Clinical trial NCT02362425
Conditions: Neuromuscular Disease
Keywords: Neuromuscular Disease Clinical Trial Ryanodine Receptor Type 1 Myopathy
Sponsor: National Institute of Nursing Research (NINR)
Start date: 2015-02-12; Completion date: 2018-05-30
Enrollment: 63 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Background:

\- Ryanodine receptor type 1-related myopathies (RYR1-RM) are the most common non-dystrophic muscle diseases that people are born with in the U.S. They affect development, muscles, and walking. Researchers want to test a new drug to help people with these diseases.

Objectives:

\- To see if the drug N-acetylcysteine decreases muscle damage in people with RYR1-RM. To see if it improves their exercise tolerance.

Eligibility:

\- People age 7 and older with a confirmed genetic diagnosis of RYR1 or a clinical diagnosis of RYR1 and a family member with a confirmed genetic diagnosis.

Design:

* Participants will be screened with a checklist of criteria. Adult participants may have a muscle biopsy. A needle will remove a tiny piece of muscle in the lower leg.
* Study visits will take several days.
* Visit 1:
* Medical history
* Physical exam
* Blood, urine, and saliva tests
* Questions about symptoms and quality of life
* Heart, lung, and walking tests
* Muscle Oxygenation Capacity Test. A blood pressure cuff around the thigh will be tightened for up to 10 minutes.
* Biodex testing, stretching the leg against resistance
* Muscle ultrasounds. A probe will be moved over the skin.
* Participants may be photographed or videotaped during procedures.
* They may have a muscle biopsy.
* Six months later, visit 2 will repeat visit 1. Participants will start taking the study drug dissolved in water or placebo three times a day for 6 months.
* Participants will stay at NIH for 2 days after starting the study drug.
* Participants will be contacted by phone during the study to monitor side effects
* Six months after starting the study drug, study visit 3 will repeat some or all of visit 1.

Detailed description:
Although genetic disorders of muscle that present at birth are rare, RYR1-related myopathies comprise the most common non-dystrophic congenital myopathy in the United States, with a prevalence of approximately 1/90,000 people (Amburgey et al, 2011). Causative mutations in the ryanodine receptor gene of skeletal muscle, RYR1, have been found in several myopathy subtypes, including central core disease and centronuclear myopathy. These mutations result in defective excitation-contraction coupling and increased mitochondrial oxidative stress. Most patients present in childhood with delayed motor milestones, extremity muscle weakness, impaired ambulation, joint contractures, progressive scoliosis, and in some cases eye movement paralysis, respiratory failure, or susceptibility to malignant hyperthermia, an allelic condition. Despite these important morbidities and the risk of early mortality, no treatments exist to date.

RYR1 encodes a homotetrameric transmembrane ion channel, RyR1, which resides on the terminal sarcoplasmic reticulum in close proximity to the T-tubule. By releasing calcium from the sarcoplasmic reticulum into the cytosol in response to muscle fiber stimulation by the motor neuron at the neuromuscular junction, it mediates excitation-contraction coupling and functions as a regulator of cellular calcium concentrations and redox homeostasis. Dowling et al. (2012) recently elucidated the latter mechanism in zebrafish and patient myotubes, showing that RYR1 mutations result in increased oxidative stress and that this is rescued in both models by treatment with N-acetylcysteine (NAC), a known anti-oxidant. NAC functions as a precursor of glutathione, an endogenous antioxidant that becomes deficient during oxidative stress. This was substantiated by a cystic fibrosis clinical trial in which low glutathione levels in neutrophils undergoing oxidative stress significantly increased with NAC administration.

Dowling et al. (2012) found significant changes post NAC treatment including increased travel distance (endurance) in zebrafish and complete protection from cell death induced by experimentally increasing oxidative stress in myotubes. Thus NAC was a successful treatment in both ex vivo and in vivo model systems. Based on these results, we plan to perform a randomized, double-blinded, placebo controlled clinical trial of NAC in a subgroup of RYR1-related myopathy patients as the first pathophysiologically based treatment for this devastating disorder.

The objectives of the study are to determine if NAC reduces oxidative stress, fatigability, and fatigue in a study population of patients with RYR1-RM. The study population includes both males and females 7 years of age and older. The study design has two phases. The first 6-month phase will be used to validate the selected outcome measures in RYR1 congenital myopathy. The second 6-month phase is a randomized, double-blinded, placebo controlled drug intervention trial. The primary outcome measures are blood glutathione for oxidative stress and six minute walk test for fatigability. Healthy volunteers will be evaluated to determine normal values of biomarkers, muscle ultrasound, and near infrared spectroscopy in this rare disease, in order to develop a comparison between healthy and RYR1-RM individuals.

Arms: arm 1: N-acetylcysteine arm 2: Placebo arm 3: Healthy volunteers who had physical exam, study biomarker, Near Infrared Spectroscopy (NIRS) testing and muscle ultrasound only, in one visit.
Interventions: intervention 1: N-acetylcysteine intervention 2: Placebo
Intervention descriptions: intervention 1: None intervention 2: None

Protocol documents:
Prot_000.pdf: 
1 
 
 
Protocol Title: Antioxidant therapy in RYR1-related congenital myopathy 
Abbreviated Title: Antioxidant in RYR1-RM 
Protocol Number: 15-NR-0072 
Date of This Submission/Version: 1/31/2018                          
 
Principal Investigator 
Suzanne 
Wingate, PhD, 
ANP-BC 
NINR 
Bldg/Rm 
10/2-1339 
Phone  
301-827-0982 
 
E-mail  
suzanne.wingate@nih.gov 
 
Adjunct Principal Investigator   
James 
Dowling, 
MD, PhD 
Hospital for 
Sick Children 
Bldg/Rm 
555 
University 
Ave 
6th Floor 
Neurology 
Toronto, ON 
M5G 1X8 
Phone  
 
(416) 813-
7654 x309090 
E-mail 
 
james.dowling@sickkids.ca 
 
Associate Investigators  
Name 
 
Katherine 
Meilleur, 
PhD, PNP-
BC 
Branch/Institute 
 
Tissue 
Injury/NINR 
Bldg/Rm 
 
60/252 
Phone  
 
301-453-1503 
E-mail  
 
meilleurk@mail.nih.gov 
 
Carsten 
Bonnemann, 
MD 
 
Irene 
Chrismer, 
BSN 
 
Diana 
Bharucha, 
MD 
 
A. Reghan 
Foley, MD 
 
 
NGB/NINDS 
 
 
 
Tissue 
Injury/NINR 
 
 
NINDS/NGB 
 
 
 
NINDS/NGB 
 
 
 
35/2A116 
 
 
 
10/2-1350 
 
 
 
12N210 
 
 
 
12N210 
 
 
60/257 
 
301-594-5496 
 
 
 
301-451-4881 
 
 
 
301-594-5796 
 
 
 
301-402-2273 
 
 
301-496-3950 
 
carsten.bonnemann@nih.g
ov 
 
 
irene.chrismer@nih.gov 
 
 
 
diana.bharucha@nih.gov 
 
 
 
reghan.foley@nih.gov 
 
 
monal.punjabi@nih.gov 


2 
 
Monal 
Punjabi, BS 
 
Minal Jain, 
PT,  DSc, 
PCS 
 
 
Melissa 
Waite, PT 
 
 
Kim 
Amburgey, 
MS, CGC 
 
Etsuko 
Tsuchiya, BS 
 
 
Sonia 
Razaqyar, 
BA 
 
Monique 
Shelton, BA 
 
Kuo, Anna, 
BA 
 
 
Todd, 
Joshua, PhD 
 
 
Allen, 
Carolyn, 
DNP, MS, 
CRNP-F 
 
Bart 
Drinkard, PT 
 
 
Tissue 
Injury/NINR 
 
Rehab 
Medicine 
Department/CC 
 
Rehab 
Medicine 
Department/CC 
 
 
Hospital for 
Sick Children 
 
 
Hospital for 
Sick Children 
 
 
Tissue 
Injury/NINR 
 
 
Tissue 
Injury/NINR 
 
Tissue 
Injury/NINR 
 
 
Tissue 
Injury/NINR 
 
 
Tissue 
Injury/NINR 
 
 
 
Rehab 
Medicine 
Department/CC 
 
 
 
Clinical 
Center 
10/1NE1469  
 
 
Clinical 
Center 
 
 
N/A 
 
 
 
N/A 
 
 
 
60/254 
 
 
 
60/254 
 
 
60/254 
 
 
 
60/254 
 
 
 
10/2-1350 
 
 
 
 
10/1-2452 
 
 
 
10/1-2452 
 
 
 
 
301-451-7566 
 
 
 
 
301-496-4733 
 
 
 
416.813-7654 
x301707 
 
 
416-813-7654 
 
 
 
202.210.7374 
 
 
 
615-740-4001 
 
 
301-451-5044 
 
 
 
301-435-1503 
 
 
 
301-827-0982 
 
 
 
 
301-496-2844 
 
 
 
210-493-7452 
 
 
 
 
mina_jain@nih.gov 
 
 
 
 
melissa.waite@nih.gov 
 
 
 
kim.amburgey@sickkids.c
a 
 
 
etsuchiya@rogers.com 
 
 
 
razaqyarms@od.nih.gov 
 
 
 
Monique.shelton@nih.gov 
 
 
anna.kuo@nih.gov 
 
 
 
joshua.todd@nih.gov 
 
 
 
carolyn.allen@nih.gov 
 
 
 
 
bdrinkard@mail.cc.nih.gov 
 
 
 
darrenmichael@hyperion 
biotechnology.com 
 


3 
 
Darren 
Michael, 
PhD 
 
 
Ami 
Mankodi, 
MD 
 
Christopher 
Grunseich, 
MD 
 
Jessica 
Witherspoon, 
PhD, DPT 
 
Tanya 
Lehky, MD 
 
Hyperion 
Biotechnology, 
Inc. 
 
 
NGB/NINDS 
 
 
 
NGB/NINDS 
 
 
 
NINR/TIB 
 
 
 
NIH/NINDS 
 
 
35/2A1002 
 
 
 
35/2A1010 
 
 
 
60/257 
 
 
 
CRC/ 7-
5680 
 
 
301-827-6690 
 
 
 
301-402-5423 
 
 
 
301-435-1503 
 
 
 
301-496-7428 
 
 
 
Ami.mankodi@nih.gov 
 
 
 
grunseichc@mail.nih.gov 
 
 
 
jessica.witherspoon@nih.g
ov 
 
 
lehkyt@ninds.nih.gov  
 
Research Contact  
Irene 
Chrismer, 
BSN 
Branch/Institute 
Tissue 
Injury/NINR 
 
Bldg/Rm 
60/259 
 
Phone 
301-451-4881 
 
E-mail 
irene.chrismer@nih.gov 
 
 
Medical Advisory Investigator  
Carsten 
Bonnemann, 
MD 
NINDS 
35/2A116 
301-451-1683 
Carsten.bonnemann@m
ail.nih.gov 
 
 
 
Human Research Protections Program Investigator and Staff Training: 
 
“Just in time” human subjects protection training courses are required for investigators and staff 
participating on this protocol: 
 
(Per SOP 25, PIs or IRBs require investigators and staff working on a protocol to complete the 
following training, as applicable. Delete those that are not applicable.  Indicate “None” if no 
training beyond the general training is required.) 
 
• Biomedical- Vulnerable Subjects - Research with Children 
• Genetic Research in Human Populations 
• CITI GCP modules 


4 
 
• International Studies- ICH Overview and ICH- Comparison Between ICH GCP E6 and 
US FDA Regulations, available to those staff who complete the CITI GCP course 
• Unanticipated Problems and Reporting Requirements in Biomedical Research 
• Unanticipated Problems and Reporting Requirements in Social and Behavioral Research 
 
Total requested accrual ceilings (separately specify planned accrual for each subject group)     
 
 
 100 Patients      (50 in study drug group, 50 in placebo group) 
 
   100 Healthy Volunteers 
 
  
Project Uses Ionizing Radiation:   x No  
 Yes  
 
IND/IDE 
 
 
 
 No   
xYes (attach FDA documentation) 
 
 
Drug/Device/#_N-acetylcysteine__125141__________________ 
 
 
Sponsor: Dr. Katherine Meilleur__ 
 
Durable Power of Attorney   
X No   
 Yes  
 
Multi-institutional Project 
 
 No   
X Yes  
 
 
 
 
 
Institution#1__Hospital for Sick Children________   FWA #  FWA00000281__ 
Date of IRB approval ___6/13/2014___________ 
 
(If NIH is the coordinating site, list each institution; attach documentation of IRB approval for each site) 
 
Data and Safety Monitoring Board  No   
X Yes  
 
Technology Transfer Agreement  
 No   
X Yes  
Agreement type and number _MTA with Dr. Dowling/Univ. Toronto NR-MTA-15-001, MTA 
with Dr. Michael/Hyperion NR-MTA-15-002; CTA with BioAdvantex Pharma NR-CTA-15-
001; MTA with Dr. Marks/Columbia Univ. NR-MTA-16-001 (muscle tissue); MTA with Dr. 
Marks/Columbia Univ. NR-MTA-16-002 (genetic mutations) 
____Expiration Date_All: 6 months after termination of protocol ______ 
 
Samples are being stored  
 
 
 No   
X Yes  
 
Flesch-Kincaid reading level of consent form (exclude boilerplate in assessing reading level): 
• Affected Adult consent: 9.0 
• Affected 14-17 year old assent: 6.2 
• Affected 7-13 year old assent: 5.0 
• Healthy volunteers consent: 8.7 
• Healthy volunteers 14-17 year old assent: 5.2 
• Healthy volunteers 7-13 year old assent: 4.6 


5 
 
Précis: 
Although genetic disorders of muscle that present at birth are rare, RYR1-related myopathies 
comprise the most common non-dystrophic congenital myopathy in the United States, with a 
prevalence of approximately 1/90,000 people (Amburgey et al, 2011). Causative mutations in the 
ryanodine receptor gene of skeletal muscle, RYR1, have been found in several myopathy 
subtypes, including central core disease and centronuclear myopathy. These mutations result in 
defective excitation-contraction coupling and increased mitochondrial oxidative stress. Most 
patients present in childhood with delayed motor milestones, extremity muscle weakness, 
impaired ambulation, joint contractures, progressive scoliosis, and in some cases eye movement 
paralysis, respiratory failure, or susceptibility to malignant hyperthermia, an allelic condition. 
Despite these important morbidities and the risk of early mortality, no treatments exist to date.  
 
RYR1 encodes a homotetrameric transmembrane ion channel, RyR1, which resides on the 
terminal sarcoplasmic reticulum in close proximity to the T-tubule. By releasing calcium from 
the sarcoplasmic reticulum into the cytosol in response to muscle fiber stimulation by the motor 
neuron at the neuromuscular junction, it mediates excitation-contraction coupling and functions 
as a regulator of cellular calcium concentrations and redox homeostasis. Dowling et al. (2012) 
recently elucidated the latter mechanism in zebrafish and patient myotubes, showing that RYR1 
mutations result in increased oxidative stress and that this is rescued in both models by treatment 
with N-acetylcysteine (NAC), a known anti-oxidant. NAC functions as a precursor of 
glutathione, an endogenous antioxidant that becomes deficient during oxidative stress.  This was 
substantiated by a cystic fibrosis clinical trial in which low glutathione levels in neutrophils 
undergoing oxidative stress significantly increased with NAC administration. 
 
Dowling et al. (2012) found significant changes post NAC treatment including increased travel 
distance (endurance) in zebrafish and complete protection from cell death induced by 
experimentally increasing oxidative stress in myotubes. Thus NAC was a successful treatment in 
both ex vivo and in vivo model systems. Based on these results, we plan to perform a 
randomized, double-blinded, placebo controlled clinical trial of NAC in a subgroup of RYR1-
related myopathy patients as the first pathophysiologically based treatment for this devastating 
disorder. 
 
The objectives of the study are to determine if NAC reduces oxidative stress, fatigability, and 
fatigue in a study population of patients with RYR1-RM. The study population includes both 
males and females 7 years of age and older and age-matched healthy volunteers. The study 
design has two phases.  The first 6-month phase will be used to validate the selected outcome 
measures in RYR1 congenital myopathy.  The second 6-month phase is a randomized, double-
blinded, placebo controlled drug intervention trial. The primary outcome measures are urine 15 
F2t-isoprostane for oxidative stress and six minute walk test for fatigability.  Healthy volunteers 
will be evaluated to determine normal values of biomarkers, muscle ultrasound, and near infrared 
spectroscopy in this rare disease, in order to develop a comparison between healthy and RYR1-
RM individuals. 
 


