Conditions: Pitt Hopkins Syndrome
Keywords: Pitt Hopkins Syndrome
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 28
Healthy volunteers: no

Brief summary:
A study of the safety, tolerability and pharmacokinetics of NNZ-2591 and measures of efficacy in children and adolescents with Pitt Hopkins Syndrome.

Detailed description:
The primary purpose of this study is to investigate the safety, tolerability and pharmacokinetics of treatment with NNZ-2591 oral solution in children and adolescents with Pitt Hopkins Syndrome. The secondary purpose is to investigate measures of efficacy. Subjects will receive treatment with NNZ-2591 oral solution (50 mg/mL) doses for a total of 13 weeks.

Arms:
arm 1: NNZ-2591 oral solution (50mg/mL) to be administered twice daily dose for 13 weeks.

Interventions: intervention 1: NNZ-2591
Intervention descriptions: intervention 1: NNZ-2591 oral solution (50mg/mL) to be administered twice daily dose for 13 weeks.

Protocol document:
Prot_002.pdf: 


Neuren Pharmaceuticals Ltd. 
Clinical Trial Protocol 
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Confidential 
Neu-2591-PTHS-001 
October 30, 2023 
 
CONFIDENTIALITY STATEMENT 
All information concerning the Investigational Medicinal Product, supplied by Neuren Pharmaceuticals 
Ltd. in connection with this study and not previously published, is considered confidential and proprietary 
information. This information includes the Investigator’s Brochure, Clinical Protocol, and Case Report 
Forms. This confidential information shall remain the sole property of Neuren Pharmaceuticals Ltd. and 
shall not be disclosed to others without prior written consent from Neuren Pharmaceuticals Ltd. and shall 
not be used except in the performance of this study. 
 
The information developed during the conduct of the clinical study is also considered confidential and 
will be used by Neuren Pharmaceuticals Ltd. in connection with the development of the Investigational 
Medicinal Product. This information may be disclosed as deemed necessary by Neuren Pharmaceuticals 
Ltd.  


Neuren Pharmaceuticals Ltd. 
Clinical Trial Protocol 
Version 3.0 
Confidential 
Neu-2591-PTHS-001 
October 30, 2023 
 
TABLE OF CONTENTS 
CONFIDENTIALITY STATEMENT ..................................................................................................... 2 
1. LIST OF ABBREVIATIONS ............................................................................................................. 7 
2. PROTOCOL APPROVAL ............................................................................................................... 11 
3. PROTOCOL SYNOPSIS .................................................................................................................. 13 
4. SCHEDULE OF ASSESSMENTS ................................................................................................... 27 
5. INTRODUCTION ............................................................................................................................. 36 
5.1 
Pitt-Hopkins Syndrome ............................................................................................................... 36 
5.2 
Current Treatments for Pitt-Hopkins Syndrome ......................................................................... 37 
5.3 
Genetic Basis of Pitt-Hopkins Syndrome ................................................................................... 37 
5.4 
Scientific Rationale for use of NNZ-2591 in Pitt-Hopkins Syndrome ....................................... 38 
5.5 
Preclinical Data ........................................................................................................................... 38 
5.5.1 
Overview of Pharmacology and Toxicology Findings ......................................................... 38 
5.5.2 
Overview of Pitt-Hopkins Mouse Model Findings .............................................................. 39 
5.6 
Clinical Data ............................................................................................................................... 40 
6. STUDY RATIONALE AND OBJECTIVES .................................................................................. 41 
6.1 
Study Rationale ........................................................................................................................... 41 
6.2 
Rationale for Dose Selection and Route of Administration ........................................................ 41 
6.2.0 
Rationale for Dose Selection ................................................................................................ 41 
6.2.1 
Rationale for Duration of Dosing ......................................................................................... 42 
6.2.2 
Rationale for Selection of Efficacy Outcomes ..................................................................... 42 
6.3 
Study Objectives and Endpoints ................................................................................................. 43 
6.3.1 
Primary Objective ................................................................................................................. 43 
6.3.2 
Secondary Objectives ........................................................................................................... 44 
7. STUDY DESIGN ............................................................................................................................... 46 
7.1 
Study Design Overview .............................................................................................................. 46 
7.1.1 
Study Design ........................................................................................................................ 47 
7.2 
Dose-levels and Titration ............................................................................................................ 47 
7.2.1 
Adjustment of the Dose for Tolerability ............................................................................... 47 
7.2.2 
Dose Escalation Review by the DSMC ................................................................................ 48 
7.3 
Treatment Assignment ................................................................................................................ 48 
7.4 
Study Timeline ............................................................................................................................ 49 
7.5 
COVID-19 .................................................................................................................................. 49 
8. PARTICIPANT POPULATION ...................................................................................................... 49 
8.1 
Inclusion Criteria ........................................................................................................................ 50 
8.2 
Exclusion Criteria ....................................................................................................................... 50 
8.3 
Abnormal Laboratory Findings .................................................................................................. 54 
8.4 
Withdrawal Criteria .................................................................................................................... 54 
8.5 
Risks and Benefits ...................................................................................................................... 54 
9. STUDY PROCEDURES ................................................................................................................... 55 
9.1 
Overview of Study Visits ............................................................................................................ 55 


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9.2 
Study Procedures by Visit ........................................................................................................... 56 
9.2.1 
Visit 1 Screening/Baseline (in-clinic) – Week -4 (r 2 days) ................................................ 56 
9.2.2 
Visit 2 Screening/Baseline (telemedicine and in-home) – 2 Weeks from 
Visit 1 (± 1 day) .................................................................................................................... 57 
9.2.3 
Visit 3 Baseline (in-clinic) – Week 0 (r 2 days) .................................................................. 58 
9.2.4 
Visit 4 Treatment Period (telemedicine and in-home) – Week 1 (r 1 day) .......................... 59 
9.2.5 
Visit 5 Treatment Period (in-clinic) – Week 2 (r 2 days) .................................................... 60 
9.2.6 
Visit 6 Treatment Period (in-home nurse) – Week 3 (r1 day) ............................................. 61 
9.2.7 
Visit 7 Treatment Period (telemedicine and in-home) – Week 4 (r1 day) ........................... 61 
9.2.8 
Visit 8 Treatment Period (in-home nurse) – Week 5 (r1 day) ............................................. 62 
9.2.9 
Visit 9 Treatment Period (in-clinic) – Week 6 (r 2 days) .................................................... 63 
9.2.10 Visit 10 Treatment Period (in-home nurse) – Week 7 (r 1 day) .......................................... 63 
9.2.11 Visit 11 Treatment Period (telemedicine and in-home) – Week 8 (r 1 day) ........................ 64 
9.2.12 Visit 12 Treatment Period (in-home nurse) – Week 9 (r 1 day) .......................................... 65 
9.2.13 Visit 13 Treatment Period (telemedicine and in-home) – Week 10 (r1 day) ....................... 65 
9.2.14 Visit 14 Treatment Period – (in-home nurse) Week 11 (r1 day) ......................................... 66 
9.2.15 Visit 15 Treatment Period (in-home nurse) – Week 12 (r day) ......................................... 67 
9.2.16 Visit 16 End of Treatment (in-clinic) – Week 13 (-3 days) .................................................. 67 
9.2.17 Visit 17 Follow-up (telemedicine and in-home) – Week 15 (+ 4 days) ............................... 68 
9.3 
Screening and Baseline Procedures ............................................................................................ 69 
9.4 
Treatment of Participants ............................................................................................................ 71 
9.4.1 
Investigational Medicinal Product ........................................................................................ 71 
9.4.2 
Investigational Medicinal Product Administration ............................................................... 71 
9.4.3 
Treatment Compliance ......................................................................................................... 72 
9.4.4 
Protocol Deviations .............................................................................................................. 72 
9.4.5 
Early Discontinuation ........................................................................................................... 72 
9.4.6 
Concomitant Therapy ........................................................................................................... 74 
9.5 
Assessments ................................................................................................................................ 75 
9.5.1 
Vital Signs ............................................................................................................................ 75 
9.5.2 
Laboratory Tests ................................................................................................................... 76 
9.5.3 
Blood sample for biomarkers and stool sample for microbiome .......................................... 80 
9.5.4 
Medical/Clinical History ...................................................................................................... 81 
9.5.5 
Physical and Neurological Exam .......................................................................................... 81 
9.5.6 
81 
9.5.7 
Ophthalmology Assessment ................................................................................................. 81 
9.5.8 
12-lead ECG and Stopping Criteria for Prolonged QT ........................................................ 82 
9.5.9 
Battery of Pitt-Hopkins Syndrome Assessment Instruments ................................................ 83 
9.5.10 Caregiver Diary .................................................................................................................... 88 
9.6 
Pharmacokinetic Measurements ................................................................................................. 88 
10. ADVERSE EVENTS ......................................................................................................................... 90 
10.1 Definition of Adverse Event ....................................................................................................... 90 
10.2 Definition of Serious Adverse Event .......................................................................................... 90 
10.3 
 ............................................................................................. 91 


