An Exploratory Single Blind Study of Ergoloid Mesylates, 5-Hydroxytryptophan, and the Combination in Adult Males With Fragile X Syndrome
Phase: PHASE2
Conditions: Fragile X Syndrome
Sponsor: Elizabeth Berry-Kravis

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
Page 1 of 42 
 
Clinical Trial Protocol 
 
Title: 
An Exploratory Single Blind Study of Ergoloid 
Mesylates, 
5-Hydroxytryptophan, 
and 
the 
Combination in Adult Males with Fragile X 
Syndrome 
 
Substance Identifier: 
Ergoloid mesylates, 5-hydroxytryptophan 
IND Number: 
Exempt 
 
Indication: 
Behavior, functional skills, cognition 
 
Phase: 
Phase 2 
 
Sponsor: 
Elizabeth Berry-Kravis, MD, PhD – Study Sponsor 
Purposeful IKE (Funding Study Drug) 
FRAXA Research Foundation (Funding Study only) 
 
Principal Investigator: 
 
Elizabeth Berry-Kravis MD PhD 
1725 West Harrison, Suite 718 
Chicago, IL 60612 
Protocol Version:                         ERG/5-HTP in FXS 
 
   
                                                       Date:  23 May 2022 
                                                       V4.0 
 
 
 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
   
Page 2 of 42 
 
SYNOPSIS 
Name of Sponsor/Company: 
Elizabeth Berry-Kravis, MD, PhD – Study Sponsor 
Purposeful IKE (Funding Study Drug) 
FRAXA Research Foundation (Funding Study only) 
Name of Investigational Product: 
Ergoloid mesylates, 5-Hydroxytryptophan 
Study Title:   
An Exploratory Single Blind Study of Ergoloid Mesylates, 5-
Hydroxytryptophan, and the Combination in Adult Males with 
Fragile X Syndrome 
Study Phase: 
Phase 2 
Study Objectives: 
 
In male Fragile X participants aged 18-45 years, inclusive, receiving standard medications: 
● 
To obtain preliminary assessments of the efficacy of Ergoloid Mesylates, 5-Hydroxytryptophan, and the 
Combination 
● 
To evaluate the safety and tolerability of Ergoloid Mesylates, 5-Hydroxytryptophan, and the Combination 
● 
To obtain biomarker (ERP and eye tracking) data on Ergoloid Mesylates, 5-Hydroxytryptophan, and the 
Combination  
 
Exploratory Efficacy Outcome Measures 
 
● 
NIH Toolbox Cognitive Battery for Intellectual Disabilities (NIH-TCB) 
● 
KiTAP executive battery – alertness, distractibility, go-nogo and flexibility subtests 
● 
Clinical Global Impression Severity – Investigator rated (CGI-S) 
● 
Clinical Global Impression Improvement – Investigator rated (CGI-I) 
● 
Visual Analog Scale (VAS) rating for function, language and behavior using participant-specific anchors 
● 
Aberrant Behavior Checklist (ABC)  
● 
Anxiety, Depression, and Mood Scale (ADAMS) 
● 
Vineland-3 Adaptive Behavior Scale 
● 
Event-Related Potentials (ERP)  
● 
Eye Tracking  
 
Safety and Tolerability Endpoints 
 
● 
Treatment-emergent Adverse events  
● 
Changes in vital signs  
● 
Clinical laboratory values for hematology and chemistry 
 
Study Design 
This is a single-center, Phase 2, single-blind, 4-period sequential study will obtain a preliminary assessment of the 
effects of ergoloid mesylates (EM) 1 mg TID and 5-hydroxytryptophan (5-HTP) 100 mg TID and the combination 
compared to a placebo period in participants with Fragile X Syndrome. The study will consist of a Screening period 
of up to 28 days prior to initial study drug administration, followed by four 4 week single-blind treatment periods.  
The screening and baseline visits may occur at the same time, provided the results of safety labs can be obtained. A 
final follow-up visit or phone contact for safety is planned one week after the conclusion of Period 4. 
 
Eligible participants will be started on EM for 4 weeks in Period 1, then will take EM and 5-HTP for 4 weeks in 
Period 2, then, 5-HTP for 4 weeks in Period 3, then placebo for 4 weeks in Period 4. Study visits will occur at baseline 
and every 4 weeks at transition times between periods, then at the end of the study.  
 
Brief cognitive and behavioral assessments will be performed during each clinic visit.  Safety and tolerability 
assessments will include adverse event monitoring, vital signs, blood chemistry and hematology, and urinalysis.   
 
 
 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
Page 3 of 42 
 
 
 
 
 
 
 
 
 
 
 
 
EM=ergoloid mesylates 1 mg TID, 5-HTP = 5-hydroxytryptophan 100 mg TID 
 
Planned Numbers of Participants 
 
Enrollment of a total of 15 eligible participants is planned. 
 
Study Duration 
The total duration of the study for each participant will be up to 21 weeks, including a maximum of 4-week screening 
period, four 4-week Single-Blind treatment Periods, and a follow-up telephone call approximately 1 week after the 
last treatment visit. 
 
 
Study Procedures: 
The Screening Visit will occur up to 28 days prior to the first study drug administration on Day 1.  During screening, 
participants and their parent/legal authorized guardian, if indicated, will review and sign an Informed Consent/Assent 
form prior to any study procedures being performed.  Following confirmation of a prior diagnosis for Fragile X, 
participants will have information collected regarding their neurological and medical/surgical history, race/ethnicity, 
social history (tobacco, alcohol, and/or drug use), and use of prescription and over-the-counter medications.  
Participants will undergo a full physical exam, and have vital signs measured.  Height, weight, and BMI will also be 
collected.  Fasting blood samples will be collected for chemistry and hematology. Urine will be collected for 
urinalysis.  The Stanford-Binet version 5 will also be administered at screening, as will an assessment of suicidality 
risk.   
 
At the Baseline visit (Period 1/Day 1), participants will receive an abbreviated physical examination including vital 
signs.  However, these will not be repeated if the Screening and Baseline visits occur on the same day.  Cognitive, 
behavioral, ERP and eye tracking assessments will be performed, and these measurements will be used as a common 
set of baseline measurements to which post-treatment assessments will be compared for all treatment Periods. 
 
During all four single-blind periods, participants will receive three times a day treatment with single-blinded study 
medication, two caps three times a day, each dose at least 4 hours apart. In the first period they will take an EM 
capsules and a placebo capsule at each dose, the second period an EM and 5-HTP caps at each dose, the third period 
a 5-HTP and placebo capsule each dose, and the fourth period 2 placebo capsules each dose. EM and 5-HTP will be 
over-encapsulated to maintain the single-blind. 
 
Participants will return to the clinic at the end of each Period at weeks 4, 8, 12, and 16.  Cognitive and behavioral 
evaluations will be repeated at Weeks 4, 8, 12, and 16 (end of Period 1, 2, 3, 4). Additionally, participants will be 
monitored for adverse events via a telephone call at the end of Week 1 of each Period, and one week following 
completion of Period 4 or following early discontinuation.  During clinic visits, adverse effects will be assessed, and 
laboratory measures and vital signs will be repeated according to the Schedule of Assessments. Suicidality risk will 
also be evaluated during the treatment periods per the Schedule of Assessments; if a concern is detected, the 
participant will be referred for further evaluation and treatment. 
 
4 Weeks 
Period 4 
4 Weeks 
Period 3 
4 Weeks 
Period 1 
4 Weeks 
Period 2 
Screen/Enroll 
EM + 5-HTP  
EM  
5-HTP  
Placebo  
N=15 participants 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
Page 4 of 42 
 
Participant Inclusion/Exclusion Criteria: 
Individuals are eligible for the study if they meet all of the Inclusion and none of the Exclusion criteria.  The criteria 
below will be assessed during Screening, which will be up to 28 days prior to first study drug administration.  
Continued participant eligibility will be verified on Baseline Day 1.  
 
Participant Inclusion Criteria   
1. Male aged 18 to 45 years, inclusive. 
2. Participant has Fragile X Syndrome with a molecular genetic confirmation of the full Fragile X Mental 
Retardation (FMR1) mutation (≥200 CGG repeats). 
3. Current treatment with no more than 3 prescribed psychotropic medications. Anti-epileptic medications 
are permitted and are not counted as psychotropic medications if they are used for treatment of seizures. 
Anti-epileptics for other indications, such as the treatment of mood disorders, count towards the limit of 
permitted medications. 
4. Permitted concomitant psychotropic medications must be at a stable dose and dosing regimen for at least 
2 weeks prior to Screening and must remain stable during the period between Screening and the 
commencement of study medication. 
5. Anti-epileptic medications must be at a stable dose and dosing regimen for 12 weeks prior to Screening 
and must remain stable during the period between Screening and the commencement of study medication. 
6. Participants with a history of seizure disorder who are currently receiving treatment with anti-epileptics 
must have been seizure-free for 3 months preceding screening, or must be seizure-free for 3 years if not 
currently receiving anti-epileptics. 
7. Behavioral and therapy treatments/interventions must be stable for 4 weeks prior to Screening and must 
remain stable during the period between Screening and the commencement of study medication, and 
throughout the study. Minor changes in hours or times of therapy that are not considered clinically 
significant will not be exclusionary. Changes in therapies provided through a school program, due to 
school vacations, are allowed. 
8. Participant must be willing to practice barrier methods of contraception while on study, if sexually active. 
Abstinence is also considered a reasonable form of birth control in this study population. 
9. Participant has a parent, legal authorized guardian or consistent caregiver. 
10. Participant and caregiver are able to attend the clinic regularly and reliably. 
11. Participant is able to swallow capsules. 
12. For participants who are not their own legal guardian, participant's parent/legal authorized guardian is 
able to understand and sign an informed consent form to participate in the study. 
13. If participant is his own legal guardian, he can understand and sign informed consent to participate in the 
study. 
14. If participant is not their own legal guardian, the participant provides assent for participation in the study, 
if the participant has the cognitive ability to provide assent. 
 
Participant Exclusion Criteria 
1. History of, or current cardiovascular, renal, hepatic, respiratory, gastrointestinal, psychiatric, neurologic, 
cerebrovascular, or other systemic disease that would place the participant at risk or potentially interfere 
with the interpretation of the safety, tolerability, or efficacy of the study medication.   
Common diseases such as mild hypertension, well-controlled type 2 diabetes mellitus (hemoglobin A1C 
[Hgb A1C] <6.5%), etc. are allowed per the investigator's judgment as long as they are stable and 
controlled by medical therapy that is constant for at least 4 weeks before randomization. 
2. Clinically significant abnormalities, in the investigator's judgment, in safety laboratory tests, vital signs, 
as measured during Screening. 
3. History of substance abuse within the past year, according to investigator assessment. 
4. Use of CYP3A4 inhibitors, beta-blockers, MAO inhibitors or triptans at any time during participation in 
the study. 
5. Significant hearing or visual impairment that may affect the participant's ability to complete the test 
procedures. 
6. Concurrent major psychiatric condition (e.g., Major Depressive Disorder, Schizophrenia or Bipolar 
Disorder) as diagnosed by the investigator. Participants with additional diagnosis of Autism Spectrum 
Disorder or Anxiety Disorder will be allowed as these are characteristics of FXS. 
 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
Page 5 of 42 
 
7. Participant has active diseases that would interfere with participation, such as acquired immunodeficiency 
disorder, hepatitis C, hepatitis B, or tuberculosis. 
8. Participant is planning to commence psychotherapy or cognitive behavior therapy (CBT) during the 
period of the study or had begun psychotherapy or CBT within 4 weeks prior to Screening. 
9. Participant has participated in another clinical trial within the 30 days preceding Screening. 
Investigational Product, Dosage, and Mode of Administration:   
In this single-blind, 4-period study, 15 participants will be enrolled and will be treated with the following during the 
4 periods of the study.  
● 
Ergoloid mesylates 1 mg TID (Medisca)* and matching placebo for 5-Hydroxytrptophan 100mg TID*** 
● 
Ergoloid mesylates 1 mg TID (Medisca) and 5-Hydroxytryptophan 100 mg TID (5-HTP, Basic Vitamins)** 
● 
5-Hydroxytryptophan 100 mg TID (5-HTP, Basic Vitamins) and matching placebo for Ergoloid mesylates 
1mg TID*** 
● 
Matching placebo for Ergoloid mesylates 1mg TID and Matching placebo for 5-Hydroxytryptophan 100mg 
TID*** 
● 
 
* 1 mg will be mixed with methyl cellulose and placed in a size 00 capsule  
** will be over-encapsulated in identical size 00 capsules  
***will be ascorbic acid powder in identical size 00 capsules 
 
All study medications will be prepared at the Rush Oak Park Compounding Pharmacy and provided to the RUMC 
Investigational Pharmacy for dispensing as identical-appearing capsules in bottles.  Two bottles of capsules will be 
provided at each visit and one capsule from each bottle will be taken 3 times a day. Doses should be taken at least 4 
hours apart. 
 
