (or mild vortexing) 
e. Aliquot 500 µl CSF per tube into 0.7 ml FluidX tubes 
 - use PP pipette tips 
f. Freeze the aliquots at -80°C. 
g. Sample aliquots are stored at -80C and shipped on dry ice in the original FluidX 
boxes (x 48 tubes). 
h. 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.   
 
2. The collected 2 ml CSF in 5 ml PP tube for dementia biomarkers will be used for analysis of 
abeta42, t-tau and p-tau181: 
a. Transfer to -80C immediately after collection. 
b. Samples are stored at -80C in freezer-compatible cardboard or plastic storage boxes.  
c. Samples are shipped on dry ice. 
d. Further handling: 

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i. Bergen will send batches of 20 samples to Akershus University Hospital for 
dementia marker analysis   
ii. Trondheim will send batches of 20 samples to Akershus University Hospital 
for dementia marker analysis 
iii. London will send this along with the rest of the CSF sample to Bergen (see 
below).   
3. Samples must never be allowed to warm or thaw except from when to be used in analyses. 
Any deviation from this must be registered. The number and date of thawing an aliquot for 
analyses must be registered.   
 
Responsible: 
Contact person for biosampling and laboratory preparation: Siri Hinteregger, MBF 
Contact person for Biobank: Hilde Kristin Garberg 
 

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4.7 
Fecal sample for biobanking 
Performed at: HUS  
 
Fecal samples from the last 24h will be collected according to standard clinical routines and stored at 
-80C. The patient must bring a 3-day diet registration from together with the fecal sample om the 
morning of each visit. For details on fecal sample collection and storage, see document: “SOP for 
fecal sample collection” (Prosedyre for avføringsprøvetaking hjemme).  
 
Responsible: 
Bergen: Birgitte Betrentsen 
 

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4.8 
Urine for biobanking 
Performed at: HUS  
 
Morning urine will be collected according to standard clinical routines, aliquoted and stored at -80C. 
Responsible: 
Bergen: Hanne Linda Nakkestad 
 

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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 
Department of Neurology 
Contact person for cells, CSF collection 
Kibret Mazinga 
90236164, kibret.yimer.mazengia@helse-bergen.no 
Brage Brakedal 
Kristoffer Haugarvoll 
Geir Olve Skeie 
55975045 / 99777962, bragebrakedal@gmail.com  
55975045 / 98266741, haugarvoll@gmail.com  
55975045 / 48103361, Geir.Skeie@uib.no  
Gry Hilde NIlsen 
Hanne Linda Nakkestad 
95274886, Gry.Nilsen@uib.no  
97571149, hanne.linda.nakkestad@helse-bergen.no  
 
  
 
 
 

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APPENDIX B - MRI AND FDG-PET – PROTOCOL, N-DOSE  
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) 
 
Bergen:  
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 (sagittal, 1x1x1 mm, 3D-BRAVO or 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. 
Total imaging time:  
25.5 min imaging time - 35 min including changing the coil for CSI. 
Specific procedures for 31P-MRS image analysis: 

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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 
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 
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(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 PD-related metabolic pattern 
(termed PDRP)17 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 PDRP 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). 
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). 

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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). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX C - CLINICAL RATING SCALES 
 
1 
GENERAL INFORMATION 
The following appendix lists the clinical rating scales used in the N-DOSE study. Where clinical rating 
scales were used in specific sequences, this has been specified. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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2 
MRS-UPDRS 
The MDS-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale 
For Part 1, part 3 and part 4, the English version was used. For part 2, the Norwegian version 
of the questionnaire was used.  
MRS-UPDRS – Page 1 
 

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3 
MDS-NMS 
International Parkinson and Movement Disorder Society – Non-Motor Rating Scale 
MDS-NMS - Page 1 
 
 
 

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4 
MODIFIED GIDS-PD 
The Gastrointenstinal Dysfunction Scale for Parkinson’s Disease modified*  
The following version of GIDS-PD has been modified by permission and license from MDS for use in 
the N-DOSE trial. The modifications in this version is that the timeframe for all questions has been 
reduced from six months to one month. Part 1b has also been omitted from the scale.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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5 
MOCA 
Montreal Cognitive Assessment 
MoCA tests were alternated to avoid learning effects. The sequence of versions for testing for each 
participant is listed in the table below: 
Visit  
Version 
1 (Week 0) 
Version 8.3 Norwegian version 
2 (Week 4) 
Version v8.1 Norwegian version 
3 (Week 8) 
Version V8.2 Norwegian version 
4 (week 12) 
Version v8.3 Norwegian version 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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MocA – Version 8.1 Norwegian version 
 
 
 
 

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6 
EQ-5D-EL 
The 5-level EQ-5D 
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