Antioxidant Therapy With N-acetylcysteine for Motor Behavior and/or Learning in Children With Neurofibromatosis Type 1
Phase: PHASE2
Conditions: Neurofibromatosis 1
Sponsor: Children's Hospital Medical Center, Cincinnati

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Antioxidant Therapy with N-acetylcysteine for Learning and 
Motor Behavior in Children with Neurofibromatosis Type 1 
 
 
 
 
 
NCT04481048 
IND 139468 
 
 
 SPONSORS: 
CCHMC - ARC RASOPATHIES PROGRAM 
U.S. Department of Defense 
 
 
 
I NVESTIGATOR INFORMATION: 
Principal Investigator: 
Donald L. Gilbert, MD MS 
Co-Director, Transcranial Magnetic Stimulation Laboratory  
Professor of Pediatrics and Neurology 
Children’s Hospital Medical Center, MLC 2015, Neurology 
 
 
 
 
 
 
 
17MAY2023 
 
 
 

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CINCINNATI CHILDREN’S HOSPITAL MEDICAL CENTER 
STUDY TITLE:   
Antioxidant therapy with N-acetylcysteine for motor behavior and/or learning in children 
with neurofibromatosis type 1 
 
SHORT TITLE: 
DoDNAC 
 
SPONSOR NAME: Department of Defense (DoD) 
Proposal Number: NF190013, Award Number: W81XWH-20-1-0139, HRPO Log Number: 
E01443.1a 
 
 
 
INVESTIGATOR INFORMATION: 
Principal Investigator’s Name:          
Donald L. Gilbert, MD MS 
 
 
 
 
 
 
 
Co-Director, Transcranial Magnetic Stimulation Laboratory      
Professor of Pediatrics and Neurology 
Cincinnati Children’s Hospital Medical Center, MLC 2015, Neurology 
3333 Burnet Avenue 
Cincinnati, OH 45229-3039 
Office 513-636-4222 
Fax 513-636-3980 
Donald.gilbert@cchmc.org 
 
Sub-investigators at Cincinnati Children’s Hospital 
Elizabeth K. Schorry, MD – Genetics 
Lindsey E. Aschbacher-Smith, MS - Genetics 
Dean W. Beebe, PhD – Neuropsychology 
Kim M. Cecil, PhD - Imaging 
Mark W. DiFrancesco, PhD – Imaging 
Leach, James, MD –Imaging  
Bin Zhang – Biostatistician  
Steve W. Wu, MD – Neurology 
Nancy Ratner, PhD – Experimental Hematology and Cancer Biology 
 
External Consultants 
Karin S. Walsh, PsyD – Neuropsychology – Children’s National Hospital 
Pediatric Neuropsychologist 
Division of Pediatric Neuropsychology | Children's National Hospital 
kwalsh@ChildrensNational.org  
Itamar Kahn, PhD – Neuroscientist – Technion – Israel Institute of Technology 
Associate Professor 
Department of Neuroscience 
kahn@technion.ac.il 
 
 

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ABSTRACT: 
Children with neurofibromatosis type 1 (NF1) commonly suffer from the effects of cognitive, 
behavioral, and motor impairments. At present, there is no specific treatment for this NF1 
complication. In this project, we will assess the safety and clinical benefit of N-acetylcysteine 
(NAC) as a pharmacological intervention in children with NF1. This drug choice is based on the 
recent findings from mouse models to study the central nervous system manifestations of NF1 at 
Cincinnati Children’s Hospital Medical Center (CCHMC). These findings revealed a role for 
myelin-forming oligodendrocytes in the control of nitric oxide synthases (NOS) and their product, 
nitric oxide (NO), in maintenance of brain structure and function, including regulation of behavior 
and motor control. Treating these mice with NAC corrected cellular and behavioral abnormalities. 
This data from animal models of NF1 along with uncontrolled clinical observations in children 
with NF1 suggest that the antioxidant compound, NAC, may reduce these impairments. Therefore, 
we propose performing a single center double-blind placebo controlled, prospective, Phase II study 
to explore safety, tolerability, and efficacy of NAC on motor behavior and/or learning in children 
with NF1 aged 8 through 16 years old. Participants will be carefully monitored for side effects. 
Primary and secondary outcome measures will be administered at baseline, follow-up, and post-
treatment. 
 
PURPOSE OF STUDY: 
This is a phase II clinical trial with the goal to explore safety, tolerability, and efficacy of NAC on 
motor behavior in children with NF1 aged 8 through 16 years old. We hypothesize that NAC therapy 
will improve motor function evaluated by the PANESS scale. This is based on studies demonstrating 
that NAC significantly improved impairments in the animal model of NF1. We will also analyze NAC 
effects on attention deficit and impulsivity in children with NF1. 
 
This study will also help develop novel predictive biomarkers of response to neurocognitive 
therapies in patients with NF1 which are needed to evaluate treatment outcomes. 
 
We will gain information in children with NF1 about possible clinical benefit of anti-oxidant 
treatment and to develop and evaluate quantitative brain-based and blood biomarkers relating to 
presence of NF1, symptom severity, and response to antioxidant therapy. Clinically, 50% of children 
with NF1 are underperforming or failing at school [1].  This frequently leads to decreased 
educational attainment and fewer opportunities as adults.  An important first step was our 
preliminary work using the PANESS scale and Transcranial Magnetic Stimulation (TMS)-evoked 
Short Interval Cortical Inhibition (rSICI) in children with NF1. We propose to develop and extend 
our understanding of NF1-related motor and learning behavior in response to antioxidant therapy 
with NAC. The purpose of the present study is to 1) evaluate tolerability, safety, and clinical benefit 
of NAC in this double-blind placebo controlled study using the motor function scale (PANESS); 2) 
to evaluate the effects of NAC on measures of NF1 neurocognitive symptomatology 
(ADHD/impulsive symptoms, executive function, working memory); and 3) to determine if TMS 
measurement (SICI) in children with NF1 will correlate with clinical effects of NAC treatment and 
evaluate utility of advanced brain imaging and spectroscopy measurements in children with NF1, 
and effects of NAC therapy. 
 
We propose to study 58 children with NF1, ages 8-16 years, at baseline and after completion of 8 
weeks of treatment with NAC, followed by a washout period of 4 weeks. 

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We believe this work has the potential to lay groundwork for future use of relevant biomarkers for 
treatment and outcomes research for NF1 as well as other biologically similar conditions, 
collectively designated the “RASopathies” (due to involvement of the RAS family of proteins) and 
ultimately to guide development of more effective treatments based on disease pathophysiology.  
 
STUDY OBJECTIVE: 
NAC Trial at Cincinnati Children’s Hospital Medical Center (CCHMC) 
We propose performing a single center randomized double-blind placebo controlled, prospective, 
Phase II study to explore safety, tolerability, and efficacy of NAC on motor behavior in children 
with NF1 aged 8 through 16 years old. 
 
Hypothesis:  
We hypothesize that NAC therapy will improve motor function evaluated by the PANESS scale. This 
is based on studies demonstrating that NAC significantly improved impairments in the animal model 
of NF1. We will also analyze NAC effects on attention deficit and impulsivity in children with NF1. 
 
Specific Aim:  
The primary outcome of this study is to characterize the effects of NAC treatment on motor function 
in children and adolescents with NF1 using the PANESS. We hypothesize that motor function scores 
rated with the PANESS scale will improve after treatment with NAC. 
 
Secondary Aims: 
1. To evaluate the effects of NAC on measures of NF1 neurocognitive symptomatology 
(ADHD/impulsive symptoms, executive function, working memory), we will use Response 
Evaluation in Neurofibromatosis and Schwannomatosis (REiNS) committee recommended 
assessments tools DuPaul ADHD rating scale (ADHD-RS), Behavioral Rating Inventory of 
Executive Function second edition (BRIEF-2), Wechsler Intelligence Scale for Children, Fifth 
Edition (WISC-V) subtests, and Test of Variables of Attention (TOVA). 
2. To determine if TMS measurement (SICI) in children with NF1 will correlate with clinical 
effects of NAC treatment. 
3. To quantify microstructural properties of brain tissue based on water diffusion, glutathione GSH 
concentrations, and γ-aminobutyric acid (GABA) concentration using brain magnetic resonance 
imaging (MRI) and magnetic resonance spectroscopy (MRS) in children with NF1. This will 
allow for regional correlation between imaging, spectroscopy and neuropsychometric outcomes. 
We will also determine if these magnetic resonance-based outcomes correlate with clinical 
effects of NAC treatment. 
4. To evaluate safety and tolerability of NAC in children with NF1. 
5. To explore motor function (PANESS) and brain-based measures (TMS, MRI-MRS, MRI-DTI) 
as biomarkers of impaired executive function (ADHD-RS; BRIEF-2; TOVA) in individuals with 
NF1.   
 