Overview of Endpoints 
 
Exploratory Efficacy Outcome Measures 
● 
NIH-TCB: Cognitive battery assessing different domains of cognition, administered using an iPad 
● 
KiTAP: Computerized executive battery with assessments of alertness (reaction time), distractibility, go/nogo 
(impulsiveness), and flexibility  
● 
Clinical Global Impression Severity – Investigator rated (CGI-S) Standardized ranking scale with 7 rankings 
● 
Clinical Global Impression Improvement – Investigator rated (CGI-I): Standardized ranking scale with 7 
rankings 
● 
Visual Analog Scale (VAS) – Parent/caregiver-rated assessment of participant-specific behavioral anchors: 
Domains of daily functioning, anxiety/irritability and language.   
● 
ABC: Parent/caregiver-rated scale with six subscales to assess irritability, social avoidance, lethargy, 
hyperactivity, inappropriate speech and social avoidance, using ABC-FX factoring system. 
● 
ADAMS:  Parent/caregiver rated scale with a total score and five sub-scores to assess manic/hyperactive 
behavior, depressed mood, social avoidance, general anxiety, and obsessive/compulsive behavior. 
● 
Vineland-3 Adaptive Behavior Scale: Clinician-administered standardized interview yielding adaptive behavior 
composite score and domain standard scores in domains of: communication (receptive, expressive, and written 
adaptive language functions), daily living skills (personal, domestic, and community skills), socialization 
(interpersonal relationships, play and leisure time, and coping abilities), and motor skills (gross and fine motor 
skills).  
● 
Event-Related Potentials (ERP) - Measure of auditory habituation based on EEG signals 
● 
Eye Tracking – Measure of gaze aversion (social anxiety) and pupilometry (autonomic function) 
 
Safety and Tolerability: The safety variables to be assessed include adverse events; clinical laboratory parameters 
(chemistry, hematology, and urinalysis); physical examinations; and vital signs (including blood pressure, heart rate, 
and respiratory rate). 
 
 
 
 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
Page 6 of 42 
 
Statistical Considerations 
 
Efficacy Analyses 
The primary efficacy population will be the intent to treat (ITT) efficacy population, which will include all 
participants who received at least one dose of treatment and returned for at least one follow-up visit. The completers 
population (CP), defined as all randomized participants who complete all 4 treatment periods with no significant 
protocol violations, will be used to evaluate the robustness of the ITT results. 
 
All efficacy parameters will be summarized at each time point collected and standard descriptive statistics provided. 
Baseline measurements are defined as those obtained on Day 1/Period 1 prior to receipt of any study medication.  For 
cognitive parameters measured at baseline, change from baseline will also be calculated and standard descriptive 
statistics provided for each time point.   
 
Safety Analyses 
The Safety population will include all participants who received at least one dose of study treatment. Adverse Events 
(AEs), including clinically meaningful laboratory abnormalities and significant behavioral changes will be tabulated. 
AE severity and relatedness to treatment will be assessed. AEs will be tabulated for placebo, EM, EM + 5-HTP and 
5-HTP treatments. Summaries will also be provided by severity and relationship to treatment. Serious Adverse Events 
(SAEs) will be summarized separately, in a manner similar to that used for Adverse Events. 
 
 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
   
Page 7 of 42 
 
1 TABLE OF CONTENTS 
1 
TABLE OF CONTENTS 
7 
2 
LIST OF ABBREVIATIONS 
9 
3 
introduction 
11 
3.1 
Background 
11 
3.2 
Rationale 
12 
3.3 
Risk/Benefit 
12 
3.3.1 
Preclinical Pharmacology 
12 
3.3.2 
Clinical Experience 
14 
3.4 
Study objectives and endpoints 
15 
3.4.1 
Study Objectives 
15 
3.4.2 
Exploratory Efficacy Outcome Measures 
15 
3.4.3 
Safety and Tolerability Endpoints 
16 
4 
investigational plan 
16 
4.1 
Overall Study Design 
16 
4.1.1 
Dose Selection 
16 
4.2 
Study Duration 
17 
5 
selection and Withdrawal of participants 
17 
5.1 
Study Population 
17 
5.2 
Participant Inclusion Criteria 
17 
5.3 
Participant Exclusion Criteria 
18 
5.4 
Participant Withdrawal 
19 
5.5 
Replacement of Participants 
19 
6 
treatment of participants 
19 
6.1 
Treatment Arms 
19 
6.2 
Study Medications 
20 
6.3 
Drug Accountability 
20 
6.4 
Blinding 
20 
6.5 
Study Drug Administration 
20 
6.6 
Prior and Concomitant Medications 
21 
6.7 
Other Therapeutic Treatments 
21 
6.8 
Dietary Guidelines 
21 
6.9 
Lifestyle Guidelines 
21 
7 
study procedures and assessments 
21 
7.1 
Informed Consent 
21 
7.2 
Medical /Surgical History 
21 
7.3 
Demographics and Social History 
22 
7.4 
Stanford Binet Intelligence Scale 
22 
7.5 
Physical Examination (Full and Abbreviated) 
22 
7.6 
Height, Weight, Body Mass Index 
22 
7.7 
Vital Signs 
23 
7.8 
Laboratory Assessments 
23 
7.9 
Management of Abnormal Clinical Laboratory Tests 
23 
7.10 Additional Safety Measures 
23 
7.11 Exploratory Efficacy Assessments 
24 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
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7.11.1 Descriptions of Efficacy Assessment Instruments 
24 
7.11.1.1 
NIH-TCB 
24 
7.11.1.2 
Test of Attentional Performance (KiTAP) 
24 
7.11.1.3 
Clinical Global Impression 
25 
7.11.1.3.1 Clinical Global Impression Severity: Investigator Rated (CGI-S) 
25 
7.11.1.3.2 Clinical Global Impression Improvement: Investigator –Rated (CGI-I) 
25 
7.11.1.4 
VAS Rating Scale 
25 
7.11.1.5 
Aberrant Behavior Checklist (ABC) 
25 
7.11.1.6 
Anxiety Depression and Mood Scale (ADAMS) 
26 
7.11.1.7 
Vineland-3 Rating Scale 
26 
7.11.1.8 
Event-Related Potentials (ERP) 
26 
7.11.1.9 
Eye Tracking 
26 
7.11.2 Timing of Efficacy Assessments 
27 
7.11.2.1 
Baseline (Period 1/Day 1) 
27 
7.11.2.2 
Week 4, 8, 12, 16 (end Periods 1 through 4) 
27 
8 
Evaluation and Reporting of Adverse Events 
27 
8.1 Adverse Events 
27 
8.2 Definitions 
28 
8.2.1 
Adverse Drug Reaction 
28 
8.2.2 
Unexpected Adverse Event/ Unexpected Adverse Drug Reaction 
28 
8.2.3 
Serious Adverse Events 
29 
8.3 Assessment of Adverse Events by the Investigator 
29 
8.3.1 
Causality/Relatedness 
29 
8.3.2 
Severity 
30 
8.3.3 
Adverse Event Monitoring and Follow-up 
30 
9 
Study procedures and assessments by visit 
31 
9.1 Schedule of Assessments 
31 
9.2 Screening (Day -28 to Day -1) 
31 
9.3 Baseline (Period 1/Day 1) 
32 
9.3.1 
Prior to Drug Dispensing 
32 
9.3.2 
Weeks 1, 5, 9, 13 (Periods 1 through 4) 
32 
9.3.3 
Week 4, 8, 12, 16 (end Periods 1 through 4) 
32 
9.3.4 
Week 17 (Period 4, Final Contact at End of Study) 
33 
9.3.5 
Early Termination 
33 
10 planned Statistical methods 
34 
10.1 
Sample Size 
34 
10.2 
Demographics 
34 
10.3 
Analysis Populations 
34 
10.4 
Efficacy: Cognitive, Behavioral and Pharmocodynamic Measures Analysis 
34 
10.5 
Safety Analysis 
34 
11 DATA MANAGEMENT 
35 
11.1 
Data Handling 
35 
11.2 
Data Entry into EDC 
35 
11.3 
Data Validation 
35 
12 INVESTIGATOR REQUIREMENTS AND QUALITY CONTROL 
35 
12.1 
Ethical Conduct of the Study 
35 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
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12.2 
Institutional Review Board (IRB) 
35 
12.3 
Informed Consent 
36 
12.4 
Record Storage and Retention 
36 
12.5 
Protocol Amendments and Deviations 
36 
13 References 
37 
14 Appendices 
41 
14.1 
Schedule of Assessments 
41 
14.2 
Clinical Laboratory Analytes 
42 
 
2 LIST OF ABBREVIATIONS  
ABC (-FX) 
Aberrant Behavior Checklist (Fragile X specific factoring system) 
ADAMS 
Anxiety, Depression, and Mood Scale 
AE(s) 
Adverse event(s) 
ADR 
Adverse drug reaction 
ALT 
Alanine transaminase 
bid 
Twice daily 
BMI 
Body mass index 
BP 
Blood pressure 
BPM 
Beats per minute 
BUN 
Blood urea nitrogen 
°C 
Degrees centigrade 
CBT 
Cognitive Behavioral Therapy 
CFR 
Code of Federal Regulations 
CGI-I 
Clinical Global Impression Improvement – Investigator rated 
CGG 
CGG trinucleotide repeating codon 
CGI-S 
Clinical Global Impression Severity – Investigator rated 
CNS 
Central nervous system 
CP 
Completers population 
CRF 
Case report form 
DCCS 
Dimensional change card sort (test) 
EDTA 
Ethylenediaminetetraacetic acid 
EEG 
Electroencephalogram 
ERP 
Event-Related Potentials 
FDA 
Food and Drug Administration 
FMR1 
Fragile X mental retardation 
FXS 
Fragile X Syndrome 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
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gm 
Gram 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
HgbA1C 
Hemoglobin A1C 
ICF 
Informed consent form 
ICH 
International Conference on Harmonization  
IRB 
Institutional Review Board 
ITI 
Inter-trial interval 
ITT 
Intent to treat (population) 
kg 
kilogram 
KiTAP 
Test of Attentional Performance 
mg 
Milligram 
mL 
Milliliter 
msec 
Millisecond 
µ 
Micro 
ng 
Nanogram 
NIH-TCB 
NIH Toolbox Cognitive Battery for Intellectual Disabilities 
PO 
By mouth (per os) 
RBC 
Red blood cell 
ROI 
Region of interest 
SAE 
Serious adverse event 
SD 
Standard deviation 
SOP 
Standard operating procedure 
VAS 
Visual analog scale 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
   
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3 
INTRODUCTION 
3.1 Background 
Mutational inactivation of the FMR1 gene encoding the Fragile X Mental Retardation protein 
(FMRP) causes a spectrum of symptoms including seizures, sleep disorders, anxiety, irritability, 
autism, mild to severe cognitive impairment and intellectual disability. The constellation of 
symptoms is known as Fragile-X syndrome (FXS).  
 