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10.4 Adverse Reactions ...................................................................................................................... 91 
10.5 Clinical Laboratory Abnormalities and Other Abnormal Assessments as AEs 
and SAEs .................................................................................................................................... 91 
10.6 Time Period, Frequency, and Method of Detecting AEs and SAEs ........................................... 91 
10.7 Recording of AEs and SAEs ....................................................................................................... 92 
10.8 Evaluating AEs and SAEs .......................................................................................................... 92 
10.8.1 Assessment of Intensity ........................................................................................................ 92 
10.8.2 Assessment of Causality ....................................................................................................... 92 
10.8.3 Follow-up of AEs and SAEs ................................................................................................ 93 
10.9 Prompt Reporting of the SAEs to the Sponsor ........................................................................... 94 
10.9.0 Completion and transmission of the SAE reports ................................................................. 94 
10.10 Deaths ......................................................................................................................................... 95 
10.11 
 ........................................ 95 
10.12 Pregnancy ................................................................................................................................... 96 
11. STATISTICAL PLAN ...................................................................................................................... 96 
11.0 Definitions .................................................................................................................................. 96 
11.0.0 Participant Populations ......................................................................................................... 96 
11.1 Sample Size Determination ........................................................................................................ 97 
11.2 Safety Analyses........................................................................................................................... 97 
11.3 Efficacy Analysis ........................................................................................................................ 97 
11.4 Pharmacokinetic Analysis ........................................................................................................... 98 
11.5 Biomarkers and Microbiome ...................................................................................................... 98 
12. STUDY DRUG MANAGEMENT .................................................................................................... 98 
12.0 Identity of Investigational Medicinal Product ............................................................................ 98 
12.1 Management of Clinical Supplies ............................................................................................... 99 
12.1.0 Drug Accountability ............................................................................................................. 99 
12.2 Overdose ..................................................................................................................................... 99 
13. QUALITY ASSURANCE AND DATA MANAGEMENT .......................................................... 100 
13.1 Data Quality Assurance ............................................................................................................ 100 
13.2 Case Report Forms.................................................................................................................... 100 
13.3 Monitoring ................................................................................................................................ 100 
13.4 Auditing .................................................................................................................................... 101 
13.5 Source Documents .................................................................................................................... 101 
13.6 Protocol Deviations ................................................................................................................... 101 
13.7 Records Retention ..................................................................................................................... 101 
14. ETHICS ............................................................................................................................................ 103 
14.1 Informed Consent ..................................................................................................................... 103 
14.2 Institutional Review Board Approval ....................................................................................... 103 
14.3 Additional Protections for Minors ............................................................................................ 104 
15. STUDY MANAGEMENT .............................................................................................................. 105 
15.1 Principal Investigators .............................................................................................................. 105 
15.2 Data Safety Monitoring Committee .......................................................................................... 105 
15.3 Publications ............................................................................................................................... 105 


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16. REFERENCES ................................................................................................................................ 106 
APPENDIX A. ALLOMETRIC SCALING TO DETERMINE PHASE 2 
EXPOSURES ................................................................................................................................... 109 
 
 
 


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1. LIST OF ABBREVIATIONS 
2MWT 
2-Minute Walk test 
AAC 
Augmentative and alternative communication 
ABC-2 
Aberrant Behavior Checklist 2 
AE 
Adverse event 
 
 
ALP 
Alkaline phosphatase 
ALT 
Alanine amino transferase 
AMSE 
Autism Mental Status Exam 
APTT 
Activated Partial Thromboplastin 
AS 
Angelman syndrome 
ASD 
Autism Spectrum Disorder 
AST 
Aspartate transaminase 
AUC 
Area under (concentration-time) curve to last time point 
AUCinf 
Area under (concentration-time) curve from time zero extrapolated to infinite time 
AUC0-24 
Area under (concentration-time) curve from time 0 to 24 hours at steady state 
AUCtau 
AUC from time zero to the end of the dosing interval 
bHLH 
Basic helix–loop–helix 
BID 
Twice daily 
BP 
BPI-SF 
Blood pressure 
Behavior Problems Inventory-Short Form 
bpm 
Beats per minute 
BR 
Bilirubin 
BSID-4 
Bayley Scales of Infant Development version 4 
BUN 
Blood urea nitrogen 
CAS 
Chemical Abstracts Service 
CBC 
Complete Blood Count 
CBD 
Cannabidiol 
CDKL5 
Cyclin-dependent kinase-like 5 
CdLS 
Cornelia de Lange syndrome 
CFR 
Code of Federal Regulations 
CGI-I 
Clinical Global Impression Scale – Improvement 
CGI-S 
Clinical Global Impression – Severity 
cGP 
Cyclic Glycine-Proline 
 
 
CLss/F 
Apparent total body clearance after oral administration  
CLtv 
True population value for clearance of the drug 
Cmax 
The maximum observed blood concentration 
CMP 
Comprehensive metabolic panel 
CoRDS 
Coordination of Rare Diseases 
COVID-19 
Coronavirus disease 2019 
CSHQ 
Child Sleep Habits Questionnaire 


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Css 
Concentration at steady-state  
Ct 
Concentration at the last measurable time point 
Ctv 
True population value for clearance of the drug 
CĲ 
Concentration at the end of the dosing interval 
°C 
Degree(s) centigrade 
CVA 
Costovertebral angle tenderness 
DBP 
Diastolic blood pressure 
dL 
Deciliter 
DNA 
Deoxyribonucleic acid 
DSM-5 
Diagnostic and Statistical Manual of Mental Disorders, 5th Edition 
DSMC 
Data Safety Monitoring Committee 
DQ 
Development quotient 
EC 
Expressive communication 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EDC 
Electronic Data Capture System 
eGFR 
Estimated glomerular filtration rate 
EL 
Expressive language 
EOT 
End of Treatment 
FDA 
Food and Drug Administration 
FM 
Fine motor 
FR 
Fluid reasoning 
FSIQ 
Full scale intellectual quotient  
g 
Gram 
GCP 
Good clinical practice 
GERD 
Gastroesophageal reflux disease 
GGT 
Gamma-glutamyl transferase 
GI 
Gastrointestinal 
GIHQ 
Gastrointestinal Health Questionnaire 
GM 
Gross motor 
GMP 
Good manufacturing practice 
GRAS 
Generally Recognized as Safe 
GSV 
Growth scale values 
Hb 
Hemoglobin 
HbA1C 
Glycated (or glycosylated) hemoglobin 
Hct 
Hematocrit 
HDPE 
High-density polyethylene 
hr, hrs 
Hour, hours 
HPF 
High power field 
HTN 
Hypertension 
IB 
Investigator’s brochure 
ICH 
International Council on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
ICND 
Impact of Childhood Neurological Disability Scale 


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IGF-1 
Insulin-Like Growth Factor 1 
IND 
Investigational new drug 
IMP 
Investigational medicinal product 
IQ 
Intelligence quotient 
IRB 
Institutional review board 
ITT 
Intent to treat 
IUD 
Intrauterine device 
 
 
kg  
Kilogram 
KN 
Knowledge 
L 
Liter 
m  
Meter 
MAO 
Monoamine oxidase 
MB-CDI 
MacArthur-Bates Communicative Development Inventory 
MCH 
Mean cell hemoglobin 
MCHC 
Mean cell hemoglobin concentration 
MCV 
Mean cell volume 
MDMA 
Methylenedioxymethamphetamine 
MedDRA 
Medical Dictionary of Regulatory Activities 
mg 
Milligram 
MIS-C 
Multisystem Inflammatory Syndrome in Children 
mL 
Milliliter 
mmHG 
Millimeter of mercury 
mmol/L 
Millimole per liter 
ms 
Millisecond 
N 
Number of participants 
NCA 
Non-compartmental analysis 
NDD 
Neurodevelopmental disorder 
NGAL 
Neutrophil gelatinase-associated lipocalin 
NHANES 
National Health and Nutrition Examination Survey 
NOAEL 
No adverse effect level 
NVIQ 
Nonverbal IQ 
ORCA 
Observer-Reported Communication Ability 
OTC 
Over-the-counter 
PHRF 
Pitt Hopkins Research Foundation 
PK 
Pharmacokinetic 
PLT 
Platelets 
PMS 
Phelan-McDermid syndrome 
popPK 
Population pharmacokinetics 
PP 
Per protocol 
PTHS 
Pitt-Hopkins syndrome 
QI-Disability 
Quality of Life Inventory-Disability 
QR 
Quantitative reasoning 
QTcF 
Fridericia’s correction factor for QT 


