Exploratory Study of Nicotinamide Riboside on Mitochondrial Function in Li-Fraumeni Syndrome
Phase: PHASE1
Conditions: Cancer, Skin Fibroblasts, Muscle Weakness
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
CLINICAL RESEARCH PROJECT 
                                     NCT03789175 
 
 
 
 
 
 
 
 
 
     12/12/2018 
 
 
 
 
 
 
 
 
 
     Protocol # P184388 
 
 
 
 
 
 
                                     IND #: NA 
(Note: Supplement, Nicotinamide Riboside, is not subject to IND regulations as covered 
by the Dietary Supplement Health and Education Act of 1994.) 
 
 
NHLBI Protocol:  Exploratory Study of Nicotinamide Riboside on Mitochondrial 
Function in Li-Fraumeni Syndrome 
 
Short Title: Nicotinamide Riboside in Li-Fraumeni syndrome 
 
Keywords:  p53, skeletal muscle, mitochondria 
 
Principal Investigator: 
*Paul M Hwang, MD, PhD (E) 
Cardiovascular Branch 
National Heart, Lung, and Blood Institute 
National Institutes of Health 
Building 10, Room 5-5-5330 
Bethesda, MD 20892 
Phone: 301-435-3068 
 
Associate Investigators: 
*Rebecca Huffstutler, CRNP 
CB, NHLBI (E) 
*Michael N. Sack, MD, PhD 
CB, NHLBI (E) 
Ping-yuan Wang, PhD 
 
CB, NHLBI (E)  
 
*Investigators authorized to obtain informed consent 
 
Research Coordinator: 
*Rebecca Huffstutler, CRNP 
CPB, NHLBI (E) 
 
Biospecimen Tracking: 
 
BSI 
Study Site: 
 
 
 
NIH 
 
Subjects in study at NIH:  
Number - 1; Gender - M/F; Age range - 18-70 years 
Multi-center trial: 
 
 
No 
Ionizing Radiation for Research: No 
Off-Site Project: 
 
 
No 
DSMB Involvement:  
 
No 
Tech Transfer: 
 
 
No 
IND/IDE: 
 
 
 
No 
  

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
Table of Contents 
  
A.    Precis …………………………………………………………………………. 3 
B. 
Background and Preliminary Results……………………………………...  3 
C. 
Hypothesis and Objectives  ………………………………………………… 6 
D. 
Recruitment and screening ………………………………………………….6 
E. 
Eligibility Assessment ………………………………………………………..7 
F. 
Study Design and Schedule ………………………………………………….7 
G. Procedures ……………………………………………………………………11 
H. Dietary Supplement ………………………………………………………….13 
I. 
Data and Biospecimen Management Plan ………………………………….14 
 
Data sharing and future use of data ………………………………………...15 
J. 
Statistical Considerations ……………………………………………………16 
K. Off Study Criteria ……………………………………………………………16 
L. 
Data and Safety Monitoring …………………………………………………16 
        Safety monitoring ……………………………………………………………..16 
Adverse event reporting ……………………………………………………...17 
M. 
Human Subjects Protections …………………………………………………20 
Rationale for subject selection ……………………………………………….19 
Rationale for the exclusion of children ……………………………………...19 
Rationale for the exclusion of pregnant women …………………………….19 
Rationale for the exclusion of cognitively impaired subjects ………………20 
Risk/benefit assessment ………………………………………………………20 
Risks and discomforts ………………………………………………………...20 
N. 
Conflict of Interest ……………………………………………………………23 
O. Reimbursement for Travel …………………………………………………...23 
P. 
Financial Compensation ……………………………………………………...24 
Q. References ……………………………………………………………………..25 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
 
A.  Precis 
 
 
We have previously reported that inherited mutations of TP53, which causes the 
premature cancer disorder Li-Fraumeni syndrome (LFS), can promote mitochondrial 
function both in patients and mouse models (1).  In the course of our follow up studies, 
we encountered a LFS patient with a long-standing history of fatigue and muscle 
weakness of unclear etiology.  Notably, we observed in vivo evidence of markedly 
decreased mitochondrial function in her leg skeletal muscle during exercise using 
noninvasive phosphorus-31 magnetic resonance spectroscopy (31P-MRS), a technique 
that has previously been used to study patients with primary mitochondrial disorders.  
The decrease in mitochondrial function was also confirmed by the patient’s skin 
fibroblasts in vitro using standard biochemical measurements. 
 
There is growing evidence that nicotinamide adenine dinucleotide (NAD+) 
homeostasis plays a significant role in maintaining the mitochondria through various 
mechanisms and that it is possible to improve mitochondrial function by dietary 
supplementation with the vitamin B3 analogue nicotinamide riboside (NR), an 
intermediate in the NAD+ salvage pathway.  Remarkably, we observed that culturing the 
LFS patient’s fibroblasts in medium containing NR rescued the severe deficit in 
mitochondrial respiration.  While continuing our investigations into the molecular 
mechanism(s) underlying the mitochondrial dysfunction observed in this patient, the in 
vitro rescue of the respiratory deficiency by NR presents a unique opportunity to 
investigate whether it can also be observed in vivo using skeletal muscle 31P-MRS. We 
propose to explore the effect of NR, currently available as a dietary supplement, on in 
vivo mitochondrial function in this LFS patient. 
 
 
B.  Background and Preliminary Results 
 
 
Mitochondrial metabolism plays an essential role in both tumorigenesis and 
cardiovascular function.  We showed that TP53, which encodes the p53 protein and is 
the most commonly mutated tumor suppressor gene in human cancers, can regulate 
mitochondrial respiration and impact aerobic exercise capacity (2) (3).  Beside 
modulating genes important for cell cycle regulation and cell death, it is now clear that 
p53 can regulate mitochondrial biogenesis both by transcriptional mechanisms and by 
direct effects within the mitochondria (4).  We subsequently provided evidence that 
patients with Li-Fraumeni syndrome (LFS), an early onset cancer disorder caused by 
germline mutations of TP53, have increased oxidative metabolism through our 
translational clinical protocol NHLBI 07-H-0030 (1).  We have continued to study 
mitochondrial metabolism in LFS patients, because insights from our translational study 
have provided impetus for testing a new cancer prevention strategy in LFS (5).  
Furthermore, LFS is caused by many different germline mutations of the TP53 gene, so 
the specific metabolic effects of each unique p53 mutation remains to be clarified. 
 
During the course of our studies, a LFS patient (Patient A) with prior history of 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
breast cancer participated in our clinical protocol with the chief complaint of life-long 
fatigue but with no clear diagnosis after medical evaluation.  Although treadmill exercise 
testing can be used as a marker of mitochondrial function, there are many factors such 
age, gender and exercise training that can contribute to aerobic fitness.  To avoid these 
potential confounders, we adapted a previously developed non-invasive technique to 
directly measure mitochondrial function in the skeletal muscle (6).  This method relies 
on the regeneration of phosphocreatine (PCr) that normally serves to shuttle high 
energy phosphate from the mitochondria to the cytosol in order to maintain skeletal 
muscle ATP levels during work performance.  The measurement of PCr regeneration 
after exercise-induced depletion using phosphorus-31 magnetic resonance 
spectroscopy (31P-MRS) has been reported to provide a sensitive measure of in vivo 
oxidative phosphorylation capacity (7).  Consistent with her symptoms, we observed a 
profound delay in the phosphocreatine recovery time constant (PCr recovery Tc) of ~80 
sec, indicating decreased mitochondrial function (Figure 1A). 
 