6 
 
Table of Contents  
 
Title Page ........................................................................................................................................ 1-4 
Précis ................................................................................................................................................. 5 
1. Introduction ................................................................................................................................ 8-9 
2. Study Objectives ........................................................................................................................... 10 
3. Subjects .................................................................................................................................. 10-12 
4. Study Design and Methods ....................................................................................................... 12-21 
5. Storage of Data and Samples .................................................................................................... 21-22 
6. Additional Considerations ............................................................................................................. 22 
7. Risks and Discomforts .............................................................................................................. 22-27 
8. Subject Safety Monitoring ........................................................................................................ 27-28 
9. Outcome Measures .................................................................................................................. 28-30 
10. Statistical Analysis .................................................................................................................. 30-33 
11. Human Subjects Protection .................................................................................................... 33-36 
12. Anticipated Benefit ..................................................................................................................... 36 
13. Classification of Risk ............................................................................................................... 36-37 
14. Consent Documents and Process ............................................................................................ 37-38 
15. Data and Safety Monitoring .................................................................................................... 38-39 
16. Quality Assurance ....................................................................................................................... 39 
17. Reporting of Unanticipated Problems, Adverse Events and Protocol Deviations ......................... 39-40 
18. Alternatives to Participation ........................................................................................................ 40 
19. Privacy ....................................................................................................................................... 40 
20. Confidentiality ............................................................................................................................ 41 
21. Conflict of Interest ................................................................................................................. 41-42 
22. Technology Transfer .................................................................................................................... 42 
23. Research and Travel Compensation ......................................................................................... 42-43 
24. References ............................................................................................................................ 43-44 
25. Attachments .......................................................................................................................... 44-45 
26. Consent Forms ............................................................................................................................ 45 
 
List of Abbreviations 
6MWT: 6 Minute Walk Test 
CPET: Cardiopulmonary Exercise Test 
DCFH: 2’,7’-dichlorofluorescein 
DHEA: Dehydroepiandrosterone 
EIM: Electrical Impedance Myography 
ELISA: enzyme-linked immunosorbent assay 
FACIT:  Functional Assessment of Chronic Illness Therapy 
GSH: glutathione 
MFI: Multidimensional Fatigue Inventory 
MFM: Motor Function Measure  
MRI: Magnetic Resonance Imaging 


7 
 
MRS: Magnetic Resonance Spectroscopy  
NAC: N-acetylcysteine 
NADPH: Nicotinamide adenine dinucleotide phosphate 
NeuroQoL:  Quality of Life in Neurological Disorders 
NIRS: Near Infrared Spectroscopy 
OM: Outcome Measure 
PROMIS:  Patient-Reported Outcomes Measurement Information System 
PTGS2: prostaglandin-dependent cyclo-oxygenase 
QoL: Quality of Life 
ROS: reactive oxygen species 
RYR1: ryanodine receptor 1-related myopathies 
 
 
 
 
 


8 
 
1.  Introduction  
 
Although genetic disorders of skeletal muscle that present at birth are comparatively rare, 
RYR1-RM comprises the most common non-dystrophic congenital myopathy in the United 
States, with a prevalence of approximately 1/90,000 (Amburgey et al., 2011). Causative 
mutations in the gene (RYR1) have been found in several myopathy subtypes, including central 
core disease and centronuclear myopathy.  
The defining histopathological feature of RYR1 mutation on muscle biopsy is the 
presence of amorphous cores, likely due to reduced oxidative enzyme activity (Jungbluth et al., 
2011). Most patients present in childhood with delayed motor milestones, extremity muscle 
weakness, impaired ambulation, joint contractures, progressive scoliosis, and, in some cases, eye 
movement paralysis, respiratory failure, or susceptibility to malignant hyperthermia, an allelic 
condition (Jungbluth et al., 2011; Bevilacqua et al., 2011; Zhou et al., 2007). Despite these 
important morbidities and the risk of early mortality, no treatments exist to date.  
RYR1 encodes a homotetrameric transmembrane ion channel, RyR1, which resides on the 
terminal sarcoplasmic reticulum in close proximity to the T-tubule. By releasing calcium from 
the sarcoplasmic reticulum into the cytosol in response to muscle fiber stimulation by the motor 
neuron at the neuromuscular junction, it mediates excitation-contraction coupling and functions 
as a regulator of cellular calcium concentrations and redox homeostasis (Bevilacqua et al., 2011, 
Brookes et al., 2004; Betzenhauser and Marks, 2010).  RYR1 gene mutations result in defective 
excitation-contraction coupling and increased mitochondrial oxidative stress (Jungbluth et al, 
2011). 
To elucidate novel pathogenic mechanisms in RYR1-RM, Dowling and colleagues 
(2012) employed expression array analysis in a zebrafish model of RYR1-RM (ryr zebrafish), 
which disclosed abnormalities in pathways associated with cellular stress. Further studies were 
completed to confirm the presence of increased oxidative stress. Beta-galactosidase, a measure of 
cellular stress in vivo, was elevated in ryr zebrafish. Ryr zebrafish also showed increased 
carbonylation by oxyblot, a marker of oxidative stress, compared to wildtype controls (Arbogast 
et al., 2009). DCFH, a measure of overall intracellular oxidant activity was tested in ryr zebrafish 
against controls and the affected zebrafish showed increased activity.  The increased oxidant 
activity appeared to be a direct result of the ryr mutation. Similarly, myotubes from patients with 
RYR1-RM showed increased oxidative stress (in DCFH assay and oxyblot analysis). Further 
studies were completed to determine if oxidative stress in RYR1-RM is due to over-abundant 
oxygen species or to insufficient antioxidants (GSH). Dowling et al. (2012) measured the 
expression of genes related to antioxidant defense.  There were no changes in the expression of 
the following antioxidant genes: catalase, superoxide dismutase 1, mitochondrial superoxide 
dismutase and extracellular superoxide dismutase.  These results indicate the cause of oxidative 
stress is not a lack of antioxidants, but rather over-abundance of reactive oxygen species (ROS). 
Possible sources of reactive oxygen species include NADPH oxidase complex (Hidalgo et al., 
2006), PTGS2, and mitochondria.  Dowling et al. (2012) tested all three potential sources, yet 
only mitochondria appeared to be the cause of the over-production of ROS. 
Given these findings, Dowling et al. (2012) subsequently tested the effect of NAC in 
cultured patient myotubes and zebrafish. N-acetylcysteine (NAC) is an antioxidant and a 
precursor to GSH.  By increasing the intracellular content of GSH, NAC augments the cell’s 
reducing potential and restores the redox imbalance (Tirouvanziam et al., 2006).  In the 
experiment, control myotubes and RYR1-related myopathy myotubes were exposed to oxidants, 


9 
 
producing a higher cell death rate in RYR1-RM myotubes.  However, when pre-treated with 
NAC, the RYR1-RM myotubes were completely protected from cell death. Ryr zebrafish 
endurance and swim velocity were also tested.  When treated with NAC, ryr zebrafish showed 
rescued endurance (represented by swim distance), matching the endurance of controls treated 
with NAC.  However, strength (represented by swim velocity) did not appear to improve for ryr 
zebrafish treated with NAC.  These findings corroborate the known mechanism of NAC, which 
functions as a precursor of GSH, an endogenous antioxidant that becomes deficient during 
oxidative stress.   
 
Based on these findings regarding NAC’s effect on RYR1-RM in zebrafish and patient 
myotubes and on evidence of improved oxidative stress (increased GSH) in NAC human trials 
for other conditions, we plan a clinical proof of concept study.  15-F2t-isoprostane likewise 
measures oxidative stress (due to lipid peroxidation) and has been reported as the most accurate 
method to assess in vivo oxidative stress status (Milne et al. 2012). Early animal studies showed 
that glutathione peroxidase (GPx), a reducing agent mediated by glutathione availability, may be 
important for combatting peroxidation of mitochondrial lipid membranes by free radicals 
(McCay et al. 1976). NAC is a direct precursor to glutathione (Schmitt et al. 2015), and RYR1-
RM preclinical studies showed that the source of oxidative stress in preclinical disease models 
was due to mitochondrially derived reactive oxygen species (Dowling et al. 2012).  
The treatment group will receive 15mg/kg/day (no more than 1800 mg/day) for the first week.  
After the first week, subjects who weigh fewer than 50kg will receive 30mg/kg/day of NAC (no 
more than 2700mg/day) divided TID over a period of six months.  To increase compliance, as 
the tablets are formulated as 900mg each, subjects who weigh greater than 50kg will receive 
2700mg/day.  This dose is within the range of study references and was approved by the FDA on 
the IND (125141) for this trial.   
This dose is scientifically acceptable. The dose of 2700mg/day is similar to that of other  
clinical trials.  A trial of NAC for cystic fibrosis, using a dose of 0.6 to 1.0g TID, significantly 
showed increased GSH levels in blood neutrophils. Furthermore, sputum elastase activity, the 
strongest predictor of CF pulmonary function, markedly decreased (P = 0.006) and the treatment 
was safe (Tirouvanziam et al., 2006). NAC is currently indicated as a mucolytic for cystic 
fibrosis and other pulmonary conditions. A pediatric study was completed in three to ten year 
olds with autism to treat behavior issues (Hardan et al, 2012).  The dosage began as 900mg once 
a day for four weeks, and worked up to 900mg TID for four weeks (entire trial was 12 weeks).  
NAC decreased irritability in the population, measured by the ABC (Aberrant Behavior 
Checklist) Irritability Score.  Side effects were related to GI issues, but there was no significant 
difference between the side effects of the control group and the treatment group.  A trial of NAC 
in patient with fibrosing alveolitis showed increased GSH levels over a period of 12 weeks 
(idiopathic pulmonary fibrosis) and is now FDA-approved for this indication (Behr et al, 1997). 
Natural history data for RYR1-RM are limited to date, but the Motor Function Measure (MFM), 
a scale that measures motor function, has been validated in this population and has shown stable 
motor function scores over a 1 year time period (Vuillerot et al., 2012). We intend to use the 
MFM as a gold-standard outcome measure to track progression of disease in the natural history 
portion of this trial.  Clinical manifestations usually improve in early childhood and may remain 
stable for some years, as RYR1-RM is not degenerative. However, natural history data observing 


10 
 
oxidative stress levels, fatigability, and fatigue are lacking.  Thus, the initial portion of the trial 
will be a study of the natural history in RyR1-RM.   
 
Summary 
A randomized, double-blinded, placebo controlled clinical trial of NAC in RYR1-RM patients 
will be performed to test the effect on oxidative stress, fatigability, and fatigue.  The primary 
outcome measure will be 15-F2t-isoprostane for oxidative stress. Success will be defined as 
finding a significant difference in 15-F2t-isoprostane concentration between the treatment and 
placebo groups at the termination of the study. If successful, this study of NAC in RYR1-RM 
patients will provide the first treatment available for this debilitating muscle disease.  
 
 
2.  Study objectives  
 
Aim 1.  To determine if NAC reduces oxidative stress in patients with RYR1-RM. 
Hypothesis: Compared to the placebo group and baseline values for each patient, N-
acetylcysteine will reduce oxidative stress in the treatment group as evidenced by: 1) decreased 
15-F2t-isoprostane in urine, 2) decreased 2’,7’-Dichlorodihydrofluorescein (DCFH) in biopsied 
muscle, and 3) increased glutathione (GSH) in blood.  Exploratory markers of anaerobic 
threshold, fraction of inspired O2, and tissue oxygenation by Near Infrared Spectroscopy (NIRS) 
will also be measured pre and post treatment. 
 
Aim 2.  To determine if NAC reduces fatigability and fatigue in patients with RYR1-RM. 
Hypothesis: Compared to the placebo group and baseline values for each patient, N-
acetylcysteine will reduce fatigability (objective construct) and fatigue (subjective construct) in 
the treatment group as evidenced by: (for fatigability) 1) the 6MWT, 2) timed function tests, 3) 
grip and pinch strength, as measured by Myotools 4)  muscle fatigability by Biodex-; and (for 
fatigue) 5) Patient-Reported Outcomes Measurement Information System (PROMIS), 6) 
Functional Assessment Chronic Illness Therapy (FACIT) fatigue scale, 7) Fatigue scales: 
Multidimensional Fatigue Inventory (MFI). 
As secondary and exploratory measures, we will also test the following pre and post treatment: 
CPET by cycle ergometry, Electrical Impedance Myography (EIM) to measure skeletal muscle 
health, muscle MRI to evaluate for subclinical muscle involvement as well as the health of 
muscle tissue, MFM for motor function, salivary fatigue biomarker index (FBI), melatonin, 
dehydroepiandrosterone (DHEA), the NeuroQoL scale, and qualitative interview for health 
related quality of life.  
 
Aim 3. To determine values of study biomarkers, NIRS, muscle ultrasound, and MRI in age-
matched healthy volunteers 
 
3.  Subjects: 
  
a.  Description of study populations  
 
This study will include patients age 7 years or older with mutations in RYR1 (confirmed 
by previous genetic testing), who are not currently taking antioxidants, and who are able and 
willing to perform all procedures at the time of enrollment.   Children under age 7 may not be 


11 
 
able to successfully follow instructions for outcome measure testing.  The accrual ceiling is 100. 
Withdrawals/dropouts will not be replaced. The target number of completers is 76. 
 
Sixty to 100 age-matched healthy volunteers will be recruited.  
NIH employees may participate.  The NIH Information Sheet on Staff Research Participation 
will be provided to any staff member wishing to participate in this study (see Appendix L). 
 
 
b.  Inclusion criteria 
Patients: 
• Have a confirmed genetic diagnosis of RYR1-related myopathy, including congenital 
myopathy, OR have a clinical diagnosis of RYR1-related myopathy and a family member 
with a confirmed genetic diagnosis of RYR1-related myopathy 
• Ambulatory. 
• 7 years of age or older.   
• Preferred:  The affected subject has a previous muscle biopsy with available report 
confirming RYR1-related myopathy histopathology. 
Healthy Volunteers: 
• Ages 7 and older (15-25 7-12 year olds, 15-25 13-17 year olds, 15-25 18-40 year olds, 
15-25 41 and older) without medical condition resulting in or complaints of muscle 
weakness and/or fatigue. 
• The ability to provide consent.  
 
c.  Exclusion criteria 
Patients: 
• Adults who cannot provide their own consent and pediatric participants who do not have a 
parent able to provide consent  
• Patients with a history of peptic ulcers, gag reflex depression, and esophageal varices. Patients 
with gastrostomy tubes may be considered for participation, in the case of gag reflex 
depression or other swallowing or feeding difficulties. 
• Patients who have a severe pulmonary dysfunction (FEV1< 40% predicted) or evidence of 
pulmonary exacerbation. Pulmonary exacerbations refer to an acute worsening of respiratory 
symptoms that result from a decline in lung function (Flume et al., 2009). Participants may 
present with increased coughing, increased dyspnea, increased haemoptysis, increased 
fatigue, decreased pulmonary function by a min of 10%, or a change in sputum color (Bilton 
et al., 2011). 
• Pregnant and breastfeeding women. 
• Consumption of antioxidants [including NAC, GSH, melatonin, Immunocal (Immunotac 
• Patients with a history of liver disease (Liver Function Tests will be collected at baseline and 
at each study visit as a precautionary measure). Liver disease is defined as moderate to severe 
hepatic impairment based on the following: 
          ALT ≥ 8x upper limit of normal (ULN) with total bilirubin 2x ULN (plus >35% ‘direct’      
          bilirubin)and/or INR >1.5  
          or 
         GGT > 2-3x ULN with bilirubin 2x ULN (plus >35% ‘direct’ bilirubin) and/or  INR  
         >1.5GGT >2-3x ULN and INR >1.5 


12 
 
Research, Vandreuil-Dorion, QC, Canada), Nacystelyn (Galephar, Brussels)] in the 4 weeks 
before recruitment. 
• Daily use of acetaminophen (including Percocet, Vicodin, Oxycodone, Excedrin, and other 
acetaminophen-containing drugs), nitroglycerine, or carbamazepine during the past 7 days. 
• Current use of Angiotensin-converting enzyme (ACE) inhibitors or Angiotensin Receptor 
Blockers (ARBs) 
• Patients who have ever used Beta2-adrenergic agonist tablets, for the purpose of increasing 
muscle mass (such as albuterol tablets). 
• For the muscle biopsy procedure only (second and third visits, if applicable): Patients who 
have taken Aspirin, Ibuprofen, Advil, Motrin, or Aleve within the 3 days prior to the muscle 
biopsy procedure, and/or patients who have taken Plavix, fresh garlic, gingko, or ginseng 5 
days prior to the muscle biopsy. 
• Participation in trials for other therapeutic investigational drugs simultaneously or 4 weeks 
before recruitment. 
Other clinically significant medical disease that, in the judgment of the investigators, would 
expose the patient to undue risk of harm or prevent the patient from completing the study.  
Examples include anemia (defined as Hgb < 8 gm/dl), an inability to walk safely without 
assistance for at least 6 minutes, and/or an inability to consume at least 6 ounces of fluid, 3 times 
a day, either orally or via G-tube. Patients with comorbidities (i.e. cancer, epilepsy) will be 
carefully assessed to determine if their comorbidity could lead to confounding or safety issues, 
should their participation continue.  
Healthy Volunteers: 
• Diagnosis of RYR1-related myopathy or other neurological disorder (by neurological 
exam, genetic testing, or muscle biopsy 
• Complaints of fatigue or weakness 
• Consumption of antioxidants [including NAC, GSH, melatonin, Immunocal (Immunotac 
• Research, Vandreuil-Dorion, QC, Canada), Nacystelyn (Galephar, Brussels)] in the 4 
weeks before recruitment. 
• Use of Beta2-adrenergic agonists.  
 
 
4.  Study Design and Methods: 
 
a.  Study overview  
The study design has two phases: first, a prospective natural history study and second, 
prospective, randomized, double-blinded, placebo-controlled phase.  Each participant will be in 
the study for about 18 months from start to finish, with a total of three visits of 4-6 days each and 
7 phone calls and/or emails. All emails containing Patient Identifying Information are to be sent 
using secure email, behind the NIH firewall (SEFT).  Participants will be given a link to SEFT 
and will be asked to send all medical records and other sensitive information through the SEFT 
system.  Visits will be outpatient at the Clinical Center, National Institutes of Health in Bethesda, 
MD. Samples will be analyzed at National Institutes of Health or at the Eicosanoid Core Facility 
at Vanderbilt University, Tennessee on a fee-for-service basis. Analyses will be overseen by the 
Core’s Director, Dr. Ginger Milne.  
In the natural history phase (Phase 1), participants will be seen at baseline, called one 
month later (for re-test reliability of fatigue and quality of life scales), and will be seen again at 


13 
 
six months (±2 weeks) to undergo procedures by an investigator trained in questionnaire 
administration. The participants will complete the scales at home, using CTSS. Sixty to one 
hundred healthy volunteers will undergo a physical exam, study biomarker, NIRS testing, and 
muscle ultrasound only, in one outpatient visit to NIH. Eight-32 of these healthy volunteers will 
also undergo a muscle MRI (2-8 people from each of the 4 age groups.  See Inclusion Criteria for 
age groups of healthy volunteers).  Outcome measures are listed and described under “Outcome 
Measures”. Since many outcome measures used in this study are already well established and 
have been previously used to measure function in other neuromuscular diseases, we will only 
validate measures novel to RYR1-RM, such as NIRS, biodex fatigue index, and salivary fatigue 
biomarkers.  The self-administered questionnaires will also be validated by analyzing 
questionnaire data on 20 participants at 0 months and 1 month. 
After data collection at six months, for each individual participant, the participant will 
immediately begin the second phase of the study when they will be randomized, using block 
randomization, to a placebo or a study drug group at the end of the 6-month visit to NIH. The 
allocation scheme is computer-generated using random permuted blocks to maintain balance of 
the two groups (placebo and control) and to maintain balance between children and adults. The 
participant will be randomized to the study drug or placebo group by an NIH pharmacist, who is 
blinded to the allocation scheme.  Study drug assignments are recorded by the pharmacist, to be 
revealed only after the study is complete.  BioAdvantex Pharma will provide oral effervescent 
NAC capsules, (PharmaNac) as well as identically packaged placebo capsules so that 
participants will not be able to distinguish the treatment from placebo.  The study drug group 
will receive 15mg/kg/day (no more than 1800 mg/day) for the first week. After the first week, 
subjects who weigh fewer than 50kg will receive 30mg/kg/day of NAC (no more than 
2700mg/day) divided TID over a period of six months.  To increase compliance, as the tablets 
are formulated as 900mg each, subjects who weigh greater than 50kg will receive 2700mg/day. 
After Phase 2 starts, participants will be observed at the NIH for 2 days.  They will then 
receive a phone call and/or email at approximately one and three weeks to monitor their status on 
the study drug or placebo by a health care professional.  After the first month, the subjects will 
be monitored by phone and/or email monthly (month 2, 3, 4, and 5). Participants will be 
expected to take their dose three times per day for about six months. Adherence will be assessed 
by a pill count at their 3-month assessment and at their post-treatment visit (third visit).  The pill 
count will be performed by the participant at home for the 3-month phone call assessment and by 
a member of our team at NIH during their third visit.   Finally, the outcome measures will be 
obtained at the final visit 6 months (± 2 weeks) after start of treatment (about 12 months into the 
study).  Six months after their third visit (± 2 weeks), the participant will be called for a post-
treatment follow up.  The participant (or parent) will be asked open-ended questions to assess 
their symptoms and current medication regime.  They will also be asked to fill out the same 
fatigue and QoL scales completed during their visits, at home on CTSS.  The participant will 
then be finished with the study. Please see Appendix A for a detailed Visits and Procedures 
Schedule. 
 
b.  Recruitment 
Although RYR1 congenital myopathies constitute a rare disease, the PIs and AIs on this protocol 
work closely with patients with this condition and with patient groups such as the Muscular 
Dystrophy Association and Cure CMD. We will advertise the study through the patient groups 
by providing information about the study and posting it on their websites (Appendix B). We will 


14 
 
also provide a letter describing the study including contact information to health care providers 
in the neuromuscular field (Appendix D). If interested, potential participants can call the research 
coordinator listed on the advertisements.  
Flyers have been created for healthy volunteer and patient populations (Appendix B).  The 
patient flyer includes contact information for the Patient Recruitment and Public Liaison office 
(PRPL).  We will distribute our flyers to the managers of the Facebook group pages entitled: 
“The RYR-1 Foundation”, which is found at this link: 
https://www.facebook.com/ryr1foundation/?fref=ts, and 
“Central Core Disease & Minicore: a place for support, learning & friends”, which is found at 
this link: https://www.facebook.com/groups/243087794204/. We will also tweet our study at 
https://twitter.com/NINR and use the web slide carousel on the NINR website 
https://www.ninr.nih.gov/researchandfunding/dir#.VzzUSRIrKRs, to advertise our study.   
 