FXS in humans is caused by a CGG triplet expansion mutation (> 200 repeats) in the 5′ untranslated 
region of the FMR1 gene located on the X chromosome, which leads to gene methylation, 
inactivation, and resultant loss of fragile X mental retardation protein (FMRP) expression. FMRP 
functions as a translational regulator, affecting synthesis of many proteins including those involved 
in synaptic pruning during development.1 Meta-analysis estimates the frequencies of individuals 
with the full mutation FXS allele to be approximately 1 in 7000 males and 1 in 11,000 females.2 
FXS is severely debilitating in males. Females generally are less affected than males due to 
mosaicism resulting from X-chromosome inactivation which occurs randomly early in 
embryogenesis.3 
 
FXS presents with a variable clinical phenotype. In affected individuals, the disease presents during 
childhood with delayed developmental milestones. Intellectual deficit can be of variable severity and 
may include problems with working and short-term memory, executive function, language, 
mathematics and visuospatial abilities. Behavioral anomalies can be mild (e.g. anxiety, mood 
instability) to severe (e.g. aggressive behavior, autism). Autistic-like behavior can include hand 
flapping, poor eye contact, hand biting, gaze avoidance, social phobia, social and communication 
deficits and tactile defensiveness. In females, intellectual and behavioral disorders are typically 
milder than in males and usually consist of shyness, social and other forms of anxiety, and variable 
learning problems, with IQ ranging from normal to intellectually impaired. About 25% of girls have 
an IQ less than 70. Attention deficit hyperactivity disorder (ADHD) is present in over 89% of males 
and 30% of females and behavioral disinhibition is very common. Recurrent otitis media (50%) and 
seizures (12%) can also be observed. Individuals with FXS display a range of neuropsychiatric 
symptoms including intellectual disability, delayed language acquisition, poor social interaction, 
features of autism spectrum disorder, anxiety, hyperarousal, hypersensitivity, repetitive behaviors, 
disrupted sleep, attention deficit hyperactivity disorder and irritability/aggression.3 These behavioral 
changes are modelled in adult male Fmr1 knockout (KO) mice which display a spectrum of 
behavioral phenotypes due to the Fmr1 gene deletion. The mutant mice show hyperarousal in the 
open field test, have impaired social interaction, are less likely to build nests when provided cotton 
batting and are less likely to bury marbles in the cage bedding. Adult male mice have been used for 
most studies with animal models, as male FXS patients typically suffer more severe symptoms than 
do female patients due to the single X chromosome. In both humans with FXS `and the Fmr1 KO 
mice, there are alterations in the density, size, shape and maturity of dendritic spines, the principle 
recipients of excitatory inputs from other neurons.1 
 
Efforts to treat FXS have included numerous investigations have not been widely successful, which 
has led to revision of trial designs and exploration for additional and new therapies.3 Management 
of FXS is currently symptom-based and requires a multidisciplinary approach. Speech, physical and 
sensory integration therapy as well as individualized educational plans and behavioral interventions 
may be combined with medication, such as stimulants for attention deficit-hyperactivity disorder; 
selective serotonin reuptake inhibitors (SSRIs) for anxiety, depression, obsessive-compulsive 
ORA: 21051102-IRB01   Date IRB Approved: 9/1/2021   Amendment Date: 6/13/2022

Protocol:  ERG/5-HTP in FXS 
 
 
    
Date:  23 May 2022  v4.0 
 
 
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disorder; and atypical antipsychotic agents for self-injury and aggressive behaviors. New targeted 
treatments for FXS are being studied. Despite this slow progress, treatment of FXS remains still an 
unmet medical need. 
 
3.2 Rationale 
FXS is a disorder in which affected individuals display intellectual disability as well as symptoms 
typical of autism spectrum disorder, due to silencing of the X-linked, FMR1 gene.  
 
Two previously untested products in FXS were predicted in silico to have beneficial activity in FXS 
and proposed for in vivo follow-up in Fmr1 KO mice, namely ergoloid mesylates (EM) and 5-
hydroxytryptophan (5-HTP). Both agents showed an interesting polypharmacological predicted 
bioactivity spectrum, which is a necessity for complex disorders, such as FXS. The most strongly 
associated targets after the statistical enrichment were HTR1A, HTR2A, DRD2 and OPRM1 for 
EM, and ESR1, HTR1A, GRIA1, and GRIK1 for 5-HTP. Both products worked in the majority of 
tests in vivo in the Fmr1 KO mouse model (see below Section 3.3.1 Preclinical Pharmacology), 
which led to the hypothesis that their serotonergic action would be additive, whilst the diversity in 
target and pathway modulation could indeed produce some synergistic effect, which was also 
confirmed in vivo, leading to the alleviation of all FXS phenotypes in the Fmr1 KO mouse.  
 
3.3 Risk/Benefit 
3.3.1 Preclinical Pharmacology 
Preclinical in vivo data are available for the suggested formulations and their combination. Blinded 
experiments were conducted to assess the effect of the two treatments and their combination in Fmr1 
KO mice (Fmr1 gene deletion). Adult male mice were used for all experiments. Fmr1 knockout mice 
recapitulate the human phenotype and represent a valuable preclinical model for assessment of 
putative drug treatments. The Fmr1 KO carries an insertion in exon 5.4 It is a protein null, although 
Fmr1 mRNA is still present.5 These mice have been backcrossed to the C57/Bl6 or the FVB strains. 
The Fmr1 KO2 is a null allele at Fmr1 generated by deletion of the promoter and first exon of Fmr1.6 
It is both protein and mRNA null. This mutation is the same as is produced by Cre-mediated excision 
of the loxP sites present in the Fmr1 cKO described below (we house these and other mice models 
of FXS). According to literature, Fmr1 KO mice show alterations in the density, size, shape and 
maturity of dendritic spines, the principle recipients of excitatory inputs from other neurons.1 
Toxicological information on the acute toxicity of 5-HTP in animals are available7, as is the 
corresponding information for EM8. The treatment of the mice with 5-HTP, EM was conducted 
according to the matrix shown below in Table 1. 
 
Table 1. Treatment matrix for EM, 5-HTP and the combination in Fmr1 KO mice.  
Group Number 
Route 
Concentration 
 Regimen 
No. animals 
1. WT 
- 
 - 
QD 
10 
2. KO 
- 
 - 
QD 
10 
3. KO 5-HTP 
IP 
80 mg/kg 
QD 
10 
4. KO EM 
PO 
4 mg/kg 
QD 
10 
5. KO EM + 5-HTP 
PO + IP 
2 mg/kg + 40 mg/kg 
QD 
10 
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The behavioural tests performed were for hyperactivity (open field), stereotypy (self-grooming), 
sociability (three chamber partition test), memory and learning (novel object recognition), anxiety 
(hyponeophagia), daily living (nest building). Concise definitions follow below.  
One-way Analysis of Variance and Dunnett’s multiple comparisons against the KO group were 
performed suggesting that both treatments as well as their combination were significantly different 
from the KO group (adjusted p-value <0.0001) whilst the actual scoring across tests for both the 
treatments and their combination was similar to the WT group. Specifically, for all six tests, the 
treatment combination scoring measurements were found to be very similar to those provided for 
the WT group. Additional in vivo experiments were conducted to assess the effect of SSRIs on 5-
HTP + EM treatment, specifically the standard of care SSRI Fluvoxamine was administered 
(40mg/kg) in another 10 diseased mice. Results confirmed that Fluvoxamine did not alter the 
effectiveness of 5-HTP + EM treatment (adjusted p-value<0.0001). 
 
Figure 1. Open field WT-V, KO-V, 5-HTP, EM and combination (EM + 5-HTP) 
 
Figure 2. Stereotypy WT-V, KO-V, 5-HTP, EM and combination (EM + 5-HTP) 
 
Figure 3. Sociability WT-V, KO-V, 5-HTP, EM and combination (EM + 5-HTP)  
 
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Figure 4. Memory and learning for WT-V, KO-V, 5-HTP, EM and combination (EM + 5-HTP) 
 
 
Figure 5. Hyponeophagia for WT-V, KO-V, 5-HTP, EM and combination (EM + 5-HTP) 
 
Figure 6. Test of daily living in WT-V, KO-V, 5-HTP, EM and combination (EM + 5-HTP) 
 
3.3.2 Clinical Experience 
Ergoloid Mesylates, trade name Hydergine, is a mixture of the methanesulfonate salts of three 
dihydrogenated ergot alkaloids, dihydroergocristine, dihydroergocornine, and alpha- and beta-
dihydroergocryptine. Ergot alkaloids are dopamine agonists which activate dopamine receptors (in 
the basal ganglia and other parts of the brain involved in motor function) and a prolactin inhibitor. 
Ergot is a strong vasoconstrictor and thus helps to reduce bleeding by narrowing of the blood vessels. 
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Ergot was perhaps first used in medicine as an oxytocic drug, to promote uterine contraction during 
childbirth. They have been used for treating migraine headaches since 1883 and also in treating 
Parkinson's disease, restless leg syndrome, Alzheimer’s disease, dementia, hyperprolactinemia and 
other purposes.9 
 
There have been 26 clinical drug trials which investigated the effectiveness of EM in geriatric 
psychopharmacology, with significant improvement in at least 50% of the studies in the areas of 
cognitive dysfunctions, mood depression, and the composite scores as measured by subjective 
clinical behavioral rating scales.10 Assessments of studies on the effect of EM in dementia have 
shown strong effects in Vascular Dementia but only a modest efficacy in Alzheimer’s Dementia, 
with a stronger potential on higher dosage,11 as well as a statistically significant improvement on all 
ten components of the Sandoz Clinical Assessment-Geriatric scale.12 Olin et al.13 assessed its overall 
effects on patients with possible dementia, and showed significant treatment effects. EM was found 
to be well tolerated in controlled studies of elderly patients with age-related cognitive decline.14  
 
5-hydroxytryptophan, also known as 5-HTP has a nutraceutical status and has never been approved 
as a drug for any indication,  but as a dietary supplement has been used extensively for several 
disorders for many years such as in the therapy of depression, fibromyalgia, obesity, insomnia and 
chronic headache. The starting dose is generally 50 mg, three times a day, which can be doubled 
after a couple of weeks, if necessary.15 
 
Despite its relevance to L-tryptophan for which there are safety concerns, 5-HTP has demonstrated 
a very good safety profile. In humans, 5-HTP has been administered in combination with  SSRIs  
and  tricyclic  antidepressants,16,17 Monoamine oxidase inhibitors (MAOIs)18,19,20 and tryptophan.21 
According to Turner and Blackwell,22 serotonin syndrome has not been reported in humans in 
association with 5-HTP, either as monotherapy or in combination with other medications. In 
clinicaltrials.gov (accessed on 5 Feb 2021) there are 8 registered clinical studies in phases 2 and 3 
involving 5-HTP in one of the arms as a treatment for various indications such as major depressive 
disorder [NCT04395183], spinal cord injuries [NCT04520178] Crohn’s syndrome [NCT03574948] 
and others.  
 