 
BACKGROUND: 
Neurofibromatosis type 1 (NF1)  

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Neurofibromatosis (NF1) is an autosomal dominant condition affecting many organs, resulting from 
mutations in the neurofibromin 1 gene [1]. Characteristically, individuals with this condition have 
cutaneous manifestations, most prominently hyperpigmented macules termed “café-au-lait” spots. 
The neurofibromin gene functions in part as a tumor suppressor, and persons with NF1 are 
susceptible to malignant and benign tumors in multiple organs. What is less well studied is that 
individuals with NF1 commonly suffer from difficulties with behavioral and emotional regulation 
including attention-deficit/hyperactivity disorder (ADHD) symptoms, learning disabilities, and 
developmental delays in motor function [2]. These cause substantial morbidity in childhood and 
throughout life. There are no effective treatments for motor behavior and/or learning difficulties for 
NF1 and other RASopathies. Lack of sensitive objective biomarkers that can evaluate motor system 
or behavioral symptoms causes difficulties in evaluating the efficacy of pharmacological 
interventions in NF1. 
 
Preliminary results regarding motor function and brain-based measures as a rationale for 
additional study. A pre-planned analysis of predictor variables for NAC response has been 
performed, prior to breaking the blind in the randomized controlled treatment study. We performed 
these analyses on measures obtained at the baseline (pre-treatment) visit. These findings are now 
published, and some additional findings will be presented at scientific meetings. The strength of 
these preliminary findings warrants an expansion of the secondary aims, in order to validate these 
measures in a larger sample. Inclusion/exclusion criteria will be minimally changed to expand 
eligibility without compromising safety or study rigor. Individuals not wishing to participate in the 
NAC vs. Placebo treatment may elect to enroll and participate in an assessment of the baseline 
biomarkers. This will allow for additional assessment of the relationship between these measures 
and the NF1 diagnosis as well as for associations with motor and cognitive/behavioral impairments. 
 
STUDY DESIGN:  
This is a prospective single-center; randomized, double-blind, placebo-controlled Phase II study 
to determine the safety and efficacy of NAC on motor behavior and/or attention deficits of children 
with NF1 aged 8 to 16 years. Participants will be randomized to 8-weeks of treatment with NAC 
(70 mg/kg/day) versus placebo. This is a collaborative study between multiple specialties at 
CCHMC and external collaborators at Children’s National Hospital and Technion Israel Institute 
of Technology. We have received IND approval from the FDA to use NAC for the application in 
this study (IND# 139468).  
 
It is plausible and ethical to employ a placebo group, as no standard therapy with established 
efficacy is being withheld. There is no crossover in this study due to a lack of data concerning the 
length of possible washout effects. Primary and secondary outcome measures will be administered 
at baseline, after 8 weeks of treatment and at follow-up, 4 weeks after cessation of treatment to 
determine any washout effects. 
 
In addition, the effect of NAC on measures of impulsivity, attention, executive function, and SICI 
and imaging biomarkers will be assessed. Participants will be carefully monitored for side effects 
by a medical safety monitor for the study. The safety of NAC will be evaluated using laboratory 
tests, clinical signs and adverse effects, which will be monitored at regular intervals over the 8-
week treatment period. 
 

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An additional cohort of age/sex-matched NF1 participants will be recruited in order to characterize 
and validate biomarkers of brain function.  
 
DURATION:  
The current plan is for the study to last for 4 years. Data analysis and completion of study 
reports/papers will occur in the last 6 months of the study. 
 
SELECTION AND RECRUITMENT OF PARTICIPANTS: 
We plan to enroll 58 children ages 8-16 years with NF1 (Figure 16). Recruitment will be primarily 
from the NF1 and RASopathies Programs at CCHMC. The multidisciplinary program at CCHMC 
follows over 1500 individuals with NF1 and over 60% are under 20 years of age. We will also 
recruit from local events (including philanthropic and education events). Advertisements may also 
be posted on the NF1-associated websites including, but not limited to the Children’s Tumor 
Foundation (CTF) website, NF1 facebook group, RASopathy facebook group, et cetera. Physical 
presence of research personnel in the clinic has been minimized in response to the covid-19 
pandemic. Video recordings may aide recruitment and explanation of study assessments/details. 
These recordings may be distributed in a similar manner to other advertisements (NF1-associated 
websites, philanthropic/educational events, through interactions related to clinic visits, et cetera). 
Examples or video content would be a short description of TMS and PANESS procedures. A 
general outline of the study (risks/benefits, procedures, timeline, et cetera) as would be described 
during informed consent would also be advantageous. 
 
An additional “Single visit, non-treatment” cohort will be recruited of 40 individuals with NF1 for 
a single “biomarker” study visit. These individuals will undergo motor function (PANESS) and 
brain-based measures (TMS, MRI-MRS, MRI-DTI) as biomarkers of impaired executive function 
(ADHD-RS; BRIEF-2; TOVA),  but will not be assigned to receive NAC/Placebo. 
 
We will use control data from age matched children ages 8-16 years, from previous studies (cohorts 
of 52 children with ADHD and 62 typically developing children). 
 
We will also use advertising materials approved through the CCHMC Institutional Review Board 
(IRB). These include emails and small flyers which are posted at CCHMC locations. The study 
will be posted in clinicaltrials.gov.  
 
Inclusion criteria ѱ:  
1. Male and females aged 8 - 16 years (up to 16 years and 6 months) at time of enrollment 
2. Meets NIH diagnostic criteria for NF1 
3. Abnormal PANESS study (score at or above the age/sex-based mean) 
4. Participants must have a full-scale intelligence quotient (IQ) of 70 or above, as determined by 
neurocognitive testing within the last 3 years or during the enrollment process 
5. Participants on stimulant or any other psychotropic medication should stay on a stable dose (no 
change in dose) for at least 30 days before entering the study. A stable dose should be maintained 
throughout the study until completion of all study visits. 

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Exclusion criteria:  
1. Participants should not be receiving chemotherapy currently, or have received chemotherapy in 
the 6 months prior to entering the study 
2. Active intracranial lesions (stable low-grade glioma is acceptable) ѱ 
3. History of seizure disorder or epilepsy. History of a single seizure that occurred more than 12 
months prior to enrollment is acceptable. History of febrile seizures if the last febrile seizure 
occurred more than 12 months prior to enrollment is acceptable. Recurrent, unprovoked seizures 
(epilepsy) is sufficient for exclusion. ѱ 
4. Major Depression, Bipolar Disorder, Conduct Disorder, Adjustment Disorder, other major 
Anxiety Disorders, or other developmental psychiatric diagnoses, based on history 
5. For females, pregnancy 
6. Implanted brain stimulator, vagal nerve stimulator, ventriculoperitoneal shunt, cardiac 
pacemaker, or implanted medication port ѱ 
7. Asthma (bronchospasm has been reported as occurring infrequently and unpredictably when 
NAC is used as a mucolytic agent) 
8. High risk of upper gastrointestinal hemorrhage. Examples: presence of esophageal varices or 
peptic ulcers 
9. Current use of MEKINIST (MEK-inhibitor) or use within 30 days 
 
ѱ Indicates Inclusion/Exclusion Criteria for the treatment- and non-treatment cohort (no 
mark indicates exclusion requirements for the treatment-cohort only). 
 
Inclusion of women and minorities: We will include males and females in numbers consistent 
with the distribution expected in the clinic populations. We will not exclude any participant based 
on race or ethnicity. 
 

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Randomization:  
Following 
the 
screening 
period, 
each participant will 
be 
sequentially 
assigned to one of the 
2 treatment groups: 
NAC or placebo. This 
randomization will be 
stratified to ensure 
balance of ADHD, 
ages 
and 
sexes 
between 
the 
intervention groups. 
Randomization will 
be implemented by 
the study statistician 
for this trial utilizing 
a permuted blocks 
approach. Password 
protection 
will 
be 
implemented 
to 
ensure 
that 
only 
certified 
clinical 
personnel are allowed 
to randomize patients 
and 
 
access 
the 
database. At the time 
of randomization, each participant will be assigned a unique medication kit identification number 
that will be used throughout the duration of the study. 
 
Masking Procedure and Labeling:  
In order to preserve the double-masking of the trial, only the investigation pharmacy will be 
unmasked. Both treatments (NAC and placebo) will be distributed from the investigational 
pharmacy. Except for the pharmacist, all staff will be blinded to the treatment sequence. 
 