 
This was in marked contrast to the PCr recovery Tc values of 30 sec and 37 sec in her 
family members (Patients B and C) carrying the same TP53 mutation, which were both 
within range of our previously published mean values of ~31 sec in a cohort of LFS 
patients and ~37 sec in non-mutation carriers (Figure 1B) (1).  Further investigations in 
the laboratory using skin fibroblasts obtained from Patient A compared with Patient B 
confirmed decreased mitochondrial respiration (oxygen consumption) using standard 
biochemical techniques (Figure 2). 
 
PCr recovery Tc = 80 s
Patient A
Patient C
Patient B
Relative PCr level
Time (s)
PCr recovery Tc = 30 s
PCr recovery Tc: 37 s
Time (s)
Time (s)
Figure 1.  Delayed PCr recovery Tc in LFS Patient A compared with Patients B and C.  
Noninvasive measurement of phosphocreatine levels in the tibialis anterior skeletal muscle by 31P-
MRS before, during and after exercise.  The arrowheads point to the derived PCr recovery curves 
after stopping leg exercise in the indicated patients.  Note the slow rise in PCr levels after exercise in 
Patient A compared with Patients B and C which were quantified by the PCr recovery Tc kinetics.  
The delayed PCr recovery Tc in Patient A was confirmed by another 31P-MRS study 6 months later. 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
 
 
A consequence of impaired mitochondrial oxidative phosphorylation is decreased 
NAD+ levels, which in turn appears to exacerbate mitochondrial dysfunction by 
inactivating the NAD+ dependent deacetylase enzymes (sirtuins).  Experimental models 
and human translational studies using NAD+ precursors (including nicotinamide 
mononucleotide (NMN) and nicotinamide riboside (NR)) to increase NAD+ levels 
through the NAD+ salvage pathway have shown that normalizing the NADH/NAD+ ratio 
and activating sirtuin enzymes can improve mitochondrial function with beneficial effects 
in various systems including the skeletal muscle (8-10).  We therefore examined the 
effect of culturing LFS Patient A’s fibroblasts in medium containing NR.  Compared with 
the fibroblasts of her sibling Patient B which were relatively unresponsive, there was a 
significant improvement in the mitochondrial respiration of Patient A fibroblasts to NR 
exposure (Figure 2B). 
 
Published human studies have shown that NR supplementation increases 
cellular NAD+ content and can be tolerated up to 2000 mg daily without adverse side 
effects (11-13).  A pharmacokinetics study (NCT02689882) of NR with dose escalation 
over 9 d up to 1000 mg twice daily increased blood NAD+ concentrations over pre-
treatment levels in every subject, on average about 2-fold (range 1.34- to 2.66- fold, P = 
0.001) (12).  This study revealed no significant side effects, and the primary pre-
specified safety data showed no changes in the levels of potassium, glucose, uric acid, 
creatine kinase or alanine aminotransferase levels.  The subjects also displayed no 
changes in hemodynamics, although the hematocrit dropped from 40 ± 3% to 39 ± 3% 
and the platelets dropped from 220 ± 40 to 200 ± 30 (x103/µL), both with P < 0.05.  The 
modest reduction in hematocrit, hemoglobin and platelet count were suspected to result 
from the repeated daily laboratory blood draws over the 9-day study.  Additional studies 
are actively recruiting human subjects to study the effects of NR on mitochondrial 
biology in aging (PI:  Dr. Christopher R. Martens, University of Colorado, Boulder) and 
in heart failure (PI:  Dr Kevin O’Brien, University of Washington, Seattle).  Based on 
their prior pharmacokinetics study, the University of Washington team is using 1000 mg 
twice daily in their Heart Failure protocol (12).  Within the NHLBI DIR, more than 30 
human subjects have received NR 1000 mg daily without adverse side effects through 
protocol 16-H-0129, and heart failure patients will received NR 2000 mg daily through 
the recently approved protocol 18-H-0107 (Dr. M. Sack, CB/NHLBI-NIH, PI on both 
protocols). 
 
The significant improvement in mitochondrial function in the fibroblasts of Patient 
Relative O2 consumption
NMN:   −
+
−
+
Patient A
Patient B
Figure 2.  Nicotinamide mononucleotide 
(NMN) rescues the decreased 
mitochondrial respiration of Patient A 
fibroblasts.  Fibroblasts obtained from the 
indicated patients were grown under 
standard tissue culture conditions with or 
without 100 μM NMN, a metabolite of NR, 
prior to measuring whole cell respiration (O2 
consumption) using a Seahorse Metabolic 
Analyzer. 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
A by NR treatment and its benign nature as a commercially available dietary 
supplement make it compelling to examine whether the improvement in fibroblast 
mitochondrial function can be replicated in vivo.  To date, this is the only LFS patient in 
whom we have observed significant mitochondrial dysfunction, representing an 
opportunity to further expand our understanding of the biology.  While continuing our 
experimental investigations into the molecular mechanism(s) underlying the 
mitochondrial dysfunction observed in Patient A, we propose to perform a translational 
study using skeletal muscle 31P-MRS to investigate whether NR can improve the in vivo 
mitochondrial function of Patient A. 
 
 
C.  Hypothesis and Objectives 
 
 
We hypothesize that NR supplementation in this LFS patient with evidence of 
mitochondrial deficiency will result in improvement of mitochondrial function in vivo. 
 
 
Primary objective 
Investigate the effect of NR supplementation on the PCr recovery Tc of skeletal muscle 
after exercise as a marker of mitochondrial oxidative phosphorylation capacity. 
 
Secondary objectives 
Examine whether NR supplementation affects the: 
1)  respiratory capacity of blood mononuclear cells; and 
2)  cardiopulmonary exercise test (CPET) performance as a measure of aerobic 
capacity. 
 
Exploratory consideration 
Self-reported symptoms of fatigue 
 
Proposed scientific/clinical innovations and advances of the study 
1)  Scientific innovation of demonstrating improved mitochondrial function in vivo with 
NR supplementation using noninvasive PCr 31P-MRS. 
2)  Clinical innovation of using novel treatments such as NR to improve the symptoms of 
LFS patients with mitochondrial insufficiency. 
3)  Advance our understanding of the molecular mechanisms of mitochondrial regulation 
in LFS patients and the effects that novel treatments may have on this regulation. 
 
 
D. Recruitment and screening 
 
 
This is a one-subject study so there are no recruitment or screening of other 
subjects. The subject has confirmed Li-Fraumeni syndrome and TP53 mutation.  The 
subject is currently enrolled in study # 07-H-0030 and is referred to this protocol by Dr. 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
Hwang.   
 
 
E. Eligibility Assessment 
 
 
The PI or AI may assess eligibility using results from Li-Fraumeni syndrome 
natural history protocol (07-H-0030). 
 