A telephone or email prescreening will be used to determine preliminary eligibility (prescreen 
questionnaire, Appendix G). The prescreening telephone call will be scheduled or the prescreen 
questions will be sent via email.  During the prescreening phase, we will request the potential 
participant send the results of their genetic testing confirming diagnosis of RYR1 (if done) and 
the results of their muscle biopsy confirming RYR1 histopathology (if done).  We will receive 
these documents via mail, fax, or secure email before their visit.  This will prevent patients who 
are clearly not eligible from making an unnecessary trip to NIH.  The records of any participant 
who subsequently does not qualify will be destroyed or returned to the participant.  
We will not request records of relatives of participants/potential participants who are 
enrolling under the criteria of a clinical diagnosis with a family member with confirmed RYR1.  
 
There is no direct solicitation of employees/staff by supervisors.  Direct solicitation by co-workers 
is considered improper. 
 
c.  Screening  
Patients: 
 
Informed consent will be obtained before any study procedures begin.  We will screen 
participants by determining whether or not they meet inclusion/exclusion criteria by using the 
eligibility checklist (Appendix E). 
At the onset of the study, individuals will be required to have a documented muscle 
biopsy confirming RYR1 diagnosis. If 20 individuals with this documentation cannot be 
recruited within the first 6 months, those without muscle biopsy confirmation will also be 
enrolled. 
Healthy Volunteers: 
Healthy volunteers will be screened by phone, email, or in-person for the presence of any 
neurological condition and/or complaints of weakness and fatigue and will be asked about 
current medications, per the eligibility criteria. 
 
d.  Study procedures 
Patients: 
Participants will travel to the NIH Clinical Center three times over the course of one year.  At 
each visit, participants will complete some or all of the outcome measure tests and up to 20 
adults who volunteer will undergo optional muscle biopsy testing at the second and third visit for 


15 
 
a maximum of two biopsies.  The muscle biopsy is the only procedure that will not be required 
of all participants due to its invasive nature, all other procedures are required unless 
contraindicated. 
 
All study procedures are for research purposes and consent will be obtained prior to any 
procedures. No radiation will be used in the study.  All visits are part of the study. Participants 
will follow up with their primary physician or neurologist for clinical care.   
 
Medical History: Participants will have access to an encrypted online survey system (Clinical Trial 
Survey System or CTSS) to enter their coded medical history information. Participants will be 
given the website and login by an investigator.  Legal guardians may enter this information for 
minors. Study physicians, nurse practitioners and/or nurses will obtain medical history information 
to supplement the online medical history if needed. 
 
Physical Examination: Height, weight, head circumference, BMI, and ulna length may be obtained 
by trained staff, including nurses, nurse practitioners, physicians, and physical therapists. Height, 
ulnar length, and head circumference will be repeated in children only at visit 2 and 3. For 
participants with contractures, height may be estimated by ulnar length obtained by a segmometer, 
which has been shown to correlate with height. If available, a sling scale will be used to weigh 
participants who are unable to stand on a scale. Study physicians or nurse practitioners will perform 
a physical examination including the evaluation of cranial nerves, reflexes, sensation, strength, 
gait, movement, motor function and coordination. The physical examination will take 
approximately 30 minutes.  
 
Cardiology: Cardiologic evaluations in individuals will include electrocardiogram (EKG), which 
is required prior to CPET. EKGs take 10 minutes or less. A cardiology consult will be requested 
only if the EKG is abnormal. Participants deemed unable to complete the CPET will not be 
excluded from the rest of the study procedures. 
 
Pulmonary Functions Tests: Each subject will perform pulmonary function testing (PFTs) to assess 
vital capacity (VC) and maximal voluntary ventilation (MVV). To measure VC the participant will 
be asked to take the deepest breath they can, and then exhale into a flow sensor. For maximal 
voluntary ventilation (MVV) the patient will be instructed to breathe as fast and deep as possible 
for 12 seconds. During the test, soft nose clips may be used to prevent air escaping through the 
nose (patients will use their own flow sensors so filters will not be needed). Measures to be 
obtained include vital capacity (VC), forced expiratory volume at 1 second (FEV1), and maximal 
voluntary ventilation. Pulmonary Function testing will take approximately 30 minutes. 
 
Cardiopulmonary Exercise Testing (CPET):  The cardiopulmonary exercise test (CPET) will be 
administered using an electronically braked cycle ergometer.  Subjects will be asked to maintain 
a pedaling cadence of at least 30 revolutions per minute (rpm) for the entire test.  Subjects will 
begin the CPET pedaling against an unloaded work rate for the first minute after which point the 
work rate will increase each additional minute.  The CPET will be stopped when the subject can 
no longer maintain the predetermined pedaling cadence despite strong verbal encouragement.   
Test duration, maximal oxygen uptake (VO2max), and Anaerobic Threshold, determined using the 


16 
 
V-slope method, will be obtained during CPET testing. CPET takes approximately 1 hour to 
complete.  
 
Electrical Impedance Myography (EIM) Assessment: Measurements of electrical impedance of 
muscle tissue will be made to determine the health of the muscles by using an electrical 
impedance investigational device. Current and sensing electrodes are placed non-invasively on 
the subject’s skin at known distances and the voltage at the sensing electrode is measured. 
Resistance, reactance, and the phase of multiple muscles are then recorded and the interpretation 
of these results is used to determine the health of the muscle tissue. 
 
Magnetic resonance imaging (MRI): An MRI scan of the leg muscles may be performed in order 
to visualize structures using a 1.5 or 3 Tesla Magnet. T2 and T1 weighted imaging and MRS will 
be performed. High resolution conventional MRI will be performed with volumetric 
reconstruction of designated regions. Time in the MRI scanner will be limited to 1 hour, in order 
to allow the subject to rest and assure the best quality of the images.  We will allot additional 
time for any delays and to better ensure comfort.  Audiovisual distraction equipment such as 
music system with headphones will be available.  Children will be given extra time to get 
adjusted to the new environment in the imaging facility, as well as in the scan room to alleviate 
anxiety and nervousness.  Sedation will not be used for any procedure.   
 
Arterial Occlusion Muscle Oxygenation Capacity Test:  A blood pressure cuff will be placed 
around the distal thigh of the dominant leg. A near infrared spectroscopy (NIRS) light source and 
receiving sensors will be placed on the leg, distal to the occlusion. Using a Hokanson® rapid 
inflation vascular testing system, the cuff will be rapidly inflated to a pressure that totally 
occludes blood flow into the gastrocnemius, typically between 60 and 80 mmHg above resting 
systolic pressure. The occlusion will be maintained for up to 10 minutes and tissue saturation 
will be measured over the time course. The tissue saturation index will be calculated as TOI= 
[O2-Hb] / Tot-Hb. The rate of decline and rise in TOI will be the main muscle tissue saturation 
measures. 
 
Physical therapy: Evaluation will include timed functional tests: 10 meter walk test, supine to 
stand, ascend 4 stairs, descend 4 stairs, and the 6 minute walk test.  Grip and pinch strength will 
also be determined with Myotools. The myogrip is a hand held dynamometer designed to 
measure grip strength, while the myopinch is a pinch gauge designed to measure finger strength. 
The patient will be seated comfortably with his/her elbow flexed to 90 degrees, with the forearm 
and wrist in neutral position. The patient is asked to squeeze the dynamometer and pinch the 
gauge. This process is repeated three times (3 trials). Time to complete: 30 min.  
 
Motor Function Measure 32 (MFM): The MFM is a generic scale which provides a measurement 
of the effects of muscle weakness in neuromuscular diseases (NMD). Assessments are based on 
posture and movements of the whole body. It is applicable to both ambulant and non-ambulant 
patients with a wide range of disease severity. The patient is asked to roll, sit, lift head from prone 
and supine position, get up from a lying position, prop on arms, kneel, crawl, stand and step. This 
test will be combined with timed tests (floor to stand, ascend/descend 4 steps). Time to complete 
both tests: approximately 60 minutes.  
 


17 
 
6 Minute Walk (6MWT): The self-paced 6MWT assesses the submaximal level of functional 
capacity. The patient is asked to walk for 6 minutes with their best effort. A clinician accompanies 
the patient during the test to ensure patient’s safety.  Heart rate and blood pressure is monitored 
before and after exercise and after 5 minutes of recovery to ensure safety of the patient.  The patient 
is allowed to stop if he feels tired.  Time to complete the entire test:  approximately 20 minutes.  
 
Biodex: Biodex will be used to test muscle groups using a dynamic sub-max fatigue test with a 
repetitive motion.  Blood pressure will be taken prior to testing to confirm the patient is not 
hypertensive (>140/90). Clinicians will assess participants for previous knee injury prior to 
testing.  The biodex test will not be performed if the participant has a history of knee injury. 
Clinicians will provide a 5-minute break immediately after the test, during which time the patient 
may not rise from the seat. Time to complete the entire test:  approximately 60 minutes.   
 
Phlebotomy, Genetic Testing, and Exome Sequencing: Phlebotomy will be performed to obtain 
blood for liver function testing at baseline and glutathione levels. Blood and blood products may 
be analyzed or stored for creatine kinase (CK) level, lactate/pyruvate ratio, cell counts, blood 
chemistries, molecular genetic analysis and DNA banking. The lab will draw blood for DNA to 
confirm a genetic mutation, for exome sequencing, and/or for future studies and other clinical lab 
testing. If confirmatory genetic testing of an enrolled family member reveals that a participant 
does not have an RYR1 mutation causative of RYR1-RM, the participant will be informed and 
withdrawn from the study.   
DNA studies may increase knowledge of the pathomechanism underlying RYR1. The target 
blood draw is 30ml. A local anesthetic cream such as Eutectic Mixture of Local Anesthetics 
(EMLA) may be used to minimize the pain for young children. Phlebotomy will take 
approximately 15 minutes.  Blood will not be drawn on participants if they are known to be 
anemic (Hgb < 8 gm/dl) per medical history, or if their medical condition causes an investigator 
to believe that blood drawing might be detrimental to the participant. For pediatric participants, 
no more than 5 mL/kg will be drawn in a single day, and no more than 9.5 mL/kg will be drawn 
over any eight-week period. Timing of blood draw will be between 8:30-10:30am and will be 
taken prior to any food consumption. Participants will be notified the night before not to 
consume food prior to the blood draw the next day. 
 
Saliva collection: For the saliva collection, patients will be asked to chew on a cotton swab for 
about 5 minutes and then deposit the cotton swab in a specimen container.  Saliva will be 
measured for exploratory salivary biomarkers of fatigue such as fatigue biomarker index, 
melatonin, cortisol, and didehydroepiandrosterone (DHEA). Saliva will be collected the same 
day as the blood draw by a health care professional between 8:30 and 10:30 am prior to any food 
consumption. Participants will be notified the night before not to consume food prior to the 
saliva collection the next day. Also, a non-fasting post-exertion saliva sample will be taken 
immediately after the participant performs the biodex test.  The saliva will be put on ice 
immediately and stored at -80.   
 
Urine collection: Participants will be asked to provide a clean catch urine sample of at least 10 
ml. Urine collection is required to detect levels of 15-isoprostane-F2 (normalized for creatinine), 
which allows for the indirect measurement of ROS. Additionally, urine samples will be used for 
pregnancy tests. 


18 
 
 
Pregnancy testing: All women and girls of childbearing potential will have a urine pregnancy test 
performed at each visit, which must be negative before proceeding further with the study. 
Contraception will be required for women and girls of childbearing age who are sexually active 
during phase 2 of the study. If a pregnancy test is positive, the study participant will be notified of 
the results.  If the study participant is a minor, she will be notified, and we will help her inform her 
parent(s) or guardian of the results. Women who become pregnant during the study will be 
followed through their pregnancy and birth. 
 
Videotaping and/or Photography: Videotaping or photography may be performed during portions 
of the physical examination to accurately document the condition. When possible, the patient’s 
face and other identifying marks will not be included in the recording or photograph in order to 
protect confidentiality. Consent will be obtained for videotaping and photography. 
 
Qualitative interviews and/or questionnaires of fatigue and quality of life: Questionnaires to be 
used include Neuromuscular Quality of Life (NeuroQOL), the Patient-Reported Outcomes 
Measurement Information System (PROMIS), FACIT (Functional Assessment of Chronic Illness 
Therapy), and MFI (Multidimensional Fatigue Inventory). Patients and/or parents will be asked to 
participate in research qualitative interviews. Parent proxy measures and patient outcome measures 
will be used to measure patient quality of life and daily function. Interviews and questionnaires 
will take approximately 2 hours. The NeuroQOL, PROMIS, FACIT, and MFI will be 
readministered at 1 month after the initial visit and will be completed by the participant in CTSS.  
Coded questionnaires may be administered online in a database such as CTSS or Assessment 
Center Lite. 
 
Muscle Ultrasound: Ultrasound will be used to evaluate skeletal muscles using standard medical 
soft tissue ultrasound. Muscle ultrasound will take approximately 30 minutes.   
 
Muscle biopsy (OPTIONAL, Adults Only): A needle muscle biopsy will be obtained twice 
during the study on up to 20 adults who voluntarily comply with this procedure at 6 months and 
12 months into the study.  An unblinded researcher, independent of Dr. Meilleur’s research team, 
will allocate an approximately equal number of subjects from each arm of the study based on the 
randomization scheme created for the entire study in advance by the NIH pharmacy for muscle 
biopsy: approximately 10 subjects from the intervention arm will be biopsied, approximately 10 
subjects from the placebo arm will be biopsied. An outside muscle biopsy performed by the 
participants’ local providers in the six months prior to the study 6-month time point will also be 
acceptable, to reduce the number of invasive procedures.   
 
This muscle biopsy will be performed on the vastus lateralis or tibialis anterior muscle.  After 
skin preparation using povidone-iodine or equivalent antiseptic, the skin and subcutaneous 
tissues will be anesthetized with 7-10 ml of 1% lidocaine without epinephrine.  For side-cut 
biopsy needles (known as Bergstrom or UCHL needles) a 3mm incision will be made in the skin 
and muscle fascia, and muscle tissue will be obtained with 1 to 3 successful passes of the biopsy 
needle.  Successful passes are defined as passes which obtain any muscle tissue.  Due to issues, 
such as fatty infiltration of muscle in RYR1-RM, one additional incision may be made to obtain 
muscle tissue. The total number of passes will be limited to 6. Biopsy specimens (5 to 40 mg) 


19 
 
will be frozen as 1 or 2 aliquots depending on tissue yield.  The aliquots will be frozen in liquid 
nitrogen within 2 minutes of removal and stored at -80°C until use. If additional tissue is 
obtained, it will be prepared as primary muscle cells and/or cell lines.  Patients will be evaluated 
on the last day of the study visit to ask about post-biopsy pain or complications.  To minimize 
variance resulting from time of day or muscle activity, the biopsy will be performed at a similar 
time of day and after a similar schedule of activities in all subjects at both study 
visits.  Participants will be provided with a “Needle Aspiration Muscle Biopsy Procedure 
Information Sheet” (Appendix I.) prior to their visit, which describes the pre-procure medication 
restrictions, procedure details, and post-procedure care instructions.  This muscle biopsy will not 
be performed in minors. 
 
 
Administration of study drug: Before participants return for their second visit, they (and/or their 
parent) will be provided with a “Medication/Placebo Information and Instructions Sheet” 
(Appendix J).  This sheet provides information about the drug, dosages, side effects, and contact 
information.  The study drug group will receive 15mg/kg /day (no more than 1800 mg/day) for 
the first week. After the first week, subjects who weigh fewer than 50kg will receive 
30mg/kg/day of NAC (no more than 2700mg/day) divided TID over a period of six months.  To 
increase compliance, as the tablets are formulated as 900mg each, subjects who weigh greater 
than 50kg will receive 2700mg/day. After the initial dose, participants will have two outpatient 
clinic visits with vital signs at the NIH. The first clinic visit will include 2 hours of observation 
after the first dose is administered.  The second clinic visit will be the following day and will 
also include vitals. 
 
Phone calls and emails: Participants will be called or emailed one month after their first visit as a 
reminder to complete the FACIT, MFI, NeuroQoL, and PROMIS scales.  Additional phone calls 
and/or emails will occur 1 week, 3 weeks and then monthly starting at 2 months after the placebo 
or drug is started to check for tolerability.  
 