PROCESS OF OBTAINING INFORMED CONSENT: 
The informed consent process will involve a member of the study team and the parent and child. 
If the individual is recruited over the telephone or by advertisements/mail/email, he or she will be 
sent a consent form and be contacted by a study coordinator to review the details of the study and 
answer questions. Interested families will then undergo screening as described in the Eligibility 
Verification document (a checklist of eligibility criteria). 
 
Prior to study entry, all children recruited will undergo an initial screening lasting up to 15 to 20 
minutes with a parent to determine their general suitability for inclusion or exclusion. This 
screening may be done over several days and can take place in person, on the phone, via email, 
Figure 16: NAC phase II double-blind placebo controlled study design. 

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videoconference, and/or via electronic surveys such as REDCap. If determined to be eligible 
during the initial screen, the patient will enter the second phase of screening as outlined in the 
Eligibility Verification document.  
 
After eligibility criteria are confirmed, the informed consent process will occur. Informed consent 
can take place in person, on the phone, via email, videoconference, and/or via electronic surveys 
such as REDCap. Following the consent process, eligibility screening will occur. The primary 
diagnostic screen will be the PANESS. Clinically administered PANESS scores performed within 
90 days of enrollment can be used to confirm eligibility and as a baseline PANESS score for this 
study. Participants whom have not had a recent (within 90 days) PANESS assessment as a part of 
clinical care will be screened with PANESS. A set of behavioral and neuropsychometric 
assessments will be administered and families will be informed if they are eligible for the next 
phase of the study. The questionnaires are outlined in Table 4.  
 
Parents/participants will deliver a teacher rating scale packet, with questionnaires/rating scales 
targeted to the classroom environment, to their child’s teacher. Teachers will return completed 
rating scales to the study team via US mail using an addressed/stamped envelope provided by the 
study team.  A letter will be included within the teacher packet iindicating that, by completing the 
questionnaires and mailing them in, they provide implicit consent. Communication with teachers 
and distribution of surveys may occur digitally via REDCap, telephone, videoconference, and/or 
email. 
 
NAC/Placebo Treatment 
 
After initial screening (visit 1) and baseline (visit 2, completed only on initial entry to the study) 
there will be approximately two on-site study visits (Table 4), involving approximately 120 
minutes of diagnostic, motor, and neuropsychological assessment, a 90-minute TMS session, and 
60 minutes of actual scan time (total = 5-6 hours). The administration of all interviews and 
cognitive testing will also be standardized with respect to personnel. A third day of testing may be 
added to accommodate the subjects’ schedules and all research evaluations. 
 
Non NAC/Placebo treatment cohort 
 
These participants will undergo the baseline studies (TMS, MRI, motor testing, cognitive 
testing), as described in the prior paragraph, but no further on-site visits. 
 
Consent 
 
A separate consent form will be signed by each participant. Subjects 11 years and older will 
provide documented assent via a signature on the consent form.   
 
Participation in this study will not change current medical treatment. Permission of the primary 
care/attending physician will not be sought.  
 
To minimize the possibility of coercion, families will be encouraged to discuss this study in private 
(without the presence of study staff) and to ask the study staff questions to ensure understanding 

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of the study parameters. The decision to enroll can be considered for the duration of the enrollment 
period (e.g. until recruitment goal is reached). 
 
For minor subjects who are brought to CCHMC by a person other than the parent or Legally 
Authorized Representative (LAR), the consent form may be sent to the parent for review. If so 
this consent process can take place over the phone, via email, videoconference, or REDCap 
eConsent. If the parent or LAR agrees to allow the participant to take part in the study, a copy of 
the signed consent will be returned and reviewed by a study coordinator before any research 
procedures occur. The consent form can be returned to CCHMC by fax, e-mail, regular mail, or 
REDCap eConsent response (which would be documented with a pdf in the participant’s e-
regulatory folder). 
 
Electronic Consent (e-Consent) and Assessments: 
To securely and efficiently maintain the data collected for this study, an electronic (eREG) binder 
will be utilized. As described below, paper regulatory documents will be scanned, verified, and 
filed in the eREG folder. To optimize the eREG process, electronic consent and assessments can 
be utilized. The format of electronic regulatory documents include pdf and/or REDCap. In addition 
to facilitating questionnaire distribution and document management, REDCap will enhance data 
analysis. Digital consent forms and questionnaires can be distributed to the participants/families 
who have reliable access to internet/email. Completion of electronic questionnaires and/or signing 
of e-Consent documents may take place during study visits using CCHMC resources (tablet, 
computer, signature pad) currently available to the study team. 
 
Partial HIPAA Waiver 
To facilitate eligibility screening of potential participants, the study team will implement a partial 
HIPAA waiver to screen, recruit, or determine eligibility of prospective subjects for a research 
study without screening-specific informed consent. As such, protected health information (PHI) 
access/retention for the purposes of identifying potential subjects is allowable. The use or 
disclosure of protected health information involves no more than a minimal risk to the privacy of 
individuals undergoing eligibility screening for this study. Eligibility screening for this study will 
not adversely affect the rights and welfare of the subjects. As with all research participants, security 
of protected health information (PHI) obtained during eligibility screening will be prioritized as 
described below.  
 
PARTICIPANT WITHDRAWAL 
A participant may withdraw from the study at any time. Participants considering withdrawal 
should contact the investigator so that the investigator can help you with a plan to stop the study 
drug safely.  
 
The person in charge of the research study or the sponsor can remove participants from the research 
study without their approval. Possible reasons for removal include: 
1. Adverse events requiring removal from protocol therapy 
2. Inability or refusal to continue the study by patient/parent/guardian 
3. Physician determines it is in patient’s best interest 
 

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In the case of participant withdrawal/removal, data already collected may not be removed from 
the study database.  The study team will request permission to collect data from your routine 
medical care. If the participant agrees, this data will be handled the same as research data.  
 
The study team will inform the participant about any new information that may affect health, 
welfare, or choice to stay in the research. 
 
STUDY PROCEDURES: 
Study Variables and Measurements - Overview: 
We propose to study all measurements at baseline, 8 weeks after treatment, and 4-week post-
treatment (washout period) to assess for carry-over effects. We will compare PANESS and SICI 
measures in 58 children with NF1 to data already acquired at CCHMC for those 2 measures 
(Figure 14). Next, these children will be treated with NAC (n=29) or placebo (n=29) as described 
above (Figure 16). 
 
Treatment with NAC:   
For this single center randomized double-blind placebo controlled, prospective, Phase II study, each 
subject will be dosed with approximately 70 mg/kg/day of NAC for 8 weeks. To facilitate drug 
compounding, three tiers of drug dose will be administered based on body weight as described in 
Table 3. 
 
Table 3: NAC Dosing 
Participant’s weight (kg) 
Dose (BID) 
< 20 
700 mg 
20-40 
1050 mg 
> 40 
1350 mg 
*Max dose not to exceed 2700mg/day (1350mg BID) 
 
We have treated 5 patients off-label with NAC at 900 mg twice daily with improved behaviors 
without side effects. NAC is a natural supplement that acts as an antioxidant and a glutamate 
modulating agent. It is widely available over the counter. We have received IND approval from the 
FDA to use NAC for the application in this study (IND# 139468). 
 
To mitigate potential non-compliance, participants will be given a ‘test dose’ of the study  placebo 
(capsule and/or powder) at the screening visit. If prescribed encapsulated drug, they will have to 
demonstrate that they can swallow the capsule. If prescribed powder, they will have to demonstrate 
that they can drink the powder without any issues. 
 
Blood Acquisition and Storage: 
Blood will be collected per institutional practice after participants are recruited and consented. 
No more than 3 ml of blood per kg of body weight will be collected during any single blood 
draw. No more than 5% of the patient’s estimated blood volume will be taken for both clinically 
indicated and research purposes within a 24-hour time period. Samples will be logged and stored 
in the Cincinnati Children’s Biobank. 
 

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Collection/use/disclosure of video recordings.  
Certain study assessments (i.e.: PANESS) will be recorded using a tripod mounted video camera 
or similar device. The recorded assessment will be reviewed by multiple trained personnel to 
standardize grading across participants and improve the precision and accuracy of the scores. 
These records may be used for purposes of study, research, and teaching. If a clinically significant 
phenotype is noted in the recording that would benefit the scientific and clinical communities, 
portions of the recorded assessment may be published in scientific journals or presented at 
scientific conferences. The participant’s/family’s name will not be used. Information about the 
intention to record a portion of the study visits will be outlined in the consent form. Authorization 
is given when the consent form is signed by the participant/family. 
 
Physical and Neurological Examination for Subtle Signs (PANESS) scale ѱ: 
At present, there are no clinical characteristics, neuropsychologic variables, phenotypic variants 
or biomarkers known to accurately predict treatment response of abnormal motor/learning 
behaviors in NF1 to NAC therapy. 
 