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RBC 
Red blood cell count 
RDW, RDW 
CV 
Red cell distribution width 
RL 
Receptive language 
rpm 
Respirations per minute 
SAE 
Serious adverse event 
SAER 
Serious adverse event report 
SAP 
Statistical analysis plan 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 
SB5 
Stanford-Binet Intelligence Scales version 5 
SBP 
Systolic blood pressure 
SCQ 
Social Communication Questionnaire 
SUSAR 
Suspected Unexpected Serious Adverse Events 
t1/2 
Terminal elimination half-life 
T3 
Triiodothyronine 
T4 
Thyroxine 
TCF4 
Transcription Factor 4 
TEAE 
Treatment emergent adverse event 
THC 
Tetrahydrocannabinol 
Tmax 
Time to maximal concentration 
TSH 
Thyroid stimulating hormone 
UA 
Urinalysis 
UK 
United Kingdom 
ULN 
Upper limit of normal 
US 
United States 
VIQ 
Verbal intelligence quotient 
VR 
Visual reception 
VS 
Visual-spatial 
WBC 
White blood cell count 
WM 
Working memory 
WTcentered 
Population value for the weight in the adult population corresponding to the derived 
CLTV 






Neuren Pharmaceuticals Ltd. 
Clinical Trial Protocol 
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3. PROTOCOL SYNOPSIS 
Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
Study Protocol Title 
An Open-Label Study of the Safety, Tolerability, and 
Pharmacokinetics of Oral NNZ-2591 in Pitt-Hopkins Syndrome 
Sponsor 
Neuren Pharmaceuticals Limited 
Investigational Product  
Research Number 
NNZ-2591 
Generic Name 
Cyclo-L-Glycyl-2-Allyl Proline (cG-2-AllylP) 
Protocol Number 
Neu-2591-PTHS-001 
NCT Number 
NCT05025332 
IND Number 
155461  
Clinical Phase (Trial Type) 
Phase 2  
Treatment Indication 
Pitt-Hopkins syndrome (PTHS) 
Primary Objective 
x 
To investigate the safety, tolerability, and pharmacokinetics 
(PK) of treatment with NNZ-2591 Oral Solution, 50mg/mL 
Investigational Medicinal Product (IMP) in children and 
adolescents with PTHS 
Secondary Objectives 
x 
To investigate measures of efficacy during treatment with 
NNZ-2591 Oral Solution, 50 mg/mL IMP in children and 
adolescents with PTHS 
Trial Design 
Neu-2591-PTHS-001 is an open-label study of the safety, tolerability, 
and PK of NNZ-2591 Oral Solution, 50mg/mL in male and female 
children and adolescents with PTHS. Participants receive treatment of 
orally administered NNZ-2591 for a total of 13 weeks. 
 
Overall Design 
There are a total of 17 study visits over the duration of the study, 5 in-
clinic visits, and 12 remote/in-home visits (see Table 1). Remote visits 
between the Investigator or designee at the clinical site and the 
participant at home are conducted via telemedicine or video facilities 


Neuren Pharmaceuticals Ltd. 
Clinical Trial Protocol 
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Neu-2591-PTHS-001 
October 30, 2023 
 
Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
as appropriate. In-home visit refers to a visit made to the participant’s 
home by a visiting nurse. 
The study will commence with an approximately 4 to 6 week 
Screening and Baseline period. During this Screening/Baseline period, 
participants will be assessed for study eligibility. Rescreening of 
participants is allowable under certain circumstances with the approval 
of the Sponsor and Medical Monitor.  
 
During the Screening/Baseline period data will also be collected to 
establish the participant’s baseline characteristics and symptom 
severity based on assessments collected during the 4 to 6 week period. 
There are two in-clinic visits and one remote/in-home visit during the 
Screening and Baseline period (Visits 1, 2, 3).  
 
Once eligibility is confirmed, participants will be dosed at a starting 
dose of 4 mg/kg twice-daily (BID) and up-titrated to a target dose of 
12 mg/kg BID of orally administered NNZ-2591. The mg/kg dosing is 
based on the participant’s weight at Baseline (Visit 3). Participants will 
receive treatment with NNZ-2591 for a total of 13 weeks. During the 
treatment period there are three in-clinic visits: Week 2 (Visit 5), Week 
6 (Visit 9) and Week 13 (Visit 16). There is a remote visit by the 
Investigator via telemedicine and a same-day in-home visit by a 
visiting nurse at Week 1, Week 4, Week 8, and Week 10. Additionally, 
there are in-home visits by a visiting nurse at Week 3, Week 5, Week 
7, Week 9, Week 11, and Week 12. 
All participants will have a combined remote Follow-Up visit with the 
Investigator via telemedicine and an in-person home visit by a visiting 
nurse approximately 2 weeks after the end of treatment (Week 15, 
Visit 17). 
 
For all visits designated as in-clinic, an in-person visit is preferred. All 
requests for off-site assessments due to extenuating circumstances, 
such as related to the Coronavirus-disease (COVID-19) health 
emergency, must be approved by the Sponsor or Medical Monitor in 
advance. 
 
Enrollment Groups 
For enrollment, the participants will be divided into three groups by 
age: 13–17-year-olds (Group 1), 8–12-year-olds (Group 2), and 3–7-
year-olds (Group 3). Enrollment will commence in the oldest age 
group (Group 1). After at least three participants in Group 1 have 
received two weeks of treatment with the IMP at the starting dose (4 
mg/kg BID), safety and tolerability data will be reviewed by the Data 
Safety Monitoring Committee (DSMC). If tolerability and safety data 
are deemed acceptable, enrollment for Group 2 will proceed and 


Neuren Pharmaceuticals Ltd. 
Clinical Trial Protocol 
Version 3.0 
Confidential 
Neu-2591-PTHS-001 
October 30, 2023 
 
Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
dosing will begin at the starting dose. When at least three participants 
in Group 2 have received the first two weeks of treatment at the 
starting dose, safety and tolerability data will be reviewed by the 
DSMC. If tolerability and safety data for Group 2 are deemed 
acceptable, enrollment for Group 3 will commence and dosing will 
begin at the starting dose. 
 
COVID-19  
As a result of the COVID-19 public health emergency, this protocol 
includes provision for alternative methods of performing safety and 
efficacy assessments in accordance with the FDA Guidance on 
Conduct of Clinical Trials of Medical Products during the COVID-19 
Public Health Emergency January 27, 2021.* Provision is made for 
off-site assessments where necessary, and all efforts have been made to 
ensure the alternative processes are consistent as much as possible with 
in-clinic visits. For visits that are designated as in-clinic, an actual in-
clinic visit is preferred. However, an in-home/remote visit may be 
conducted for a scheduled in-clinic visit with the prior approval of the 
Sponsor and Medical Monitor. Clinical investigators are to document 
the reason for any contingency measures implemented. If at any time 
in the study, the participant or his or her caregiver tests positive for 
VHYHUH DFXWH UHVSLUDWRU\ V\QGURPH FRURQDYLUXV  6$56-CoV-2) or 
becomes ill with symptoms of COVID-19, the family should be 
instructed to contact the site as soon as possible. Once informed, the 
Investigator should contact the Medical Monitor. 
Study Population 
Approximately 10-20 male and female participants between the ages of 
3 and 17 years are anticipated to complete the study.  
The participants will be categorized by age in three groups: 13 to 17-
year-olds (Group 1), 8 to 12-year-olds (Group 2), and 3 to 7-year-olds 
(Group 3). Enrollment will be balanced to the extent possible by sex 
and age with a minimum of 3 males and 3 females across all age 
groups, and a minimum of 3 participants in each age group. 
Inclusion Criteria 
To be eligible for this study, participants must meet all of the inclusion 
criteria and none of the exclusion criteria. Inclusion criteria include: 
1. Clinical diagnosis of PTHS with a documented disease-
causing genetic etiology for the disorder.  
2. Males or females ages 3 to 17 years at screening, inclusive. 
 