 
Pill count:  Participants will be instructed to count their pills and give the number of unused pills 
to the licensed care provider, during their 3-month post intervention assessment.  This number 
will be compared to their expected number of remaining pills. Participants will also be instructed 
to save all leftover pills and bring them to their post-intervention (third) visit.  During that visit, 
the leftover pills will be counted and this number will be compared to the expected number of 
remaining pills.  All participants should have some remaining pills as all are given extra pills, as 
a precaution.  If it is discovered that a participant has missed >20% of their expected number of 
doses during their 3-month post intervention assessment, the participant will be informed that 
they need to increase their compliance, in order to remain in the study.  If it is discovered that a 
participant has missed >20% of their expected number of doses after returning for their third 
visit, the participant will be withdrawn from the study due to non-compliance. 
 
 
The overall study flow is depicted in the diagram below. 
 
 


20 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Healthy Volunteers: 
Phase 1 only 
 
Physical Examination: Height, weight, head circumference, BMI, and ulna length may be obtained 
by trained staff, including nurses, nurse practitioners, physicians, and physical therapists. Study 
physicians or nurse practitioners will perform a physical examination including the evaluation of 
cranial nerves, reflexes, sensation, strength, gait, movement, motor function and coordination. The 
physical examination will take approximately 30 minutes.  
 
Videotaping and/or Photography: Videotaping or photography may be performed during portions 
of the physical examination. When possible, the participant’s face and other identifying marks will 
not be included in the recording or photograph in order to protect confidentiality.  Consent will be 
obtained for videotaping and photography. 
 
Phlebotomy: Phlebotomy will be performed to obtain blood for liver function testing at baseline 
and glutathione levels. Blood and blood products may be analyzed or stored for creatine kinase 
(CK) level, lactate/pyruvate ratio, cell counts, blood chemistries. The target blood draw is 30ml. 
A local anesthetic cream such as Eutectic Mixture of Local Anesthetics (EMLA) may be used to 
minimize the pain for young children. Phlebotomy will take approximately 15 minutes.  Blood 
will not be drawn on participants if they are known to be anemic (Hgb < 8 gm/dl) per medical 
history, or if their medical condition causes an investigator to believe that blood drawing might be 
 
PHASE 1 
PHASE 2 
 
           
 
 
 
 
 
 
 
 
 
 
 
 
 
        Randomization 
 
 
 
 
 
 
        to study drug/placebo 
      
 
 
 
 
 
        2 day NIH observation 
Start of study 
 
 
 
 
 
 
 
 
 
         End of study 
 
OM-Outcome measures, MBX=Muscle biopsy on select adults 
*Participants will then be phoned or emailed monthly after the start of Phase 2 
1 Month 
Phone 
call/email to 
retest scales 
6 Months 
All OM 
(MBX) 
3 weeks out: 
Phone  
call/email to 
check 
tolerability*
0 Months 
All OM 
12 Months: 
All valid OM 
(MBX) 
1 week out: 
Phone 
call/email to 
check 
tolerability of 
NAC 
6 months 
post-
treatment: 
Phone call to 
assess 
symptoms 
and retest 
scales 


21 
 
detrimental to the participant. For pediatric participants, no more than 5 mL/kg will be drawn in a 
single day, and no more than 9.5 mL/kg will be drawn over any eight-week period. Timing of 
blood draw will be between 8:30-10:30am and will be taken prior to any food consumption. 
Participants will be notified the night before not to consume food prior to the blood draw the next 
day. 
 
Saliva collection: For the saliva collection, participants will be asked to chew on a cotton swab 
for about 5 minutes and then deposit the cotton swab in a specimen container.  Saliva will be 
measured for exploratory salivary biomarkers of fatigue such as fatigue biomarker index, 
melatonin, cortisol, and didehydroepiandrosterone (DHEA). Saliva will be collected the same 
day as the blood draw by a health care professional between 8:30 and 10:30 am prior to any food 
consumption. Participants will be notified the night before not to consume food prior to the 
saliva collection the next day. The saliva will be put on ice immediately and stored at -80.   
 
Urine collection: Participants will be asked to provide a clean catch urine sample of at least 10 
ml. Urine collection is required to detect levels of 15-isoprostane-F2, which allows for the 
indirect measurement of ROS.  
 
Arterial Occlusion Muscle Oxygenation Capacity Test:  A blood pressure cuff will be placed 
around the distal thigh of the dominant leg. A near infrared spectroscopy (NIRS) light source and 
receiving sensors will be placed on the leg, distal to the occlusion. Using a Hokanson® rapid 
inflation vascular testing system, the cuff will be rapidly inflated to a pressure that totally 
occludes blood flow into the gastrocnemius, typically between 60 and 80 mmHg above resting 
systolic pressure. The occlusion will be maintained for up to 10 minutes and tissue saturation 
will be measured over the time course. The tissue saturation index will be calculated as TOI= 
[O2-Hb] / Tot-Hb. The rate of decline and rise in TOI will be the main muscle tissue saturation 
measures. 
 
Muscle Ultrasound: Ultrasound will be used to evaluate skeletal muscles using standard medical 
soft tissue ultrasound. Muscle ultrasound will take approximately 30 minutes 
 
Magnetic resonance imaging (MRI): Only 2-8 people from each of the 4 age groups will be 
asked to undergo an MRI.  An MRI scan of the leg muscles may be performed in order to 
visualize structures using a 1.5 or 3 Tesla Magnet. T2 and T1 weighted imaging and MRS will 
be performed. High resolution conventional MRI will be performed with volumetric 
reconstruction of designated regions. Time in the MRI scanner will be limited to 1 hour, in order 
to allow the subject to rest and assure the best quality of the images.  We will allot additional 
time for any delays and to better ensure comfort.  Audiovisual distraction equipment such as 
music system with headphones will be available.  Children will be given extra time to get 
adjusted to the new environment in the imaging facility, as well as in the scan room to alleviate 
anxiety and nervousness.  Sedation will not be used for any procedure. 
 
 
e. End of participation 
Patients: 


22 
 
Participants will remain under the care of their local physicians and/or primary neurologist while 
under this study.  Participants will receive the results of any clinically relevant findings during 
the study. 
Healthy Volunteers: 
Healthy volunteers will have one visit only during Phase 1 and then their participation will be 
complete. 
 
5.  Storage of Data and Samples 
Patients: 
If permitted by the participants in the consent process, samples and data will be retained, 
including samples (blood, DNA, urine) and data (results of rehabilitation medicine 
Department testing, questionnaires, and qualitative interviews). This protocol is subject to 
the Genomic Data Sharing Policy. De-identified exome data will be submitted to a 
database (to be decided) through unrestricted access within 1 year of obtaining the data. 
Exome data will be obtained by sending de-identified DNA samples to a fee-for-service 
company to perform the exome sequencing. To begin, we will send up to 10 exomes for 
exome sequencing. The location of storage for the samples will be the laboratory of the 
National Institute of Nursing Research.  Data will be stored in the offices of Dr. 
Meilleur’s team on the NIH campus.. De-identified samples will be sent to Emory 
University for analysis only and will not be stored at Emory. MTAs have been obtained 
to transfer coded muscle tissue samples and coded genetic mutations to Dr. Andy Marks 
at Columbia University. Mutations identified by exome sequencing will be shared with 
Dr. Andy Marks (NR-MTA-16-002) by sending an excel file by SEFT to him and/or 
designated his lab members.  A Certificate of Confidentiality is not needed. 
 
Samples will be sent to Dr. Milne at Vanderbilt University and Dr. Tirouvanziam at 
Emory University for analysis on a fee-for-service basis. These entities may also assist in 
interpretation of de-identified data for their respective analyses. Samples and data will be 
shared with Dr. Michael at Hyperion (non-genomic data) and Dr. Andy Marks at 
Columbia University (including genomic data).   
 
Data and samples may also be shared with collaborating laboratories at NIH or outside of 
NIH and/or submitted to NIH-designated repositories and databases if consent for sharing 
was obtained. Repositories receiving data and/or samples from this protocol may be 
open-access or restricted access.  
 
Samples and data will be stripped of identifiers and may be coded (“de-identified”) or 
unlinked from an identifying code (“anonymized”). When coded data is shared, the key 
to the code will not be provided to collaborators, but will remain at NIH.  Data and 
samples may be shared with investigators and institutions with an FWA or operating 
under the Declaration of Helsinki (DoH) and reported at the time of continuing review.  
Analyses at Vanderbilt and Emory Universities will be conducted on a fee-for-service 
basis. Sharing with investigators without an FWA or not operating under the DoH will be 
submitted for prospective IRB approval.  Submissions to NIH-sponsored or supported 
databases and repositories will be reported at the time of Continuing Review.  


23 
 
Submission to non-NIH sponsored or supported databases and repositories will be 
submitted for prospective IRB approval. 
 
Required approvals from the collaborating institution will be obtained and materials will 
be shipped in accordance with NIH and federal regulations.    
 
Healthy Volunteers: 
If permitted by the participants in the consent process, samples and data will be retained, 
including samples (blood, urine, saliva). The location of storage for the samples will be 
the laboratory of the National Institute of Nursing Research.  Data will be stored in the 
office of Dr. Meilleur on the NIH campus. De-identified samples will be sent to 
Vanderbilt and Emory Universities for analysis and will not be stored at either site. 
 
6.  Additional Considerations 
 
a.  Research with investigational drugs or devices 
N-acetylcysteine (NAC) requires an IND, and the FDA approved IND number is: 125141.  The 
company providing NAC is BioAdvantex Pharma.  The study sponsor is Dr. Katherine Meilleur 
at NIH. 
 
7.  Risks and Discomforts 
 
Medical history: There may be some psychological distress associated with providing medical and 
family history. Frequent breaks or even cessation of procedure may be taken to reduce risk of 
distress. 
 
Physical examination: There is minimal medical risk and discomfort from the physical 
examination. This risk also applies to healthy volunteers. 
 
Cardiology evaluation: There are no known complications associated with a cardiology consult or 
the use of echocardiography. Patients may experience slight discomfort from laying still. There 
are no known risks for the EKG, however patients may experience discomfort with removal of the 
electrodes or a topical reaction to the adhesive on the electrodes. There are no known risks for 
Holter monitoring however prolonged application of the adhesive electrode patches may cause 
skin breakdown irritation at the application site. 
 
Pulmonary Function Tests:  There minimal risks. Participants may feel tired after the procedures.  
 
CPET: Decreased risk when screened and monitored with 12 lead EKG. 1/20,000 deaths in 
adults with cardiac disease (Paridon et al., 2006).  There is literature to suggest that patients with 
mutations in RYR1 are susceptible to exertional rhabdomyolysis or heatstroke with extreme 
exercise. We will not be having the patients do extreme exercise, but will take precautions to be 
ready to prevent this. See “Subject Monitoring” for further details.  
 
EIM: This procedure is non-invasive, but there may be slight discomfort (pressure) with the 
placement of the electrode on the skin.  


24 
 
 
MRI: Participants are at risk for injury from the MRI magnet if they have pacemakers or other 
implanted electrical devices, brain stimulators, some types of dental implants, aneurysm clips 
(metal clips on the wall of a large artery), metallic prostheses (including metal pins and rods, heart 
valves, and cochlear implants), permanent eyeliner, implanted delivery pump, or shrapnel 
fragments. Welders and metal workers are also at risk for injury because of possible small metal 
fragments in the eye of which they may be unaware. Individuals will be screened for these 
conditions before having any scan, and if they have any, they will not receive an MRI scan.  In 
addition, all magnetic objects (for example, watches, coins, jewelry, and credit cards) must be 
removed before entering the MRI scan room. This risk also applies to healthy volunteers. 
 
Participants with fear of confined spaces may become anxious during an MRI. Those with back 
problems or significant contractures may have pain or discomfort from lying in the scanner. The 
noise from the scanner is loud enough to damage hearing, especially in participants who already 
have hearing loss. Everyone having a MRI scan will be fitted with hearing protection. There are 
no known long-term risks of MRI scans. Participants may learn pregnancy status through 
required pregnancy testing. 
 
Physical therapy: 6 Minute Walk, Motor Function Measure32, timed tests, and Myotools tests 
are patient self-limiting tests with minimal risk and discomfort.  Participants may become 
fatigued.  
 
Biodex: Biodex may produce strain on the heart, muscles, and joints. The following 
contraindications will be ruled out prior to proceeding with Biodex: history of CAD and/or 
valvular disease, hypertension (>140/90), aneurysm, severe osteoporosis (BMD >-2.5 standard 
deviations and history of pathological fracture), acute sprain or strain, unstable joint/bone, knee 
pain, quadriceps tendonitis, moderate-severe joint effusion, extremely limited joint range of 
motion, severe pain, soft tissue healing constraints, unable to follow commands appropriately, 
unable to perform desired motion.  BP would be taken immediately prior to testing to confirm 
appropriate parameters. 
 
Arterial Occlusion Muscle Oxygenation Capacity Test:  The only known risk associated with 
arterial occlusion is discomfort. This procedure will not be performed on individuals who have 
disorders predisposing them to thrombosis, as a precaution. Arterial occlusion has been 
performed in patients with various myopathies, including Duchenne muscular dystrophy, and 
there were no reported adverse events (Sander et al., 2000). There are no published data to our 
knowledge regarding the incidence of a clot being formed or dislodged due to this procedure. 
However, due to the nature of the procedure, this is a theoretical risk.  Thus, we are excluding 
subjects with disorders predisposing them to thrombosis, as a precaution.  This risk also applies 
to healthy volunteers. 
 
Phlebotomy: Participants may have some discomfort and bruising at the site of needle entry. There 
is a very small risk of fainting. Infection in the area of the needle insertion is rare. This risk also 
applies to healthy volunteers. 
 
Genetic Testing: Risks of sharing genomic data may include the possibility of re-identification. 


25 
 
 
Saliva collection: There are minimal risks associated with saliva collection. This also applies to 
healthy volunteers. 
 
Urine collection: There are minimal known risks associated with urine collection.  This also applies 
to healthy volunteers. 
 
Urine pregnancy test: there is a risk that a pregnancy test will be positive and this will be 
upsetting to the participant or the parent of the participant, if a minor.  A clinician will be present 
the results to the participant and be available for any questions.  
 
Photographs/videos: Risks are minimal. The patient may be identified if a face is featured. When 
possible, the patient’s face and other identifying marks will not be included in the recording or 
photograph in order to protect confidentiality. This also applies to healthy volunteers. 
 
Qualitative interviews and/or questionnaires: The interviews and questionnaires are not harmful, 
but they may be frustrating or stressful. There may be some psychological distress associated 
with answering personal questions.  None of the questionnaires query thoughts of harm to self or 
others.  However, some of the questions ask the participants about depression, anxiety and anger 
and these responses may not be viewed by the investigators for several days. If upon review of 
the answers, the investigators feel there exists a possibility of a psychiatric diagnosis, the adult or 
parent, in the case of a minor, will be contacted in order to inform them of the possibility and 
they will be given a referral to a psychiatric professional in their area.   
 
Muscle ultrasound: Muscle ultrasound is not harmful. Participants may experience some slight 
discomfort from having to stay still during the procedure or may experience discomfort from the 
coldness of the gel.  This risk also applies to healthy volunteers. 
 
Muscle biopsy: The muscle punch-biopsy procedure has potential risks including infection, 
bleeding, pain, and post-procedure anesthesia or paresthesia if a cutaneous nerve is damaged. The 
risk of these events is minimized by having the procedure performed only by experienced 
neuromuscular licensed care providers under sterile conditions with adequate local anesthesia. 
 
Drug Administration:  Please see next section. 
 
Phone calls and email correspondence: Minimal risk. 
 
Pill count: Minimal risk.  
  
Provide the incidence/frequency of adverse effects, if known 
 
When administered as oral effervescent tablets, NAC has few severe side effects, providing a 
favorable risk benefit ratio to the proposed trial (Tirouvanziam et al, 2006; Hardan et al., 2012). 
Side effects of PharmaNAC effervescent tablets, which we plan to administer, include diarrhea, 
nausea, abdominal pain, vomiting, constipation, and flatulence, particularly when used 
chronically in high doses up to 6.9g/day.  The dosage (2700mg/day) for our study is within the 


26 
 
range of dosages previously used in clinical trials and is considered high. If the participants, at 
any point in the study, should need to take antibiotics, they will be informed to take their 
antibiotic medication (penicillins, tetracyclines, cephalosporins, and aminoglycosides) at least 
two hours prior to taking NAC. Other drugs that interact with NAC and GSH include, 
nitroglycerine, carbamazepine for NAC, and acetaminophen for GSH. Use of these medications 
is exclusionary, and participants cannot have taken them within seven days of the start of the 
study. Participants will not stop taking prescription medications for the purposes of this study 
unless they can be safely discontinued.  In this case, the discontinuation will be done under the 
care of the prescribing physician. It is important to note that high levels of NAC administration 
have not been tested in pregnant or lactating women, therefore, pregnancy is an exclusion 
criteria. As mentioned previously, if women become pregnant during this study, we will follow 
up with these women during their pregnancy and at birth.  
 Side effects reported when NAC is inhaled or injected include rhinorrhea, stomatitis, 
generalized urticarial/facial flushing, rash, pruritis, headache, drowsiness, clamminess, chest 
tightness, bronchoconstriction, and hypotension. Anaphylaxis is a rare but possible side effect in 
IV administered NAC in response to acetaminophen overdose. However, we will not be 
administering NAC in the inhaled or IV routes, and there have been no reports of anaphylaxis 
with oral administration. 
Reported side effects for FDA-approved prescription medications differ from those 
reported for over-the-counter oral supplements.  For example, the FDA-approved intravenous 
formulation of NAC has a side effect of prolonged bleeding (delayed blood clotting).  However, 
this side effect has not been reported for the dietary supplement of NAC.  Other side effects 
related to prescription formulations of NAC, especially those that are intravenous, inhaled or 
injected, may be found in the literature and on the internet, but may not be applicable to the 
dietary supplement utilized in this study.  Participants are informed that they may find 
information about side effects related to NAC, which may in fact only be related to other 
prescription formulations of the drug.  Participants are expected to inform study investigators 
about any concerns they have regarding side effects for NAC found in outside sources.  
In a pediatric study by Tirouvanziam et al. (2006), 18 patients took the approximate dose 
in our proposed study using the same administration we plan to use (PharmaNAC effervescent 
tablets). 7/18 of these patients had mild symptoms of heartburn (4 patients), bad taste (1 patient), 
flatulence (1 patient), and nausea (1 patient).  None of these symptoms required clinical 
monitoring. In another pediatric study by Hardan et al (2012) also using the PharmaNAC 
effervescent tablets, but through a higher dose range, thirty-three subjects (31 male subjects, 2 
female subjects; aged 3.2–10.7 years) were randomized to receive NAC or placebo for 12 
weeks. Subjects were initiated on NAC at 900 mg daily for 4 weeks, then 900 mg twice daily 
for 4 weeks and 900 mg three times daily for 4 weeks. This was a 12-week double blind, 
randomized, placebo-controlled trial in children with autistic disorder. Based on the result of the 
study, NAC resulted in significant improvements on an irritability subscale compared to placebo. 
The most common adverse effects observed during the study were gastrointestinal and included 
nausea/vomiting, constipation, increased/decreased appetite and diarrhea (Hardan et al., 2012). 
We expect the same side effects in our pediatric population given that NAC is a precursor to the 
endogenous antioxidant GSH. We do not suspect any specific side effects in myopathies given 
this mechanism of action. 
During the course of this study, one participant experienced rash and chest tightness with 
coughing, one participant experienced hives, one participant experienced tachycardia, two 


27 
 
participants experienced hypotension, and some participants experienced dehydration, dizziness, 
and/or headaches.  One participant had a benign ovarian cyst, which grew during the course of 
the study, including during the drug phase.  It is not known if these participants were on NAC or 
placebo.  
 