Therefore, the primary outcome of the proposed study is motor function rated with the PANESS, 
a validated scale that consistently demonstrates significant impairments in children with ADHD, 
and which our preliminary data suggest may demonstrate more extreme problems in children with 
NF1. 
 
The PANESS test measures timed movements, lateral preference, motor overflow, dysrhythmia, 
coordination, gait, balance, and motor persistence. The PANESS will be administered by staff, 
which has already been trained in PANESS administration. Our team has performed this 
examination as part of multiple studies. Administration of this measure is approximately 30 
minutes.  
 
When possible, the PANESS assessment will be recorded using a device such as a tri-pod mounted 
video camera. Digital files will be saved to  a secure network drive to protect participant privacy. 
PANESS recordings can be reviewed by multiple personnel to improve precision and accuracy of 
the assessment and ensure reliability of the scores. 
 
We will correlate the PANESS measurements with ADHD/behavioral symptoms using well 
established neuropsychological assessments. Thus, we will assess ADHD attention and 
hyperactive/impulsive symptoms with the DuPaul, BRIEF-2, and TOVA clinical rating tests. 
Parent symptom ratings are precise and appear to be quite sensitive to change and capture real-
world functional change. 
 
Psychometric Measures 
All primary measures will first be administered at screening. We will use the Response Evaluation 
in Neurofibromatosis and Schwannomatosis (REiNS) Neurocognitive committee reviewed and 
recommended assessment tools to assess symptomatology in NF1. The length of time required 
for the child to complete the baseline neuropsychological assessment is estimated to be 
approximately 90 minutes, including self-report questionnaires. Parent questionnaires can be 
completed simultaneously while the child is being evaluated and can be completed in about 30 

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minutes. All neuropsychological assessments that will be used in this study are published tests that 
have normative data and established reliability and validity for the age range in this study. These 
tests are all extensively used in clinical and/or research settings. All tests have standardized 
instructions for administration ensuring there will be no site variation in administration.  
To minimize any test-retest effects for neuropsychological outcome measures, alternate forms will 
be used where possible, to minimize potential practice effects. Furthermore, the use of a placebo 
group will allow us to identify any potential practice effects and distinguish whether any changes 
in the treatment group are larger than those accounted by practice effects. In addition, statistical 
analysis of the data will be conducted to account for confounding variables such as practice effects. 
 
The WISC-V testing is optional for individuals who will not be receiving NAC/Placebo. However, 
the PANESS and the neuropsychometric testing below will be performed in all “single-visit, non-
treatment” participants. 
 
DuPaul ADHD-Rating Scale,(ADHD-RS) Home (parent) form ѱ 
This 18 question scale is based on the DSM-5 diagnostic criteria and is widely used in clinical 
trials and routine care. The ADHD-RS rated high in the REiNS committee for assessment of 
ADHD symptomatology in NF1. Administration of this measure is approximately 15 minutes. The 
ADHD-RS has high internal consistency (alpha>0.90) and good test-retest reliability (r=0.85). The 
rating scale has excellent convergent and discriminant validity. 
 
Behavioral Rating Inventory of Executive Function Second Edition (BRIEF-2) ѱ 
The Behavioral Rating Inventory of Executive Function (BRIEF-2) is a parent-reported 
symptom rating form that is used regularly in NF1 research and is recommended by the REiNS 
Neurocognitive Committee for the assessment of executive functions in NF clinical trials. This 
assessment measures executive function behaviors at home and at school for children and 
adolescents (age 5–18). This scale would give a sense of if any changes are occurring in real-world 
executive function. The Parent Form is completed by the child’s parent or guardian. It contains 86 
items, with eight clinical scales and two validity scales. Behavior descriptors of a child are rated 
on a three-point Likert scale: “never,” “sometimes,” “always.” Items were developed to capture 
everyday behaviors associated with executive functions, and tap eight domains including: initiate, 
inhibit, shift, plan, organize, self-monitor, emotional control, and working memory. These scales 
are combined to form two broader indices (Behavioral Regulation and Metacognition) and a 
Global Executive Composite score. Administration of this measure is approximately 15 minutes. 
The BRIEF-2 has high internal consistency, with a reliability coefficient of 0.90, and good test-
retest reliability (r=0.82). This measure has well established convergent validity with other 
measures of behavior and intelligence, and good discriminant validity has been demonstrated 
against measures of emotional and behavioral functioning. 
 
Test of Variables of Attention (TOVA) ѱ 
The TOVA is a computerized, objective measure of attention and inhibitory control normed by 
gender for ages 4 to 80+. The test is a 22-minute test administered to the participant that provides 
objective data on response time, inattention, and impulsivity. Test-retest reliability of the TOVA 
is good, ranging from .74 - .87 with a 1-week interval [66]. The TOVA has also demonstrated 
good convergent and discriminant validity. 

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Wechsler Intelligence Scale for Children, Fifth Edition (WISC-V) (ѱ optional) 
Working Memory Index. This index of the WISC-V consists of two scales – Digit Span and Picture 
Span, which assess auditory and visual working memory in school-aged children. This objective 
measure of working memory has been recommended by the REiNS Neurocognitive Committee 
(specifically Digit Span) as an outcome measure for working memory in NF clinical trials. This 
is a standardized assessment tool used very frequently in clinical care and clinical research. 
Administration takes approximately 10 minutes and produces age-referenced standardized scores 
for each subtest as well as the Index score.  
 
Processing Speed Index. This index of the WISC-V consists of two scales – Coding and Symbol 
Search, which assess processing and response speed in school-aged children. This is a standardized 
assessment tool used very frequently in clinical care and clinical research. Administration takes 
approximately 10 minutes and produces age-referenced standardized scores for each subtest as 
well as the Index score. 
 
The WISC-V has high internal consistency, with reliability coefficients for subtests, primary index 
scores, and FSIQ ranging from 0.80 to 0.96. It also has good test-retest reliability, with coefficients 
ranging from 0.80 to 0.94 on subtests, primary indexes, and FSIQ. The WISC-V has high criterion 
validity and good convergent and discriminant validity. 
 
We will provide a referral for clinical evaluation to potential participants that receive an 
ineligible IQ rating (<70) 
Cognitive Function: Kaufman Brief Intelligence Test, Second Edition (K-BIT-2) ѱ 
Each child will be administered a psychological screening battery by trained researchers under the 
supervision of a licensed psychologist, to assist in evaluation of inclusion and exclusion criteria, 
and to describe the sample. Within the constraints of limited time and resources, we have selected 
the following test to evaluate cognitive functioning and learning problems. Participants will 
undergo a brief IQ screener at baseline using the Kaufman Brief Intelligence Test-2 (KBIT2). The 
K-BIT-2 has high internal consistency (a ranging from 0.89 to 0.96) and composite test-retest 
reliability (r=0.90). The measure also has moderate to high correlations for construct and 
concurrent validity. 
 
We will provide a referral for clinical evaluation to potential participants that have incidental 
findings noted in the MRI/MRS 
The Imaging Research Core (IRC) at Cincinnati Children’s Hospital will assign a radiologist to 
review MRI/MRS data and create a report to outline any incidental findings. The incidental finding 
report is essentially a safety report where the radiologist documents if there are abnormalities 
separate from their underlying diagnosis. Upon notification of incidental findings, the study team 
will review the report within 3 business days. The study team will determine whether a clinical 
referral and/or correspondence with the family is necessary. If the study team determines that the 
family should be notified (with or without the need for a clinical referral), a member of the study 
team will contact the family to schedule a call with the study doctor or designee. 
 

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At the study doctor’s discretion, we may provide reports or referrals for clinical evaluation to 
families regarding anxiety and depression assessments 
Pediatric Anxiety Rating Scale (PARS) [21] 
The PARS is a clinician rated scale that is completed following interview with the parent and child. 
The PARS has two sections, a symptom checklist and severity items, each of which is linked to 
the past week. We chose PARS over several possible self-report scales because young children 
may not be able to complete a self-report scale. Administration of this measure is approximately 
10 minutes. The PARS has adequate test-retest reliability (ICC=0.55) and fair internal consistency 
(a=0.64). This scale also has adequate convergent and divergent validity. 
 
Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS) 
The KSADS is compatible with the DSM-5 and has been validated in multiple research and 
treatment settings. The KSADS cross-cutting measure (DSM-5 CC-SM) is an abbreviated version 
of the extended form. The abbreviated measure is a reliable way to recognize mood, anxiety, 
psychotic, and disruptive behavior disorders and is a useful screen for eligibility of potential 
participants. The DSM-5 CC-SM are designed to be self-report measures, completed by the parent 
and child.  The assessment typically is completed in 10 minutes.  
 