Inclusion criteria: 
• 
At least 18-years of age and able to give informed consent  
•          Have delayed PCr recovery time constant >45 sec by 31P-MRS testing and a 
history of fatigue symptoms 
• 
Ability to undergo study procedures, including scheduled visits, blood draws and 
skeletal muscle exercise NMR spectroscopy 
• 
Have Li-Fraumeni syndrome and confirmed TP53 mutation by genetic testing 
• 
Committed to using reliable contraception which may include abstinence during 
study participation 
•          Female participants of child-bearing ability and potential willing to commit to 
reliable contraception while participating in the study 
 
 
Exclusion criteria:  
• 
Current systemic treatment for cancer 
• 
Unable to perform required study visits or procedures 
• 
MRI incompatible hardware 
• 
Pregnant or breastfeeding women 
• 
History of intolerance to NR precursor compounds, including niacin or 
nicotinamide 
 
 
F.  Study Design and Schedule 
 
Participant with documented LFS meeting study criteria will have baseline tests 
performed only after providing consent. The study design is an open label exploratory 
pilot study with the subject serving as own control with testing at baseline, week 6 and 
week 12 of NR supplementation.  The research subject will maintain routine diet and not 
make changes in dietary supplements for the duration of the study.  If the primary 
endpoint of improved skeletal muscle mitochondrial function using 31P-MRS is met with 
NR supplementation, there will be a 6-wk washout period followed by re-testing to 
confirm the specificity of the NR effect.  If the primary endpoint is not met or there is no 
self-reported improvement in fatigue symptoms, the patient may choose to continue NR 
supplementation for a 12-wk extension period.  If the primary objective is met after this 
extension period, it will be followed by a washout period.  The outline of the protocol is 
shown in tabular form. 

8 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
 
 
Week 
0 
1 
6 
7 12 18 24 
30 
Visit 
1 
 
2 
 
3 
4* 4** 5** 
Subject contact***  
 
X 
 
X 
 
 
 
 
History 
X 
 
X 
 
 
X 
X 
X 
Physical exam 
X 
 
X 
 
 
X 
X 
X 
Start NR supplementation 
X 
 
 
 
 
 
 
 
NR dose adjustment 
 
X 
X 
X 
 
 
 
 
Stop NR supplementation 
 
 
 
 
X* 
 
X 
 
Urine pregnancy test 
X1 
 
X1 
 
X1 X1 
X1 
X1 
Clinical blood tests 
X 
 
X 
 
X 
X 
X 
X 
P-31 MR spectroscopy (31P-MRS) 
X 
 
X 
 
X 
X 
X 
X 
CPET (cardiopulmonary exercise test) 
X 
 
 
 
X 
 
X 
 
Echocardiogram 
X2 
 
 
 
 
 
 
 
Research blood 
X 
 
X 
 
X 
X 
X 
X 
Skin biopsy 
X3 
 
 
 
 
 
 
 
SF-36 
X 
 
X 
 
X 
X 
X 
X 
AE assessment 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
X 
NR compliance 
 
X 
X 
X 
X 
 
X 
 
 
X1 For subject with childbearing potential only 
X2 For subject who has not had a prior echocardiogram in past 5 years 
X3 For subject who does not have skin fibroblasts available 
*   For subject who meets primary endpoint at Week 12 
**  For subject who does not meet primary endpoint at Week 12 and elects treatment extension 
(For scheduling flexibility, the visits and contacts may be performed ± 5 working days.) 
*** Subject contact can be done by phone, email or other electronic means 
 
Week 0/Visit 1 (Screening and Baseline) 
The following will be obtained at the screening/baseline visit. 
 
Screening 
• 
Demographic information, medical history and physical examination 
• 
      Pregnancy test if indicated 
 
Baseline 
• 
Clinical blood tests:  chemistry panel, CBC+Diff, hsCRP, HbA1C, uric acid, ALT, 
CK, lactate and pyruvate 
• 
Research blood draw 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
• 
Skeletal muscle exercise NMR spectroscopy 
• 
Cardiopulmonary exercise test 
• 
Echocardiogram (if a baseline study is not available) 
• 
Administer SF-36 
• 
NR Supplement will be dispensed for the study duration.  
 Week 0: 250 mg twice a day (Level 1, 500 mg daily, po, to initiate dietary 
supplementation in evening after Visit 1) 
 Weeks 1-5: 500 mg twice a day (Level 2, 1000 mg daily, po, to initiate on 
Week 1) 
 Week 6: 750 mg twice a day (Level 3, 1500 mg daily,  po, to initiate after 
Visit 2 if tolerated) 
 Weeks 7-12: 1000 mg twice a day (Level 4,2000 mg daily, po, to initiate 
on Week 7 if tolerated). 
  
Week 1 
• 
Level 2 dose escalation 
• 
Patient contact through telephone call or secure email for AE assessment, 
concomitant treatments or interventions, and NR compliance 
 
Week 6/Visit 2 
• 
Medical history and physical examination 
• 
Skeletal muscle exercise NMR spectroscopy 
• 
Clinical blood tests:  chemistry panel, CBC+Diff, hsCRP, HbA1C, uric acid, ALT, 
CK, (lactate and pyruvate only if baseline levels were abnormal) 
• 
Research blood draw 
• 
Pregnancy test if indicated 
• 
AE assessment 
• 
Record of concomitant treatments or interventions 
• 
NR compliance 
• 
Communicate the initiation of Level 3 NR after Visit 2 
 
Week 7 
• 
Level 4 dose escalation 
• 
Patient contact through telephone call or secure email for AE assessment, 
concomitant treatments or interventions, and NR compliance 
 
Week 12/Visit 3 
• 
Medical history and physical examination 
• 
Clinical blood tests:  chemistry panel, CBC+Diff, hsCRP, HbA1C, uric acid, ALT, 
CK (lactate and pyruvate only if baseline levels were abnormal) 
• 
Research blood draw 
• 
Pregnancy test if indicated 
• 
Skeletal muscle exercise NMR spectroscopy 
• 
Cardiopulmonary exercise testing 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
• 
SF-36 form 
• 
AE assessment 
• 
Record of concomitant treatments or interventions 
• 
NR compliance  
• 
The results of the PCr recovery Tc will be discussed with the subject and whether 
the 12 week NR extension period is a consideration. 
 
Week 18/Visit 4* 
• 
NR washout visit if primary endpoint is met at Visit 3 
• 
Medical history and physical examination 
• 
Clinical blood tests:  chemistry panel, CBC+Diff, hsCRP, HbA1C, uric acid, ALT, 
CK (lactate and pyruvate only if baseline levels were abnormal) 
• 
Research blood draw 
• 
Pregnancy test if indicated 
• 
Skeletal muscle exercise NMR spectroscopy 
• 
SF-36 form 
• 
AE assessment 
• 
Record of concomitant treatments 
 
Week 24/Visit 4** 
• 
Extension period visit if primary endpoint is not met at Visit 3 and the subject 
elected to do the extension of NR supplementation for 12 wk 
• 
Medical history and physical examination 
• 
Clinical blood tests:  chemistry panel, CBC+Diff, hsCRP, HbA1C, uric acid, ALT, 
CK (lactate and pyruvate only if baseline levels were abnormal) 
• 
Research blood draw 
• 
Pregnancy test if indicated 
• 
Skeletal muscle exercise NMR spectroscopy 
• 
Cardiopulmonary exercise testing 
• 
SF-36 form 
• 
AE assessment 
• 
Record of concomitant treatments or interventions 
• 
NR compliance  
 
Week 30/Visit 5** 
• 
Extension period NR washout visit 
• 
Medical history and physical examination 
• 
Clinical blood tests:  chemistry panel, CBC+Diff, hsCRP, HbA1C, uric acid, ALT, 
CK (lactate and pyruvate only if baseline levels were abnormal) 
• 
Research blood draw 
• 
Pregnancy test if indicated 
• 
Skeletal muscle exercise NMR spectroscopy 
• 
SF-36 form 
• 
AE assessment 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
• 
Record of concomitant treatments 
 
Dispensing of NR supplement 
NR supplement will be stored and dispensed by the NIH pharmacy. 
 