* FDA Guidance on Conduct of Clinical Trials of Medical Products During the COVID-19 Public Health 
Emergency: Guidance for Industry, Investigators, and Institutional Review Boards (Center for Biologics 
Evaluation and Research; Center for Devices and Radiological Health; Center for Drug Evaluation and 
Research; Office of the Commissioner; Office of Clinical Policy and Programs; Office of Clinical Policy; Office 
of Good Clinical Practice) (2021). 


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Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
3. Body weight of 12 kg or higher at Screening. Weight 
measurements may be repeated during the Screening period in 
consultation with the Medical Monitor. 
4. Participants with a Clinical Global Impression – Severity 
(CGI-S) score of 4 or greater at the Screening visit. 
5. Not actively undergoing regression or loss of skills, defined as 
no persistent loss of previously acquired developmental skills 
for a period within 3 months of the Screening visit. 
a. Previously acquired skills include any skill established 
and used on a daily basis for at least 3 months. 
b. Variations in use of skills that is part of a general 
pattern of ups and downs, as determined by the 
investigator, are not counted as regression. 
c. Skills may include language, non-verbal 
communication, fine motor skills, ambulation 
(including gait, coordination, independence of 
walking/standing), social skills, play, self-help.  
6. Each participant must be able to swallow the IMP provided as 
a liquid solution. 
7. The participant’s caregiver(s) must have sufficient English 
language skills to complete caregiver assessments in English. 
Exclusion Criteria 
To be eligible for this study, participants must meet all of the inclusion 
criteria and none of the exclusion criteria. Exclusion criteria include: 
Laboratory and Screening Values 
 
1. Body weight < 12 kg at Screening. Weight measurements may 
be repeated during the Screening period in consultation with 
the Medical Monitor. 
2. HbA1c values above 7% at the Screening visit. 
3. Clinically significant abnormalities (as determined by the 
Investigator) in safety laboratory values, or vital signs, as 
measured at the Screening visit. Laboratory testing may be 
repeated during the Screening period in consultation with the 
Medical Monitor. 
4. Positive pregnancy test at the Screening visit. 
5. QTcF Exclusions (any of the following): 
a. Screening QTcF interval of >450 ms assessed using 
Fridericia’s correction factor based on 12-lead 
electrocardiogram (ECG) obtained as a continuous 
ECG of at least 50 seconds. 
b. Hypokalemia (Serum potassium at the Screening visit 
<3.0 mmol/L or mEq/L). Serum potassium may be 
repeated during the Screening period with the 
agreement of the Medical Monitor. 








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Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
35. Seizure medication whose dosing regimen is not stable 8 
weeks prior to commencing IMP. Adjustments for changes in 
weight or age are allowable. If the treatment was discontinued, 
the discontinuation must have occurred no fewer than 2 weeks 
before the Screening visit. 
36. Psychotropic medications or any other medication used for a 
chronic illness (not including antibiotics, pain relievers, anti-
diarrheals, and laxatives) whose doses and dosing regimen 
have not been stable for at least 4 weeks prior to the Screening 
visit. If the treatment was discontinued, the discontinuation 
must have occurred no fewer than 2 weeks before the 
Screening visit. 
a. This includes nutraceutical treatments and 
cannabinoids (tetrahydrocannabinol [THC] and 
cannabidiol [CBD]) which are allowable but also must 
meet the stability conditions above. 
b. Pain relievers, laxatives, and anti-diarrheals may be 
used as needed. 
c. Standard of care immunizations and vaccinations 
(including COVID-19) are allowed and are not subject 
to the above stability conditions. 
d. The Investigator should consult with the medical 
monitor if a participant is being treated with ocular 
toxic, hepatotoxic, nephrotoxic or antibiotic 
medications.  
e. If the Investigator is unsure if a medication is 
permitted, the Medical Monitor should confirm if the 
medication is allowed and will provide written 
documentation to support the use of the medication, if 
allowed. 
 
Regression of Skills 
 
37. Actively undergoing regression or loss of skills defined as a 
persistent loss of previously acquired developmental skills 
within 3 months of the Screening visit.  
a. Previously acquired skills include any skill established 
and used on a daily basis for at least 3 months. 
b. Variations in use of skills that is part of a general 
pattern of ups and downs (as determined by the 
Investigator) are not counted as regression. 
c. Skills may include language, non-verbal 
communication, fine motor skills, ambulation 
(including gait, coordination, independence of 
walking/standing), social skills, play, self-help.  
 


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Seizures 
 
38. Seizure profile that is not stable 8 weeks prior to commencing 
IMP. The overall pattern of seizure activity for the participant 
should be consistent with their typical pattern of seizures and 
be stable within the 8-week period in terms of type, intensity, 
duration, and frequency as determined by the clinical 
Investigator.  
 
Medical Conditions 
39. Currently pregnant, lactating or breastfeeding. 
40. Current clinically significant (as determined by the 
Investigator) cardiovascular, gastrointestinal, respiratory, or 
endocrine disease or clinically significant organ impairment. 
The Investigator may consult with the Medical Monitor as 
needed if he/she is uncertain if symptoms are exclusionary. 
41. Current clinically significant (as determined by the 
Investigator) hypo- or hyperthyroidism. If the participant has 
hypo- or hyperthyroidism that is controlled with medication, 
the Investigator should consult with the Medical Monitor to 
confirm if the participant is eligible.  
42. Type 1 or Type 2 diabetes mellitus requiring insulin (whether 
well controlled or uncontrolled), or uncontrolled Type 1 or 
Type 2 diabetes.  
43. Has planned surgery during the study. 
44. History of, or current, cerebrovascular disease or brain 
trauma. 
45. History of, or current catatonia or catatonia-like symptoms. 
46. History of, or current, malignancy. 
47. Current major or persistent depressive disorder (including 
bipolar depression). 
48. Significant uncorrected hearing impairment. 
 
Other Criteria 
49. Allergy to strawberry. 
50. The participant is unable to take the IMP orally and swallow 
the IMP provided as a liquid solution. Administration of the 
IMP by nasogastric tube, feeding tube, thickened liquid, or 
gastrostomy tube is not allowed. 
51. Has participated in another interventional clinical study within 
30 days prior to the Screening visit.  
52. Prior enrollment in this clinical trial.  
a. Individuals who leave the study during the Screening 
period and have not entered the study may be able to 
rescreen pending review and approval of the Sponsor 
and Medical Monitor.  
b. Individuals who were deemed eligible and entered the 
study (enrolled) who discontinue the study may not re-
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Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
53. The participant is judged by the Investigator or the Medical 
Monitor to be inappropriate for the study for any reason. 
 
Investigational Product 
NNZ-2591 Oral Solution, 50mg/mL will be administered open-label at 
the target dose of 12 mg/kg BID. The mg/kg dosing is based on the 
participant’s weight at Baseline (Visit 3). Participants will be up-
titrated as outlined in the table below.  
Dosing Schedule 
 
Week 1 
and 
Week 2 
Week 3 
Week 4 
and 
Week 5 
Week 6 
Week 7 
and 
Week 8 
Week 9 
Week 10 
to Week 
13 
4 mg/kg 
BID  
(8 mg/kg 
daily) 
4 mg/kg 
BID  
(8 mg/kg 
daily)1 
8 mg/kg 
BID  
(16 
mg/kg 
daily) 
8 mg/kg 
BID  
(16 
mg/kg 
daily)1 
12 
mg/kg 
BID  
(24 
mg/kg 
daily) 
12 
mg/kg 
BID  
(24 
mg/kg 
daily) 
 
12 
mg/kg 
BID  
(24 
mg/kg 
daily) 
 
DSMC 
Reviews 
Data 
Prior To 
Up-
titration
2 
DSMC 
Reviews 
Data 
Prior To 
Up-
titration
2 
DSMC 
Reviews 
Data 
After 2 
Weeks 
on 12 
mg/kg 
to 
Confirm 
Safety2 
BID = twice daily; DSMC = Data Safety Monitoring Committee 
1Participants will stay on their current dose until the data is reviewed by the DSMC. 
2Expected time for DSMC review will be one week. 
The dose titration schedule may be modified based on updated 
pharmacokinetic modelling analyses including data from the study. 
 