Children’s Depression Rating Scale (CDRS) [22] 
The CDRS is a clinician-completed scale that rates presence and severity of depressive symptoms, 
based on interviews with the parent and child separately. There are 17 behavior items of affective 
state, rated dimensionally normed for children 6-12 years. Administration of this measure is 
approximately 10 minutes. The CDRS has good test-retest reliability (a=0.86). The scale has a 
good validity correlation (0.87) and high convergent validity. 
 
Suicide assessment: 
Per FDA guidance we will screen for Suicidal Ideation and Behavior at each visit. The Columbia 
Suicide Severity Rating Scale (C-SSRS) is a fast assessment of current and past suicide ideation 
that is used throughout Cincinnati Children's Hospital. The C-SSRS is administered by a trained 
researcher and takes approximately 5 minutes to complete. 
 
Review of anxiety, depression, and C-SSRS responses and referral for clinical evaluation: 
Study doctor (or designee) will verbally administer and then review each suicide assessment. The 
‘Columbia-Suicide Severity Rating Scale Screen with Triage Points’ document outlines the 
planned course of action based on the participant’s response. In particular, if a participant/family 
discloses any suicide ideation at any point in this study, the study staff will contact the participant’s 
mental health provider or primary care provider and/or Psychiatric Intake Response Center (PIRC) 
at CCHMC for further assessment and guidance within 24 hours. 
 
The Pediatric Intake Response Center (PIRC) manages psychiatric patient consultation 
and intake for Cincinnati Children’s Hospital Medical Center. Staff members are 
available 24 hours a day, seven days a week at 513-636-4124 or 
psychiatryresponse@cchmc.org . 
 

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Rationale for Imaging and Neurophysiology Studies – Overview: 
As an additional secondary endpoint, we will use TMS and DTI, because quantitative biomarkers 
of white matter structures may help measure and predict behavior in NF1 and facilitate 
interventions into clinical practice. DTI, including at least one of its derived parameters, FA, 
shows global changes in NF1 brains which are consistent with white matter dysfunction [11]. In 
other brain disorders, DTI indices have shown sensitivity to changes after treatment [67-69]. It is 
clear that white matter properties can also be evaluated using neurophysiological techniques, 
including Transcranial Magnetic Stimulation. TMS studies are generally brief (30 to 60 minutes), 
inexpensive, readily repeatable, and do not require sedation. TMS may be helpful for identifying 
clinically useful, specific brain biomarkers and assessments related to motor and learning 
difficulties in NF1 and, ultimately, other conditions involving RAS pathways. Specifically, we 
will test motor system inhibitory physiology (SICI), measured using TMS. Preliminary measures 
in our NF1 population show abnormalities similar to established findings in ADHD obtained in 
our TMS laboratory at CCHMC. 
 
Transcranial Magnetic Stimulation (TMS) ѱ:  
Paired pulse TMS will be performed using a Magstim 200R stimulator (Magstim Co., New York, 
NY, USA) connected through a BistimR module to a 90 mm circular coil. The coil is placed with 
its center near the vertex in the optimal position and orientation for producing a motor evoked 
potential (MEP) in the right first dorsal interosseous (FDI) muscle. The electromyogram (EMG) 
is recorded from the right (or dominant) FDI with surface electrodes, amplified, and filtered 
(100/1000 Hz) (Coulbourn Instruments, Allentown, PA) before being digitized at 2 kHz and stored 
for analysis using SignalR software and a Micro1401 interface (Cambridge Electronic Design, 
Cambridge, UK). 
 
The resting MEP threshold (RMT) is measured using a method similar to that described elsewhere 
[70] by setting the stimulator to 20 % of maximum output and increasing by 10 % increments until 
a MEP is obtained. The intensity is then decreased in increments of 1 % of maximum stimulator 
output until an intensity is reached where 10 averaged stimuli failed to produce a MEP. Active 
MEP threshold (AMT) is determined by having the participant moderately contract the abductor 
pollicis brevis with auditory feedback to maintain a constant level of EMG. The stimulator output 
is decreased in increments of 1 % of maximum output from the RMT until 10 averaged stimuli fail 
to show a MEP above background. 
 
SICI inhibition is measured in resting muscle with a paired-pulse paradigm using paired vs. single 
pulse conditions[71]. The first condition is a single test pulse, delivered at an intensity that 
consistently produces a 500- 1500 mV MEP. In the second condition, a subthreshold stimulus 
(60% of RMT) is followed after by a test pulse. Twenty trials are performed for the single-pulse 
condition, and 20 each for the other interstimulus intervals 3 milliseconds (ms), 10 ms. Relative to 
the single pulse response, the 3 ms interval is inhibitory (SICI) and the 10 ms interval is excitatory 
(intracortical facilitation). The order of the intervals is varied randomly, and the interval between 
trials varies randomly by <20% around a mean of 6 seconds. SICI is expressed as a ratio of the 
mean MEP amplitude after the conditioned pulses divided by the mean MEP amplitude after the 
single pulses. A detailed review of systems will be obtained before and after TMS, and participants 
will be provided the number of the investigator and study coordinator to call if there are concerns 
about possible side effects after the laboratory visit. 

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Magnetic Resonance Imaging (MRI) ѱ:  
The MRI protocol will be comprised of un-sedated imaging sequences providing high-resolution 
detail of brain tissue structure with a primary focus on diffusion FA. A three-dimensional T1-
weighted volume will be acquired with 1 mm isotropic resolution and strong contrast between gray 
matter and white matter tissue. This image volume will be used for anatomic reference and may 
also serve to explore voxel-level volumetric impact of NF1. A DTI sequence (≤ 2 mm isotropic 
resolution) will follow, which is directionally sensitive to the rate of water diffusion in brain tissue. 
This sequence will be designed to measure diffusion rates in 32 or more directions for two or more 
levels of diffusion weighting (e.g., b = 1000 s/mm2 and 2000 s/mm2) to allow voxel-wise 
determination of not only FA, our primary DTI marker, but also additional microstructural indices 
that may be sensitive to NF1 and changes with NAC [72, 73]. The DTI data are unique in this 
study’s context in that they are brain-wide: this assessment will be done per voxel, or, alternatively, 
per predefined white matter Region of Interest (ROI).  These additional indices, as well as the 
potential for regional volumetric assessments, will be exploratory and allow us to address 
contingencies. DTI will be especially focused on white matter, where restricted diffusion of axonal 
structures leads to a high degree of anisotropy that reflects tissue integrity.  
 
The T1-weighted image will be processed using one of the commonly available software packages 
for MRI. Processing will include segmentation into gray matter, white matter, and cerebrospinal 
fluid tissue types and normalized by transformation into a common spatial template [74]. DTI 
volumes will have been aligned to each other and with the T1 anatomic image and normalized to 
the same template space before processing in software to extract FA on a voxel-wise basis. In 
healthy white matter, diffusion will be greatest along axonal fibers and FA describes the degree to 
which axial diffusion exceeds transverse diffusion. Besides FA, this processing will provide 
estimates of mean diffusivity (MD) and the radial (RD) and axial (AD) diffusivities, which have 
shown changes with NF1 [11, 75, 76] and its treatment [54]. Increased FA with decreased RD 
might suggest improvement in myelin. Images may be processed in other ways to extract 
additional structural information. The T1 segmentations may be analyzed as tissue probability 
maps for voxel-based morphometry [77]. The DTI data may allow axonal fiber tractography to 
ascertain structural connectivity between brain regions pertinent to NF1, including tracts of the 
cingulum and corpus callosum. Our primary imaging outcomes in white matter will be voxel-wise 
comparison of FA between NAC and Placebo groups and regional correlation between FA and 
neuropsychometric outcomes pre and post-NAC. We have healthy reference DTI (FA) data for 
children 8 to 16 years for comparison to NF1. Since all images will have been normalized to the 
same space, voxel-wise comparisons of indices across subjects will be possible. 
 
Magnetic Resonance Spectroscopy (MRS) ѱ:  
We will use MRS to quantify glutathione (GSH) and GABA levels in children with NF1 to assess 
effects of NAC therapy. This will allow for regional correlation between MRS and 
neuropsychometric outcomes. The HERMES MRS sequence (echo time 68 ms, repetition time 
2000 ms) requires 11 minutes per 27 mL location of interest sampled in the brain. MRS is 
performed within the medial frontal cortex including dACC [78, 79]. MRS detects neurochemical 
concentrations within key neural elements (neurons, axons, glia) reflecting structural composition 
such as N-acetyl aspartate, creatine with phosphocreatine, total choline, and myo-inositol and 
functioning with glutamate, GSH and GABA. The HERMES method provides GSH, GABA and 

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GLX; the GABA-off and GSH-off portion of the HERMES experiment provides a spectrum for 
standard metabolites. MRS data are postprocessed with LCModel and Gannet software and 
adjusted for tissue composition and neurochemical relaxation, to produce concentrations. 
 