3.4 Study objectives and endpoints  
3.4.1 Study Objectives 
In male Fragile X participants aged 18-45 years, inclusive, receiving standard medications:  
● To obtain a preliminary assessment of the efficacy of ergoloid mesylates (EM) 1 mg TID, 5-
hydroxytryptophan (5-HTP) 100 mg TID, and the combination  
● To evaluate the safety and tolerability of EM, 5-HTP, and EM + 5-HTP in fragile X syndrome 
● To obtain pharmacodynamic (ERP, eye tracking) data on EM, 5-HTP, and EM + 5-HTP 
3.4.2 Exploratory Efficacy Outcome Measures 
The following instruments will be used to assess the exploratory efficacy endpoints:  
● 
NIH Toolbox Cognitive Battery Modified for Intellectual Disabilities (NIH-TCB) 
● 
Test of Attentional Performance (KiTAP) 
● 
Clinical Global Impression Severity – Investigator rated (CGI-S) 
● 
Clinical Global Impression Improvement – Investigator rated (CGI-I) 
● 
VAS Rating Scale using participant-specific behavioral anchors 
● 
Aberrant Behavior Checklist (ABC)  
● 
Anxiety, Depression, and Mood Scale (ADAMS) 
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● 
Vineland-3 Rating Scale 
● 
Event-Related Potentials (ERP)  
● 
Eye Tracking  
 
3.4.3 Safety and Tolerability Endpoints 
The following safety assessments will be conducted during the study. 
● Treatment-emergent Adverse events  
● Changes in vital signs  
● Clinical laboratory evaluations (chemistry, hematology, urinalysis)  
 
4 
INVESTIGATIONAL PLAN 
4.1 Overall Study Design 
This is a single center, Phase 2, single-blind, 4 period study to obtain preliminary assessment of the 
effects of ergoloid mesylates (EM), 5-hydroxytryptophan (5-HTP), and the combination in 
participants with Fragile X Syndrome. As schematic display of the study design is shown in Figure 
7.  
 
The study will consist of a Screening period of up to 28 days prior to initial study drug 
administration, followed by four 4-week single-blind treatment periods.  The screening and baseline 
visits may occur at the same time, provided the results of safety labs can be obtained.  No washout 
period will be utilized between treatment periods. A final follow-up phone contact for safety is 
planned one week after the conclusion of Period 4. 
 
Eligible participants will be started on over-encapsulated EM 1 mg TID during Period 1, then will 
take EM 1 mg TID and 5-HTP 100 mg TID in Period 2, then 5-HTP 100 mg TID in Period 3, and 
placebo in Period 4. Throughout all 4 periods, participants will take two identical capsules three 
times a day. If only taking one over-encapsulated drug, they will take one over-encapsulated placebo 
pill with the drug at each dose, and when in period 4 they will take two over-encapsulated placebo 
pills at each dose.  
 
Cognitive and behavioral assessments will be performed during each clinic visit.  Safety and 
tolerability assessments throughout the study will include adverse event monitoring, vital signs, 
blood chemistry, hematology, and urinalysis.   
Figure 7      Study Design Schematic 
 
 
 
 
 
 
 
 
 
 
 
 
4.1.1 Dose Selection 
The doses of EM and 5-HTP were chosen as these are standard doses used in clinical management. 
4 Weeks 
Period 1 
4 Weeks 
Period 2 
4 Weeks 
Period 4 
4 Weeks 
Period 3 
Screen/Enroll 
EM + 5-HTP 
EM  
5-HTP 
Placebo  
N=15 participants 
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EM label for elderly demented patients suggests 1 mg tablets, 3 times a day, according to the product 
label23. 5-HTP labels commonly suggest 1-4 tablets totaling 200mg per day, however for depression, 
most commonly, 150-800 mg daily is taken for 2-6 weeks.24 Given this range, as well as the duration 
of the study, 100mg TID is suggested for 5-HTP.  
 
Furthermore, when compared to the mouse doses tested in vivo (2 mg/kg for EM and 40 mg/kg for 
5-HTP), if these are extrapolated to humans by allometric scaling, i.e. assuming that metabolic rate 
scales proportionally to body weight raised to the ¾ power, the proposed doses of 1 and  100 mg 
TID for EM and 5-HTP, respectively, are safely lower. Namely the 2 mg/kg mouse dose scales to 
about 20 mg daily dose for a typical 70 kg male, while for 5-HTP. a 40 mg/kg mouse dose scales to 
about 400 mg daily dose.25  
 
4.2 Study Duration 
The total duration of the study for each participant will be up to 21 weeks, including a maximum of 
a 4-week screening period, four 4-week single-blind Treatment Periods, and a follow-up call 
approximately 1 week after last treatment. 
 
5 
SELECTION AND WITHDRAWAL OF PARTICIPANTS 
5.1 Study Population 
Individuals are eligible for the study if they meet all of the inclusion and none of the exclusion 
criteria.  The criteria below will be assessed at the Screening visit which should within 28 days prior 
to first study drug administration.  The Screening and Baseline Visits may be combined if site 
procedures allow. 
 
5.2 Participant Inclusion Criteria 
1. Participant is male aged 18 to 45 years, inclusive. 
2. Participant has Fragile X Syndrome with a molecular genetic confirmation of the full 
Fragile X Mental Retardation 1 (FMR1) mutation (≥200 CGG repeats). 
3. Current treatment with no more than 3 prescribed psychotropic medications. Anti-epileptic 
medications are permitted and are not counted as psychotropic medications if they are used 
for treatment of seizures. Anti-epileptics for other indications, such as the treatment of 
mood disorders, count towards the limit of permitted medications. 
4. Permitted concomitant psychotropic medications must be at a stable dose and dosing 
regimen for at least 2 weeks prior to Screening and must remain stable during the period 
between Screening and the commencement of study medication. 
5. Anti-epileptic medications must be at a stable dose and dosing regimen for 12 weeks prior 
to Screening and must remain stable during the period between Screening and the 
commencement of study medication. 
6. Participants with a history of seizure disorder who are currently receiving treatment with 
anti-epileptics must have been seizure-free for 3 months preceding screening or must be 
seizure-free for 3 years if not currently receiving anti-epileptics. 
7. Behavioral and therapy treatments/interventions must be stable for 4 weeks prior to 
Screening and must remain stable during the period between Screening and the 
commencement of study medication, and throughout the study. Minor changes in hours or 
times of therapy that are not considered clinically significant will not be exclusionary. 
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Changes in therapies provided through a school program, due to school vacations, are 
allowed. 
8. Participant must be willing to practice barrier methods of contraception while on study, if 
sexually active. Abstinence is also considered a reasonable form of birth control in this 
study population. 
9. Participant has a parent, legal authorized guardian or consistent caregiver. 
10. Participant and caregiver are able to attend the clinic regularly and reliably. 
11. Participant is able to swallow tablets and capsules. 
12. For participants who are not their own legal guardian, participant's parent/legal authorized 
guardian is able to understand and sign an informed consent form to participate in the 
study. 
13. If participant is his/her own legal guardian, he/she can understand and sign informed 
consent to participate in the study. 
14. If participant is not their own legal guardian, the participant provides assent for 
participation in the study, if the participant has the cognitive ability to provide assent. 
 
5.3 Participant Exclusion Criteria 
The following Exclusions apply to findings during Screening or at Baseline (Day 1): 
 
1. History of, or current cardiovascular, renal, hepatic, respiratory, gastrointestinal, 
psychiatric, neurologic, cerebrovascular, or other systemic disease that would place the 
participant at risk or potentially interfere with the interpretation of the safety, tolerability, 
or efficacy of the study medication.   
Common diseases such as mild hypertension, well-controlled type 2 diabetes mellitus 
(hemoglobin A1C [HgbA1C] <6.5%), etc. are allowed per the investigator's judgment as 
long as they are stable and controlled by medical therapy that is constant for at least 4 
weeks before randomization. 
2. Clinically significant abnormalities, in the investigator's judgment, in safety laboratory 
tests, vital signs, or ECG, as measured during Screening. 
3. History of substance abuse within the past year, according to investigator assessment. 
4. Use of CYP3A4 inhibitors, beta-blockers, MAO inhibitors or triptans at any time during 
participation in the study.  
5. Significant hearing or visual impairment that may affect the participant's ability to 
complete the test procedures. 
6. Concurrent major psychiatric condition (e.g., Major Depressive Disorder, Schizophrenia 
or Bipolar Disorder) as diagnosed by the investigator. Participants with additional 
diagnosis of Autism Spectrum Disorder or Anxiety Disorder will be allowed. 
7. Participant has known or suspected human immune deficiency virus-positive status or has 
diseases such as acquired immunodeficiency disorder, hepatitis C, hepatitis B, or 
tuberculosis. 
8. Participant is planning to commence psychotherapy or cognitive behavior therapy (CBT) 
during the period of the study or had begun psychotherapy or CBT within 4 weeks prior to 
Screening. 
9. Participant has participated in another clinical trial within the 30 days preceding Screening. 
 
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5.4 Participant Withdrawal  
All participants have the right to withdraw from this study at any time.  In addition, it is the right of 
the Investigator to remove participants from the study as a result of adverse reactions, a protocol 
violation, or non-compliance, or any other reason.  Participants must be discontinued for the 
following reasons: 
● Participant withdraws consent or requests discontinuation from the study for any reason; 
● Occurrence of a medical condition or circumstance that exposes the participant to substantial 
risk and/or does not allow the participant to adhere to the requirements of the protocol as 
determined by the Investigator; 
● Any serious adverse event (SAE), clinically significant adverse event, severe laboratory 
abnormality, concomitant illness, or other medical condition which indicates to the Investigator 
that continued participation is not in the best interest of the participant; 
● Requirement for prohibited concomitant medication; 
● Termination of the study by the Sponsor or the regulatory authority. 
 
Should a participant withdraw after administration of EM in Period 1, or should the Investigator 
decide to withdraw the participant, all efforts will be made to complete and report the protocol-
stipulated observations up to the time of withdrawal.  Week 16 procedures (see Section 10.3.5 and 
Section 14.1 Schedule of Study Assessments) will be completed at the time of the participant’s 
withdrawal and an explanation provided as to why the participant is withdrawing or being withdrawn 
from the study. 
 
In a case of participants lost to follow-up, attempts to contact the participant must be made and 
documented in the participant’s medical records. 
 
The Investigator must document the primary reason for discontinuation of a study participant on the 
appropriate case report form (CRF).  If the reason for withdrawal is a clinical AE, monitoring will 
continue until the AE resolves or the Investigator assesses the AE as chronic and stabilized. 
 
5.5 Replacement of Participants 
Participants who do not meet eligibility criteria at Baseline will not receive any study drug and will 
be considered screen failures.  Participants who withdraw from the study for any reason will not be 
replaced.   
 
6 
TREATMENT OF PARTICIPANTS 
6.1 Treatment Arms 
In this single-blind, 4-period study, 15 participants will be enrolled and will be treated with the 
following during the 4 periods of the study.  
● Ergoloid mesylates 1 mg TID (Medisca)* and matching placebo for 5-Hydroxytryptophan 
100mg TID*** 
● Ergoloid mesylates 1 mg TID (Medisca) and 5-Hydroxytryptophan 100 mg TID (5-HTP, 
Basic Vitamins)** 
● 5-Hydroxytryptophan 100 mg TID (5-HTP, Basic Vitamins) and matching placebo for 
Ergoloid mesylates 1mg TID*** 
● Matching placebo for Ergoloid mesylates 1mg TID and Matching placebo for 5-
Hydroxytryptophan 100mg TID *** 
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* 1 mg will be mixed with methyl cellulose and placed in a size 00 capsule  
** will be over-encapsulated in identical size 00 capsules  
***will be ascorbic acid powder in identical size 00 capsules 
 
All study medications will be prepared at the RUMC research pharmacy and provided as identical-
appearing capsules in bottles.  Two bottles of capsules will be provided at each visit and one capsule 
from each bottle will be taken 3 times a day. Doses should be taken at least 4 hours apart. 
 
6.2 Study Medications 
The Ergoloid Mesylate is provided as powder manufactured by Medisca and 5-HTP 100 mg capsules 
available to the RUMC pharmacy are manufactured by Basic Vitamins.  All study medications and 
their matching placebos will be compounded and over-encapsulated at the Rush Oak Park 
Compounding Pharmacy. 
 