Steps taken to minimize risk 
 
In our proposed study, after participants receive drug or placebo effervescent tablets at 
the 6-month visit, they will be monitored at NIH for 2 consecutive days as an extra precaution to 
rule out rhinorrhea, stomatitis, urticarial/facial flushing, rash, pruritus, hives, coughing, 
headache, drowsiness, clamminess, chest tightness, bronchoconstriction, and hypotension, which 
are the most severe potential side effects of inhaled or injected NAC. This monitoring will 
include being observed in the outpatient clinic for 2 hours after their initial dose, including 
having vitals signs measured. The second day of observation at NIH will include an outpatient 
clinic visit with vital signs. If at any other time, the participant notices any symptoms, they will 
be directed to contact our onsite nurses. A Licensed Care Provider will then call them at one and 
three weeks to monitor adverse reactions. We will then monitor participants monthly (2, 3, 4, and 
5 months) by phone and/or email, primarily for gastrointestinal symptoms, which are the main 
symptoms of PharmaNAC effervescent tablets. If a patient reports any of the side effects that 
have already been reported (rash, chest tightness with coughing, hives, or hypotension), they will 
be advised to stop taking the study medication immediately. They will only restart the study 
medication after discussing their symptoms with the PI.  Patients are also asked, in the consent 
form, to limit their alcohol consumption to no more than 2 alcoholic drinks per day as excess 
alcholol consumption may interact with glutathione levels. Participants are informed to call study 
investigators immediately if they have any problems or side effects and are provided with contact 
information.  They are instructed to visit the emergency room if they have a possible allergic 
reaction or a serious or life-threatening emergency.  They are expected to tell the ER staff that 
they are in a research study and ask the emergency personnel call the research nurse.  
Participants are instructed to continue under the care of their local clinicians throughout 
participation.  Participants are provided with a “NAC Information Letter for Clinicians” 
(Appendix F) to share with their home clinicians.  This letter provides study drug information 
and information about possible side effects. 
If a patient reports Grade 3 side effects for diarrhea, nausea, vomiting, constipation, or 
abdominal pain, or any other Grade 3 side effect, according to the NCI Common Terminology 
Criteria for Adverse Events v.3.0, we will discontinue their participation. If Grade 1 side effects 
are reported, we will monitor them by phone and/or email and keep them on the drug.  If Grade 2 
side effects are reported, we will discontinue drug until the side effect resolves and then resume 
administration with careful observation for reoccurrence. Monitoring for grades 1 and 2 side 
effects will include weekly phone calls and/or emails until symptoms subside. If symptoms 
worsen subjects will visit their primary care physician. Subjects who experience grade 3 
symptoms will receive an assessment by our healthcare provider and will be advised to schedule 
a prompt appointment with their local healthcare provider. We will also encourage them to 
contact us at any time with questions or concerns. 
We will offer an evaluation at NIH for any participant who would like to return for an 
evaluation of side effects, such as, but not limited to, local participants.  This evaluation will 
include a history and physical examination, discussion with the medical advisory investigator, 


28 
 
and appropriate consults, for example a GI consult. 
An informational letter (Appendix F) to the participant’s physician will be given to each 
subject to give to their primary care provider. 
 
8.  Subject Safety Monitoring 
For CPET, there is literature to suggest that participants with mutations in RYR1 are susceptible 
to exertional rhabdomyolysis or heatstroke with extreme exercise. We will not be having the 
patients do extreme exercise, but will take precautions to be ready to prevent these 
susceptibilities just in case.  The precautions include having the CPET performed in a cool room 
with a fan blowing on the patient, while monitoring the patient’s temperature.  We will ask the 
patient if they are experiencing cramping, stiffness, or pain throughout the procedure.  If the 
answer is yes, or if their temperature exceeds 101 degrees Fahrenheit, we will stop the exercise 
immediately. A STAT blood draw will be done to check serum creatine kinase and potassium 
levels and urine will be collected for myoglobin. If the patient is medically unwell, the patient 
will be admitted to the day hospital and an IV will be started immediately for hydration. If the 
CK, potassium and urine myoglobin levels are elevated suggestive of rhabdomyolysis, they will 
be treated with sodium bicarbonate and monitored until the rhabdomyolysis is safely resolved.  
 
Investigators will evaluate participants for any foreseeable safety concerns before study 
procedures are performed.  Investigators will allow participants to miss individual procedures 
(other than the two primary outcome measures— urine collection for 15 F2t-isoprostane levels 
and 6MWT), at one or all study visits, if safety concerns arise. Examples include: If a participant 
has a recent history of knee dislocation or knee injury, that participant will be excluded from the 
Biodex procedure.  If a participant has an increased risk of blood clotting, that participant will 
not perform the arterial occlusion test.  If a participant is at NIH and recently recovered from a 
rhabdomyolysis crisis, that participant will not perform CPET at that visit, but may perform 
CPET at following visits, if deemed safe at that time.  If a participant has certain types of metal 
in their body, that participant will not perform the MRI procedure at any visit. Investigators will 
ask participants about potential safety concerns for each procedure and participants are also 
asked to inform study investigators about potential risk factors in the consent.  Investigators will 
have a participant miss certain non-primary outcome measure procedures, due to safety concerns, 
at their discretion.  Participants will remain enrolled in the study if they are able to safely 
perform the two primary outcome measures and are also able to safely consume the study 
medication/placebo, given the expected side effects.  Dr. Meilleur can withdraw a participant, at 
any time, if she believes continuation is not in their best medical interest, or if the participant is 
unable to comply with the study. 
 
For monitoring for NAC administration, please see previous section under “Steps Taken to 
Minimize Risk”. 
 
For other individual monitoring plans, please see previous section under “Steps Taken to 
Minimize Risk.” 
 
Criteria for stopping procedures in an individual include: 
If a patient reports Grade 3 side effects for diarrhea, nausea, vomiting, constipation, or 
abdominal pain, or any other Grade 3 side effect, according to the NCI Common Terminology 


29 
 
Criteria for Adverse Events v.3.0, we will discontinue their participation.  
 
Criteria for individual subject withdrawal from the study 
If Grade 1 side effects are reported, we will monitor them by phone and/or email and 
keep them on the drug.  If Grade 2 side effects are reported, we will discontinue drug until the 
side effect resolves and then resume administration with careful observation for reoccurrence.  
Please see Section 7 under “Steps Taken to Minimize Risks” for further details. 
 
9.  Outcome Measures 
 
Outcome Measures 
Aim 1=Oxidative Stress 
Aim 2=Fatigability, Fatigue 
Primary 
urine 15-F2t-isoprostane 
6MWT 
Secondary 
DCFH assay in muscle 
biopsy (6 and 12 months 
only), blood GSH 
Timed function tests; Biodex; 
PROMIS, FACIT, MFI, MRI, 
MFM, NeuroQoL scales, 
Myotools 
Exploratory 
Anaerobic threshold, NIRS; 
secreted ER calcium 
monitoring proteins 
(SERcaMPs) assay in blood 
and muscle 
CPET, EIM, salivary FBI, 
melatonin, cortisol and 
DHEA; QoL qualitative 
interview 
 
 
Aim 1 
Blood and urine samples will be collected at each visit. Samples will be analyzed at NIH or 
shipped to Dr. Milne for urine isoprostane analysis at the Vanderbilt University Ecosinoid Core 
Facility. De-identified blood samples will also be shipped to Emory University to assay 
glutathione via mass spectrometry.  Needle muscle biopsy will be performed at six and twelve 
months on up to 20 willing adult participants only.  
 
GSH: Mass spectrometry, fluorescent, and colorimetric assays will be used to measure levels of 
reduced (GSH) and oxidized (GSSG) glutathione in plasma and whole blood to observe changes 
in oxidative stress.  These assays include deproteinization, derivatization, and/or catalytic 
reactions.  
 
DCFH: As in the preliminary studies above, DCFH will be used on participant muscle biopsies 
to analyze intracellular oxidant activity [H2DCFDA (H2-DCF, DCF)].  
 
15-isoprostane-F2: Urine will be assayed for 15-isoprostane-F2, which is formed when 
arachidonic acid reacts with ROS.  A validated GC-MS/MS method will be used to quantify 14-
isoprostane-F2. 
 
Anaerobic threshold: Anaerobic threshold and time to anaerobic threshold will be extrapolated 
from data obtained during CPET.  
 


30 
 
NIRS: Percentage of local muscle oxygen and oxygen consumption as determined by tissue 
oxygen index (TOI) and hemoglobin. 
 
SERcaMPs: Measurement of protein level of Gaussia luciferase-based secreted ER calcium-
monitoring protein (SERCaMP) in blood, primary muscle cells and/or cell lines. (Hendersen et 
al. 2015) 
 
Aim 2 
All procedures for Aim 2 will be performed at the NIH Clinical Center at each visit.   
 
CPET: VO2 peak, peak power, test duration of entire test, time to AT, work efficiency (VO2 
work rate slope), and ventilator equivalent (VE/VO2) will be obtained. 
 
EIM: An EIM will be used to measure skeletal muscle health. Resistance, reactance and phase of 
multiple muscles will be measured. 
 
6MWT: Meters walked in 6 minutes will be recorded (to the nearest meter).  Distance in meters 
will be recorded at each minute interval. Speed will be calculated. 
 
Timed tests: Participants will be timed in the performance of the following: 10 meter walk test, 
supine to stand, ascend 4 stairs, descend 4 stairs. They will be graded on an ordinal scale. 
 
Myotools: Force required for participants to squeeze the grip dynamometer and pinch the pinch 
gauge. The best effort of 3 trials will be recorded for grip and pinch strength. 
 
Biodex:  Biodex will be used to test muscle groups using a dynamic sub-max fatigue test with a 
repetitive motion.  Blood pressure will be taken prior to testing to confirm the patient is not 
hypertensive (>140/90).  Outcome measures will include peak torque measured in newton meters 
and number of repetitions to 50% decline in peak torque. 
 
Arterial Occlusion Muscle Oxygenation Capacity Test:  The tissue saturation index will be 
calculated as TOI= [O2-Hb] / Tot-Hb. The rate of decline and rise in TOI will be the main 
muscle tissue saturation measures. 
 
MFM: The final outcome is a percentage based score of each domain and the total score. 
 
PROMIS, FACIT, MFI, NeuroQoL:  Questionnaires will be scored based on each scale’s 
requirements. 
 
MRI:  The outcomes will be muscle mass via T1, fat composition via T1 to T2 comparison, 
water composition via T1 to T2 comparison and chemical composition (lipids) via MRS. 
 
Saliva collection: FBI, melatonin, cortisol and DHEA levels will be obtained.   
 
Qualitative interview: Responses to the interview will be obtained and major themes will be 
identified. 


31 
 
 
10.  Statistical Analysis 
 
a.  Analysis of data/ study outcomes 
 
Natural history study (Phase 1 of protocol): 
 
During the natural history phase of the study, we will take one look at the data prior to the final 
analysis with the following two approaches:  
1) After 30 participants complete their baseline visit, we will compare their baseline 
outcome measure values against healthy controls. For the primary outcomes, 15-F2t-
isoprostane and 6MWT distance will be compared against healthy  individuals. This 
analysis will be repeated when all baseline visits are completed. 
 
 
Hypothesis: 15-F2t-isoprostane will be significantly increased in RYR1-RM myopathy 
patients compared to healthy individuals. 
 
Hypothesis: 6MWT distance will be significantly decreased in RYR1-RM patients 
 
compared to healthy individuals.  
2) After 30 participants complete their 2nd visit at 6 months (prior to starting study 
drug/placebo), disease progression will be assessed using paired t-tests to determine 
changes between 0 and 6 month visits for each outcome measure. For the primary 
outcome measures, 15-F2t-isoprostane and 6MWT will be assessed for change between 0 
and 6 months. This analysis will be repeated when all 6 month visits are completed. 
 
 
Hypothesis: 15-F2t-isoprostane concentration will not change significantly between 
 
0 and 6 months in RYR1-RM myopathy patients. 
 
Hypothesis: 6MWT distance will not change significantly between 0 and 6 months in 
 
RYR1-RM patients.  
 
The purpose of the above interim look analyses (under #1) is to confirm that the affected subjects 
are different from healthy individuals. Because of the rolling nature of the study and the length 
of time involved to complete the first phase of the study, this allows evaluate whether these 
outcomes are different from healthy individuals, as expected, in order to confidently proceed 
with the study. The purpose of the above interim look analysis (under #2) is to characterize the 
natural history and variability of the RYR1-RM patients for these specific outcome measures and 
to contribute novel data to the field of RYR1 as soon as possible for future trial design, given 
that an N of 30 is a large number in rare disease. 
Optimal measures for fatigability in RYR1-RM will be determined using paired t-tests for 
6MWT distance and speed, time for graded functional tests, and fatigue index for biodex fatigue 
test.  Correlative analysis (Pearson correlation and/or Spearman rho) will be used to examine the 
linear relationship between subjective fatigue measures (FACIT and MFI) and the salivary 
fatigue biomarker to the objective fatigability measures mentioned above as well as to compare 
forced vital capacity to slow vital capacity. Spearman rho correlation, Pearson correlation and or 
Bland Altman analysis will also be used to examine the relationship between slow vital capacity 
and forced vital capacity. 
Reliability will be assessed for PROMIS, FACIT, MFI, MFM, and NeuroQoL by Cronbach’s 
alpha for internal consistency and by Intraclass correlation coefficient for test-retest. If the test-


32 
 
retest analysis yields a poor correlation (ICC<0.40), then the scales will not be used in the study 
since they are not primary outcome measures. However, if they yield moderate to high 
correlation, we will use the scales throughout the entire study. 
 
Randomized, double-blind, placebo controlled, drug trial (Phase 2 of protocol): 
Student’s t test will be used to compare treatment to control groups. As mentioned in the 
Introduction, under “Summary”, success of the trial will be defined as finding a significant 
difference in the primary oxidative stress measure of of urine 15-F2t-isoprostane  between the 
treatment and placebo groups at the termination of the study (significantly decreased urine 15-
F2t-isoprostane concentration in treatment group compared to placebo group).  A sub group 
analysis will be performed on adults versus children given the difference in dosing. A paired t 
test will be used to compare subjects to their baseline values. A student’s t test will be used to 
compare the difference in glutathione between 6 months and 12 months, between the two groups. 
General linear modeling will be used to control for possible confounders such as gender, age, 
and baseline values. Log transformed values may be used if there is no bell curve. We will use an 
alpha of 0.05 to determine significance.  
 
We will use the Grounded Theory Method (GTM) of data analysis to analyze the semi- 
structured interview data. GTM methodically organizes unique individual experiences collected 
during interviews into comparable categories so that it becomes possible to find commonalities 
as well as variations in personal experiences. We use GTM because it is particularly effective for 
analyzing qualitative interview data and because it is rigorous. A GTM approach is appropriate 
when there is limited knowledge about a substantive area, such as the fatigue and quality of life 
of individuals with RYR1-related myopathy. 
 
Select patients will undergo exome sequencing as, occasionally, patients with RYR1-related 
myopathies with identical RYR1 mutations have different phenotypes. Exome sequencing may 
help identify the cause of this difference. Because the RyR1 protein interacts with numerous 
other proteins, the genes encoding these other proteins may be modifiers of severity.  Patients 
will be selected for exome analysis if they have the same mutations but have a difference of 
greater than 10% on the MFM32 score. A candidate gene approach will be taken to sequence the 
genes encoding these other proteins, to evaluate whether they have SNPs or mutations that affect 
severity. Using the exome, additional mutations in RYR1 as causative for the phenotypic 
difference will be ruled out prior to looking at other candidate genes. Genes that encode proteins 
without a known function in relation the RyR1 protein will not be interrogated so as to avoid 
unintentional incidental findings. 
 
Healthy volunteer data: 
In the first phase of the study, results will be obtained from healthy controls for the 
biomarkers, NIRS, muscle ultrasound, and MRI.  An age-matched analysis will be performed to 
compare healthy volunteer data to patient data for these outcome measures. We will use an alpha 
of 0.05 to determine significance.  For the comparison to healthy volunteers, age-matched 
participants will be used.  Please see inclusion criteria under healthy volunteers for age grouping. 
 
b.  Power analysis  
Sample size: 


33 
 
This is a randomized controlled trial with two groups: NAC and Control. For sample size 
planning purposes, we have used glutathione data, pre and post NAC treatment, from an HIV 
population (Muller et al. 2000). Assuming the NAC treatment group will have a delta (post-
treatment glutathione – pre-treatment glutathione) of 1.7 and delta standard deviation of 2.34, 
and the control group will have a delta of 0 and delta standard deviation of 2.80, for a 2-sided 
alpha of 0.05 and 80% power we will need 38 patients per group (2-group Satterthwaite t-test of 
equal means and unequal variances).  
We need 76 participants, per group, based on the current sample size. We expect 24 drop outs, 
per group.  Therefore, our accrual ceiling for all participants is 200.  
 
The primary analysis will be an intent-to-treat (ITT) analysis, i.e. all randomized participants will 
be included in the analysis. The mechanism for missing data will be evaluated. If missing data 
are unrelated to the intervention or primary outcome (i.e. missing completely at random or 
missing at random), the analysis will be based on multiple imputations (20-40 imputed datasets, 
depending on the extent of the missing data) with a pooled average of the 20-40 imputations 
included in the final dataset and used in hypothesis-testing analyses. Minimum and maximum 
value constraints for imputed data will be determined using per protocol averages.  
 