 
Table 4: Schedule of Assessments 
 
Visit 1 
Visit 2 
 
 
 
Visit 3 
 
Visit 4 
 
Screenin
g 
Baseline 
Abbreviated 
Remote 
monitor and 
safety 
Remote 
monitor 
and safety 
Abbreviated 
Remote 
monitor and 
safety 
End 
Intervention 
Remote 
monitor 
and safety 
Follow Up 
Task 
Week 0 
Week 1 
Week 2 
Week 4 
Week 6 
Week 8 
Week 10 
Week 12 
Review Eligibility Criteria ѱ 
X 
 
 
 
 
 
 
 
KBIT-2 (Kaufman Breif Intelligence 
Test, Second Edition) ѱ 
X 
 
 
 
 
 
 
 
KSADS (MedHis) 
X 
 
 
 
 
 
 
 
Cognitive Function Screening ѱ 
X 
 
 
 
 
 
 
 
Test dose 
X 
 
 
 
 
 
 
 
Informed Consent ѱ 
X 
 
 
 
 
 
 
 
Brain Imaging (MRI/MRS) ѱ 
 
X 
 
 
 
X 
 
X 
Urine Pregnancy (Females only) 
X 
X 
 
 
 
 
 
 
Vitals* 
 
X 
 
 
 
X 
 
X 
Safety labs*  
 
X 
 
 
 
X 
 
X 
Physical Exam* 
 
X 
 
 
 
X 
 
X 
ADHD-RS (DuPaul) ѱ 
 
X 
 
X 
 
X 
X 
X 
KSADS (CC) 
 
X 
 
X 
 
X 
X 
X 
PARS (Pediatric anxiety rating scale) 
 
X 
 
X 
 
X 
X 
X 
CDRS (Children’s depression rating 
scale) 
X 
X 
 
X 
 
X 
X 
X 
C-SSRS (Columbia Suicide Severity 
Rating Scale) 
X 
X 
 
X 
 
X 
X 
X 
Wechsler Intelligence Scale for 
Children, Fifth Edition (WISC-V) ѱ 
X 
X 
 
 
 
X 
 
X 
Behavioral Rating Inventory of 
Executive Function (BRIEF-2)  ѱ 
 
X 
 
 
 
X 
 
X 
Test of Variables of Attention 
(TOVA) ѱ 
 
X 
 
 
 
X 
 
X 
PANESS scale ѱ 
 
X 
 
 
 
X 
 
X 
TMS assessment ѱ 
 
X 
 
 
 
 
X 
 
 
X 
NAC/Placebo Prescription 
 
X 
 
 
 
 
 
 
Med Log 
 
X 
X 
X 
X 
X 
X 
X 
AE assessment (MedDRA) 
 
X 
X 
X 
X 
X 
X 
X 
Follow-up plan 
 
 
 
 
 
 
 
X 
 * Indicates clinical procedures performed as standard of care 
 ѱ Indicates research procedures to be completed in both treatment and non-treatment cohorts 
 
DATA ANALYSIS/METHODS: 
NAC Trial at CCHMC 

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We propose performing a single center randomized double-blind placebo controlled, prospective, 
Phase II study to explore safety, tolerability, and efficacy of NAC on motor behavior and/or 
attention deficit in children with NF1 aged 8 through 16 years old. 
 
Determination of sample size:  
No data exist on the effects of NAC in children with NF1. Clinical improvement will be evaluated 
as a change score (PANESSpost-NAC – PANESSbaseline). Those subjects whose severity PANESS 
score decreased by at least 20% from baseline to week 8 will be considered clinically meaningful. 
The sample size calculation was based on a two-sided t-test to detect the difference in change 
scores between two groups. We assume the PANESS score does not change for patients in placebo 
group. A sample size of 26 participants per group (52 in total) achieves 81% power to detect an 
effect size of 0.8 with significance level of 0.05 [80]. To allow for a 10% dropout rate prior to the 
12-week point, a total of 58 participants will be enrolled. 
 
Randomization:  
Following screening period, each participant will be sequentially assigned to one of the 2 treatment 
groups: NAC or placebo. This randomization will be stratified to ensure balance of sexes and 
ADHD between the intervention groups. Randomization will be implemented by the study 
statistician for this trial utilizing a permuted blocks approach. Password protection will be 
implemented to ensure that only certified clinical personnel are allowed to randomize patients and 
access the database. At the time of randomization, each participant will be assigned a unique 
medication kit identification number that will be used throughout the duration of the study. 
 
Statistical analysis:  
All analysis will be performed with SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). 
Spearman’s correlation coefficient will be used to evaluate the univariate association between 
primary outcome (improvement of PANESS scores) and other neurophysiology and brain imaging 
measurements. Multivariate linear regression model will be used to assess the correlation between 
the outcomes and the risk factors adjusting for all possible confounders. Model selection criterions 
such as stepwise method will be employed when necessary. A p-value less than 0.05 will be 
considered statistically significant. 
 
1. We will assess behavior vs. neurophysiology correlations:  
1) two-month PANESS change (PANESSpost-NAC–PANESSbaseline) vs. SICI change (SICIpost-
NAC – SICIbaseline).  
2) two-month DuPaul change (DuPaulpost-NAC–DuPaulbaseline) vs. SICI change (SICIpost-NAC – 
SICIbaseline).  
3) two-month BRIEF change (BRIEFpost-NAC–BRIEFbaseline) vs. SICI change (SICIpost-NAC – 
SICIbaseline). Paired t test comparisons of single pulse TMS responses (resulting from pTMS) 
will be performed to test whether, at each session, single pulse TMS responses differed. The 
mean single pulse TMS responses for each individual constitute the denominators for the SICI 
and ICF ratios and need to remain consistent for changes in SICI to be interpretable. 
2. We will assess primary behavior (PANESS) vs. neuroimaging correlations:  
1) two-month PANESS change (PANESSpost-NAC – PANESSbaseline) vs. Fraction Anisotropy 
(FA) change (FApost-NAC – FAbaseline).  

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2) twomonth PANESS change (PANESSpost-NAC – PANESSbaseline) vs. MRS GSH change 
(GSHpost-NAC – GSHbaseline).  
3) two-month PANESS change (PANESSpost-NAC – PANESSbaseline) vs. MRS GABA change 
(GABApost-NAC – GABAbaseline). The current HERMES method data analysis of MRS can 
observe GSH concentration changes in the brain of over 20%. We will determine if NAC dose 
changes GSH in brain and correlates with the primary outcome of study (PANESS). 
 
For analyses of change over time, mixed models which include subject-specific random effects 
will be used to account for the correlation of multiple measurements on each subject and capture 
between-subject variation in rates of change. Unless otherwise noted, the Type I error rate for 
hypothesis tests and confidence intervals will be controlled at a=0.05. To assist interpretation, the 
size of effects of interest in each model will be represented by R2 (proportion of outcome variation 
explained by predictors) for linear regression models and/or Cohen’s d (difference in group means, 
in standard deviation units) for two sample t-tests [81]. 
 
Exploratory and descriptive data analysis will precede all inferential analyses to ensure that 
distributional assumptions are satisfied. In addition to computation of basic descriptive sample 
statistics (measures of central tendency, variability, and bivariate association), graphical tools such 
as histograms and scatterplots will be used to assess, e.g., approximate normality, linearity, and 
presence of influential outlying observations. If substantial violations of parametric assumptions 
are detected, we will implement non-parametric alternatives, including classical rank-based 
procedures as well as computationally-intensive sample re-use procedures such as the 
nonparametric bootstrap. Non-parametric procedures generally have less power than parametric 
procedures when the assumptions of the latter are met, but often have markedly better properties 
when the assumptions fail. 
 
In the event that a participant is lost to follow-up, an incomplete series of observations can still be 
analyzed using the mixed-model methodology, provided that the dropout can be assumed to be 
missing at random in the sense that the probability that data are missing depends only on observed 
covariates (e.g., prior values of the outcomes, treatment assignment), rather than the unobserved 
values themselves. This assumption cannot be directly verified from the data. When it holds, 
however, mixed-model procedures yield the most efficient unbiased estimates, and implicitly 
impute the missing data using a subject-specific linear trend estimated from the non-missing 
observations. This effectively assumes that observed trends would have continued, in contrast to 
the “last observation carried forward” assumption that future observation would remain at a 
constant level equal to the last value. Neither assumption can be empirically proven correct. As a 
sensitivity analysis, therefore, the last available observation will be substituted for the protocol-
defined last observation, provided that the participant had received at least one dose of study 
medication prior to the last observation. It is possible to determine whether participants who drop 
out are systematically different from those who do not with respect to observed data (e.g., baseline 
values, treatment assignment, demographic and clinical covariates). Logistic regression analyses 
will be used to explore whether such variables are predictive of dropout. 
 