 
G.  Procedures 
 
Skeletal muscle phosphocreatine P-31 magnetic resonance spectroscopy (31P-
MRS) (primary endpoint): 
 
To assess whether NR enhances mitochondrial function in skeletal muscle, 31P-
MRS will be performed at baseline, 6 wk and 12 wk after NR supplementation using a 
protocol developed at the NIH (1).  If the patient meets the primary objective of 
demonstrating improved PCr recovery Tc after NR supplementation, the test will be 
repeated 6 weeks after NR washout to determine the specificity of its effect. 
 
A foot-exercise apparatus is employed to deplete PCr levels in the tibialis 
anterior, a muscle in the superficial anterior lateral aspect of the leg mainly composed of 
mitochondria-enriched oxidative type I and type IIA fibers.  The participant engages in a 
submaximal exercise by dorsiflexing one foot against 30% of the maximum weight lifted 
before testing.  The phosphocreatine level will be measured using 31P-MRS during the 
following sequence of 3-minute rest, 2-minute exercise, and 6-minute recovery periods.  
The 31P spectra will be obtained during these periods and analyzed with the use of 
SAGE 7 (GE Healthcare) and IDL, version 6.4 (Exelis Visual Information Solutions), 
software.  The single exponential recovery time constant (Tc) is calculated from the 
post-exercise recovery period data. 
  
Cardiopulmonary exercise testing: 
 
As the metabolic response to exercise can reflect mitochondrial function, 
metabolic exercise testing will be conducted with 12-lead EKG monitoring at baseline 
and at the end of 12 weeks of NR administration.  Cardiopulmonary exercise testing will 
be performed using a conservative ramping protocol while wearing a facemask enabling 
breath-by-breath analysis of inspired and expired air (SensorMedics, CA) as previously 
performed (14) (15).  EKGs are obtained at rest, after each minute of exercise, at end-
exercise, and at each minute for 5 minutes after exercise. Blood pressure will be 
measured at rest, at the end of each stage of exercise (3 minutes in duration), at end-
exercise, and at 3 minutes after exercise. 
 
The exercise will be symptom-limited (fatigue, shortness of breath or chest pain) 
and supervised by an experienced nurse and a LIP investigator of this study.  Exercise 
will be terminated prior to this end-point if clinically indicated per testing criteria or if the 
patient wishes to stop exercise.  Analysis of VO2max and the anaerobic threshold will be 
measured and compared to each subject at baseline and after completion of the NR 
supplementation protocol.  Subject will be asked not to significantly modify activity levels 
for the duration of the study. 
 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
Echocardiography:  
 
Cardiac function is a major determinant of aerobic exercise capacity, so a resting 
clinical echocardiogram may be performed if no baseline studies are available.  Cardiac 
morphology and function will be assessed through standard clinical ultrasound imaging 
and doppler techniques. 
 
Blood and skin biopsy sample collection: 
 
In addition to the clinical blood samples (estimated 40 mL), subject will have 
research bloods drawn within the blood withdrawal volume limits established by the 
Clinical Center.  Blood NAD intermediate levels, respiration measurements, 
metabolomics, and cytokine profiling may be performed to assess the effect of NR on 
metabolism and overall systemic response to improved mitochondrial function.  If skin 
fibroblasts are not already available from the subject, a skin biopsy sample may be 
obtained at the beginning of the study.  This procedure is performed using local 
anesthetic.  After washing the skin with alcohol and numbing the skin, a 4 mm or 
smaller circle of skin is removed sterilely and the wound is dressed.  Mitochondrial 
biogenesis and function will be assessed in the primary skin fibroblasts to correlate with 
the 31P-MRS data. 
 
SF-36 Health Survey: 
 
The Short Form (36) Health Survey is a well-validated 36-item, patient-reported 
survey of patient health.  It involves completing is a set of generic, coherent, and easily 
administered quality-of-life measures.  It includes one multi-item scale that assesses 
eight health concepts:  1) limitations in physical activities because of health problems; 2) 
limitations in social activities because of physical or emotional problems; 3) limitations in 
usual role activities because of physical health problems; 4) bodily pain; 5) general 
mental health (psychological distress and well-being); 6) limitations in usual role 
activities because of emotional problems; 7) vitality (energy and fatigue); and 8) general 
health perceptions.  We will use it to monitor perceived health status before and at the 
end of treatment with nicotinamide riboside and following the washout period. 
 
 
H.  Dietary Supplement 
 
 
Niagen (trademark) is a commercially-available form of nicotinamide riboside 
(NR). The nucleoside NR is a single chemical moiety containing nicotinamide and 
ribose (16).  NR is a form of vitamin B3 present in trace amounts in foods like milk, 
yeast extract and beer.  It is also postulated that NR is generated in the gastrointestinal 
tract as part of dietary NAD+.  Thus, humans are constantly exposed to NR from the 
diet, albeit at low levels.  Since 2013, Niagen has been sold as a dietary supplement in 
the United States.  Labeling guidelines recommend consumers to limit their intake to 2 
capsules/day (250 mg/day).  This recommended level is the equivalent of 3.8 mg/kg 
body weight/day, which is 1000-fold less than the highest dose determined to be safe 
and well tolerated in rats, and there is no limit on the duration of ingestion. 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
 
There are extensive literature on the beneficial effects of NR in animal models 
(17,18), and multiple lines of non-clinical data suggest that it should be well tolerated in 
human subjects.  In an acute toxicology study, rats that were given a single oral dose 
(5000 mg/kg) of NR did not show clinical signs of toxicity or mortality (unpublished 
results).  The NR dose proposed for this study is within the doses tested in mice for up 
to 4 months and is 300-fold below the daily dose that was given in rats in a 14-day dose 
range finder study.  There are also accumulating data on the pharmacokinetics and 
safety of orally administered NR in humans.  Recently, a 2 x 6-week randomized, 
double-blind, cross-over study was performed to assess the tolerability of chronic NR 
supplementation and its efficacy for increasing NAD+ bioavailability by investigators at 
the University of Colorado, Boulder (13).  They demonstrated that the oral 
administration of NR 500 mg twice daily (total 1000 mg daily, Niagen, ChromaDex, Inc.) 
was well tolerated, readily absorbed, and detectable in human plasma, white blood 
cells, and urine.  In a pharmacokinetic study of 8 healthy volunteers at the University of 
Washington, Seattle, the oral dose of NR dose was escalated from 250 mg daily to 
1000 mg twice daily over a 9 day period with a resultant 100% increase in blood NAD+ 
content that highly correlated to NR levels (12).  No adverse events were associated 
with NR treatment in either of these two published human studies.   
 