Because of uncertainties regarding the true effect size of NAC treatment in our patient 
population, and the rare nature of this disease possibly limiting accrual, we plan to perform a 
sample size reevaluation and interim analysis once 30 patients have completed the trial, i.e., 
when 40% of our potentially achievable accrual reach Month 12. 
 
For the sample size reevaluation, and based on the observed data so far, we will calculate the 
following: the control group delta and its standard deviation and the standard deviation of the 
delta for the active arm. Based on these observed parameter values for urine 15-F2t-isoprostane, 
we will re-calculate the sample size and compare it to the current N=76. 
 
Based on O’Brien-Fleming boundary calculations (O'Brien and Fleming, 1979), the interim 
analysis will result in one of 3 actions:  
1. If there is strong indication of no benefit of NAC in decreasing 15 F2t-isoprostane 
levels, i.e. if the observed standardized treatment effect is less than -3.36, we will 
consider stopping the study early for futility. 
2. If there is no clear indication of benefit of NAC decreasing 15 F2t-isoprostane levels, 
i.e. if the observed standardized effect size is between -3.36 and 3.36, we will 
continue the study and analyze the data at the completion of the study. 
3. If there is strong indication that the NAC treatment decreases 15 F2t-isoprostane 
levels, i.e. if the observed standardized treatment effect is greater than 3.36, we will 
consider stopping the study early for efficacy.  
Stopping the trial early when there is strong evidence for futility or efficacy will reduce patient 
burden, decrease excess costs, and provide a potential treatment sooner for this rare disease, 
which currently has no treatment. 
 
The primary outcome measure is being changed, initially due to pipette calibration error in the 
lab at NIH. Additional review of our data at Emory after sending a second batch of samples and 


34 
 
further literature review identified further justification for changing the primary oxidative stress 
biomarker from glutathione (GSH) to 15-Ft2-isoprostane: 
 
--The processing of GSH and its oxidized form GSSG often results in methodological 
artifacts. For instance, sample acidification for protein precipitation leads to an increase 
in GSSG levels thus decreasing GSH:GSSG ratio (Marrocco et al. 2017). The assay is 
sensitive to exposure to air/time and sample volume and is therefore prone to error. The 
aforementioned methodological artifacts are not unique to this protocol and have been 
described previously (McGill et al. 2015). 
 
--During the current protocol, such methodological issues arose at Emory in addition to 
the pipette calibration issue at NIH. For example, when the second sample for each 
patient was rerun at Emory, including samples unaffected by the pipette error, the 
results between the first patient and second patient sample were unreliable. These 
methodological errors in GSH as a biomarker and the recurrent, significant time delays 
for processing by Emory, in addition to the pipette error, prompted a change in primary 
outcome measure from GSH to 15-Ft2-isoprostane. 
 
--15-Ft2-isoprostane was collected on each individual at each visit as a secondary 
outcome measure for oxidative stress throughout the life of the protocol. The change 
from GSH to 15-Ft2-isoprostane is being made prior to treatment allocation un-blinding 
and prior to statistical analyses, which is the optimal time to change a primary endpoint 
when such a change is required (Evans, 2007). Changes in primary endpoints are 
reported in approximately 30% of clinical trials (Ramagopalan, 2014). 
 
--Elevated oxidative stress is a hallmark pathological feature in RYR1-RM and has been 
identified in several pre-clinical models of the disease (Durham et al. 2008, Dowling et 
al. 2012, Lee et al. 2017, Michelucci et al., 2017). In particular, lipid peroxidation of 
mitochondrial membranes has been implicated as a central pathomechanism that leads 
to severe mitochondrial dysfunction and/or death in the RYR1Y522S/W mouse model 
(Durham et al. 2008). Notably, treatment of the aforementioned mice with N-
acetylcysteine (NAC), (ad lib via drinking water for 8 weeks) rescued lipid 
peroxidation and concomitantly prevented previously observed decrements in force 
generation (Durham et al., 2008). These findings have recently been supported by a 
longer-term study of NAC treatment in the same mouse model in which NAC treatment 
led to a decreased number of structural cores and improved skeletal muscle function 
(Michelucci et al., 2017).  
 
--15-F2t-isoprostane has been reported as the most reliable in vivo biomarker of oxidative 
stress and is a measure of lipid peroxidation (Milne et al. 2007, van 't Erve et al., 2017). 
Moreover, a typical 15-F2t-isoprostane concentration has been established through 
substantial data collection from otherwise healthy volunteers (n= 1881) allowing 
comparisons to be made to disease populations (van 't Erve et al., 2017). Finally, when 
our participants’ baseline and 6 month (untreated) isoprostane levels were compared, 
the findings were not significantly different, suggesting reliability of this biomarker in 
our population, in contrast to our GSH data analysis of the same time points. 
 


35 
 
--In a recent crossover trial, conducted in 36 individuals with low resting GSH 
concentrations, NAC supplementation (2 × 600 mg, twice daily, for 30 days) led to a 
22% reduction in 15-F2t-isoprostane concentration and improved exercise performance 
(Paschalis et al. 2017). These findings are pertinent for this protocol as it has been 
demonstrated pre-clinically that RYR1-RM models have low resting GSH 
concentrations, and both 15-F2t-isoprostane concentration and improved exercise 
performance are being tested in our trial. Additional antioxidant clinical trials have also 
shown a beneficial effect of treatment on 15-F2t-isoprostane concentration. In 
particular, an α-tocopherol (vitamin E) antioxidant trial in smokers (n= 81 placebo; n= 
82 vitamin E) demonstrated that consuming 400 IU vitamin E daily resulted in a 
statistically significant (21%) difference in corrected urinary F2-isoprostane 
concentration compared to placebo following 32-months treatment (Guertin et al. 
2016). 
 
           In combination, these findings strengthen the rationale for using 15-F2t-isoprostane 
concentration as the primary outcome measure for this protocol in place of GSH. The sample 
size recalculation for 15-NR-0072 was therefore performed using 15-F2t-isoprostane data from 
our study. Dr. Paul Wakim, Clinical Center Statistician, was unblinded for the sample size 
recalculation. All investigators remain blinded until the study is complete.  Dr. Wakim used the 
current study data on isoprostane levels from participants in the treatment vs. placebo groups to 
calculate the N required to complete the study.  The N achieved was 182, which is not feasible in 
this rare disease population and the study is being closed to data analysis.   
 
 
11.  Human Subjects Protection 
 
a.  Subject selection 
Subject selection will be equitable as much as possible due to the rarity of RYR1-related 
myopathy. All ages within the specified range, races, and both genders will be accepted. 
Individuals with a genetic mutation in RYR1 will be prioritized to decrease the rate of screening 
failures.  Individuals with a muscle biopsy showing central cores will be prioritized to decrease 
the variability in the sample. 
Subject selection for healthy volunteers will be equitable. 
 
b.  Justification for inclusion of children  
Children age 7 years old and older are affected with this disease and are affected with fatigue. 
This drug in this route of administration has a good safety profile in children at this dose for 
other indications. Children <7 years of age will be excluded as they will not be able to follow the 
commands in order to perform all of the outcome measures. 
Healthy volunteer children are needed to provide age-matched groups as controls for biomarkers, 
NIRS, muscle ultrasound, and MRI. 
 
c.  Justification for exclusion of other vulnerable subjects  
Those unable to provide consent due to cognitive issues will be excluded, due to the requirement 
to be able to respond to questionnaires such as PROMIS and FACIT. 
 


36 
 
d.  Justification of sensitive procedures (use of placebo) 
The use of a placebo is necessary to determine the effects of NAC on this population.  Without 
the placebo, we would not be able to determine if NAC has a significant effect on participants 
with RYR1-RM.  Use of a placebo will allow us to draw stronger conclusions about the effects 
of NAC than if we conducted the study without a placebo. 
 
e.  Safeguards for vulnerable populations (e.g. DPA, pregnancy testing, contraception use, 
ethics consult, HSPU involvement) 
Safeguards:  Protections for employees and staff participating in this study include 1) assuring 
that the participation or refusal to participate will have no effect, either beneficial or adverse, on 
the subject’s employment or position at the NIH, 2) giving employees and staff who are 
interested in participating the “NIH Information Sheet on Employee Research Participation 
(Appendix L)” prior to obtaining consent, 3) assuring that there will be no direct solicitation of 
employees or staff, and, 4) Independent consent monitoring will be provided by the NIH HSPU.  
 
This study collects sensitive information on pregnancy status, which will be in the participant’s 
NIH medical record. The PI will train study staff regarding obtaining and handling potentially 
sensitive and private information about co-workers through staff discussions and written 
branch/section procedures.  All research activities will be conducted in as private a setting as 
possible, limiting personnel present.  
 
For pediatric patients, we will have parental consent and minor assent. Pediatric staff and 
pediatric equipment will be used. During blood draws, a local anesthetic cream such as Eutectic 
Mixture of Local Anesthetics (EMLA) may be used to minimize the pain for young children. For 
pediatric participants, no more than 5 mL/kg will be drawn in a single day, and no more than 9.5 
mL/kg will be drawn over any eight-week period.  
 
Blood will not be drawn on participants if they are known to be anemic (Hgb < 8 gm/dl) per 
medical history, or if their medical condition causes an investigator to believe that blood drawing 
might be detrimental to the participant.  
 
Parents can accompany their child to the MRI suite and watch their child through a window 
during the procedure. During the procedures, there will be music and/or video to distract the 
child.  All participants will be able to communicate with the MRI staff at all times. 
 
For women of childbearing age, we will do pregnancy testing to exclude women who are 
pregnant at the beginning of the study. We will also exclude women who are breastfeeding. 
Women who are sexually active will require contraception. Acceptable contraception includes 
barrier methods (contraceptive sponge, diaphragm, female and male condoms), hormonal 
methods (oral contraceptives, the patch, shot/injection, and vaginal ring), implantable devices 
(implantable rods and intrauterine devices), and permanent birth control methods (sterilization 
implant and surgical sterilization).  
 
f.  Qualifications of investigators 


37 
 
The Principal Investigator has verified that all individuals working on this protocol required to 
take HRPP training under OHSRP SOP 25 (Training requirements for the NIH Human Research 
Protections Program) have completed all required training. 
 
Suzanne Wingate, PhD, ANP-BC is a board certified adult nurse practitioner and the Clinical 
Director of NINR. When the study was changed to Data Analysis only, she began to provide 
oversight for the study, but there is no clinical or medical management required for subjects in 
this phase. 
 
Katherine Meilleur, Ph.D., PNP-BC is an assistant clinical investigator in the Tissue Injury 
Branch/NINR. She is a board certified pediatric nurse practitioner involved in study design, data 
analysis, interpretation of results and all procedures.  She is an investigator authorized to obtain 
informed consent from participants. 
 
James Dowling, MD, PhD is an assistant professor at the University of Toronto and is a Medical 
Doctor, licensed and board certified pediatric neurologist. He is involved in study design, data 
analysis, interpretation of data, and is an investigator authorized to obtain informed consent. 
 
Carsten G. Bonnemann, M.D. is the Chief of the Neuromuscular and Neurogenetic Disorders of 
Childhood Section of the NGB/NINDS. He is a Medical Doctor and a licensed and board 
certified pediatric neurologist. He is involved in study design, data analysis, interpretation of 
data, and is an investigator authorized to obtain informed consent from participants. 
 
Irene Chrismer, BSN, RN has been a research nurse in the NINR for 1 year. Prior to that she 
worked at the Children’s National Medical Center cardiac unit as a staff nurse. She is a board 
certified nurse and will be involved in eligibility screening and patient care.  Due to her 
experience over that past year and dedicated involvement on this protocol, she is an investigator 
authorized to obtain informed consent from participants. 
 
Diana Bharucha, MD, is a pediatric neurologist and clinical research fellow for NGB/NINDS.  
She has several years of experience with RYR1 patients and will be involved with data 
collection.  She is authorized to obtain informed consent from participants.  
 
Reghan Foley, MD, is a clinical Research Fellow for NGB/NINDS.  She has previous experience 
at the Dubowitz Neuromuscular Centre, Institute of Child Health at the University College of 
London.  She will be involved in patient referral, study design, data analysis, and interpretation 
of results.  She is an investigator authorized to obtain consent from participants. 
 
Monal Punjabi is a PharmD student, Class 2015, at Rosalind Franklin University of Medicine 
and Science. She is a summer student and special volunteer.  She will assist with the IND 
application and assist as need.  She is not authorized to obtain informed consent. 
 
Minal Jain, PT, DSc, PCS, Melissa Waite, PT, and Bart Drinkard, PT are licensed physical 
therapists in the Rehabilitation Medicine Department of the Clinical Center. They will administer 
outcome measures (fatigue and fatigability).  They are not authorized to obtain informed consent. 
 


38 
 
Tanya Lehky, MD, is a physician at the Clinical Center.  She will perform the EIM procedure.  
She is not authorized to obtain informed consent. 
 
Kimberly Amburgey, MS, CGC, is a board certified genetic counselor at the University of 
Toronto.  She is involved in the recruitment of patients and study design. She is an investigator 
authorized to obtain informed consent. 
 
Ami Mankodi, MD, and Christopher Grunseich, MD, are licensed and board certified 
neurologists.  They will be involved in patient evaluation and muscle biopsy procedures. They 
are investigators authorized to obtain informed consent from participants. 
 
Sonia Razaqyar, BA, is a postbac at the NINR.  She earned a BA with honors in biochemistry.  
She will be involved with data entry, data analysis, and patient coordination.  She is an 
investigator not authorized to obtain informed consent from participants. 
 
Monique Shelton, BA, is a postbac at the NINR.  She earned a BA in biology and Spanish.  She 
will be involved with data entry, data analysis, and patient coordination.  She is an investigator 
not authorized to obtain informed consent from participants. 
 
Anna Kuo, BA, is a postbac at NINR.  She earned a BA in neuroscience and behavior.  She will 
be involved with data entry, data analysis, and patient coordination.  She is an investigator not 
authorized to obtain informed consent from participants. 
 
Joshua Todd, PhD, is a postdoc at NINR.  He earned a PhD in biomedical sciences and a BS in 
sports science.  He will be involved in data collection, data entry, data analysis, and 
dissemination of results.  He is an investigator authorized to obtain informed consent. 
 
Allen, Carolyn, DNP, MS, CRNP-F, is a Family Nurse Practitioner at NINR. She will be 
involved in data collection, data entry, data analysis, interpretation of results and procedures, and 
dissemination of results.  She is an investigator authorized to obtain informed consent.  
 
Jessica Witherspoon, PhD, DPT, is a postdoc at the NINR.  She will be involved in data 
collection, data entry, data analysis, and dissemination of results.  She is an investigator 
authorized to obtain informed consent. 
 
Etsuko Tsuchiya, BS, is a research coordinator at U. Toronto and will coordinate samples and 
data between the 2 sites. She is an investigator not authorized to obtain informed consent. 
 
Darren Michael, PhD, is a scientist who has developed a fatigue biomarker in saliva and works at 
Hyperion Biotechnology Inc. He will oversee and participate in the processing and analysis of 
samples, as well as the interpretation and publication of the results. He is an investigator not 
authorized to obtain informed consent. 
 
12.  Anticipated Benefit 
This study may offer a temporary direct benefit to adult and pediatric participants in the 
study drug group, such as reducing oxidative stress, fatigue, and fatigability. Reduced 


39 
 
oxidative stress decreases cellular necrosis and improves energy efficiency. In turn, this 
would allow the participants to improve their energy level. (Hybertson et al., 2011) 
Additionally, improved energy efficiency translates to reducing fatigability, thereby 
improving endurance, which was noted by Dowling et al., in the RYR1 zebrafish model 
with NAC administration.  
There will be no benefit for healthy volunteers. 
 
13.  Classification of Risk (for the study as a whole) 
 
a.  For adults: More than minimal risk 
 
b.  For Adults without consent capacity (not applicable)  
 
c.  For Children (Patients): 45CFR46.405 Research involving greater than minimal risk but 
presenting the prospect of direct benefit to the individual child 
For Children (Healthy Volunteers): 45CFR46.406 Research involving no greater than a minor 
increment over minimal risk and no prospect of direct benefit but likely to yield generalizable 
knowledge  
 
d.  Overall risk and benefit consideration 
The risks are reasonable in relation to anticipated direct benefit. 
 
14.  Consent Documents and Process 
 
a.  Designation of those obtaining consent 
Study investigators, designated as able to obtain consent in section 11f above, will obtain 
informed consent.  All NIH study investigators obtaining informed consent have completed the 
NIMH HSPU “Elements of Successful Informed Consent” training.  Assent will be obtained 
from minor subjects. 
 
b.  Consent procedures  
All participants will receive a verbal explanation in terms suited to their comprehension of the 
purposes, procedures, and potential risks of the study and of their rights as research participants. 
Participants will have the opportunity to carefully review the written consent form and ask 
questions regarding this study prior to signing.  Written assent will be obtained, when possible, 
from minors age 7 and older.  Minors who reach age 18 while enrolled will be consented as 
adults for continued participation. 
 
If eligible, the subjects will undergo an in-person written consent at NIH. During the consent 
process, the child and custodial parent will sign the consent form.  The original consent form will 
be filed in the participant’s NIH chart. A copy of the completed signed consent will be returned 
to the participant. The consenting investigator will document the consent procedure in the NIH 
medical record.  
 
For pediatric patients, if the parents are married, written consent may be obtained from one 
parent only.  For pediatric healthy volunteers, consent from both parents is required, even if 
parents are married. If the parents are not married, written consent will be obtained from: 1) the 


40 
 
custodial parent if only one parent has legal custody, or 2) from both parents if they share legal 
custody for medical decision-making. For unmarried parents, signature of one parent will suffice 
if the other parent is deceased, unknown, incompetent, or not reasonably available. When 
signature of both parents is required, written consent will be obtained in-person from at least one 
parent. When the second parent is unable to attend the consent process conference in person, the 
following telephone process to obtain written consent will be used.   
 
Telephone Consent Procedures: The unavailable parent will be provided with a copy of the 
consent form, usually by fax, email, or hard copy mail.  Once the consent form is received, an 
Investigator authorized to obtain consent will arrange for a telephone call with the parent in the 
presence of a witness to review study and the consent form and to answer any questions. If the 
parent cannot arrange for a witness in his/her location, the consenting investigator will have a 
witness present for the teleconference at NIH.  Once the parent agrees to his/her child’s 
participation, the parent and witness (if present with the parent) will sign and date their copy of 
the consent form. The Investigator will enter a note documenting the consent process in the 
Medical Record. The 2nd parent will return their signed copy to the Investigator. Once the copy 
with the parent signature is received, the Investigator and witness (if present at NIH) will sign 
and date the 2nd parent consent form, place the original copy with all signatures in the Medical 
Record, retain a copy for research records, and mail a copy to the parent.   
 