6.3 Drug Accountability 
The Investigator or designated study personnel will be responsible for keeping accurate records of 
the study drugs (and other components) used in this study.  These records will include documentation 
of receipt, inventory, and disposition to participant.  The records should include dates, quantities, 
batch/serial numbers, expiration dates (if applicable), and any unique code numbers assigned (if 
applicable). 
 
All drug returned will be disposed of by the study team after medication reconciliation to determine 
compliance. Amount of drug returned and disposed will be recorded in the participant study binders. 
In accordance with Good Pharmacy Practices, gloves will always be worn by study personnel when 
handling study drug. 
 
6.4 Blinding 
This is a single-blind study, meaning the participant and his family/caregivers will not know what 
drug or combination the participant is receiving. The investigator and study coordinator will know 
what drug the participant is taking. The remainder of the study team performing assessments such 
as the Vineland, eye tracking, ERP, Toolbox, and the KiTAP will not know the type of treatment the 
patient is on when testing is done. 
 
The PI or designee will be responsible for maintaining the single blind, consistent with protocol 
design, throughout the study, except in the case of an emergency need to unblind the participant for 
medical reasons. Blinding is not to be broken during the study unless considered necessary by the 
Investigator for emergency situations for reasons of participant safety. When the blind is broken, the 
reason must be fully documented. 
 
6.5 Study Drug Administration 
Two capsules will be taken orally in the morning, afternoon and evening. Doses should be taken at 
least 4 hours apart.  
 
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6.6 Prior and Concomitant Medications 
A prior medication is defined as any psychotropic or anti-epileptic medication taken by the 
participant for more than 28 days during the 6 months prior to first dose of study drug (Day 1), which 
was subsequently discontinued within that same 6 month period. 
 
A concomitant medication is defined as any medication taken 14 days prior to the first dose of study 
drug (Day 1) and during the study through the Week 14 Follow-up Visit. Concomitant medications 
should be recorded with indication, daily dose, and start and stop dates of administration. 
 
Current treatment with no more than 3 prescribed psychotropic medications is allowed. Anti-
epileptic medications are permitted and are not counted as psychotropic medications if they are used 
for treatment of seizures. Anti-epileptics for other indications, such as the treatment of mood 
disorders, count towards the limit of three permitted medications. Permitted concomitant 
psychotropic medications must be at a stable dose and dosing regimen for at least 2 weeks prior to 
the commencement of study medication. 
 
Anti-epileptic medications must be at a stable dose and dosing regimen for 12 weeks prior to 
Screening and must remain stable during the period between Screening and the commencement of 
study medication. 
 
6.7 Other Therapeutic Treatments 
Behavioral and therapy treatments/interventions must be stable for 4 weeks prior to Screening and 
must remain stable during the period between Screening and the commencement of study 
medication, and throughout the study. 
 
6.8 Dietary Guidelines 
There are no dietary restrictions. Participants should follow their usual eating behaviors. 
 
6.9 Lifestyle Guidelines 
There no lifestyle restrictions or guidelines associated with this study.  
 
7 
STUDY PROCEDURES AND ASSESSMENTS 
The study will be conducted in accordance with the CFRs (21 CFR § 11, 50, 54, 56, and 312 Subpart 
D), which originate from the ethical principles laid down in the current revision of the Declaration 
of Helsinki, Good Clinical Practice (GCP),26,27 and the policies and procedures as outlined by the 
ethical requirements for IRB review and informed consent form.   
 
7.1 Informed Consent 
Informed consent will be obtained prior to the participant entering into the study and before any 
protocol-directed procedures are performed. 
 
7.2 Medical /Surgical History  
Medical/Surgical history will be recorded at the Screening Visit as specified in Section 10.2. 
Participant eligibility will be evaluated to determine all inclusion and none of the exclusion criteria 
are met. The Investigator will inquire with the participant on Period 1/Day 1 (Baseline, prior to 
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randomization) to determine if there have been any changes in the participants’ s health affecting 
eligibility or requiring an update to their medical and surgical history. 
 
7.3 Demographics and Social History 
Demographics (sex, ethnicity, race) and social history (tobacco, alcohol, and/or drug use) will be 
recorded at the Screening Visit as specified in the Schedule of Assessments. 
 
7.4 Stanford Binet Intelligence Scale 
The Stanford Binet Intelligence Scale – Version 5 will be administered at Screening in order to 
support characterization of the study cohort and to determine whether cognitive and behavioral 
responses are dependent on baseline level of cognitive functioning. The scale will be scored 
according to the Hessl z-deviation method to prevent floor effects.28  
 
7.5 Physical Examination (Full and Abbreviated) 
The full physical examination will be conducted at the Screening as specified in the Schedule of 
Assessments and will include: 
● General appearance 
● Skin 
● Eyes, ears, nose, and throat (EENT) 
● Head/neck 
● Extremities 
● Musculoskeletal examination 
● Respiratory examination 
● Cardiovascular assessment including rhythm and presence of cardiac abnormalities 
● Abdominal examination 
● Basic neurologic examination  
● Any additional assessments necessary to establish baseline status or evaluate symptoms or 
adverse experiences 
 
An abbreviated physical examination will be conducted at Period 1/Day 1 (if not the same day as 
Screening) and at the visit at the end of each Period and will include: 
● Any changes noted since the initial physical examination 
● General appearance 
● Skin 
● Extremities 
● Respiratory examination 
● Cardiovascular assessment including rhythm and presence of cardiac abnormalities 
● Abdominal examination 
 
Significant findings prior to the start of dosing will be recorded on the Medical History page of the 
CRF.  Only changes from baseline physical examination findings that meet the definition of a 
treatment-emergent AE will be recorded as an AE. 
 
7.6 Height, Weight, Body Mass Index  
Body weight and height are to be measured at Screening.  Body weight will be repeated at the visit 
at the end of each Period as specified in the Schedule of Assessments.  Participants will wear indoor 
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clothing and remove their shoes prior to the measurements.  Body Mass Index (BMI) will be 
calculated and recorded.   
 
7.7 Vital Signs 
Vital signs (pulse rate, blood pressure, respiration rate, and temperature) will be measured at 
Screening, during the Treatment Visits, and Follow-Up as specified in the Schedule of Assessments. 
Blood pressure and heart rate will be measured using a calibrated, fully automated machine with a 
cuff that is appropriate to the size of the upper arm.  If a fully automated machine is not available, 
blood pressure may be measured manually.  For each participant, measurement on the same arm 
(right or left) using the same method (either automated or manual) should occur throughout the 
study.   All blood pressure measurements should be obtained with the participant in the sitting 
position, after the participant is at rest for 5 minutes. 
 
Additional vital signs measurements may be performed as deemed medically necessary by the 
Investigator. 
 
7.8 Laboratory Assessments 
Appendix 14.2 Clinical Laboratory Analytes provides a list of the clinical laboratory tests that will 
be performed according to the collection schedule provided in the Schedule of Assessments.   
Blood samples for chemistry and hematology and urine samples will be collected in accordance with 
acceptable laboratory procedures.   
 
Since this is a single-site study, an accredited local laboratory will be used to analyze the clinical 
laboratory samples. 
 
7.9 Management of Abnormal Clinical Laboratory Tests 
It is the Investigator’s responsibility to review the results of all lab tests as they become available 
and to document their review by signing and dating the lab report.  For each lab test outside of the 
laboratory normal range, the Investigator must ascertain if this is a clinically significant change from 
baseline for the individual participant.  This determination does not necessarily need to be made the 
first time an abnormal lab is observed.  The Investigator may repeat the lab test or request additional 
tests to verify the results of the original lab test. 
 
All clinically significant laboratory abnormalities occurring during the study that were not present 
at baseline should be followed and evaluated with additional tests if necessary, until determination 
of the underlying cause or adverse event resolution. 
 
7.10 Additional Safety Measures 
Participants and caregivers will be instructed to inform the study physician and/or research personnel 
of any AEs, including significant behavioral changes not within the typical variation for the 
participant, that occur at any time during the study. 
 
Procedures will be completed as specified in this protocol unless contraindicated due to a reported 
AE. 
 
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If, in the judgement of the Investigator, additional testing is required to assess an adverse event or 
otherwise clinically significant event, this testing should be discussed first with the sponsor unless 
needed urgently.   
 
7.11 Exploratory Efficacy Assessments 
7.11.1 Descriptions of Efficacy Assessment Instruments 
Several assessment tools for cognitive and behavioral measures will be utilized in this exploratory 
study.  Raw scores (not standardized) will be collected for all assessments, except for the Vineland 
-3 composites where age-equivalent scores will also be collected. 
 
7.11.1.1 NIH-TCB 
The NIH-TCB, a component of the NIH Toolbox for Assessment of Neurological and Behavioral 
Function, was developed by a team of more than 300 scientists from nearly 100 academic institutions 
as part of the NIH Blueprint for Neuroscience Research to standardize evaluations in specific clinical 
populations for investigations of neurological development and change, disease recovery, and 
therapeutic interventions.29,30,31 The NIH-TCB is a battery of extensively validated computer-
administered cognitive tests with utility across childhood and adolescence, early adulthood, and old 
age. The NIH-TCB assessments were designed to minimize floor and ceiling effects which often are 
present in testing batteries designed for the general population. Downward extensions of many of 
the NIH-TCB tests have been created to allow feasibility down to a mental age of 3. Therefore, there 
is good reason to believe that the assessments are appropriate for individuals with intellectual 
disabilities. In fact, a pilot study has been published showing good feasibility and test-retest 
reliability in FXS and Down syndrome, and the instructions and testing protocol has been adjusted 
(without changing construct validity) for individuals with intellectual disabilities based on issues 
identified in the pilot study.32 Further validation of the adjusted measure was published 
subsequently.33 
 
The NIH-TCB includes 7 evaluations: Dimensional change card sort test (DCCS, measure of 
cognitive flexibility), Flanker inhibitory control and attention test (measure of inhibition and visual 
attention), Picture sequence memory test (measures episodic memory), List sorting working memory 
test (measures immediate recall and sequencing of different visually and orally presented stimuli), 
Pattern comparison test (measures processing speed), Oral reading recognition test (measures 
recognition of letters and words), Picture vocabulary test (measures receptive vocabulary). 
 
7.11.1.2 Test of Attentional Performance (KiTAP) 
The KiTAP is a computerized executive function battery that consists of eight nonverbal subtests 
measuring different basal as well as higher-order components of attention and executive 
functioning.34  Each subtest can be assessed separately. Four subtests will be utilized in this study: 
alertness, distractibility, go-nogo, and flexibility. To ensure optimal motivation and compliance, all 
subtests are designed in the form of short games with an enchanted castle theme. The four subtests 
chosen for use in this study have been utilized in FXS populations and shown to be feasible, 
reproducible with evidence of clinical validity when scores are compared with standardized 
behavioral questionnaires addressing relevant functions. The KiTAP has shown evidence of 
response to pharmacological intervention in clinical trials of AFQ056 and metadoxine in FXS.35,36  
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7.11.1.3 Clinical Global Impression  
The CGI-I (and CGI-S) are gold standard global measures of severity and change with treatment in 
placebo-controlled pharmacotherapy trials in developmental disabilities and have been used 
extensively in prior clinical trials in FXS.37,38  
 
7.11.1.3.1  Clinical Global Impression Severity: Investigator Rated (CGI-S) 
The Clinical Global Impression−Severity (CGI-S) is a global measure to provide a clinical judgment 
of a participant’s overall condition based on a trained clinician’s assessment of cognition, behavior 
and activities of daily living.39 The assessment of severity will be made with a 7-point scale: 1, not 
ill; 2, very mild;  3, mild; 4, moderate; 5, marked; 6, severe;  7, extremely severe. The comparison 
will be made with respect to the overall experience of the clinician with individuals of the same age 
and sex.  
 