FACILITIES and PERFORMANCE SITES:  
Cincinnati Children’s Hospital Medical Center 
1) Division of Human Genetics  

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2) Division of Neurology; TMS laboratory 
3) Imaging Research Center 
 
External Collaborators (data analysis): 
1) Neuropsychology, Children’s National Hospital 
2) Neuroscience, Technion – Israel Institute of Technology 
 
POTENTIAL BENEFITS: 
The purpose of this study is to evaluate whether participants with NF1 will benefit from improved 
thinking/learning, behavior, and movement in response to eight (8) weeks of treatment with N-
Acetylcysteine (NAC).  There is a potential for direct benefit by participating in this study if the 
treatment improves the symptoms of motor behaviors and learning associated with NF1. However, 
there is a possibility of no direct benefit to the participant. 
 
In addition to possible benefits of the study drug, medical records and/or study assessments will 
be reviewed by our multidisciplinary team that includes geneticists, neurologists, 
neuropsychologists, and radiologists. Any findings that may possibly impact the participant’s 
health and well-being will be reviewed with the parent/caregiver and appropriate referrals for 
further testing or management will be made by a physician on the study team.  Returned results 
will be based solely on the baseline assessment, when the most complete information is available, 
and when any practice effects have not yet occurred that could distort norm-referenced 
interpretations. This will be of use to help tailor other interventions to individual participants.  
 
When we finish the research, we expect that we will know more about NF1 and the associated 
cognitive (thinking) and motor (movement) concerns. This may help participants in this study as 
well as other children with NF1.  
 
POTENTIAL RISKS, DISCOMFORTS, INCONVENIENCES AND PRECAUTIONS: 
Known and potential discomforts or hazards of single and paired pulse TMS: 
Single and paired pulse TMS has been used at CCHMC under Dr. Gilbert’s direction since 2001 
for research only. Potential discomforts are mild and transient. In a prior study of 40 healthy and 
ADHD children, Garvey et al asked children to rank TMS compared to other childhood activities. 
TMS was ranked preferable to 1) a “shot”; 2) going to the dentist; and 3) a long car ride. The 
following mild, transient effects were reported in our prior study of 35 children and adults: scalp 
discomfort (12%), hand weakness (9%), headache, neck pain, arm pain, and arm tingling (6%), 
hand pain, decreased hand dexterity, hearing changes, and tiredness (3%). All of these had resolved 
by the following day. There were no physical findings after TMS supporting the subjective 
descriptions of loss of strength or dexterity. A prior common concern about use of TMS was the 
risk of seizures [28]. We follow recommended guidelines [28] and have seen no seizures in 
children or adults studied at our center. In addition, more recent studies even in children with 
epilepsy suggest that the risk of TMS inducing seizures is extremely low.  
Known and potential discomforts or hazards of MRI/MRS: 
There are no known risks injury associated with a brain MRI/MRS. A few common less serious 
risks have been reported and are described here. Participants susceptible to claustrophobia may 
experience anxiety during the scan. The noise generated by the MRI/MRS machine can be 

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uncomfortable for some participants. Ear protection will be available to minimize the noise 
exposure. Participants may experience mild muscle/nerve stimulation that may feel like a twitching 
sensation. 
Participants with a medical implant or external accessory device may experience complications 
with that device while undergoing an MRI/MRS. One potential complication is that the device 
may move in response to the magnet. Implants, external and accessory devices may heat up in 
response to the magnet, which could lead to burns. A third potential complication is the potential 
that electrochromic implants, external and accessory devices may malfunction in response to the 
magnet. To mitigate the risk of harm, we have included the use of such implants/devices as an 
exclusion factor for this study. 
 
Known and potential discomforts or hazards of NAC 
NAC is FDA approved for treatment of acetaminophen overdose, respiratory disease due to 
mucous obstruction in acute and chronic settings in children and adolescent. Common side effects 
include pruritus, rash, urticaria, diarrhea, nausea, and vomiting. NAC may not be effective for 
every participant, therefore participants may experience a continuance or worsening of their 
condition, including school performance or social interaction, while in the study.  
 
Precautions, risk minimization: 
The protocols include a number of precautions to minimize risk.  Standard of care will be followed 
for all procedures performed to decrease the risk of infection, pain, bleeding, and bruising.  The 
TMS laboratory was established in 2001 in consultation with Dr. Eric Wassermann, an 
internationally known researcher in transcranial magnetic stimulation. A trained and TMS-
administration certified neurologist will be present during TMS. All subjects will wear 34 NRR 
earplugs or headphones during TMS if the head is stimulated at an intensity of over 90%. Standard 
exclusion criteria are applied for participation in this study. Detailed questioning for any adverse 
events will occur after the experiment and the next day after the study. Access to the Magstim 
Stimulator is Limited – these TMS devices are kept in room 4 of the EEG lab on the seventh floor 
of the A tower. The laboratory is continually monitored by CCHMC during business hours and is 
locked at all other times. 
 
The effects the study drug and procedures would have on a fetus are unknown. When deemed 
appropriate by the study doctor or designee, pregnancy testing will be utilized before study 
procedures will take place. Females that test positive for pregnancy will be excluded or withdrawn 
from the study. Risks to a fetus conceived while on study drug will be reviewed with all parents. 
A conversation about risks and the importance of contraception while in this study will also be 
conducted with participants when deemed appropriate by the study doctor or designee.  Consent 
to participate in this study includes promise of compliance with appropriate contraceptive 
measures. Females will use an effective means of birth control (i.e. abstinence, hormonal, or 
intrauterine device) during the study and for 30 days after taking the last dose of study drug. Males 
will use an effective means of birth control (i.e. abstinence or double-barrier contraception) during 
the study and for 90 days (3 months) after taking the last dose of study drug. 
 
The method of monitoring study conduct:  

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Adverse events will be reviewed using direct questions with a detailed review of systems on the 
day of the study. The principal investigator or designee is responsible for reporting adverse events 
to Cincinnati Children's Hospital Medical Center Institutional Review Board. Unanticipated events 
will be reported to both IRBs, according to the individual IRBs’ requirements. 
 
DoD representatives can independently review and inspect research associated with this study. 
They can prohibit research that is determined to present unacceptable hazards or is non-compliant 
with DoD regulatory requirements. 
 
Methods for maintaining data quality and confidentiality:  
Data are maintained in case report folders identified only by an anonymous subject ID. They will 
be kept in a secured area of Cincinnati Children’s Hospital. All data kept in computerized files are 
in computers or a server with restricted, password-protected access. Only the primary investigator 
and designated study staff within his laboratory have access to case report folders and computer 
passwords (restricted by access level). A participant will be identified throughout the central 
database by his or her unique subject identification number (SID).  Information which could 
identify a subject, such as name, address or social security number, will either not be stored 
electronically. A database audit trail of all data element changes over the life of the study will be 
maintained.  
 
Data will be kept in a separate data base without demographic and clinical information and will be 
merged during the analytic stage of the study. Data entry, verification and validation will be carried 
out using our proprietary clinical data management system.  We have a fire-wall-protected and 
local-network-based database management system for paper-based data collection, reports 
processing and information handling.  
 
The study PI is responsible for overseeing execution of the research. Oversight responsibilities 
apply to all human subjects’ research, whether or not it is determined to be exempt from the 
regulations. 
 
RISK/BENEFIT ANALYSIS: 
In the opinion of the investigative team, this study, based on our experience with NAC and with 
administering TMS as well as the published literature on NAC and TMS, involves minimal risk, 
with potential for direct benefit.  
DATA SAFETY AND MONITORING: 
Data Safety Monitoring Plan:  
Steps to be taken to assure the accuracy of the study data: The study team will review case report 
forms, source data, and spreadsheet entries once annually to ensure accuracy of data entry.  
 
Adverse events and unanticipated events will be classified based on severity and attributed to 
individual components of study participation. For example, if a subject reports nausea at the end 
of the TMS baseline visit, this will be classified based on severity rating from the subject (mild, 
moderate, severe in the case report forms) and relationship to study tasks will be ascertained as 
well as possible (related to TMS, related to study medication, related to both, related to neither, 
unsure) on a case report form. 

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An external safety monitor will not be utilized for this minimal risk study. The study is deemed 
minimal risk because of the unrestricted availability of the study drug.  
 