The IMP will be administered at a starting dose of 4 mg/kg BID for a 
total of approximately three weeks. After two weeks of dosing at the 
staring dose, the data will be reviewed by the DSMC, and the 
participants will stay on the starting dose while the data are reviewed.  
If safety and tolerability are deemed acceptable, participants will be 
up-titrated to 8 mg/kg BID and be dosed for a total of approximately 
three additional weeks. After two weeks of dosing at 8 mg/kg, the data 
will be reviewed by the DSMC, and the participants will stay on the 8 
mg/kg dose while the data are reviewed. If safety and tolerability are 
deemed acceptable, participants will be up-titrated to12 mg/kg BID 
and remain on this dose for seven weeks, The DSMC will determine if 
dose escalation can proceed based on pre-specified stopping rules 
detailed in the body of the protocol. The total administration period for 
NNZ-2591 will be 13 weeks.  For staggered enrollment, the 


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Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
participants will be divided into three groups by age: 13 to 17-year-
olds (Group 1), 8 to 12-year-olds (Group 2), and 3 to 7-year-olds 
(Group 3). The DSMC will review and approve the start of enrollment 
in the younger age groups. 
 
NNZ-2591 is provided as a ready-to-use strawberry flavored liquid for 
oral administration. The IMP should not be mixed with water, liquid 
thickeners, or food for administration. The dose can be taken from a 
cup or dosing syringe. Doses should be given on an empty stomach (2 
hours after the previous meal or snack and 30 minutes prior to the 
next). 
 
The first dose of IMP will be administered in the clinic after all 
Baseline assessments are completed, or, if the Investigator judges that 
it is too late in the day, on the following day. The participant should be 
monitored for at least three hours after the first dose. If the first dose is 
administered the day following Baseline, vital signs should be 
collected before the dose is administered. Day 1 of dosing is defined 
by the date when the first dose is taken. At this visit, the site will 
provide IMP for the caregiver to dispense at home. Enough IMP will 
be provided to cover the interval until the next in-clinic visit when the 
caregiver will be re-supplied from the site. 
 
The protocol includes procedures for dose titration and dose 
adjustments, if any are necessary (Section 7.2), and pre-specified 
stopping criteria (i.e., discontinuation of IMP) consistent with the 
exclusion criteria, including QTcF, renal, ocular, and hepatic, 
exclusion criteria (Section 9.4.5). 
Endpoints 
Primary Endpoints 
 
Safety: 
The incidence of AEs and SAEs from commencement of IMP 
administration through to the time of the last study visit will be 
evaluated. Vital signs, laboratory measures, urine output history, 
physical exam results (neurological, ophthalmologic, and general), 
, and ECGs will also be evaluated from 
screening/baseline through the end of the study. 
 
Tolerability will be assessed based on caregiver reports and the 
Caregiver Diary, required dosing adjustments or discontinuations, 
dosing compliance, and AEs related to participant experience. 
 
Pharmacokinetics: 
Pharmacokinetic blood samples will be collected according to the age 
appropriate sampling schedule for each participant (See Table 2 and 
Table 3). Pharmacokinetic concentrations and exposure parameters for 
NNZ-2591 will be derived by sparse non-compartmental analysis 


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(NCA), and may include maximum concentration (Cmax), area under 
the concentration-time curve from time 0 to the end of the dosing 
period (AUCtau) and other parameters. 
 
Efficacy: 
x 
Overall Improvement: 
o PTHS-specific Clinical Global Impression Scale–
Improvement (CGI-I), Overall Improvement Scores; 
clinician-assessed 
o PTHS-specific Clinical Global Impression Scale – Domain 
Improvement Scores; clinician-assessed 
o Caregiver Global Impression Change Scores; caregiver-
assessed 
x 
PTHS Symptom Severity: 
o PTHS-specific Clinical Global Impression Scale–Severity 
(CGI-S)–Overall; clinician-assessed 
o PTHS-specific Clinical Global Impression Scale–Severity 
(CGI-S)– Domain; clinician-assessed 
o Caregiver Top 3 Concerns Likert Scale Scores; caregiver-
assessed 
x 
Communication:  
o MacArthur-Bates Communicative Development Inventory 
(MB-CDI); caregiver-assessed 
o Observer-Reported Communication Ability Measure 
(ORCA); caregiver-assessed 
x 
Maladaptive Behavior:  
o Aberrant Behavior Checklist-2; caregiver-assessed  
o Behavioral Problems Inventory-Short Form (BPI-SF); 
caregiver-assessed 
x 
Sleep: Child Sleep Habits Questionnaire (CSHQ) Scores; 
caregiver-assessed 
x 
GI: Gastrointestinal Health Questionnaire (GIHQ); caregiver-
assessed  
x 
Adaptive Behavior: Vineland Adaptive Behavior Scales-3, 
Comprehensive Interview version; clinician-assessed 
x 
Motor: 
o Bayley Scales of Infant Development 4 (BSID-4) motor 
scale, clinician-assessed (for participants who are assessed 
on the BSID-4) 
o Modified two-minute walk test; clinician-assessed 
x 
Quality of Life (Child and Caregiver) 
o Quality of Life Inventory-Disability (QI-Disability); 
caregiver-assessed 
o Impact of Childhood Neurological Disability (ICND)-
Overall quality of life rating; caregiver-assessed  
 


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Protocol Neu-2591-PTHS-001 Phase 2: Pediatric Safety, Tolerability, and Pharmacokinetic Study 
Biomarkers and Microbiome Samples 
Blood and stool samples will be collected for analysis of biomarkers 
and gut microbiome. The analysis will be detailed in a separate 
analysis plan but will not include DNA sequencing. Samples will be 
collected at Screening (Visit 1), Baseline (Visit 3, before dosing) and 
at the End of Treatment (EOT) visit, or upon early termination. 
 
Sample Size Estimation 
Sample sizes were estimated for this open-label study using within-
participant change as the measure of improvement. Given the limited 
data from interventional trials in these patient populations, and that this 
is the first clinical study of NNZ-2591 IMP in this population, 
treatment change estimates were based on CGI-I data from completed 
clinical trials in other rare, neurodevelopmental disorders. Based on the 
information provided in Glaze et al. 2019 (Double-blind, randomized, 
placebo-controlled study of trofinetide in pediatric Rett syndrome), the 
standard deviation of the CGI-I is approximately 0.7 at a single 
timepoint.1 For a sample size of 10 participants, a two-sided 
significance level of 0.05, and a Wilcoxon signed-rank test, the study 
will be able to detect a difference of 0.87 units of change in the CGI-I 
at a power of 0.90. For a power of 0.80, 0.85, and 0.90, the detectable 
difference would be 0.75, 0.80, and 0.87 or larger, respectively. The 
effect size for a power of 0.80, 0.85, and 0.90 is 1.07, 1.14, and 1.24, 
respectively. If the observed standard deviation is larger, the detectable 
differences would also be larger. For example, if the standard deviation 
is 1.5, the detectable difference for a power of 0.80, 0.85, and 0.90 is 
1.61, 1.71, and 1.86 or larger, respectively. For a sample size of 20 
participants, a two-sided significance level of 0.05, and a Wilcoxon 
signed-rank test, the study will be able to detect a difference of 0.55 
units of change in the CGI-I at a power of 0.90. For a power of 0.80, 
0.85, and 0.90, the detectable difference would be 0.48, 0.51, and 0.55 
or larger, respectively. 
Statistical Methods 
Safety  
Adverse events and other safety data will be summarized as 
frequencies and percentages, described in terms of severity and 
potential attribution. Objective safety assessments will be summarized 
over time on study; shift tables at time points of interest may be 
generated.  Details of the safety analyses will be specified in the SAP. 
 
Efficacy 
Efficacy will be assessed by looking at within-participant changes 
from the Baseline period to the EOT. The participant’s baseline 
characteristics and symptom severity will be established based on 
assessments collected during the 4 to 6 week Screening/Baseline 
period. Baseline values will be based on the average values of visits 1, 
2, and3 for assessments collected at more than one visit. A Wilcoxon 


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signed rank test will be used to test for improvement and a significance 
level will be computed.  
 