Niacin is a form of vitamin B3 and has been used to treat hypercholesterolemia 
and pellagra for many years.  Niacin administration can lead to undesirable effects such 
as spontaneous flushing, but based on its pharmacology, NR would not be predicted to 
give this side effect (19,20).  Indeed, the incidence of flushing side effect with NR 
treatment was not different compared with placebo in the double-blind cross-over study 
(13).  Nicotinamide, an expected metabolite of NR, is considered to be of low toxicity in 
food by several regulatory agencies including the United States Food and Drug 
Administration (FDA) and the European Food Safety Authority (EFSA).  Clinical trials of 
nicotinamide up to 3000 mg daily and 3 years in patients with or at risk of developing 
Type 1 diabetes have not reported significant adverse effects (21,22).  In addition, 
doses of 25 and 42 mg/kg body weight per day had no effect on a variety of biochemical 
parameters including those assessing liver and kidney function. 
 
In summary, careful analysis of the available information on NR does not reveal 
any potential serious toxicity that would preclude its use in LFS subjects.  Given the 
safety and tolerability of NR in published studies at two extramural institutions 
(University of Colorado and University of Washington) and the favorable experience 
with NR in Dr. Michael Sack’s NHLBI DIR protocol (16-H-0129, discussed in 
Background section) (12,20), we propose to test the effect of NR in LFS patients at a 
dose of 1000 mg twice daily, a dose that is currently approved for use in other active 
clinical studies of the aforementioned groups. 
 
Common name:  Nicotinamide Riboside Chloride 
Product name:  Niagen 
Chemical name:  3-(Aminocarbonyl)-1-β-D-ribofuranosyl-pyridinium chloride (1:1) 
Daily dose:  500 mg x 1 wk, 1,000 mg x 5 wk, 1500 mg x 1 wk, up to 2000 mg as 
tolerated x 5 wk (plus an optional extension period of 12 wk) 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
Route of administration:  oral 
Dosing instructions:  twice daily dosing in 250 mg increments per dose 
Supply:  supplements will be purchased from ChromaDex, Irvine, CA 
Toxicology:  none known 
Drug Interactions:  none known 
 
Off-label use:  Considering the clinical investigation is designed to study the 
relationship between a dietary supplement’s effect on structure or function in humans or 
to characterize the mechanism by which a dietary supplement acts to maintain such 
structure or function, this study would not need to be conducted under an IND.  Under 
the Dietary Supplement Health and Education Act of 1994, a dietary supplement is not 
considered a drug and is not subject to the premarket approval requirements for drugs if 
the intended use for which it is marketed is only to affect the structure or any function of 
the body (i.e., not intended to be used for a therapeutic purpose).  Similarly, whether an 
IND is needed for a clinical investigation evaluating a dietary supplement is determined 
by the intent of the clinical investigation.  If the clinical investigation is intended only to 
evaluate the dietary supplement’s effect on the structure or function of the body, an IND 
is not required. 
 
 
I.  Data and Biospecimen Management Plan  
 
 
Primary research data will be coded by replacing personally identifiable 
information (such as name) with a code that will enable the investigator to readily 
ascertain the identity of the subject through the use of a code-key, but will not reveal the 
identity of subject to parties not authorized to have access to individual subject 
identifiers. 
 
Biospecimen management at the NIH: 
 
Samples will be de-identified prior to storage on the 5th floor of Building 10 in the 
laboratory of the principal investigator following current NIH sample storage guidelines.  
Samples and data will be stored, using codes assigned by the investigators or their 
designee(s).  Research samples will be stored using BSI in accordance with NHLBI DIR 
Biospecimen policy.  Data will be kept on the NHLBI P:drive, accessible through 
password-protected computers. Only the members of the research team will have 
access to the samples and data. Coded biospecimens may be sent to collaborators 
outside of the NIH with IRB approval in accordance with applicable NIH and DIR Policy 
for sharing research resources, including an executed material transfer agreement. 
 
End of study procedures: 
 
Data retained by the NHBLI will be stored in a password-protected database in 
conformity with NHLBI DIR policy until they are no longer of scientific value.  Destruction 
of research data collected on this protocol will be consistent with NIH policy and upon 
permission of the Clinical Director. 

15 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
 
Breach of confidentiality: 
 
PIs will report any breach of subject confidentiality or trial data to the clinical 
director and IRB per NIH policy, including NIH HRPP SOP 16 - Reporting Requirements 
for Unanticipated Problems, Adverse Events and Protocol Deviations. 
 
Data sharing and future use of data: 
 
Research data may be shared with qualified non-collaborator recipients following 
publication of the primary research results after removal of PII and IRB approval (coded 
data) or Office of Human Subjects Research Protections (OHSRP) approval (unlinked 
data).  In both situations, a data use agreement between the sender and the recipient 
will be executed.  Future research use of data not defined in the research protocol may 
occur only after IRB review and approval or a determination from the NIH OHSRP.  
Refusal of a research subject participant to permit future use of data-will be honored. 
Human Data Sharing Plan: De-identified human data generated for use in future and 
ongoing research will be shared through a NIH-funded or approved repository 
(ClinicalTrials.gov) and BTRIS. At the completion of data analysis, data will be 
submitted to ClinicalTrials.gov either before publication or at the time of publication or 
shortly thereafter. 
 
 
Future use of biospecimens: 
 
Following analyses of biospecimens for primary research purposes as described 
in the protocol, remaining samples suitable for future research will be stored in manner 
that conforms with DIR policy (such as BSI) or in a publicly accessible research 
biospecimen repository following IRB approval.  Biospecimens may be destroyed only 
when permitted by the clinical director and approved by the IRB.  Any future research 
use of biospecimens not defined in the research protocol will occur only after IRB review 
and approval, if the research holds the key that identifies research subjects, or 
determination from OHSRP (non-collaborative research). 
 
Biospecimens will not be sent outside of the NIH for future research use without 
IRB approval and an executed agreement. Refusal of a research subject participant to 
allow for future use of biospecimens will be honored. 
 
Loss or destruction of samples: 
 
Should we become aware that a major breech in our plan for tracking and 
storage of samples has occurred, the IRB will be notified. 
 
 
 
J.  Statistical Considerations 
 
Primary objective: 
 
This is a pilot study translating experimental observations made in one LFS 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
patient.  It is novel in nature with no prior data available on the effect of NR on in vivo 
mitochondrial function, so statistical considerations are not feasible.  The primary 
endpoint of this study will be intra-subject comparison of baseline to 12 wk NR 
supplementation PCr recovery Tc measurements using the 31P-MRS skeletal muscle 
submaximal exercise.  This is a one-subject study so there are no statistical 
considerations. 
 
Additional analyses: 
 
Baseline and 12 wk respiratory capacity of blood mononuclear cells, 
cardiopulmonary exercise test performance, and self-reported fatigue symptoms will be 
analyzed. 
 
 
K.  Off Study Criteria 
 
1)  Subject taking less than 75% of the supplement 
2)  Completion of study visits in pre-specified time windows 
3)  Subject found to be pregnant or wishes to breastfeed during the study will 
automatically be withdrawn 
4)  Any other severe medical symptoms that may or may not be related to the NR 
supplement and as determined by contact with the physician.  
5) If subject no longer wish to participate 
6) Subject’s non-compliance 
 
 
L.  Data and Safety Monitoring 
 
Safety monitoring 
Principal investigator:  Accrual and safety data will be monitored by the PI. 
 