PRIVACY AND CONFIDENTIALITY: 
Privacy of the individuals participating in this proposed study will be maintained through non-
identifying subject ID codes (SID), locked storage and password protected files.  External 
collaborators will have access to de-identified research records only.  Distribution of participant 
medical records to external collaborators will be done via secure email transmissions.  The study 
log will be maintained in a password protected folder on the desktop computer of the principal 
investigator.   Case reports and spreadsheets will refer to subjects by number only.  Subjects will 
be informed that, if necessary, the IRB or FDA may review the data. 
 
DoD representatives will be allowed to independently review and inspect the awardee’s research. 
This may include access to identifiable information or protected health information. Participants 
will be informed about this risk to confidentiality. 
 
COST OF PARTICIPATION: 
There will be no charges to participants or to third party payers related to study visits or procedures 
in this study. NAC is an FDA approved medication. NAC will be provided for the 8 week duration 
of the study to all participants. 
 
COMPENSATION/PAYMENT FOR PARTICIPATION: 
Families will be compensated for time, effort, and travel as described in the table below. For the 
first 3 study visits, each participant will receive $25. Study visit 4 has additional conditions (i.e.: 
return of drug diary and unused drug), therefore, each participant will receive $100 for that visit. 
Total compensation for this study per participant is $175. Reimbursement  will be distributed 
within 90 days of study completion or withdrawal. 
 
 
Payment Schedule 
Visit 1 ѱ 
Visit 2 
Visit 3 
Visit 4 
$25 
$25 
$25 
$100 
 
ѱ Indicates treatment- and non-treatment cohort payment 
 
COMPLIANCE AND SAFETY MONITORING: 
Monitoring and Managing Adherence to Treatment:   
Pill/Sachet counts and/or review of the medication log will be used to estimate compliance, along 
with queries about missed doses.  We will require a standard of four days out of seven on treatment 
to obtain a rating but will counsel the participant and family about the importance of adherence in 
such a situation. We will require a minimum of 5 days per week on treatment in each of the final 
two weeks of each treatment block, so that the end-of-treatment ratings are an accurate reflection 
of treatment. For the final study visit and pTMS assessment, subjects must have 100% compliance 
for the two days prior to testing or testing will be rescheduled within a reasonable 3-4 day window 
so that compliance can be assured.   
 
Communication: 

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To facilitate data collection, safety monitoring, and accommodate preferences of participants, 
communication between study staff and the research participant/family/teachers will utilize a 
variety of technologies. Modes of communication may include phone calls, videoconference, 
electronic mail (e-mail), REDCap surveys (via e-mail link or Twilio text message), and in person 
meetings. Responses to safety assessments will be reviewed by a member of the study team 
within 48 hours. As described elsewhere, if a participant/family discloses any suicide ideation or 
significant health concerns at any point in this study, the study staff will contact the participant’s 
mental health provider or primary care provider and/or Psychiatric Intake Response Center 
(PIRC) at CCHMC for further assessment and guidance within 24 hours. 
 
Video recordings may aide recruitment and explanation of study assessments/details. These 
recordings may be distributed in a similar manner to other advertisements (websites, 
philanthropic/educational events, through interactions related to clinic visits, distributed via 
REDCap correspondence, et cetera). Examples or video content would be a short description of 
TMS and PANESS procedures. A general outline of the study (risks/benefits, procedures, timeline, 
et cetera) as would be described during informed consent would also be advantageous. 
 
Medical/safety assessments: 
During screening, children will receive a review of systems and physical examination, laboratory 
measures (CBC, basic chemistries with liver transaminase, thyroid function test, urinalysis), and 
pregnancy test (if relevant). Pregnancy tests will be administered when deemed appropriate by the 
study doctor or designee.  Results of the pregnancy test will be provided to the parent/guardian of 
the participant.  If an uninterpretable urine pregnancy test is obtained or if the participant 
cannot/refuses to provide a urine sample, then a serum pregnancy test will be performed.  All 
values outside the reference range will be assessed by the investigator, Dr. Gilbert, for clinical 
relevance. 
 
All MRI’s will undergo a safety review by a radiologist in order to determine whether there are 
any clinically relevant incidental findings that require referral for appropriate clinical follow-up. 
Referrals for clinical follow-up will be provided and that an individual with appropriate clinical 
qualifications will inform the research subjects of the need for the clinical follow-up. 
 
Measurement and Management of Adverse Effects:   
Participants and parents/guardians will be given the opportunity to report AEs to the investigator 
spontaneously in response to a general prompt (e.g. “Any changes in your health since the last 
visit?”). Terminology for adverse events (AEs) will follow the Medical Dictionary for Regulatory 
Activities (MedDRA) version 20.1 (https://www.meddra.org) (Table 6). If AEs occur, there 
investigator will rate the seriousness of the event, whether the event was anticipated, and the 
likelihood that it is related to NAC. Serious events will be reported to the IRB within 24 hours of 
awareness. All other adverse events will be reviewed quarterly by the study team.  
 
Table 6: Medical Dictionary for Regulatory Activities (MedDRA) 
MedRA System organ class 
MedDRA preferred term 
Nervous system disorders 
Headaches, Dyskinesias, Disturbances in 
initiating and maintain sleep,  

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Psychiatric Disorders 
Anxiety symptoms, decreased or increased 
physical activity levels, depressive symptom 
Gastrointestinal Disorders 
Nausea, vomiting, constipation 
 
Dose Modification: 
Definition of Dose-Limiting Toxicity (DLT) 
DLT will be defined as any of the following events that are at possibly, probably or definitely 
attributable to therapy. Adverse events will be graded using CTCAE v5.0. 
 
Dose Modification 
Dose reductions and/or withholding of doses must be based on the following: 
• Dose reduction to half dose (35mg/kg/day) to be determined by study doctor or designee 
(Table 7) 
• General dose modification guidelines for clinically significant toxicities considered related 
to treatment are provided in Table 8 
• Local medical practice and regional regulatory guidance. 
 
 
Table 7: NAC Dose Levels 
Dose Level 
Dose Schedule 
0 
70 mg/kg/day 
-1 
35 mg/kg/day 
 
Justification for Dose Modification 
Previous studies have shown oral NAC administration to humans at 35 mg/kg and 70 mg/kg 
produced CSF concentrations above the required biological activity in the brain. Therefore, it has 
been established that at 35mg/kg/day, NAC passes the blood brain barrier and would be efficacious 
as a neurocognitive therapy. 
 
Table 8: General Dose Modification Guidelines 
CTCAE Grade Action and Dose Modification 
Grade 1 
• Continue treatment at current dose level 
• Monitor closely 
• Provide supportive care according to institutional standards 
Grade 2 
• Interrupt treatment if clinically indicated 
• Monitor closely 
• Provide supportive care according to institutional standards 
• When toxicity resolves to grade 1 or baseline, restart treatment at current 
dose level 
Grade 3 
• Interrupt treatment  
• Monitor closely 
• Provide supportive care according to institutional standards 
• When toxicity resolves to grade 1 or baseline, restart treatment reduced by half 
Grade 4 
• Interrupt treatment  
• Monitor closely 
• Provide supportive care according to institutional standards 

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• Restart with treatment reduced by half once toxicity resolves to grade 1 or baseline 
• If the grade 4 toxicity recurs, permanently discontinue treatment 
 
The occurrence of adverse events should be sought by non-directive questioning of the patient at 
each visit during the program. Adverse events also may be detected when they are volunteered by 
the patient during or between visits or through physical examination, laboratory test, or other 
assessments. All adverse events must be recorded in an agreed-upon format, such as on the 
Adverse Event Form, which will be provided with the following information: 
1. Severity grade (mild, moderate, severe) 
2. Relationship to the drug(s) of interest (suspected/not suspected) 
3. Duration (start and end dates or if continuing at final exam) 
4. Whether it constitutes a serious adverse event (SAE) 
 
ELECTRONIC MAINTENANCE OF THE RESEARCH REGULATORY BINDER 
To securely and efficiently maintain the data collected for this study, an electronic (eREG) binder 
will be utilized. All data kept in computerized files are in computers or a server with restricted, 
password-protected access. The CCHMC network server is regularly backed up. Essential 
regulatory documents provided in paper format will be scanned into electronic format and filed 
appropriately in the eREG binder.  
 
A study team member will verify that the resulting copy contains all the original attributes and 
information found in the original document and certify, after verification, the electronic version is 
an accurate and complete reflection of the original. Verified electronic images will be considered 
“substitutes” or “certified copies” of the original documents. Original documents will be destroyed 
in a manner that ensures the confidentiality of the records and renders the information no longer 
recognizable as CCHMC records. 
 
The Principal Investigator or designee will provide approval for document conversion from paper 
to electronic. A Conversion Process Documentation Template will be used to document this 
process.