Potentially influential factors will be considered and addressed with a 
linear regression model. This model may include covariates such as 
age, sex, NVIQ or NVIQ equivalent, genotype, history of regression 
(yes/no), ASD diagnosis yes/no with no more than two covariates 
included in any model. Descriptive statistics and corresponding 
graphics will be used to present the results. The efficacy analysis 
details will be specified in the SAP. 
 
Pharmacokinetics: 
NNZ-2591 PK concentrations will be listed by participant and time 
point for each dose level with actual time they were acquired. The 
concentrations will be summarized by time, dose level and age group. 
For the concentrations measured at trough points dose, relationship 
will be examined statistically with age group and/or body weight of 
participant as covariate. 
Overall exposure parameters for NNZ-2591 such as Cmax and AUCtau 
may be derived by sparse non-compartmental analysis (NCA). 
Additionally, PK concentrations of NNZ-2591 measured in pediatric 
participants will be used to refine a population PK model developed 
from adult NNZ-2591 PK data and extrapolated to the pediatric 
population. After refinement, complete profiles for each participant 
will be simulated using range of variability of model parameters and 
individual covariate effects. A standard NCA approach will be used for 
simulated profiles to calculate parameters Cmax, AUCtau, etc. for 
characterization of NNZ-2591 exposure in pediatric participants. 
The PK analysis details will be specified in the PK Statistical Analysis 
Plan (PK SAP). 
 
Biomarkers and Microbiome: 
Biomarker analysis will be detailed in the SAP and will not include 
DNA sequencing. 
 
Microbiome analysis will be described in a separate analysis plan. 
Trial Sites 
Approximately 4-7 sites in the US. 










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diary must be completed daily during the entire Screening/Baseline period whether it is 4 weeks or 6 weeks. The Screening visit (Visit 1) and 
Baseline visit (Visit 3) may be conducted over three consecutive days. It is preferrable that ECGs, safety labs and biomarker sample collection are 
conducted on the same day. All assessments must be completed within the window for the visit (visit day ± 2 days).  For Visit 3, all assessments 
must be completed before administration of the IMP. 
 
b The End of Treatment visit is conducted when the participant is still on IMP treatment. For early termination, in addition to study completers, 
participants who have received IMP for longer than 2 weeks and discontinue prematurely any time after Visit 5 should return to the investigational 
site for final safety and efficacy assessments as scheduled for the EOT visit (Visit 16) and complete the study exit form. 
c At Visit 3, the first dose of IMP will be administered in the clinic after all Baseline assessments are completed, or, if the Investigator judges that 
it is too late in the day, on the following day. The participant should be monitored for at least three hours post first dose. If the first dose is 
administered the day following Baseline, vital signs (except height) should be collected before the dose is administered. Day 1 of dosing is defined 
by the date when the first dose is taken. At the Baseline visit, the site will dispense an adequate supply of IMP for dispensing by the caregiver at 
home. Dosing at the end of Baseline counts as Day 1 whether one or two doses are given. Dosing may not exceed 91 days including Day 1, and all 
efficacy assessments for the EOT visit must be completed on or before Day 91 of treatment. 
 
d A clinic visit is preferred but an off-site visit may be conducted for a scheduled in-clinic visit in extenuating circumstances due to the COVID-19 
health crisis. All requests for off-site visit assessments due to extenuating circumstances must be approved in advance by the Sponsor or Medical 
Monitor. The in-clinic visits may be conducted over three consecutive days. All assessments including caregiver-completed assessments must be 
done within the window for the visit. 
e This is a combined remote and in-person nurse visit. The remote visit is conducted by the site via telemedicine or video facilities as appropriate. 
Any caregiver-completed assessments should be completed the same day of the visit or no earlier than the day before the visit (except the 
Caregiver Diary which is completed daily) and reviewed by the Investigator. A modified version of the physical and neurological exam will be 
done at the remote visits based on the guidance documents provided to the Investigator. At the Week 4, the site should confirm with the caregiver 
that the child has started the new dose if they were approved for dose titration. These combination remote/in-home visits will be conducted 
simultaneously whenever possible, or they will ideally be conducted on the same day. If that is not possible, the telemedicine visit and the nurse 
visit may be conducted over two consecutive days. A combined remote visit may be conducted in-clinic by the study staff if agreed upon by the 
Investigator and the caregiver.See Section 9.1 and Section 9.2. 
 
f In-home visits will be conducted by a visiting nurse. At every nurse visit, the nurse will collect urine, vital signs including weight, and blood 
samples for a comprehensive metabolic panel (CMP). The nurse will also conduct a targeted physical exam assessing general appearance, 
abdominal/CVA tenderness, and edema. A suprapubic exam (bladder palpation) to assess for urinary retention may be performed. The visiting 
nurse will also review the dosing diary, query oral intake, and do a review of tolerability. Blood samples for CBC with differential, and 
coagulation will be collected at Weeks 4, 8, 10 and Week 15 (indicated by “N” in the Schedule of Assessments). At the post-treatment follow-up 
visit at Week 15, the visiting nurse will also collect ECG, in addition to the above assessments. At Week 7, the site will also have a phone call with 
the caregiver to confirm that the child has started the new dose if they were approved for dose titration. The site will confirm the visiting nurse has 


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been informed so the dosing schedule can be reviewed with the family during the in-home visit. A nurse-only in-home visit may be conducted in-
clinic by the study staff if agreed upon by the Investigator and the caregiver. See Section 9.1and 9.2. 
 
g  The in-clinic physical exam will include an abdominal exam and an assessment of CVA. A suprapubic exam (bladder palpation) to assess for 
urinary retention may be performed.  General awareness will be captured as part of the neurological exam. For in-home nursing visits, the nurse 
will do a Targeted Physical exam assessing general appearance (e.g., alertness), an abdominal/CVA, and edema. A suprapubic exam (bladder 
palpation) to assess for urinary retention may be performed.  
 
h A modified version of the neurological and physical exam will be done for the remote visit based on the guidance documents provided to the 
Investigator.  
 
 
j The visiting nurse will query any changes in tolerability during the in-home visit. Any changes will be noted and provided to the Investigator. The 
Investigator will also query changes in tolerability and note any changes in the remote assessment. The Investigator is responsible for determining 
and reporting any changes that qualify as Adverse Events. 
 
k The caregiver fills out the diary daily from the first day of screening through two weeks after the EOT Visit. X denotes review of the Caregiver 
Diary including the daily summary log, medication log, non-medicinal treatments log, seizure log and new/changes in symptoms log. All visits: 
The Investigator (or appropriately credentialed designee) is responsible for reviewing for ALL visits. In-home visits: At the in-home nurse visit, 
the nurse will review the Caregiver Diary to flag and check any issues that could be reported as AEs. The nurse will notify the site about any 
urgent issues the day of the visit and note anything else in the source documents. The nurse does not sign the diary. The source documents and the 
diary will be sent to the site. The Investigator (or appropriately credentialed designee) will review the diary and source documents collected at the 
in-home visit. 
Note: There are two rows for Caregiver Diary review procedures (to emphasize the importance of thorough and complete dosing 
documentation): this row (Caregiver Diary) denotes that the caregiver fills out the diary daily and a designated clinician reviews it for 
safety/tolerability; a second row (Review dosing compliance documentation in diary, see footnote “l”) is intended to ensure that the 
caregiver is administering IMP/documenting dose properly in the Caregiver Diary. 
 
l The daily dosing log in the Caregiver Diary will be reviewed at all post-baseline visits through EOT. If any doses are missed or mis-dosed, this 
should be queried with the caregiver and appropriate feedback given to the family. This feedback should be documented in the source documents.  
At the in-home nurse visit, the nurse will review the caregiver diary to check any issues and provide feedback to the family at the visit. The nurse 
will notify the site about any urgent issues the day of the visit and note anything else in the source documents. The nurse does not sign the daily 
dosing log/summary page. The source documents and the diary will be sent to the site. The Investigator (or appropriately credentialed delegee) 
will review the diary and source documents collected at the in-home visit. 
 
m Coagulation includes prothrombin time, activated partial thromboplastin (aPTT), and international normalized ratio (INR). 