The telephone consent process described above may be used to obtain written re-consent from 
participants who are already enrolled. Written informed consent will be obtained from minor 
participants when they reach the age of consent for continued use of their samples or data.  The 
telephone consent procedure detailed above may be used for such consent. 
 
For NIH employees: Consent may be obtained by a co-worker.  If so, independent consent 
monitoring will be provided by the NIH HSPU. 
 
c.  Consent documents  
The consent contains all required elements.  The consents attached to the protocol include adult 
patient and healthy volunteer populations (or parent of minor participants) and two different 
minor assents (ages 7-13 and 14-17), both for patients and healthy volunteers.   
 
15.  Data and Safety Monitoring 
       An independent monitoring committee will be put into place for Phase 2 of the study, 
including three experts in the field of neurology. 
 
This committee will meet once every 3 months after the start of Phase 2 and is comprised of:  
 
Andrew Mammen, MD, PhD, NIAMS, Muscle Disease Unit Leader, specializes in patients with 
myositis and autoimmune toxic myopathies and muscular dystrophies. 
 
Ronald Cohn, MD, University of Toronto, is the Chief of Clinical and Metabolic Genetics at the 
Hospital for Sick Children and specializes in neuromuscular diseases. 
 
Joan Austin, PhD, University of Indiana, Professor of Nursing at U. Indiana and specializes in 
pediatric epilepsy and chronic illnesses in children.  She has served on Data and Safety 
Monitoring Boards for 2 national trials and a number in Indiana. 


41 
 
 
a.  Data and safety monitor 
 
This study will be monitored by the PI for Phase 1 of the study and by an independent 
monitoring committee for Phase 2. 
 
b.  Data and safety monitoring plan 
Parameters to be monitored include liver function tests at the baseline visit and gastrointestinal 
symptoms throughout the 2nd phase of the study. 
 
Liver Function Testing (LFT) in patients with RYR1-RM: LFTs are often elevated in our patient 
population.  In patients with neuromuscular disease, the following criteria must be met, in order 
to determine impaired liver function.  These guidelines will be used for determining impaired 
liver function in patients with RYR1-RM.  
Evidence of moderate to severe hepatic involvement in this patient population is defined as: 
 
ALT ≥ 8x upper limit of normal (ULN) with total bilirubin 2x ULN (plus >35% ‘direct’ 
bilirubin) and/or INR >1.5  
 or  
GGT > 2-3x ULN with bilirubin 2x ULN (plus >35% ‘direct’ bilirubin) and/or  INR >1.5 
 
An increase to >3 times above the upper limit of normal in ALT, AST, ALP and/or GGT, not 
observed pre-drug/placebo, will be reported. A change in the transaminases (ALT, AST, 
ALP or GGT) from normal elevation for our population per its natural history, which is < 3X 
ULN,  plus any increase in INR>1.5 or total bilirubin>2X ULN will trigger the action of 
immediate consultation with the MAI, Dr. Bonnemann regarding a decision of withdrawal 
of the patient. If Dr. Bonnemann is unavailable, Dr. Traynor, Dr. Grunseich, Dr. Mankodi or 
Dr. Fischbeck will be consulted. If medical consultation results in the decision to withdraw, 
the patient will be withdrawn immediately.  
 
c.  Criteria for stopping the study or suspending enrollment or procedures 
 
For the first drug-related SAE, the study will be stopped in that subject. The SAE will be 
reported immediately (as soon as the PI becomes aware) to the IMC and IRB, and the PI will 
follow their instructions on what research activities can proceed or need to be stopped.  For the 
second same related SAE, the study will be temporarily stopped, including research activity with 
enrolled participants, until IRB review, unless the IRB specifically permits certain research 
activity to continue. 
 
The IMC, in consultation with the IRB, will review the data, unblinded if necessary, and will 
recommend whether to restart the intervention.  
 
16.  Quality Assurance 
 
16.1        Quality assurance monitor 


42 
 
As per the “National Institute of Nursing Research Intramural Research Program Policy on Data 
and Safety Monitoring (3/1/12)” Section IV D #5, the NINR Clinical Director will designate 
trained staff to monitor the progress of a clinical protocol. 
 
16.2        Quality assurance plan 
All study staff will maintain current training in good clinical practice and protection of human 
subjects, and will complete other protocol- and role-specific training, as needed.  
This Minor Increase over Minimal Risk Study will follow guidelines noted in the “National 
Institute of Nursing Research Intramural Research Program Policy on Data and Safety 
Monitoring (3/1/12)” Section IV H #1 and be monitored at the following intervals: within 3-6 
months of first subject enrollment, at least annual interim monitoring, and as needed. 
 
17.  Reporting of Unanticipated Problems, Adverse Events and Protocol Deviations 
 
The Principal Investigator is responsible for detecting, documenting, and reporting unanticipated 
problems, adverse events (AEs), including serious adverse events (SAEs), and deviations in 
accordance with NIH policy, IRB requirements, and federal regulations. Relatedness to the 
research of all serious adverse events will be determined by the PI in consultation with the CD. 
 
An electronic spreadsheet (Appendix K) is saved on the investigators’ secure shared drive, which 
tracks and records when each AE occurred, when the PI became aware of the event, whether the 
event is unexpected (with regard to information in the protocol, consent forms, and literature), 
whether the event is at least possibly related to the research, whether the event increased the risk 
to participants beyond that previously known or undermined the integrity of study data, how the 
event was managed, whether/when and how the event resolved, and required reporting for the 
event.  Each AE will be reviewed with the IMM, Bryan Traynor, MD.  Bryan J. Traynor, MD, 
PhD, is a tenured investigator in the Neurogenetics Branch of the National Institute of Aging 
(NIA) and is an adult neurologist. He is currently the chief of the Neuromuscular Diseases 
Research Section of the NIA.  Dr. Traynor serves as the Independent Medical Monitor (IMM) 
for this protocol and will review each AE with the PI.  He is an investigator not authorized to 
obtain informed consent.  Further investigation of AEs will be directed to the MAI, as needed.  
AEs will be reported to the IMC, regardless of PI classification.  All adverse events will also be 
submitted to and reviewed immediately by the NINR Clinical Director.  All events will be 
submitted to the IMC for review. Communication to and from the medical monitors will be in 
writing. 
 
Serious unanticipated problems, serious adverse events (including deaths) that are not 
unanticipated problems, and serious protocol deviations will be reported to the IRB and CD as 
soon as possible and in writing not more than 7 days after the PI first learns of the event, unless 
immediate reporting is waived for specific serious adverse events as noted below.  Not serious 
unanticipated problems and not serious deviations will be reported to the IRB and CD as soon as 
possible and in writing not more than 14 days after the PI first learns of the event.  Written 
reports will be submitted in PTMS. 
 
To avoid deviation reports for anticipated missed visits/procedures 
 
It is anticipated that participants in this study will occasionally miss or fail to complete an assessment or 


43 
 
procedure, due to issues such as discomfort, a medical contraindication, or failure to complete a procedure 
or visit within protocol-specified time frames.  Omissions such as these of non-primary outcome 
measures will be considered expected events and not protocol deviations provided they are infrequent and 
do not include data needed to assess safety or the primary study outcome.  Cumulative proportions of 
these missed events in the study population will be presented to the IRB annually. In addition, the rate of 
omissions will be monitored by the Investigators. If an individual misses more than 15% of the required 
assessments/procedures or if more than 15% of the participants miss completion of the same assessment 
or procedure, it will be considered a deviation and a deviation report will be sent to the IRB within two 
weeks.  
 
If the total number of expected items (study visits, study assessments/procedures) is less than 16, then two 
or more missed items are reportable. If the total number of expected items is greater than 16, then, if more 
than 15% are missed, it is reportable.  
 
All adverse events, deviations, and unanticipated problems will be summarized and reported at 
the time of Continuing Review. 
 
The PI will immediately report SAEs to the Sponsor according to the requirements of 21 CFR 
312.64(b) and as agreed upon with the sponsor. The PI will record nonserious AEs and report 
them to the Sponsor within 14 days. 
 
18.  Alternatives to Participation  
Because this study is a clinical trial to determine the effects of NAC on patients with RYR1-
related myopathy, it is necessary for subjects in the trial to take NAC or placebo. Patients may 
not take NAC prescribed by their own physician during the study. For patients who do not wish 
to take NAC or placebo or who wish to take it by their own physician, the alternative is to not 
participate in the trial.   There are no therapeutic alternatives. 
For healthy volunteers, the alternative is to not participate. 
 
19. Privacy 
All research activities will be conducted in as private a setting as possible, limiting personnel 
present.  
 
 20.  Confidentiality 
 
a.  For research data and investigator medical records: see below for research data.  Medical 
records will be stored in CRIS and CTDB behind the NIH firewall. For photographs and videos, 
when possible, the patient’s face and other identifying marks will not be included in the 
recording or photograph in order to protect confidentiality. Electronic records, videotapes, 
photographs, and other data, such as MRI scan data, will be stored on encrypted, password 
protected servers only accessible by authorized NIH associate investigators.  Coded medical 
history will be stored in CTSS.  PROMIS and NeuroQol data will be entered directly by 
participants into Assessment Center Lite, which is also behind the NIH firewall. Samples sent to 
other universities, such as U. Toronto and Emory, will be de-identified. Samples sent to other 
universities for fee-for-service analyses, such as Vanderbilt and Emory Universities, will be de-
identified. The PI will train study staff regarding obtaining and handling potentially sensitive and 
private information about co-workers through staff discussions and written branch/section 
procedures.   


44 
 
 
b.  For stored samples: Stored samples will be kept behind a double lock or will be coded and 
kept behind a single lock. 
 
c.  Special precautions  
Samples and data will be stored using codes that we assign. Data will be kept in password-
protected computers.  Samples will be kept in locked storage. Only study investigators will have 
access to the samples and data. 
 
For hard copy data/records: Hard copies of questionnaires will be kept in locked file 
cabinets in locked offices at NINR as well as a participant identification key.  All participant data 
will be coded for the purpose of data analysis and sharing between listed investigators. 
 
For samples without identifiers (coded or unlinked):  single lock will be used.  
  
 
For electronic data with identifiers or recognizable facial images: password protected 
files on secured servers will be used. Quantitative data in the form of results of rehabilitation 
medicine testing and responses to questionnaires will be stored in excel, SPSS, and/or Mplus 
files on encrypted computers at the same sites. Qualitative data (digital recordings) in the form of 
responses to open ended questions will be collected and retained on encrypted computers at NIH 
on Nvivo software (offices of Dr. Meilleur’s team, NINR). Recordings of qualitative data will be 
destroyed once the study is completed. Data will be submitted to an online database behind the 
NIH firewall (CTDB). 
 
Only investigators listed on the protocol will have access to records, data, and samples.  If 
sponsors, monitors or auditors outside of study investigators will need access, they will have 
access to coded data.  Results will be posted on cctrials.gov 
 
21. Conflict of Interest 
 
a.   Distribution of NIH Guidelines 
NIH guidelines on conflict of interest have been distributed to all investigators.   
 
b.  Conflict of interest  
There are no conflicts-of-interest to report for NIH investigators.  Non-NIH investigators will 
abide by the conflict-of-interest policies of their own institutions. We have consulted with DEC 
and there are no conflicts of interest for Dr. Michael or Hyperion. 
 
c.  Role of a commercial company or sponsor  
BioAdvantex Pharma Inc will provide complementary drug (PharmaNAC) and placebo oral 
effervescent pills to NIH for the RTC.  Under a non-disclosure agreement, the company will also 
disclose information regarding description and composition of the drug product and placebo, 
certificate of analysis and the master production document certificate that contains information 
about manufacturing, packaging and quality assurance for both NAC and placebo. BioAdvantex 
has granted permission to cross reference drug master file for NAC and environmental analysis 
requirement certification. NIH and University of Toronto will provide the protocol and the results 
of the study to BioAdvantex.  Personal identifiers of participants will not be shared with the 
sponsor. 
 


45 
 
22. Technology Transfer 
An MTA (MTA-15-002) is in place with Dr. Michael at Hyperion Biotechnology Inc., which 
states that deidentified data will be owned by NINR, but the salivary fatigue biomarker will be 
processed at Hyperion. MTAs have been obtained to transfer de-identified muscle tissue samples 
and de-identified genetic mutations to Dr. Andy Marks at Columbia University. 
A CTA (NR-CTA-15-001) has been started with BioAdvantex Pharma. The CTA states that 
BioAdvantex Pharma will provide the drug, but the NINR is responsible for sponsoring the 
study, and Dr. Meilleur is the sponsor of the IND. An MTA is not required for sending to 
samples on a fee-for-service basis to Vanderbilt and Emory Universities. 
 
 
23. Research and Travel Compensation  
Patients: 
Lodging, meals and travel will be provided to patients only by the NINR.  In the case of minors 
or adults who have difficulty traveling alone due to their myopathy, NINR will also pay for 
travel and lodging for one caregiver. In the case of minors, the caregiver will be the legal 
guardian. There will be no other payment or reimbursement.  Meal vouchers will be provided to 
the adult patient or the parent of a minor (for the child’s meals). 
For patients who are staying in a hotel off campus, complementary shuttles are available between 
their hotel and NIH.  If patients are at NIH after these routine shuttles have stopped for the day, 
taxis will be provided for them, and covered under the protocol.  NINR staff will arrange the 
taxi.  Research study staff will be present after late-day procedures, if needed, to assure 
transportation needs are addressed. 
NIH employees or staff who participate during work hours must have permission from their 
supervisor. NIH employees or staff must either participate outside of work hours or take leave in 
order to receive compensation. 
 
Healthy Volunteers: 
Travel and meals will be provided to volunteers by the NINR.  In the case of minors, NINR will 
also pay for travel and meals for one legal guardian. Only healthy volunteers will be paid for 
their participation. Each volunteer will be provided with $50 compensation.  For those 
undergoing an MRI, an additional $25 will be provided.  Only 2-8 people from each age group 
will be asked to undergo an MRI.  If the participant must stay the night in order to participate, 
one night of lodging will be provided to the participant and their legal guardian (in the case of 
minors).   
For volunteers who are staying in a hotel off campus, complementary shuttles are available 
between their hotel and NIH.  If volunteers are at NIH after these routine shuttles have stopped 
for the day, taxis will be provided for them, and covered under the protocol.  NINR staff will 
arrange the taxi.  Research study staff will be present after late-day procedures, if needed, to 
assure transportation needs are addressed. 
NIH employees or staff who participate during work hours must have permission from their 
supervisor. NIH employees or staff must either participate outside of work hours or take leave in 
order to receive compensation. 
 
24. References 


46 
 
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Taratuto, A., Laquerrière, A., Claeys, K., Marty, I., Fardeau, M., Guicheney, P., Lunardi, J., 
Romero, N. (2011). Recessive RYR1 mutations cause unusual congenital myopathy with 
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Brookes, P., Yoon, Y., Robotham, J., Anders, M., & Sheu, S. (2004). Calcium, ATP, and ROS:  
A mitochondrial love-hate triangle.  Amer J of Physiol Cell Physiol; 287:  C817-C833. 
 
Dowling, J., Arbogast, S., Hur, J., Nelson, D., McEvoy, A., Waugh, T., Marty, I., Lunardi, J., 
Brooks, S., Kuwada, J., & Ferreiro, A. (2012). Oxidative stress and successful antioxidant 
treatment in models of RYR1-related myopathy.  Brain; 135(Pt. 4):  1115-1127.  
 
Durham WJ, Aracena-Parks P, Long C, Rossi AE, Goonasekera SA, Boncompagni S, et al. 
RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 
knockin mice. Cell 2008; 133(1): 53-65 
 
Evans, S. (2007). When and how can endpoints be changed after initation of a randomized 
clinical trial? PLOS Clinical Trials. E18:1-3. 
 
Guertin KA, Grant RK, Arnold KB, Burwell L, Hartline J, Goodman PJ, Minasian LM, Lippman 
SM, Klein E, Cassano PA. Effect of long-term vitamin E and selenium supplementation on urine 
F2-isoprostanes, a biomarker of oxidative stress. Free Radic Biol Med. 2016; 95:349-56 
 
Hardan, A., Fung, L., Libove, R., Obukhanych, T., Nair, S., Herzenberg, L., Frazier, T., 
Tirouvanziam, R. (2012).  A randomized controlled pilot trial of oral N-acetylcysteine in 
children with autism. Biol Psychiatry, 71:  956-61. 
 
Henderson, M.J., Wires, E.S., Trychta, K.A., Yan, X., Harvey, B.K. (2015). Monitoring 
Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP. J Vis 
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47 
 
 
Herzenberg, L.A., De Rosa, S.C., Dubs, J.G., Roederer, M., Anderson, M.T., Ela, S.W., 
Deresinski, S.C., Herzenberg, L.H. (1997). Glutathione deficiency is associated with impaired 
survival in HIV disease. PNAS. 94: 1967-1972. 
 
Hidalgo, C., Sanchez, G., Barrientos, G., Aracena-Parks, P. (2006). A transverse tubule NADPH 
oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S-
glutathionylation.  J Biol Chem; 281:  26473-82.   
 
Jungbluth, H., Sewry, C., & Muntoni, F. (2011). Core myopathies.  Semin Pediatr Neurol.; 
18(4):  239-249.  
 
Kelly, G. (1998). Clinical applications of N-acetylcysteine.  Alt Med Rev; 3(2): 114-127. 
 
Lee CS, Hanna AD, Wang H, Dagnino-Acosta A, Joshi AD, Knoblauch M, et al. A chemical 
chaperone improves muscle function in mice with a RyR1 mutation. Nature communications 
2017; 8: 14659 
 
Marrocco I, Altieri F, Peluso I. Measurement and Clinical Significance of Biomarkers of 
Oxidative Stress in Humans. Oxid Med Cell Longev; 2017:6501046 
 
McGill MR, Jaeschke H. A direct comparison of methods used to measure oxidized glutathione 
in biological samples: 2-vinylpyridine and N-ethylmaleimide. Toxicology mechanisms and 
methods. 2015;25(8):589-595 
 
Michelucci A, De Marco A, Guarnier FA, Protasi F, Boncompagni S. Antioxidant Treatment 
Reduces Formation of Structural Cores and Improves Muscle Function in RYR1(Y522S/WT) 
Mice. Oxidative medicine and cellular longevity 2017; 2017: 6792694 
 
Milne GL, Sanchez SC, Musiek ES, Morrow JD. Quantification of F2-isoprostanes as a 
biomarker of oxidative stress. Nature protocols 2007; 2(1): 221-6 
 
Milne GL, Yin H, Hardy KD, Davies SS, Roberts LJ. Isoprostane Generation and 
Function. Chemical reviews. 2011;111(10):5973-5996. doi:10.1021/cr200160h. 
 