 
Adverse event reporting 
 
Definitions: 
Adverse Event (AE):  Any untoward medical occurrence in a human subject, including 
any abnormal signs (e.g., abnormal physical exam or laboratory finding), symptom, or 
disease, temporally associated with the subject’s participation in the research, whether 
or not considered related to the subject’s participation in the research. 
 
Abnormal laboratory values:  A moderate and severe abnormal laboratory value will 
be considered an AE if the laboratory abnormality is characterized by any of the 
following: 
• 
Results in discontinuation from the study 
• 
Is associated with clinical signs or symptoms 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
• 
Requires treatment or any other therapeutic intervention 
• 
Is associated with death or another serious adverse event, including 
hospitalization 
• 
Is judged by the Investigator to be of significant clinical impact 
• 
If any abnormal laboratory result is considered clinically significant, the 
investigator will provide details about the action taken with respect to the subject’s 
outcome. 
 
Serious Adverse Event (SAE):  A serious adverse event that: 
• 
results in death; 
• 
is life-threatening (places the subject at immediate risk of death from the event as 
it occurred); 
• 
results in in-patient hospitalization or prolongation of existing hospitalization; 
• 
results in a persistent or significant incapacity; 
• 
results in a congenital anomaly/birth defect; or 
• 
based upon appropriate medical judgment, may jeopardize the subject’s health 
and may require medical or surgical intervention to prevent one of the other outcomes 
listed in this definition. 
 
Unanticipated Problem (UP):  Any incident, experience, or outcome that meets all of 
the following criteria: 
1)  unexpected in terms of nature, severity, or frequency in relation to 
a)  the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents; and 
b)  the characteristics of the subject population being studied; and 
2)  related or possibly related to participation in the research; and 
3)  places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized. 
 
Unanticipated Problem that is not an Adverse Event:  An unanticipated problem that 
does not fit the definition of an adverse event, but which may, in the opinion of the 
investigator, involves risk to the subject, affect others in the research study, or 
significantly impact the integrity of research data. For example, report occurrences of 
breaches of confidentiality, accidental destruction of study records, or unaccounted-for 
study drug. 
 
Protocol Deviation (PD):  Any change, divergence, or departure from the IRB 
approved research protocol. 
 
Non-compliance:  The failure to comply with applicable NIH HRPP policies, IRB 
requirements, or regulatory requirements for the protection of human research. 
Noncompliance may be further characterized as: 
 
Serious non-compliance: Non-compliance that: 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
a. 
Increases risks, or causes harm, to participants. 
b. 
Decreases potential benefits to participants. 
c. 
Compromises the integrity of the NIH HRPP. 
d. 
Invalidates the study data. 
 
Continuing non-compliance:  Non-compliance that is recurring.  An example may be a 
pattern of non-compliance that suggests a likelihood that, absent an intervention, non- 
compliance will continue. Continuing noncompliance could also include a failure to 
respond to IRB requests to resolve previous allegations of non-compliance. 
 
Minor (non-serious) non-compliance:  Non-compliance that, is neither serious nor 
continuing. 
 
Adverse event management 
 
The principal investigator or designee will be responsible for assessing adverse 
events. Information on adverse events will be solicited from subjects through questions 
from study personnel and information volunteered by the subject.  Adverse events will 
be captured from the start of the first pill taken (Day 0) until the completion of the 
washout phase of the study.  The AEs will be attributed (unrelated, unlikely, possibly, 
probably or definitely) to study supplement and/or disease.  This study will utilize the 
Common Terminology Criteria for Adverse Events version 5.0 (CTCAE v5.0) for toxicity 
and adverse event reporting.  A copy of the CTCAE v5.0 can be downloaded from the 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
AEs will be recorded, verified, and followed until satisfactory resolution.  In the event of 
any treatment-related SAEs, enrollment will be suspended until discussed with the IRB 
and Clinical Director. 
 
Grading and attribution of adverse events 
Severity definitions found in the CTCAE v5.0 will be used for grading the severity 
(intensity) of AEs: 
  
1)  Mild:  Asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated. 
2)  Moderate:  Minimal, local or noninvasive intervention indicated; limiting 
age-appropriate instrumental ADL* 
3)  Severe:  Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
ADL**. 
4)  Life- threatening:  Life-threatening consequences; urgent intervention indicated. 
5)  Death:  Death related to AE. 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc. 
**Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, 
taking medications, and not bedridden. 

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Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
 
Attribution of Adverse Events 
Relationship 
Attribution 
Description 
Unrelated to 
intervention 
Unrelated 
The AE is clearly NOT related to the 
intervention 
Unlikely 
The AE is doubtfully related to the intervention 
Related to 
intervention 
Possibly 
The AE may be related to the intervention 
Probably 
The AE is likely related to the intervention 
Definitely 
The AE is clearly related to the intervention 
 
 
NIH-IRB and CD reporting 
 
Serious events 
Reports to the IRB and CD:  The PI must report Serious UPs, and Serious PDs to the 
IRB and Clinical Director (CD) as soon as possible but not more than 7 days after the PI 
first learns of the event. 
 
Non-serious events 
Reports to the IRB and CD:  The PI must report all UPs that are not Serious to the IRB 
and CD, and PDs that are not Serious to the IRB, not more than 14 days after the PI 
first learns of the event. 
 
Deaths 
Deaths possibly, probably, or definitely related to study procedures and interventions 
will be reported to the CD within 7 days after the PI first learns of the event. 
  
Reports at the time of continuing IRB review 
At continuing review, the PI will provide to the IRB a summary of: 
• 
All UPs 
• 
All PDs 
• 
All AEs (except for those granted a waiver of reporting) 
 
Waiver of reporting 
The following adverse events will be listed in the consent and not reported to the 
IRB: 
• 
Vasovagal symptoms during blood draws (expected frequency 50%). 
• 
Transient bruising at the site of blood draws (expected frequency 50%). 
The following adverse events will not be recorded or reported to the IRB: 
• 
Grade 1 adverse events 
• 
Grade 2 laboratory abnormalities not associated with clinical signs or symptoms 
except for grade 2 elevated liver enzymes, which will be recorded and reported. 
 
Reporting of pregnancy:  In the event a subject becomes pregnant while on study, this 

20 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
event will be reported to the IRB and Clinical Director as a protocol deviation.  
Monitoring of the pregnancy will continue until conclusion of the pregnancy, and then 
subject will be taken off study. 
 
M.  Human Subjects Protections 
 
Rationale for subject selection 
As described in the Background section, one subject has self-referred to us for inclusion 
in this study. There will be no racial, ethnic, or gender discrimination.  This subject is not 
cognitively impaired, institutionalized, or non-English speaking. 
 
Rationale for the exclusion of children 
The one subject is over 18 years of age.  We have no experience with 31P-MRS in 
children and the volumes of blood drawn exceed minimal risk for children. 
 
Rationale for the exclusion of pregnant women 
The subject must not be pregnant or actively seeking pregnancy in order to participate 
in this study.  NR has not been determined to be safe in pregnancy or breastfeeding 
women.  A recognized form of contraception must be used by subjects while enrolled.  
Contraception use will be determined during telephone screening and confirmed at the 
screening visit. 
 
Rationale for the exclusion of cognitively impaired subjects 
Subjects with cognitive impairment will not be considered for inclusion because there is 
no direct benefit from participating in this study, follow the instructions. 
 