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n A Comprehensive Metabolic Panel will be assessed every post-enrollment visit (see Section 9.5.2). If abnormal findings are observed on renal or 
liver function tests, additional tests as is clinically indicated may be conducted as described in Sections 9.5.2.1 and 9.5.2.3). 
 
o Both urinalysis and a urine screen for drugs of abuse will be evaluated at Screening (See Section 9.5.2). 
 
p For female participants who have reached menarche. 
 
q See Table 2 and Table 3 for PK blood collection times. 
 
r The Bayley is done for participants who cannot attain a basal score on SB5 or for whom the SB5 is developmentally inappropriate. 
 
s The study exit form should also be completed if the participant terminates the study early. 
 


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Table 2 
Pharmacokinetic Blood Sampling Schedule (Participants Ages 7 to 17 Years Old) 
Week 
Visit No. 
Treatment  
Timing of Sample  
(relative to start of dosing)a 
Sample Time 
Point 
2 
5 
  
Pre dose (-120 to -15 minutes)b 
1 
  
  
  
1-3 hours post dosec 
2 
 
 
 
4-7 hours post dosec 
3 
6 
9 
Mid-treatment 
Pre dose (-120 to -15 minutes) b 
4 
  
  
 
1-3 hours post dosec 
5 
  
  
 
4-7 hours post dosec 
6 
13 
16 
End of Treatment  Pre dose (-120 to -15 minutes)b 
7 
  
  
  
1-3 hours post dosec 
8 
  
  
  
4-7 hours post dosec 
9 
a All post-dose windows are determined from the first dose of the visit (morning dose) and all PK samples must be 
collected before the second dose (PM dose).  
 
 
b Pre dose counts as both T0 and 12 hours post dose from previous evening (near trough). The previous evening’s 
dose should be taken in the evening as late as possible, preferably before bedtime. 
 
c Post-dose sample collection can be +/- 15 minutes of the target time and must be at least 30 minutes from the 
previous sample (samples can be no closer than 30 minutes apart). The actual collection time must be recorded. 
 


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Table 3 
Pharmacokinetic Blood Sampling Schedule (Subects Ages 3 to <7 Years Old) 
Week 
Visit No. 
Treatment 
Collection 
Window 
Timing of Sample 
(relative to start of dosing)a 
Sample Time 
Point 
Comment 
2 
5 
  
  
 
Pre dose (-120 to -15 
minutes)b 
1 
 
  
  
 
A 
1-3 hour post dosec 
2  
Sample 2 can be taken at 
either collection window 
A or Bd 
 
 
 
OR 
OR 
 
 
 
B 
4-7 hours post dosec 
6 
9 
Mid-treatment 
 
Pre dose (-120 to -15 
minutes) b 
3 
 
  
  
 
A 
1-3 hours post dosec 
4 
Sample 4 can be taken at 
either collection window 
A or Bd 
 
 
 
OR 
OR 
  
  
 
B 
4-7 hours post dosec 
13 
16 
End of Treatment 
 
Pre dose (-120 to -15 
minutes)b 
5 
 
  
  
 
A 
1-3 hours post dosec 
6 
Sample 6 can be taken at 
either collection window 
A or Bd 
 
 
 
OR 
OR 
  
  
 
B 
4-7 hours post dosec 
a All post-dose windows are determined from the first dose of the visit (morning dose) and all PK samples must be 
collected before the second dose (PM dose).  
 
 
b Pre dose counts as both T0 and 12 hours post dose from previous evening (near trough). The previous evening’s 
dose should be taken in the evening as late as possible, preferably before bedtime. 
 
c Post-dose sample collection can be +/- 15 minutes of the target time and must be at least 30 minutes from the 
previous sample (samples can be no closer than 30 minutes apart). The actual collection time must be recorded. 
 
d For the 3 to <7 year-old age range, two sampling timepoints are taken at each visit. The post-dose sample may be 
taken at either collection window A or collection window B. Ideally, it is better that the second sample is not taken 
at the same collection window at each visit. So, an attempt should be made to vary the collection time of the second 
sample as is feasible. For example, if on visit 5 the second sample is taken in collection window B, at visit 9 or 16, 
the second sample should ideally be taken in collection window A.  
 
 
 


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5. INTRODUCTION 
5.1 
Pitt-Hopkins Syndrome 
Pitt-Hopkins syndrome (PTHS) is a rare, neurodevelopmental disorder characterized by developmental 
delays, moderate to profound intellectual disability, absent/limited speech, distinctive facial 
dysmorphology, and breathing problems such as episodic hyperventilation and/or breath-holding while 
awake. Other common symptoms may include repetitive behaviors and social communication 
impairments, constipation, severe near-sightedness (myopia), sleep disturbances, and seizures.2, 3  
Individuals with PTHS have distinct facial features that are important elements of the diagnostic criteria, 
including a square face with narrow forehead, thickened helix, wide nasal bridge, beaked nose 
characterized by down-turned nasal tip, flared nasal alae and wide nasal tip, full cheeks, wide mouth, full 
lips and a cupid bow upper lip.3-7 Myopia is observed in the majority of patients (48-88%) and strabismus 
and astigmatism are also observed.4, 6-11  
Individuals with PTHS demonstrate severe developmental delay in motor milestones including delayed 
ambulation and delayed, or ultimately absent, speech.8 While moderate to profound intellectual disability 
has been reported across the literature, most individuals have severe to profound cognitive impairments.  
Mental age equivalents for development level that have been reported as between 3.5 to 36 months for 
individuals between 32 months and 32 years old.9, 12 The majority of individuals (68%) have absent or 
limited speech (less than 5 words) and all have impaired speech.4, 6, 8, 9 While between 46-70% of 
individuals are reported to be independently ambulatory, the majority of individuals have gait 
abnormalities including a wide-based ataxic gait, as well as impaired motor coordination and hypotonia.4, 
6, 8-11, 13, 14 Adaptive function for individuals with PTHS is low with developmental equivalent scores of 20 
or less.12  
Breathing abnormalities are a characteristic feature and affect 45%-86% of individuals with PTHS, in 
particular hyperventilation or hyperventilation with apnea (46%) when awake.3, 6-11, 15 GI problems are 
also highly prevalent, and can be severe, with around 35% needing corrective treatment.13 Chronic 
constipation, with an onset as early as infancy,8, 9 is particularly common (67%-85% of patients).6-11, 15  
Seizures present another challenge for 30%-51% of individuals with PTHS.4, 6, 8-11, 13, 15 The range of 
seizure types includes generalized tonic-clonic, atonic, focal onset, and infantile spasms.6, 8, 10, 16-18 
Additionally, between 18%-42% of individuals with PTHS experience some type of sleep disturbance, 
typically insomnia or night awakenings.6, 8  
Individuals with PTHS face a range of behavioral challenges.3, 4, 6-8, 10 Compared to Angelman Syndrome 
(AS) and Cornelia de Lange syndrome (CdLS), other genetically based disorders with similar early signs 
and features, the behavioral phenotype of PTHS is particularly characterized by the following: 1) a high 
level of Autism Spectrum Disorder (ASD) symptomatology 2) social communication difficulties, 3) 
atypical sensory processing, and 4) high levels of physical aggression and self-injury.13 Aggression and 
aggressive behavior are observed in 40%-54% of individuals,8, 11-13 and in 38% of individuals, this has 
included destruction of property.13 Between 60-71% of individuals demonstrate self-injurious behaviors.6, 
9, 11 Anxiety (81%) and agitation (88%) are also common symptoms.6 
Up to 95% of individuals met the cut off score for possible ASD on the Social Communication 
Questionnaire (SCQ) screener for ASD.13 Individuals have also been shown to score high on the Autism 
Diagnostic Interview measure.12 On the SCQ, they demonstrated more problems with communication and 
reciprocal social interaction than individuals with AS. They also showed lower sociability with both 