7.11.1.3.2  Clinical Global Impression Improvement: Investigator –Rated (CGI-I) 
The Clinical Global Impression – Improvement (CGI-I) assessment is a 7-point scale that requires 
the clinician to assess how much the participant's illness has improved or worsened relative to a 
baseline state at the beginning of the intervention, and rated as: 1, very much improved; 2, much 
improved; 3, minimally improved; 4, no change; 5, minimally worse; 6, much worse; or 7, very 
much worse.39 
 
Completion of this scale requires the clinician to rate how much the participant’s illness (including 
behavioral and adaptive functioning) has improved or worsened relative to a baseline state.  The 
CGI-I must be administered by the same rater for a given participant at all applicable visits 
throughout the trial.  
   
7.11.1.4  
VAS Rating Scale 
In an attempt to measure the level of behavioral difficulty experienced by the parent/caregiver with 
respect to the child with FXS, the VAS will allow parents to mark on a visual line measuring 10 cm 
with one side marked “worst behavior” and the other side marked “best behavior.”  The caregiver 
will rate the participant’s behavior with respect to three domains: daily functioning, 
anxiety/irritability and language; with guidance form the investigator, the caregiver will select a 
behavior that is a problem affecting function within each domain that will be assessed throughout 
the study. The horizontal marks are measured in centimeter distance where they call from the “good 
behavior” side so that improvements or worsening of behavior over the treatment period can be 
evaluated. The VAS has been successfully used in multiple clinical trials conducted in the FXS 
population.40 The caregiver completing the assessment should remain the same at all applicable visits 
throughout the trial. 
 
7.11.1.5 Aberrant Behavior Checklist (ABC) 
The Aberrant Behavior Checklist- Community Edition (ABC) is a 58-item parent/caregiver rating 
scale used to assess behaviors across five dimensions or subscales: irritability, hyperactivity, 
lethargy/withdrawal, stereotypy, and inappropriate speech.41 Items are evaluated on a four-point 
Likert scale ranging from 0 (not at all a problem) to 3 (the problem is severe in degree).  This scale 
has been used extensively in FXS in clinical trials and other projects. The ABC will be scored using 
the FXS-specific factoring system (ABC-FX). The caregiver completing the assessment should 
remain the same at all applicable visits throughout the trial. 
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7.11.1.6 Anxiety Depression and Mood Scale (ADAMS) 
The ADAMS (Anxiety, Depression, and Mood Scale) is a 28-item behavior-based informant 
instrument rated by the parent/caregiver and designed to assess anxiety, depression and mood 
disorders in individuals with intellectual disability.42 Items are rated on a scale of 0 (“behavior has 
not occurred, or is not a problem”) to 3 (“behavior occurs a lot, or is a severe problem”). The scale 
is composed of 5 factors which address: Manic/Hyperactive Behavior, Depressed Mood, Social 
Avoidance, General Anxiety and Obsessive/Compulsive Behavior. A caregiver identified upon 
enrollment of participant should have intimate knowledge of the participant’s situation and level of 
impairment to be able to provide accurate information as required to complete the ADAMS. The 
caregiver completing the assessment should remain the same at all applicable visits throughout the 
trial. 
 
7.11.1.7 Vineland-3 Rating Scale 
The Vineland Adaptive Behavior Scales, Third Edition (Vineland-3)43 assesses adaptive behavior in 
five domains, each with subdomains:  
1. Communication 
o Subdomains: Receptive, Expressive, Written 
2. Daily Living Skills 
o Subdomains: Personal, Domestic, Community 
3. Socialization 
o Subdomains: Interpersonal Relationships, Play and Leisure, Coping Skills 
4. Motor Skills 
o Subdomains: Fine Motor, Gross Motor 
5. Maladaptive Behavior 
o Internalizing, Externalizing, Critical Items 
 
The Vineland-3 will be administered as a standardized interview to a parent/caregiver by trained 
study staff trained and authorized to perform the interview. The Vineland-3 must be administered 
by the same rater for a given participant at all applicable visits throughout the trial. The caregiver 
completing the assessment with the study staff member should also remain the same at all applicable 
visits throughout the trial. 
 
7.11.1.8 Event-Related Potentials (ERP)  
Event-Related Potentials (ERPs) enable extraction of neural responses associated with specific 
sensory, cognitive, or motor events from an overall EEG.44,45 Auditory stimuli are presented and 
EEG events assessed in relation to timing of the stimuli. For this study, an ERP protocol developed 
to measure resting state delta and alpha power, alpha responses to eye closure, CHIRP responses,46 
and processing of oddball stimuli relative to standards47 will be used. This protocol has shown 
feasibility in prior studies. The EEGs will be read by Dr. Lauren Ethridge who has developed 
multiple ERP protocols and has extensive experience in ERP evaluation. 
 
7.11.1.9 Eye Tracking   
Eye tracking has been successfully used to assess social gaze in FXS.48 Testing will be conducted in 
a quiet room with the lights turned off. The eye tracker (Tobii) will be calibrated for each participant 
at the beginning of each session. Following calibration, participants will view pictures shown on the 
screen. Each assessment begins with presentation of a scrambled face image for 1 s followed 
immediately by its matched face image for 3 s. An inter-trial interval (ITI) containing a uniform grey 
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screen is shown for 0.5, 1, or 2 s, randomly determined. The order of face presentation is 
pseudorandomized and each eye tracking session lasts approximately 6 min. Measurements include 
looking time to the eye region of interest (ROI), and number of fixations to the eye ROI, as well as 
pupil dilatation by pupilometry. These measurements have been shown to have good test-retest 
reproducibility. All measurements are analyzed offline by a trained specialist. 
 
7.11.2 Timing of Efficacy Assessments 
7.11.2.1 Baseline (Period 1/Day 1) 
The following exploratory efficacy assessments will be performed at Baseline (Period 1/ Day 1, prior 
to randomization): 
● NIH Toolbox Cognitive Battery for Intellectual Disabilities (NIH-TCB) 
● Test of Attentional Performance (KiTAP) 
● Clinical Global Impression Severity – Investigator rated (CGI-S) 
● VAS Rating Scale using participant-specific behavioral anchors 
● Aberrant Behavior Checklist (ABC)  
● Anxiety, Depression, and Mood Scale (ADAMS) 
● Vineland-3 Rating Scale 
● Event-Related Potentials (ERP)  
● Eye Tracking 
 
7.11.2.2 Week 4, 8, 12, 16 (end Periods 1 through 4) 
The full battery of cognitive, behavioral and pharmacodynamic tests will be performed at Week 4 
of each Period: 
● NIH Toolbox Cognitive Battery for Intellectual Disabilities (NIH-TCB) 
● Test of Attentional Performance (KiTAP) 
● Clinical Global Impression Severity – Investigator rated (CGI-S) 
● Clinical Global Impression Improvement – Investigator rated (CGI-I) 
● Visual Analog Scale (VAS) assessment of participant-specific behavioral anchors 
● Aberrant Behavior Checklist (ABC)  
● Anxiety, Depression, and Mood Scale (ADAMS) 
● Event-Related Potentials (ERP) 
● Eye Tracking  
● Vineland-3 Rating Scale 
 
8 EVALUATION AND REPORTING OF ADVERSE EVENTS  
8.1 Adverse Events 
An adverse event is defined as any untoward medical occurrence in a clinical investigation 
participant administered a pharmaceutical product, which does not necessarily have a causal 
relationship with this treatment. An adverse event can therefore be any unfavorable and/or 
unintended sign (including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of an investigational medicinal product, whether or not related to the 
investigational medicinal product. All adverse events, including observed or volunteered problems, 
complaints, or symptoms, are to be recorded on the appropriate CRF. 
 
Adverse events, which include abnormal and clinically significant clinical laboratory test variables, 
will be monitored and documented from the time Informed Consent until study participation is 
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complete (the Follow-up Visit). Participants should be instructed to report any adverse event that 
they experience to the Investigator. Beginning with the signing of the informed consent until the 
time of the first dose of study drug, investigators should make updates to medical history and record 
any pre-existing medical condition or signs or symptoms that changes in severity, frequency, or 
seriousness in the medical history. Serious adverse events that occur prior to the first dose of study 
drug should be reported as an update to medical history as well as be reported on the appropriate 
adverse event CRF.  
 
Beginning with the first dose of study drug, investigators should make an assessment for adverse 
events at each visit and record all adverse events, non-serious and serious, on the appropriate adverse 
event CRF. 
 
Wherever possible, a specific disease or syndrome rather than individual associated signs and 
symptoms should be identified by the Investigator and recorded on the CRF. However, if an observed 
or reported sign or symptom is not considered a component of a specific disease or syndrome by the 
Investigator, it should be recorded as a separate adverse event on the CRF. Additionally, the 
condition that led to a medical or surgical procedure (e.g., surgery, endoscopy, tooth extraction, or 
transfusion) should be recorded as an adverse event, not the procedure. Concomitant procedures 
should be recorded as such on the appropriate CRF.  
 
Any medical condition already present prior to the participant taking the first dose of study drug 
should be reported in the medical history. Any SAEs occurring prior to the first dose of study drug 
should be reported as an update to medical history as well as an adverse event. Any pre-existing 
medical condition or signs or symptoms that changes in severity, frequency, or seriousness after the 
participant takes the first dose of study drug and through the Follow-up Visit should be reported as 
an adverse event. 
 
Clinically significant abnormal laboratory values or other examinations (e.g., ECG) that are detected 
after the first dose of study drug and worsen during the study should be reported as adverse events. 
An abnormal laboratory result that is not verified by repeat testing does not necessitate reporting as 
an adverse event. The Investigator will exercise his or her medical, scientific, and clinical judgment 
in deciding whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant. Clinically significant abnormal laboratory values occurring during the clinical study will 
be followed until repeat tests return to normal, stabilize, or are no longer clinically significant. Any 
abnormal test that is determined to be an error does not require reporting as an adverse event. 
Treatment of any adverse reactions will be evaluated and managed by a physician. 
 
8.2 Definitions 
8.2.1 Adverse Drug Reaction 
For adverse events with a causal relationship to study drug, follow-up by the Investigator will be 
required until the event or its sequelae resolve or stabilize to a level acceptable to the Investigator. 
 
8.2.2 Unexpected Adverse Event/ Unexpected Adverse Drug Reaction 
An unexpected AE/unexpected suspected adverse reaction is an AE or suspected adverse reaction 
that is not listed in the Investigator Brochure  or is not listed at the specificity or severity that has 
been observed; or, if an Investigator Brochure is not required or available, is not consistent with the 
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risk information described in the general investigational plan or elsewhere in the current application, 
as amended. 
 
8.2.3 Serious Adverse Events  
An adverse event or adverse reaction is considered serious if, in the view of either the Investigator 
or Sponsor, it results in any of the following outcomes: 
● Death; 
● A life-threatening adverse event; 
● NOTE: An adverse event or adverse reaction is considered “life-threatening” if, in view of 
either the Investigator or Sponsor, its occurrence places the participant at immediate risk of 
death. It does not include an event that, had it occurred in a more severe form, might have 
caused death. 
● Requires hospitalization or prolongation of existing hospitalizations; 
NOTE: Any hospital admission with at least one overnight stay will be considered an 
inparticipant hospitalization. An emergency room visit without hospital admission will not 
be recorded as a SAE under this criterion, nor will hospitalization for a procedure scheduled 
or planned before signing of informed consent. However, unexpected complications and/or 
prolongation of hospitalization that occur during elective surgery should be recorded as 
adverse events and assessed for seriousness. Admission to the hospital for social or 
situational reasons (i.e., no place to stay, live too far away to come for hospital visits) will 
not be considered inparticipant hospitalizations.  
● A persistent or significant disability/incapacity or substantial disruption of the ability to 
conduct normal life functions; 
● A congenital anomaly/birth defect; or 
● An important medical event. 
NOTE: Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical judgment, 
they may jeopardize the participant and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition. Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in an emergency room or at 
home, blood dyscrasias or convulsions that do not result in in participant hospitalizations, or 
the development of drug dependency or drug abuse. 
 