Risk/benefit assessment 
This is a single subject supplement study (interventional) which involves greater than a 
minimal risk procedure (skin biopsy), therefore research involves greater than minimal 
risk to subjects, with no prospect of direct benefit, but is likely to yield generalizable 
knowledge (45 CFR 46.102). 
 
Risks and discomforts 
Phlebotomy:  Standard precautions for obtaining human blood samples will be taken. 
Transient discomfort and minor bruising may occur at the phlebotomy site. Vasovagal 
symptoms can occur during blood drawing. Blood samples will be obtained by 
venipuncture. Blood samples will be obtained by a nurse, physician, or other skilled 
individual. The quantities of blood to be drawn for research purposes will be less than 
300 mL, which is consistent with the CC policy as provided in Medical Administrative 
Series (MAS) 95-9 (revised 05/29/2012): for adults, no more than 10.5 mL/kg or 550 mL 
(whichever is smaller) will be drawn for research purposes over any 8-week period. 
 
Urine collection:  There is no risk associated with this procedure. 
 

21 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
Nicotinamide riboside:  NR is a dietary supplement that is currently available for 
commercial use with no safety concerns noted to date.  A recent PK study of 8 healthy 
volunteers at University of Washington gave incremental doses up to 1000 mg twice 
daily over 9 d.  The study showed no significant side effects or change in safety data.  
Longer duration of NR 1000 mg twice daily supplementation has not been reported in 
humans although there is currently an ongoing study at the University of Colorado using 
1000 mg daily for 6 wk.  Based on the structural similarities between NR and 
nicotinamide, and that nicotinamide is a downstream metabolite of NR digestion, it is 
assumed that any unexpected adverse effects of NR may be similar to those associated 
with nicotinamide intake.  The safety profile of nicotinamide has been well established in 
multiple species and provides reasonable certainty that the administration of doses of 
NR up to 1000 mg will not result in an adverse health effect.  No significant adverse 
effects have been reported in clinical trials which have used doses up to the equivalent 
of 3000 mg/day for up to 3 years to evaluate the possible benefits of nicotinamide 
administration to patients with or at risk of developing Type 1 diabetes (21) (22).  In 
addition, doses of 25 and 42 mg/kg/day had no effect on a variety of biochemical 
parameters, such as those assessing liver and kidney function. 
  
Skin biopsy: The entire procedure takes approximately 5 minutes.  Discomfort at the 
biopsy site is usually mild and transient.  This can be treated with minor analgesics.  
Normally, the risks include a reaction to the local anesthetic and the slight possibilities 
of local bleeding or infection.  Scarring always occurs at the biopsy site. 
 
MR spectroscopy:  MRI uses non-ionizing radiation and is safe when used on subjects 
that are appropriately screened for the procedure.  Subjects with any exclusionary 
criteria (pregnancy, aneurysm clip, implanted devices such as neural stimulators, 
cardiac pacemaker, defibrillator, cochlear implant, foreign body, such as, metal 
shavings, or insulin pumps) will be excluded from the study. 
 
Potential risks of MRI relate to effects of the main, static magnetic field, the 
applied radiofrequency (RF) power, and the rapidly switching magnetic field gradients. 
The risks associated with each of these are described below.  We will stay within all the 
FDA guidelines for the applied RF power, the rate of switching magnetic fields and the 
noise levels that these switching gradients produce (see Guidance for Magnetic 
Resonance Diagnostic Devices – Criteria for Significant Risk Investigations, issued July 
14, 2003. http://www.fda.gov/cdrh/ode/guidance/793.pdf). 
 
Non-Significant Risk (NSR) device determination:  The use of the MRI scanner 
constitutes a non-significant risk device study, because it is performed within the FDA 
approved limits of main static magnetic field (< 8T), specific absorptions rate (SAR), 
gradient field rates of change, and sound pressure level 22 (Appendix 1).  This protocol 
is therefore eligible for abbreviated IDE requirements of 21 CFR 812.2(b), in which IRB 
review constitutes the IDE. 
 
The use of the MRI research coils, research pulse sequencing, and research 
image processing constitute the research component of this device which are not FDA 

22 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
approved. All research surface coils undergo safety testing and review by the NIH NMR 
Center Safety Committee.  There is no potential for serious risk to the health, safety, or 
welfare of the subjects using the MRI scanner in these ways. 
 
Magnetic field:  There is no data that shows any significant adverse effects of 
exposure to static magnetic fields. There are well known minor adverse effects 
associated with high magnetic fields.  These include nausea, metallic taste, and 
detection of flashes of light.  All of these are associated with moving too rapidly in the 
magnetic field.  To avoid these the subjects will be asked to walk slowly to the patient 
table and the table will be moved slowly into the magnet.  Experiences at 3T show that 
these precautions eliminate these minor adverse reactions.  Current FDA guidance 
allows for main static magnetic field less than or equal to 8T. 
 
Radiofrequency power deposition:  The MRI will have safeguards which monitor and 
limit RF deposition within FDA guidelines.  Only one adverse effect has been observed 
on the 3T MRI at the NIH in the more than 5000 subjects that have been examined over 
the last 3 years.  This one event was temporary eye discomfort reported by a normal 
volunteer.  The eyes are sensitive to RF irradiation and the FDA guidelines take this into 
account. 
 
Switching magnetic field gradients:  In addition to the large, static magnetic field, 
MRI relies on rapidly switching magnetic field gradients of much lower strength than the 
main static magnetic field.  Subjects who participate in MRI examinations may 
experience peripheral nerve stimulation in the form of involuntary skeletal muscle 
contractions and/or twitching due to these rapidly switching gradients.  This peripheral 
nerve stimulation has also been described as a creeping sensation along the back or 
twitching of the nose or feelings of electrical shocks and has ranged from imperceptible 
to mildly painful sensations. 
 
Current FDA guidelines do not specify a strict limit that magnetic field gradients 
can be switched in MRI studies.  FDA guidelines state that the switching gradients 
should not cause severe discomfort or painful nerve stimulation.  Currently, the FDA 
approved 3T MRI can maximally switch field gradients up to 12 mT in 0.260 msec at a 
position 30 cm from isocenter.  No adverse events related to nerve stimulation have 
been reported. 
 
Noise levels:  The switched gradients also generate noise in the scanner.  Peak sound 
power produced in the magnet will be less than 140 dB and 119 dBA.  The FDA limits 
constant noise exposure to 140 dB and 99 dBA for two hours.  To stay within FDA 
guidelines on dBA limits all subjects will wear hearing protection in the form of ear plugs 
and/or headphones.  This is the procedure that is routinely done on the 1.5T and 3T 
MRI, and it is well established to decrease noise levels by 20 dbA.  There has been only 
one reported adverse effect on hearing from noise in close to 20 years of experience at 
the NIH NMR center.  In the past 3 years more than 5000 MRI subjects have been 
examined.  The one recent adverse event was temporary hearing loss reported by a 

23 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
patient after a MRI scan on a 3T MRI.  It is not clear what caused this hearing loss.  The 
patient had a previous stroke and an absent ear reflex to loud noises.  Based on these 
experiences, the risk of damage to hearing is very low. 
 