Muller, F., Svardal, M., Nordoy, Il., Berge, R.K., Aukrust, P., Froland, S.S. (2000). Virological 
and immunological effects of antioxidant treatment in patients with HIV infection. European J of 
Clinical Investigation; 30: 905-14.  
 
O'Brien, P.C. and Fleming, T.R. 1979. A multiple testing procedure for clinical trials. 
Biometrics, 35, pp. 549-556. 
 
Paridon, S.M., Alpert, B.S., Boas, S.R., Cabrera, M.E., Caldarera, L.L., Daniels, S.R., Kimball, 
T.R., Knilans, T.K., Nixon, P.A., Rhodes, J., Yetman, A.T. (2006). Clinical stress testing in the 
pediatric age group: A statement from the American Heart Association council on cardiovascular 


48 
 
disease in the young, committee on atherosclerosis, hypertension, and obesity in youth. 
Circulation; 113:1905-1920. 
Paschalis V, Theodorou AA, Margaritelis NV, Kyparos A, Nikolaidis MG. N-acetylcysteine 
supplementation increases exercise performance and reduces oxidative stress only in individuals 
with low levels of glutathione. Free Radic Biol Med. 2018; 115:288-297 
 
Ramagopalan, S., Skingsley, A.P., Handunnetthi, L., Klingel, M., Magnus, D., Pakpoor, J., 
Goldacre, B. (2014). Prevalence of primary outcome changes in clinical trials registered on 
ClinicalTrials.gov: a cross-secitonal study. F1000Research; 3:77 Last updated:27 JUN 2014. 
 
 
Schmitt B, Vicenzi M, Garrel C, Denis FM. Effects of N-acetylcysteine, oral glutathione (GSH) 
and a novel sublingual form of GSH on oxidative stress markers: A comparative crossover 
study. Redox Biology. 2015;6:198-205. doi:10.1016/j.redox.2015.07.012. 
 
Tirouvanziam, R., Conrad, C.K., Bottiglieri, T, Herzenberg, LA., Moss, R.B., Herzenburg, LA. 
(2006). High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in 
cystic fibrosis. PNAS; 103(12): 4629-4633. 
 
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MFM Study Group.  Responsiveness of the motor function measure in neuromuscular diseases. 
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M., Brockington, M., Kinali, M., Manzur, A., Robb, S., Appleton, R., Messina, S., D’Amico, A., 
Quinlivan, R., Swash, M., Mü ller, C., Brown, S., Treves, S., & Muntoni, F. (2007). Molecular 
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2024-2036.  
  
25.  Attachments/ Appendices 
A. Visits and Procedures Schedule  
B. Recruitment materials (combined patient wording, patient flyer, HV flyer) 
C. NIH recruitment advertisement 
D. Recruitment letter  
E. Eligibility checklist 
F. Clinician information letter 
G. Pre-screen Questionnaire  
H. Healthy volunteer recruitment flyer 
I. Needle Aspiration Muscle Biopsy Procedure Information Sheet 
J. Medication/Placebo Information and Instructions Sheet 
K. Adverse Event Spreadsheet 
L. NIH Information Sheet on Staff Research Participation 
M. Cover Letter for Continued Use for Minors who turn 18 


49 
 
 
26.  Consent Forms 
Adult subject consent 
Child Subject assents (ages 7-13 and 14-17) 
Healthy Volunteer: Adult Consent 
Healthy Volunteer: Child assents (ages 7-13 and 14-17) 
Consent for Continued Use for Minors who turn 18 
 
 

SAP_001.pdf: 
Date: July 2, 2018 
 
RE: Statistical Analysis Plan for Protocol 15-NR-0072 Antioxidant 
therapy in RYR1-related congenital myopathy 
 
Primary outcome measures 
Where to find data (variables) for the below analyses in locked excel files: 
1) AE_Spreadsheet_Demographics_LOCKED_2018.3.29 
2) UrineIsoprostane_6MWT_LOCKED_2018.3.29 
3) PillCount_PlasmaRedox_SalivaFBI_SMOxidativeStressDCFH_LOCKED_2018.3
.29 
 
Descriptive statistics 
• Assess data distribution (i.e. basic descriptive statistics) for demographics (sex, 
ethnicity, smoking_status, alcohol_consumption, age_month0, height_month0, 
weight_month0, bmi_month0) 
 
• Assess data distribution for urine isoprostane (all variables) 
 
 
• Assess data distribution for six-minute walk test 
(predicted_distance_walked_month0, min_6_distance_month0, 
predicted_distance_walked_month6, min_6_distance_month6, 
predicted_distance_walked_month12, min_6_distance_month12). 
 
• Assess data distribution for skeletal muscle oxidative stress (all variables),  
 
• If possible, also assess data distribution for plasma redox (GSH_GSSG_month0, 
GSH_GSSG_month6, GSH_GSSG_month12, CYS_CYSS_month0, 
CYS_CYSS_month6, CYS_CYSS_month12). 
 
• Assess data distribution for pill count (percent_compliance_month12). 
 


• Descriptive statistics (frequency, mean, median, SD, SEM, IQR, range) for all the 
above for entire cohort at baseline (month 0) for demographics. 
 
• Repeat abovementioned descriptive statistics split by treatment group (NAC and 
placebo) at baseline. 
 
• Assess the frequency of AEs by system organ class (System Organ Class (SOC) 
(AE only) and by Preferred Term (Preferred Term (AE only)). 
Natural history phase analyses 
During the natural history phase of the study (month 0 to month 6), we will take one look 
at the data prior to the final analysis with the following two approaches:  
1. After 30 participants complete their baseline visit, we will compare their baseline 
outcome measure values against healthy controls. The mean values for the 
primary outcome measures (corrected 15-F2t-isoprostane concentration and 
6MWT distance) will be compared against existing data for otherwise healthy 
individuals using summary independent t-tests and/or standardized mean 
difference (i.e. Hedge’s g which is a measure of effect size between two means, 
weighted by n). This analysis will be repeated when all baseline visits are 
completed. 
 
 
Null Hypothesis (H0) 
There will be no difference in mean corrected 15-F2t-isoprostane concentration or 
6MWT distance, between the RYR1-RM and otherwise healthy individuals. 
Alternative Hypothesis (H1) 
Corrected 15-F2t-isoprostane concentration will be significantly increased and 6MWT 
total distance will be significantly decreased in RYR1-RM myopathy patients when 
compared to otherwise healthy individuals.  
 
2. After 30 participants complete their 2nd visit at 6 months (prior to starting study 
drug/placebo), disease progression will be assessed using paired t-tests to 
determine changes between 0 and 6-month visits for each outcome measure. 
For the primary outcome measures (corrected 15-F2t-isoprostane concentration 
and 6MWT) change between 0 and 6-month time points will be assessed. This 
analysis will be repeated when all 6-month visits are completed. 
 
 
 


Null Hypothesis (H0) 
Corrected 15-F2t-isoprostane concentration and 6MWT total distance will not change 
significantly between 0 and 6 months in RYR1-RM myopathy patients. 
Alternative Hypothesis (H1) 
There will be a statistically significant difference between 0 and 6-month values for 
corrected 15-F2t-isoprostane concentration and/or 6MWT total distance in RYR1-RM 
myopathy patients. 
Intervention phase analyses (month 6 to month 12) 
• Use an independent t-test (or non-parametric equivalent) to compare pre-
intervention (month 6) values for demographics, urine isoprostane and six-minute 
walk test between NAC versus placebo groups. 
 
• Using multiple imputation (x40 datasets and minimum/maximum value 
constraints from per protocol data) impute missing month 12 data. 
 
• The following GLM should be run both per protocol and with ITT datasets. 
 
1. Run a general linear model comparing month 12 month corrected 15-F2t-
isoprostane concentration (corrected_f2-isop_month12, corrected_f2-
isop_PGF2alpha_ratio_month12) between NAC and placebo groups controlling 
for pre-intervention value. Run this model again controlling for established a priori 
confounders including age, sex, height, weight, BMI, smoking status, alcohol 
status. Variables that do not contribute significantly to the model may be 
removed. (Exclude RYR023, RYR024, and all healthy volunteers). 
Null Hypothesis (H0) 
In RYR1-RM myopathy patients, there will be no statistically significant difference in 
corrected 15-F2t-isoprostane concentration and/or corrected 15-F2t-Isop:PGF2α ratio 
between NAC and placebo groups at month 12, after controlling for established a priori 
confounders. 
Alternative Hypothesis (H1) 
At month 12, in RYR1-RM myopathy patients allocated to NAC treatment, there will be 
significantly decreased corrected 15-F2t-isoprostane concentration and/or 15-F2t-
Isop:PGF2α ratio compared to those allocated to placebo, after controlling for 
established a priori confounders. 
 


2. Run a general linear model comparing month 12 6MWT total distance 
(min_6_distance_month12) between NAC and placebo groups controlling for 
baseline value. Run this model again controlling for established a priori 
confounders including age, sex, height. Variables that do not contribute 
significantly to the model may be removed. 
Run this model again and control for one additional hypothetical confounder (post 
hoc): FVC % predicted. 
Null Hypothesis (H0) 
In RYR1-RM myopathy patients, there will be no statistically significant difference in 
6MWT total distance between NAC and placebo groups at month 12, after controlling 
for established a priori confounders. 
Alternative Hypothesis (H1) 
At month 12, in RYR1-RM myopathy patients allocated to NAC treatment, there will be 
significantly increased 6MWT total distance compared to those allocated to placebo, 
after controlling for established a priori confounders. 
 
Adverse Event Analysis 
The 3 categories for AEs in the locked data are: 
1. AE 
2. UP/SAE 
3. Non-UP/SAE 
The other categories do not apply as they are not AEs. 
Please use these categories in column F: “IRB determination of category”. 
Exclude HVs (healthy volunteers) and only use RYR numbers. Exclude RYR023 and 
RYR024. 
For the remaining AE events (i.e. in the 3 categories delineated above) all have 
individual RYR IDs. 
  
Assess: 
1. Which AEs occurred in NAC group and which occurred in Placebo group 
(descriptive statistics) (see attached p.1 Mock Table 1) 


2. Were there more specific AEs (by preferred term, Column H on excel) of any kind 
in NAC vs. Placebo groups? Or were AEs equally distributed between groups? 
Both Chi-square test and Fisher’s exact test extended to general RxC tables, aka 
the Freeman-Halton test, will be used to compare AE frequencies between NAC 
and Placebo groups.    
3. For those in NAC group, what was event causality (see AE excel column Q) and 
what was event severity (AE excel column R)? (for an example of this table, see 
p. 7 of attached, first table on p. 7). Capture the “body system” from our AE excel 
column G (AKA “system organ class”). 
  


Secondary Outcome Measures 
Natural History Phase (month 0 to month 6): 
Aim 1: plasma GSH:GSSG ratio; plasma CYS:CYSS ratio, (corrected DCF-
fluorescence intensity (month 6 only)) 
1. We will compare baseline outcome measure values against healthy controls, 
when possible, using a summary independent t-test (or non-parametric 
equivalent). Hedge’s g will be used to assess standardized mean differences 
between RYR1-RM and otherwise healthy population groups. 
Null Hypothesis (H0) 
There will be no difference in mean plasma GSH:GSSG ratio; plasma CYS:CYSS ratio,  
or corrected DCF-fluorescence intensity, between RYR1-RM and otherwise healthy 
individuals. 
Alternative Hypothesis (H1) 
Plasma GSH:GSSG, and CYS:CYSS ratios will be significantly decreased and 
corrected DCF-fluorescence intensity will be significantly increased in RYR1-RM 
myopathy patients when compared to otherwise healthy individuals.  
 
2. We will compare baseline outcome measure values against values obtained at 
month 6 using paired t-tests (or non-parametric equivalent) to determine changes 
between 0 and 6-month visits for each outcome measure. 
Null Hypothesis (H0) 
Plasma GSH:GSSG, and CYS:CYSS ratios and corrected DCF-fluorescence intensity 
will not change significantly between 0 and 6 months in RYR1-RM myopathy patients. 
Alternative Hypothesis (H1) 
There will be a statistically significant difference between 0 and 6-month plasma 
GSH:GSSG, and/or CYS:CYSS ratios and/or corrected DCF-fluorescence intensity in 
RYR1-RM myopathy patients. 
 
Aim 2: Timed function tests; Biodex; Myotools, MFM, MRI, PROMIS, FACIT, MFI, 
NeuroQoL scales. 
1. We will compare baseline outcome measure values against healthy controls, 
when possible, using a summary independent t-test (or non-parametric 


equivalent). When sufficient normative data is available, Hedge’s g may be used 
to calculate the standardized mean difference between groups. 
Null Hypothesis (H0) 
There will be no difference, in graded functional tests (time to completion); Biodex 
(fatigue indices); Myotools values (grip and pinch strength), MFM (D1, D2, D3 and total 
% of maximum score), MRI (intramuscular fatty infiltration), PROMIS (scale scores), 
FACIT (total score), MFI-20 (total score), NeuroQoL (scale scores), between RYR1-RM 
and otherwise healthy individuals. 
Alternative Hypothesis (H1) 
In RYR1-RM patients, graded functional tests (time to completion); Biodex (fatigue 
indices); Myotools values (grip and pinch strength), MFM (D1, D2, D3 and total % of 
maximum score), PROMIS (scale scores), FACIT (total score), NeuroQoL (scale 
scores) will be significantly lower and MRI (intramuscular fatty infiltration), MFI-20 (total 
score) significantly higher when compared to otherwise healthy individuals. 
 
2. We will compare baseline outcome measure values against values obtained at 6 
months using paired t-tests (or non-parametric equivalent) to determine changes 
between 0 and 6-month visits for each outcome measure. 
Null Hypothesis (H0) 
Graded functional tests (time to completion); Biodex (fatigue indices); Myotools values 
(grip and pinch strength), MFM (D1, D2, D3 and total % of maximum score), MRI 
(intramuscular fatty infiltration), PROMIS (scale scores), FACIT (total score), MFI-20 
(total score), NeuroQoL (scale scores), will not change significantly or clinically between 
0 and 6 months in RYR1-RM myopathy patients. 
Alternative Hypothesis (H1) 
There will be a statistically significant and/or clinically meaningful difference between 0 
and 6-month graded functional tests (time to completion); Biodex (fatigue indices); 
Myotools values (grip and pinch strength), MFM (D1, D2, D3 and total % of maximum 
score), PROMIS (scale scores), FACIT (total score), NeuroQoL (scale scores), MRI 
(intramuscular fatty infiltration), MFI-20 (total score) in RYR1-RM myopathy patients. 
 
3. Additionally, the following will be analyzed: 
 


Optimal measures for fatigability in RYR1-RM will be determined using paired t-tests for 
6MWT distance and speed, time for graded functional tests, and fatigue index for biodex 
fatigue test. 
Spearman rho correlation, Pearson correlation and/or Bland Altman analysis will also be 
used to examine the relationship between slow vital capacity and forced vital capacity (L 
and % predicted). Descriptive statistics and change between baseline and 6 months will 
be assessed for FVC and SVC (L and % predicted). 
Reliability will be assessed for PROMIS, FACIT, MFI, MFM, and NeuroQoL by 
Cronbach’s alpha for internal consistency and by Intraclass correlation coefficient for 
test-retest. 
Intervention phase analyses (intent-to-treat) 
• Use an independent t-test (or non-parametric equivalent) to compare baseline 
values for all secondary outcomes above between NAC versus placebo groups. 
General linear modeling will be used to control for possible confounders such as 
such as but not limited to baseline values, gender, age, and baseline values. Log 
transformed values may be used if there is no bell curve. We will use an alpha of 
0.05 to determine significance. Variables that do not contribute to the model may 
be removed. 
 
• Using multiple imputation (x40 datasets and minimum/maximum value 
constraints from per protocol data) impute missing month 12 data. 
 
• The following GLM should be run both per protocol and with ITT datasets. 
 
• General linear model comparing month 12-month data between NAC and 
placebo groups (controlling for possible confounders such as but not limited to: 
baseline value, age, sex, height, weight, BMI, smoking status, alcohol status).  
 
Exploratory Outcome Measures 
Aim 1: Anaerobic threshold, NIRS; secreted ER calcium monitoring proteins 
(SERCaMP) assay in blood and muscle 
Anaerobic threshold using NIRS will be evaluated for patterns in individuals with RYR1-
RM compared to healthy controls. From this evaluation, the tissue saturation index will 
be calculated as TOI= [O2-Hb] / Tot-Hb. The rate of decline and rise in TOI will be 
compared to healthy controls. NIRS data will also be obtained during CPET and Biodex 


testing and may be used to generate new hypotheses regarding oxidative metabolism in 
RYR1-RM. 
 
SERcaMP levels will be tested in an exploratory fashion in patient-derived myotubes to 
assess whether Ca++ levels are normal in the sarcoplasm and/or cytosol. This will 
generate new hypotheses regarding Ca++ dysregulation in RYR1-RM. 
 
Aim 2: CPET, EIM, salivary FBI, melatonin, cortisol and DHEA; QoL qualitative 
interview 
Correlative analysis (Pearson correlation and/or Spearman rho) will be used to examine 
the linear relationship between subjective fatigue measures (FACIT and MFI) and the 
salivary biomarkers (FBI, melatonin, cortisol, DHEA) to the objective fatigability 
measures mentioned above, such as CPET. This data may be used to generate new 
hypotheses fatigue and fatigability in RYR1-RM. 
 
The QoL interviews will be mined for common themes across participants. Interviews 
will also be mined for any comments about differences in QoL before and after 
treatment. This data may be used to generate new hypotheses regarding QoL in 
individuals with RYR1-RM. 
All data may be used for additional exploratory analyses, particularly for the natural 
history phase of the study. These additional analyses which will not include or alter 12-
month data/results of the intervention phase analysis. This is due to the rare nature of 
this disease and thus the substantial value of this data to contribute findings in this 
population. 
 
Katy Meilleur, PhD, PNP-BC  
NIH/NINR/TIB NIH/NINR/TIB 
1 Cloister Court, MSC 4733  
Building 60, Room 252  
Bethesda, MD 20814-9692  
Phone: (301) 435-1503  
E-mail: meilleurk@mail.nih.gov