8.3 Assessment of Adverse Events by the Investigator 
8.3.1 Causality/Relatedness 
The relationship of an adverse event to the administration of the study drug is to be assessed by the 
Investigator according to the following definitions: 
 
Not Related (unlikely related, unrelated, not related, no relation) – The time course between the 
administration of study drug and the occurrence or worsening of the adverse event rules out a causal 
relationship and/or another cause (e.g., medical history, concomitant drugs, therapies, and 
complications) is suspected. 
 
Related (possibly related, related) – The time course between the administration of study drug and 
the occurrence or worsening of the adverse event is consistent with a causal relationship and no other 
cause (e.g., medical history, concomitant drugs, therapies, and complications) can be identified. 
 
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The definition implies a reasonable possibility of a causal relationship between the event and the 
study drug. This means that there are facts (evidence) or arguments to suggest a causal relationship.   
 
The following factors should also be considered: 
• 
The temporal sequence from study drug administration - The event should occur after the 
study drug is given. The length of time from study drug exposure to event should be 
evaluated in the clinical context of the event. 
• 
Underlying, concomitant diseases (medical history) -  Each report should be evaluated in 
the context of the natural history and course of the disease being treated and any other 
disease the participant may have. 
• 
Concomitant drug - The other drugs the participant is taking or the treatment the 
participant receives should be examined to determine whether any of them might be 
recognized to cause the event in question.  
• 
Known response pattern for this class of study drug - Clinical and/or preclinical data may 
indicate whether a particular response is likely to be a class effect. 
• 
Exposure to physical and/or mental stresses - The exposure to stress might induce adverse 
changes in the recipient and provide a logical and better explanation for the event. 
• 
The pharmacology and PK of the study drug - The known pharmacologic properties 
(absorption, distribution, metabolism, and excretion) of the study drug should be 
considered. 
 
8.3.2 Severity 
The Investigator is responsible for assessing the severity (intensity) of each adverse event as mild, 
moderate, or severe according to the following definitions: 
 
Mild – An event that is easily tolerated and generally not interfering with normal daily activities. 
Moderate – An event that is sufficiently discomforting to interfere with normal daily activities. 
Severe – An event that is incapacitating with inability to work or perform normal daily activities. 
It should be noted that a severe AE need not be serious and that a serious adverse event (SAE) need 
not, by definition, be severe. 
8.3.3 Adverse Event Monitoring and Follow-up 
Participants who experience AEs will be monitored with relevant clinical assessments and laboratory 
tests, as determined by the Investigator.  In accordance with good medical practice, all AEs must be 
followed to satisfactory resolution or stabilization of the event(s), or, if a chronic condition, until 
fully characterized.  Any actions taken and follow-up results must be recorded either on the 
appropriate page of the CRF, as well as in the participant’s source documentation.  Follow-up 
laboratory results should be filed with the participant’s source documentation.   
 
For all AEs that require the participant to be discontinued from the study, relevant clinical 
assessments and laboratory tests must be repeated at appropriate intervals until final resolution or 
stabilization of the event(s). 
 
Participants who have non-serious AEs that are ongoing at study completion or study withdrawal 
must be followed until resolution of the AEs or for 30 days after the last dose of study drug, 
whichever comes first.  Participants who have an ongoing SAE at study completion or study 
withdrawal must be followed until the event resolves or the event or sequela stabilizes. 
 
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SAE documentations will include: 
Investigator details (name, phone, fax, e-mail) 
Participant number 
Participant demographics (age, date of birth, sex, weight) 
Clinical event 
o Description 
o Date of onset 
o Treatment (drug, dose, dosage form) 
o AE relationship to study drug 
o Action taken regarding study drug in direct relationship to the AE 
If the AE was fatal or life-threatening 
Cause of death (whether or not the death was related to study drug) 
Autopsy findings (if available) 
 
The Investigator must continue to follow the participant until the SAE has subsided or until the 
condition becomes chronic in nature, stabilizes (in the case of persistent impairment), or the 
participant dies. Within 24 hours of receipt of follow-up information, the Investigator must update 
the Sponsor and submit any supporting documentation (e.g., participant discharge summary or 
autopsy reports). 
 
Any new SAE that occurs within one month after the study period and is considered to be possibly 
related to the investigational product should be recorded and reported immediately to the Sponsor.   
The clinical research site will be responsible for reporting SAEs to the Institutional Review Board 
(IRB) per FDA regulations.   
 
9 STUDY PROCEDURES AND ASSESSMENTS BY VISIT 
9.1 Schedule of Assessments 
The study timetable in Section 14.1 shows the schedule of planned study procedures.  Every effort 
should be made to adhere to this procedure schedule. 
9.2 Screening (Day -28 to Day -1) 
The study will consist of a Screening period of 28 days or less.  Screening and Baseline visits may 
be combined. 
 
Each potential study participant will have the following assessments completed by the Investigator 
or designee up to 28 days prior to the first dose of study medication:  
● Obtain informed consent; 
● Obtain medical/surgical history; 
● Obtain demographics and social history (tobacco, alcohol, and/or drug use); 
● Obtain prior/concomitant medication use; 
● Conduct full physical exam; 
● Obtain height and weight; 
● Obtain vital signs; 
● Draw blood samples for Chemistry and Hematology; 
● Collect urine for safety urinalysis; 
● Complete Stanford-Binet assessment; 
● Evaluate inclusion and exclusion criteria. 
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9.3 Baseline (Period 1/Day 1) 
Participants will return to the clinic for the Period 1/Day 1 Visit (Baseline) following successful 
screening. If clinic processes allow, the Screening and Baseline visits may be combined; in this case, 
it is not necessary to repeat vital signs nor to conduct the abbreviated physical exam.   
 
9.3.1 Prior to Drug Dispensing  
The following assessments will be completed:  
● Conduct abbreviated physical exam (not to be repeated if the Screening and Baseline visits 
occur on the same day); 
● Obtain vital signs (not to be repeated if the Screening and Baseline visits occur on the same 
day); 
● Record any updates to medical/surgical history; 
● Record any updates to prior/concomitant medication use; 
● Re-evaluate inclusion and exclusion criteria based on preceding activities;  
● Verify continued eligibility; 
● Complete baseline cognitive, behavioral and pharmocodynamic assessments: 
o NIH Toolbox Cognitive Battery for Intellectual Disabilities (NIH-TCB) 
o Test of Attentional Performance (KiTAP) 
o Clinical Global Impression Severity – Investigator rated (CGI-S) 
o Visual Analog Scale (VAS) assessment of participant-specific behavioral anchors 
o Aberrant Behavior Checklist (ABC) 
o Anxiety, Depression, and Mood Scale (ADAMS) 
o Vineland-3 Rating Scale 
o Event-Related Potentials (ERP) 
o Eye Tracking  
 
If participant remains eligible for the study: 
● Dispense study medication. 
 
9.3.2 Weeks 1, 5, 9, 13 (Periods 1 through 4) 
The following assessments will be completed at the Week 1 Visit during each Period: 
● Telephone call to: 
o Record any updates to concomitant medication use 
o Assess and record any adverse events. 
 
9.3.3 Week 4, 8, 12, 16 (end Periods 1 through 4) 
The following assessments will be completed at the Week 12 Visit during Periods 1 and 2: 
● Collect weight; 
● Collect vital signs; 
● Draw blood samples for Chemistry and Hematology; 
● Collect urine for safety urinalysis; 
● Conduct the following cognitive, behavioral and pharmocodynamic tests: 
o NIH Toolbox Cognitive Battery for Intellectual Disabilities (NIH-TCB) 
o Test of Attentional Performance (KiTAP) 
o Clinical Global Impression Severity – Investigator rated (CGI-S) 
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o Clinical Global Impression Improvement – Investigator rated (CGI-I) 
o Visual Analog Scale (VAS) assessment of participant-specific behavioral anchors 
o Aberrant Behavior Checklist (ABC)  
o Anxiety, Depression, and Mood Scale (ADAMS) 
o Vineland-3 Rating Scale 
o Event-Related Potentials (ERP) 
o Eye Tracking  
● Collect study medication and record capsule count remaining in bottle to assess dosing 
compliance; 
● Record any updates to concomitant medication use; and 
● Assess and record any adverse events. 
 
9.3.4 Week 17 (Period 4, Final Contact at End of Study) 
● Telephone call to: 
o Record any updates to concomitant medication use, 
o Assess and record any adverse events, and 
o Record final participant disposition. 
 
9.3.5 Early Termination 
It is hoped that all participants can be followed through to the conclusion of the study at Period 
4/Week 17.  However, if an early termination occurs, the Week 16 procedures should be completed 
at the time of the participant’s withdrawal (if possible) and an explanation provided as to why the 
participant is withdrawing or being withdrawn from the study.  Procedures to be completed early 
termination: 
● Collect weight; 
● Collect vital signs; 
● Draw blood samples for Chemistry and Hematology; 
● Collect urine for safety urinalysis; 
● Conduct the following cognitive tests: 
o NIH Toolbox Cognitive Battery for Intellectual Disabilities (NIH-TCB) 
o Test of Attentional Performance (KiTAP) 
o Clinical Global Impression Severity – Investigator rated (CGI-S) 
o Clinical Global Impression Improvement – Investigator rated (CGI-I) 
o Visual Analog Scale (VAS) assessment of participant-specific behavioral anchors 
o Aberrant Behavior Checklist (ABC)  
o Anxiety, Depression, and Mood Scale (ADAMS) 
o Vineland-3 Rating Scale 
o Event-Related Potentials (ERP) 
o Eye Tracking  
● Collect study medication and record capsule count remaining in bottle to assess dosing 
compliance; 
● Record any updates to concomitant medication use; 
● Assess and record and adverse events; and 
● Record final participant disposition. 
 
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10 PLANNED STATISTICAL METHODS 
10.1 Sample Size 
Given the exploratory nature of this study, a definitive sample size calculation is not possible.  
 
10.2 Demographics 
Summary statistics [number (n), mean, standard deviation (SD), minimum, median, and maximum] 
will be tabulated for the observed values for all continuous demographic parameters.  Frequencies 
and percentages will be tabulated for categorical data. 
 
10.3 Analysis Populations 
The primary efficacy population will be the intent to treat (ITT) efficacy population, which will 
include all randomized participants who received at least one dose of treatment and returned for at 
least one follow-up visit. The completers population (CP), defined as all randomized participants 
who completed all treatment periods with no significant protocol violations, will be used to evaluate 
the robustness of the ITT results.  
 
The Safety population will include all randomized participants who received at least one dose of 
study treatment. 
 
10.4 Efficacy: Cognitive, Behavioral and Pharmocodynamic Measures Analysis 
All cognitive and behavioral parameters will be summarized at each time point collected and 
standard descriptive statistics provided. Baseline measurements are defined as those obtained on 
Day 1/Period 1 prior to receipt of any study medication.  For cognitive parameters measured at 
baseline, change from baseline will also be calculated and standard descriptive statistics provided 
for each time point. 
 
10.5 Safety Analysis 
Safety analysis will be based on all participants receiving at least one dose of study medication.  
Treatment emergent AEs will be summarized based on the frequency of AEs and their severity for 
all dosed participants.  Adverse Events (AEs), including clinically meaningful laboratory 
abnormalities and significant behavioral changes, will collected. AE severity and relatedness to 
treatment will be assessed. AEs will be tabulated for placebo and BPN14770 treatments using 
preferred terms and system organ classes. Summaries will also be provided by severity and 
relationship to treatment. Serious Adverse Events (SAEs) will be summarized separately, in a 
manner similar to that used for Adverse Events. 
 