Exercise stress test:  Stress tests are safe (a serious complication occurs in less than 
1 in 10,000 tests), especially when the level of monitoring during exercise (ECG, blood 
pressure, physician supervision) proposed in the present study is employed.  Shortness 
of breath and fatigue are often felt at the end of the exercise, and occasionally subjects 
may experience chest discomfort.  Abnormal heart rhythms can be triggered by 
exercise, but these are rarely persistent or severe.  The mask used to measure exhaled 
gas is worn tightly on the face and may cause discomfort. 
 
Consent processes and documentation for research subjects 
Informed consent shall be documented using the current IRB-approved consent form, 
which may be downloaded from the NIH Clinical Center active consent website. 
When consent is obtained, the consent document(s) must be signed and dated by the 
subject, and the person obtaining consent. For research conducted at the NIH Clinical 
Center a witness is also required to sign the document. Any adult other than the person 
obtaining or providing consent may serve as a witness. The witness attests only to the 
validity of the signature or mark (i.e., that the research subject signed the consent 
document), not to the validity or quality of the consent.  
The original, signed informed consent document will be placed in the medical record, 
and the subject will receive a signed copy of the informed consent document. 
Documentation of informed consent and the signed consent form will be maintained in 
CRIS. 
 
N.  Conflict of Interest 
 
None of the members of the research team report a potential conflict of interest.  The 
National Institutes of Health reviews NIH staff researchers at least yearly for conflicts of 
interest.  The following link contains details on this process. 
http://ethics.od.nih.gov/forms/Protocol-Review- Guide.pdf. 
 
O.  Reimbursement for Travel 
 
Reimbursement for travel, food, and lodging will be provided according to NHLBI policy 
and rates. 
 
 
P.  Financial Compensation 
 
The subject will  be compensated for some of the procedures that are performed at the 
NIH since they may not provide direct benefit to the subject. 
 

24 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
 
 
Procedures 
Inconvenience 
Units 
Compensation 
per procedure 
Frequency 
Total 
Compensation 
Blood draws 
             1 
              $10 
         6 
$60 
NMR spectroscopy (P-31 MRS) 
10 
$100 
5 
$500 
CPET (cardiopulmonary exercise test) 
3 
$30 
3 
$90 
Skin biopsy 
5 
$50 
1 
$50 
Maximum Compensation: 
$700 
 
 
 

25 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
Q.  References 
 
1. 
Wang PY, Ma W, Park JY, Celi FS, Arena R, Choi JW, et al. Increased oxidative metabolism in 
the Li-Fraumeni syndrome. N Engl J Med 2013;368:1027-32 
2. 
Matoba S, Kang JG, Patino WD, Wragg A, Boehm M, Gavrilova O, et al. p53 regulates 
mitochondrial respiration. Science 2006;312:1650-3 
3. 
Park JY, Wang PY, Matsumoto T, Sung HJ, Ma W, Choi JW, et al. p53 improves aerobic 
exercise capacity and augments skeletal muscle mitochondrial DNA content. Circ Res 
2009;105:705-12, 11 p following 12 
4. 
Kamp WM, Wang PY, Hwang PM. TP53 mutation, mitochondria and cancer. Curr Opin Genet 
Dev 2016;38:16-22 
5. 
Wang PY, Li J, Walcott FL, Kang JG, Starost MF, Talagala SL, et al. Inhibiting mitochondrial 
respiration prevents cancer in a mouse model of Li-Fraumeni syndrome. J Clin Invest 
2017;127:132-6 
6. 
Chance B, Im J, Nioka S, Kushmerick M. Skeletal muscle energetics with PNMR: personal views 
and historic perspectives. NMR Biomed 2006;19:904-26 
7. 
Argov Z, Lofberg M, Arnold DL. Insights into muscle diseases gained by phosphorus magnetic 
resonance spectroscopy. Muscle Nerve 2000;23:1316-34 
8. 
van de Weijer T, Phielix E, Bilet L, Williams EG, Ropelle ER, Bierwagen A, et al. Evidence for a 
direct effect of the NAD+ precursor acipimox on muscle mitochondrial function in humans. 
Diabetes 2015;64:1193-201 
9. 
Traba J, Kwarteng-Siaw M, Okoli TC, Li J, Huffstutler RD, Bray A, et al. Fasting and refeeding 
differentially regulate NLRP3 inflammasome activation in human subjects. J Clin Invest 
2015;125:4592-600 
10. 
Katsyuba E, Auwerx J. Modulating NAD(+) metabolism, from bench to bedside. EMBO J 
2017;36:2670-83 
11. 
Trammell SA, Schmidt MS, Weidemann BJ, Redpath P, Jaksch F, Dellinger RW, et al. 
Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Nat Commun 
2016;7:12948 
12. 
Airhart SE, Shireman LM, Risler LJ, Anderson GD, Nagana Gowda GA, Raftery D, et al. An 
open-label, non-randomized study of the pharmacokinetics of the nutritional supplement 
nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers. PLoS 
One 2017;12:e0186459 
13. 
Martens CR, Denman BA, Mazzo MR, Armstrong ML, Reisdorph N, McQueen MB, et al. 
Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD(+) in healthy 
middle-aged and older adults. Nat Commun 2018;9:1286 
14. 
Arena R, Humphrey R, Peberdy MA, Madigan M. Predicting peak oxygen consumption during a 
conservative ramping protocol: implications for the heart failure population. J Cardiopulm 
Rehabil 2003;23:183-9 
15. 
Arena R, Myers J, Abella J, Peberdy MA. Influence of heart failure etiology on the prognostic 
value of peak oxygen consumption and minute ventilation/carbon dioxide production slope. Chest 
2005;128:2812-7 
16. 
Chi Y, Sauve AA. Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on 
energy metabolism and neuroprotection. Curr Opin Clin Nutr Metab Care 2013;16:657-61 
17. 
Canto C, Menzies KJ, Auwerx J. NAD(+) Metabolism and the Control of Energy Homeostasis: A 
Balancing Act between Mitochondria and the Nucleus. Cell Metab 2015;22:31-53 
18. 
Yoshino J, Baur JA, Imai SI. NAD(+) Intermediates: The Biology and Therapeutic Potential of 
NMN and NR. Cell Metab 2018;27:513-28 

26 
Protocol: P184388                                                                                     
Version date: October 23, 2018 
Paul M. Hwang, MD, PhD. 
 
19. 
Benyo Z, Gille A, Kero J, Csiky M, Suchankova MC, Nusing RM, et al. GPR109A (PUMA-
G/HM74A) mediates nicotinic acid-induced flushing. J Clin Invest 2005;115:3634-40 
20. 
Canto C, Houtkooper RH, Pirinen E, Youn DY, Oosterveer MH, Cen Y, et al. The NAD(+) 
precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-
induced obesity. Cell Metab 2012;15:838-47 
21. 
Pozzilli P, Visalli N, Signore A, Baroni MG, Buzzetti R, Cavallo MG, et al. Double blind trial of 
nicotinamide in recent-onset IDDM (the IMDIAB III study). Diabetologia 1995;38:848-52 
22. 
Lampeter EF, Klinghammer A, Scherbaum WA, Heinze E, Haastert B, Giani G, et al. The 
Deutsche Nicotinamide Intervention Study: an attempt to prevent type 1 diabetes. DENIS Group. 
Diabetes 1998;47:980-4