Overall safety and tolerability will be assessed by clinical and/or statistical review of all safety 
parameters including adverse experiences, laboratory values, vital signs, physical exams, and ECG 
data.  General clinical and laboratory adverse events will be presented in frequency tables.  Summary 
statistics (n, mean, SD, median, and range (minimum, maximum) will be tabulated for the observed 
values and the change (or percent change) from baseline for all continuous parameters for vital signs 
and laboratory tests. 
 
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11 DATA MANAGEMENT 
11.1 Data Handling 
Data will be recorded at the site on CRFs. All corrections or changes made to any study data will be 
noted on the CRFs and dated appropriately.  
 
11.2 Data Entry into EDC 
Data will be collected and processed in RedCap. Data will be recorded in RedCap as the study is in 
progress. All site personnel must log into the system using their secure user name and password in 
order to enter, review, or correct study data. All passwords will be strictly confidential. 
 
The Investigator has ultimate responsibility for the accuracy, authenticity, and timely collection and 
reporting of all clinical, safety, and laboratory data entered on the CRFs and any other data collection 
forms.   
 
11.3 Data Validation 
Validation checks programmed within RedCAP, as well as supplemental validation performed via 
review of the downloaded data, will be applied to the data in order to ensure accurate, consistent, 
and reliable data. Data identified as erroneous, or data that are missing, will be corrected. 
 
The paper CRFs must be reviewed and signed by the Investigator.   
 
12 INVESTIGATOR REQUIREMENTS AND QUALITY CONTROL 
12.1 Ethical Conduct of the Study 
The Investigator agrees to adhere to the instructions and procedures that are described in the protocol 
and to conduct the study in accordance with the CFRs (21 CFR § 11, 50, 54, 56, and 312 Subpart 
D), which originate from the ethical principles laid down in the current revision of the Declaration 
of Helsinki, Good Clinical Practice (GCP),26,27 and the policies and procedures as outlined by the 
ethical requirements for IRB review and informed consent form.   
The Investigator agrees to allow inspection by the FDA or other appropriate regulatory authorities 
as needed.   
 
The Investigator will ensure proper implementation and conduct of the trial, including those study-
related duties that are delegated to other appropriately qualified individuals.  The Investigator will 
ensure that study staff cooperates with monitoring and audits and will demonstrate due diligence in 
recruiting and screening study participants. 
 
12.2 Institutional Review Board (IRB) 
Before initiation of the study, the Investigator will obtain approval or favorable opinion of the 
research protocol, informed consent form, and any advertisement for participant recruitment from 
an IRB complying with the provisions specified in 21 CFR §56 and applicable pertinent state and 
federal requirements of each participating location including International Conference on 
Harmonization (ICH) and GCP guidelines. 
 
Institutional review boards must be constituted according to the applicable laws.  It is the 
responsibility of each investigational site to submit the protocol, Investigators’ Brochure, participant 
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Page 36 of 42 
 
informed consent, participant recruitment materials (if applicable), and other documentation as 
required by the IRB to their IRB for review and approval.   
 
The study site will adhere to all requirements stipulated by their respective IRB.  This includes 
notification to the IRB regarding: protocol amendments, updates to the participant informed consent, 
recruitment materials intended for viewing by participants, serious and unexpected AEs, reports and 
updates regarding the ongoing review of the trial at intervals specified by the respective IRB, and 
submission of final summaries to IRB. 
 
The Investigator will promptly inform his/her IRB of all unexpected SAEs considered possibly 
related to the treatment or other critical safety information reported by the clinical site. 
 
12.3 Informed Consent 
The informed consent form (ICF) and any changes to the ICF made during the course of the study 
must be agreed to by the IRB prior to its use and must be in compliance with all ICH GCP, provisions 
specified in 21 CFR §50, and applicable pertinent state and federal requirements.  
 
The Investigator, or a person delegated the responsibility by the Investigator, will ensure that each 
study participant (or legally acceptable representative) is fully informed about the nature and 
objectives of the study and possible risks associated with participation and must ensure that the 
participant has been informed of his/her rights to privacy. The Investigator or delegate will allow 
the participant adequate opportunity to read the written informed consent and ask any questions. The 
Investigator will obtain written informed consent from each participant before any study-specific 
activity is performed and should document in the source documentation that consent was obtained 
prior to any study-specific activity. The original signed copy of the ICF must be maintained by the 
Investigator and is participant to inspection by the IRB/EC and/or regulatory agencies. A copy of 
the signed ICF will be given to the participant. 
 
12.4 Record Storage and Retention 
Records of participants, source documents, monitoring visit logs, CRFs, inventory of study product, 
regulatory documents, and other Sponsor correspondence pertaining to the study must be kept in the 
appropriate study files at the site. Source data is defined as all information in original records and 
certified copies of original records of clinical findings, observations, or other activities in a clinical 
study necessary for the evaluation and reconstruction of the clinical study. Source data are contained 
in source documents (original records or certified copies). These records will be retained in a secure 
file for 10 years, prior to transfer or destruction of the records. 
 
The Investigator will keep records, including the identity of all participating participants (sufficient 
information to link records, e.g., CRFs and hospital records), all original signed ICFs, copies of all 
CRFs, SAE forms, source documents, and detailed records of treatment disposition. The records 
should be retained by the Investigator according to specifications in the ICH guidelines or local 
regulations. If the Investigator relocates, retires, or for any reason withdraws from the study, the 
study records will be transferred to an acceptable designee, such as another Investigator.  
 
12.5 Protocol Amendments and Deviations 
All protocol amendments will undergo the same review and approval process as the original 
protocol. A protocol amendment may be implemented only after it has been approved by the IRB, 
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Page 37 of 42 
 
unless immediate implementation of the change is necessary for participant safety. In this case, the 
situation must be documented and reported to the IRB within 5 working days. 
 
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26. Declaration of Helsinki (Fortaleza, October 2013) Seventh revision, 64th World Medical 
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35. Berry-Kravis E, Sansone S, Crestodina C, McLennan Y, Chin J, Joshi R, Schneider A, Leigh 
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37. McCracken JT, McGough J, Shah B, et al. Risperidone in children with autism and serious 
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39. Guy, William (1976). "Clinical Global Impressions". ECDEU Assessment Manual for 
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40. Berry-Kravis E, Hessl D, Rathmell B, Zarovecs P, Cherubini M, Walton-Bowen K, 
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(arbaclofen) on neurobehavioral function in children and adults with fragile X syndrome: a 
randomized, controlled, phase 2 trial. Sci Transl Med 2012 Sep 19;4(152):152ra127. 
41. Sansone SM, Widaman KF, Hall SS, et al. Psychometric Study of the Aberrant Behavior 
Checklist in Fragile X Syndrome and Implications for Targeted Treatment. Journal of 
Autism and Developmental Disorders. 2012;42(7):1377-1392. doi:10.1007/s10803-011-
1370-2. 
42. Lisa Cordeiro, Elizabeth Ballinger, Randi Hagerman and David Hessl. Clinical assessment 
of DSM-IV anxiety disorders in fragile X syndrome: prevalence and characterization. J 
Neurodevelop Disord. 2010; 3:9067. https://doi.org/10.1007/s11689-010-9067-y. 
43. Vineland Adaptive Behavior Scales, Third Edition (Vineland-3). Sara S. Sparrow, PhD, 
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44. Luck S. J. An Introduction to the Event-Related Potential Technique. 2005. Cambridge, MA: 
MIT Press. 
45. Knoth IS, Lippé S. Event-related potential alterations in fragile X syndrome. Frontiers in 
Human Neuroscience. 2012;6:264. doi:10.3389/fnhum.2012.00264. 
46. Ethridge et al. Neural synchronization deficits linked to cortical hyper-excitability and 
auditory hypersensitivity in fragile X syndrome. Molecular Autism 2017;8:22 DOI 
10.1186/s13229-017-0140-1. 
47. Ethridge L, Thaliath A, Kraff J, Nijhawan K, Berry-Kravis E. Development of neural 
response to novel sounds in fragile X syndrome: potential biomarkers. Am J Intellect Dev 
Disabil. 2020;125:449-464.  
48. Farzin, F., Scaggs, F., Hervey, C. et al. Reliability of eye tracking and pupillometry measures 
in individuals with Fragile X Syndrome.  J Autism Dev Disord. 2011; 41:1515. 
https://doi.org/10.1007/s10803-011-1176-2. 
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14 Appendices 
14.1 Schedule of Assessments 
 
 
PERIOD 1 
PERIOD 2 
PERIOD 3 
PERIOD 4 
 
Screening 
Days -28 
thru 0 
P1 
Day 1 
P 1 
Wk1 
(±1) 
P 1 
Wk4 
(±5) 
P2 
Wk5 
(±1) 
P2 
Wk8 
(±5) 
P3 
Wk9 
(±1) 
P3 
Wk12 
(±5) 
P4 
Wk13 
(±1) 
P4 
Wk16 
(±5) 
P4 
Wk16 
(ETa) 
(±5) 
P4 
Wk17 
(±1) 
Clinic Visit 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
Telephone Call  
 
 
X 
 
X 
 
X 
 
X 
 
 
X 
Informed Consent X 
 
 
 
 
 
 
 
 
 
 
 
Eligibility   
X 
Xb 
 
 
 
 
 
 
 
 
 
 
Medical /Surgical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam  
X  
 
 
 
 
 
 
 
 
 
 
 
Abbreviated PE 
 
Xb 
 
X 
 
X 
 
X 
 
X 
X 
 
Ht, Wt, BMI 
X 
 
 
Xc 
 
Xc 
 
Xc 
 
Xc 
Xc 
 
Vital Signsd 
X 
Xb 
 
X 
 
X 
 
X 
 
X 
X 
 
Chemistry 
and 
Hematology 
X 
 
 
X 
 
X 
 
X 
 
X 
X 
 
Urinalysis 
X 
 
 
X 
 
X 
 
X 
 
X 
X 
 
Suicidality 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
Dispense 
Study 
Medication 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
1st 
Dose 
of 
Medication 
 
X 
 
 
 
 
 
 
 
 
 
 
Stanford-Binet 
X 
 
 
 
 
 
 
 
 
 
 
 
NIH-TCB 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
KiTAP 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
CGI-S 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
CGI-I 
 
 
 
X 
 
X 
 
X 
 
X 
X 
 
VAS 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
ABC 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
ADAMS 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
Vineland-3 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
ERP 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
Eye Tracking 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
IP accountability 
 
 
 
X 
 
X 
 
X 
 
X 
X 
 
Con Meds 
X 
Xb 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Final Disposition  
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Note: Screening and Period 1 Day 1 visits may be combined 
a If early termination occurs, the Week 16 procedures should be completed at the time of the participant’s withdrawal (if possible) 
b Do not need to repeat if screening and baseline done at same visit 
c Weight only 
d Vital signs include pulse rate, blood pressure, respiration rate, and temperature 
 
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14.2 Clinical Laboratory Analytes 
Standard Chemistry  
Alanine aminotransferase (ALT)  
Albumin 
Alkaline phosphatase 
Aspartate aminotransferase (AST) 
Bicarbonate  
Blood urea nitrogen (BUN) 
Calcium 
Chloride 
Creatinine 
Glucose 
Potassium 
Sodium 
Total bilirubin 
Total protein 
 
Hematology  
Hematocrit 
Hemoglobin 
Platelet count 
Red blood cell (RBC) 
Mean corpuscular hemoglobin concentration (MCHC) 
Mean corpuscular hemoglobin (MCH) 
White blood cell count and differential (basophils, 
eosinophils, lymphocytes, monocytes, and neutrophils) 
Mean corpuscular volume (MCV) 
. 
Urinalysis 
Bilirubin 
Blood 
Glucose 
Ketones 
Nitrite 
pH 
Protein 
Specific Gravity 
Urobilinogen  
Leukocyte esterase 
 
If protein, urine occult blood, nitrite, or leukocyte esterase values are out of range, the microscopic examination will be reported.  
 
 
 
 
 
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