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familiar and unfamiliar people compared to individuals with AS, and with familiar people compared to 
individuals with CdLS.13 Individuals with PTHS also have atypical sensory processing. In response to 
behaviors in social contexts, the majority of individuals are hyporesponsive, but are hyperresponsive to 
sensory input.13  
Stereotypic behaviors are highly prevalent (54%-94%)3, 6, 8, 10, 12, 13 and include object stereotypy,3, 13 body 
stereotypies such as body rocking (54%-78%) and hand and finger stereotypies (48-85%) including 
clapping, flapping (up to 80%), hand-wringing/hand-washing (45%), hand mouthing or biting (54%), 
finger movements (54%), and wrist movements (45%).6-9, 12, 13 Head shaking and head banging have also 
been reported.6, 9  
5.2 
Current Treatments for Pitt-Hopkins Syndrome 
No methods exist to prevent the onset of PTHS, nor is there any cure or approved medication for 
treatment for PTHS.  
Prescribed medications for individuals with PTHS are for the management of specific symptoms such as 
seizures or GI problems,3, 8 and do not address the underlying dysfunction of the disorder. GI problems of 
constipation and GERD are treated using standard treatments such as laxatives, stool softeners and protein 
pump inhibitors.3, 8 Seizures are commonly treated with standard antiepileptics such as valproic acid, 
levetiracetam, lamotrigine, and carbamazepine.3, 9, 19 There are no systematic recommendations for the use 
of psychotropic medications to treat behavioral problems, but caregivers report using methylphenidate, 
clonidine and benzodiazepine for agitation/irritability and anti-psychotics for challenging behaviors.3, 8 
Mood-stabilizing medications and anti-epileptics have been used for treating breathing abnormalities,8 
and melatonin has been used for sleep issues.3, 8  
Non-medication strategies for the treatment of PTHS are typically multimodal and require the 
involvement of multiple clinicians with diverse areas of expertise.2, 3 In centers that specialize in the 
treatment of individuals with PTHS, the standard of care includes physical, occupational, behavioral and 
speech therapies, as well as targeted approaches to orthopedic concerns and gastrointestinal dysfunction 
and seizures, as is reflected in recently published international guidelines.3 Establishment of an effective 
long-term care plan that can be implemented across settings, which includes pediatric medical follow-up 
and a transition plan for adult care, is a recommended approach to the treatment of individuals with 
PTHS.2, 3, 20  
5.3 
Genetic Basis of Pitt-Hopkins Syndrome 
Pitt-Hopkins syndrome is caused by loss of function of the transcription factor 4 (TCF4) gene due to 
heterozygous hypomorphic or null mutations, or up to 12 Mb interstitial deletions on chromosome 18q 
including the TCF4 gene, which result in TCF4 haploinsufficiency.3, 6, 10, 11, 14, 18, 21, 22 The majority of 
disease-causing genetic changes are de novo.2, 3, 11  
TCF4 is expressed in neurons and glial cells and it encodes a basic helix–loop–helix (bHLH) transcription 
factor that is known to heterodimerize with several other bHLH transcription factors that play important 
roles in neurogenesis and neuronal migration in the brain, regulating glial cell differentiation, and 
regulating immune cell function.14   
TCF4 is highly expressed during embryonic and early postnatal development18 and has particularly high 
expression in the hippocampus.23-25 It is also expressed in adult brain, lymphocytes, fibroblasts, gut, 
muscle, and myenteric plexus.18, 21, 26, 27  Recent cognitive and imaging studies have also shown that TCF4 
is important for normal brain function.25, 28  






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phenotype of fmr1 knockout mice and corrected impairment of AKT/ERK activation and abnormal 
dendritic morphology. 
A comprehensive battery of safety, pharmacology, and toxicology studies conducted in mice, rats and 
dogs has also been conducted, providing support for the progression of the drug into a Phase 2 clinical 
trial in a pediatric population with PTHS. Summaries of these studies are presented in the NNZ-2591 IB.  
5.6 
Clinical Data 
NNZ-2591 has been studied in 28 healthy male and female adult volunteers. Study Neu-2591-HV-001 
was a combined Phase 1 study, with an open-label single dose stage and a double-blind, randomized, 
multiple ascending dose-escalation stage with 12 participants enrolled in 2 open-label single-dose cohorts 
and 16 participants enrolled in 2 randomized, double-blind, placebo-controlled multiple dose cohorts. The 
single ascending dose stage of the Phase 1 study evaluated doses of 12.5 mg/kg and 50 mg/kg. The two 
cohorts comprising the multiple ascending dose stage of the Phase 1 study evaluated twice-daily dosing at 
3 mg/kg for 1 day followed by 6 mg/kg for 6 days (Cohort M1) and 6 mg/kg for 1 day, then 9 mg/kg for 
1 day followed by 12 mg/kg for 5 days (Cohort M2).   
 
Twice daily dosing for 5 days at 12 mg/kg following up-titration for 1 day at 6 mg/kg and 1 day at 
9 mg/kg was found to be safe and generally well-tolerated. There were no deaths, Serious Adverse Events 
(SAEs), or severe Treatment Emergent adverse events (TEAEs) reported during the conduct of this study. 
All adverse events (AEs) were mild or moderate. No clinically significant laboratory, ocular, physical 
examination, vital sign, or cardiac abnormalities were reported. There was a single discontinuation due to 
moderate AEs of somnolence and ataxia after the administration of one dose. The participant’s two 
TEAEs resolved after treatment was withdrawn. 
 
The most common TEAEs deemed related to drug were a cluster of apparently related neurological 
symptoms that included somnolence/drowsiness, blurred vision/difficulty focusing, ataxia/unsteady gait, 
dizziness, and vertigo. The majority of these TEAEs occurred within 2-3 hours of a participant’s first 
dose. All resolved spontaneously without requiring medical intervention, most within 24 hours from 
onset. This pattern suggests that these TEAEs are an acute phenomenon temporally correlated with initial 
Tmax rather than being associated with cumulative exposure and that participants possibly develop 
tolerance to these neurological Aes. There were no clinically significant findings on the laboratory tests or 
electrocardiograms (ECGs), and no renal or hepatic findings were observed. 
 
PK appeared to be linear and predictable, with peak and total exposures (as measured by Cmax and area 
under the plasma concentration-time curve [AUC], respectively) appearing to increase in a generally dose 
proportional manner after both the morning and evening doses. 
 
A summary of the study is provided in the NNZ-2591 IB.  
In addition to this study, NNZ-2591 is being assessed in Phase 2 studies in Phelan McDermid syndrome, 
Angelman Syndrome, and Prader-Willi Syndrome. In these open label studies participants receive BID 
treatment for a total of 13 weeks. The dose is up-titrated from 4 mg/kg (three weeks of treatment) to 8 
mg/kg (three weeks of treatment) to 12 mg/kg (7 weeks of treatment). As of the date of the last annual 
report to FDA, a total of 16 participants had been dosed across this study, (PTHS-001) and the Angelman 
Syndrome and Phelan McDermid Syndrome studies. There were no SAEs or Adverse Events of Special 
Interest (renal, hepatic or ocular) reported. There have been two discontinuations. 


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6. STUDY RATIONALE AND OBJECTIVES 
6.1 
Study Rationale 
Pitt-Hopkins syndrome (PTHS) is a rare genetic neurodevelopmental disorder caused by the loss of 
function of the TCF4 gene, and the resulting reduction in the number and morphology of dendritic spines 
and the synaptic plasticity in mature neurons.29 Characterized by developmental delays, moderate to 
profound intellectual disability, absent/limited speech, distinctive facial dysmorphology, and breathing 
problems,2, 3 PTHS causes lifelong difficulties for the individuals affected by the condition and their 
families. There is currently no approved treatment for PTHS.  
In pre-clinical pharmacodynamic studies, NNZ-2591 has demonstrated the ability to improve impaired 
synaptic structure and signaling as well as rescuing the neurobehavioral phenotype. NNZ-2591 is a 
synthetic analogue of cGP that inhibits neuroinflammation and restores synaptic function and plasticity. 
NNZ-2591 has better stability than cGP, readily crosses the blood-brain barrier and has high 
bioavailability when administered orally. Therefore, the purpose of this study is to investigate the safety, 
tolerability, and pharmacokinetics (PK) of treatment with oral NNZ-2591 Oral Solution, 50 mg/mL IMP 
in children and adolescents with PTHS. 
6.2 
Rationale for Dose Selection and Route of Administration 
6.2.0 
Rationale for Dose Selection  
Selection of the dose schedule of NNZ-2591 IMP that will be utilized in this study is informed by: 
x 
Results of toxicity studies which assessed the effects of oral NNZ-2591 in dogs and juvenile rats 
with 13 weeks of dosing and a 4-week recovery period. These studies, summarized in the IB, 
found that the No Adverse Effect Level (NOAEL) of NNZ-2591 was 200 mg/kg/day in juvenile 
rats and 120 mg/kg/day in adult dogs. At the NOAEL, no NNZ-2591-related clinical signs of 
toxicity considered adverse, or mortality occurred and no non-reversible effects on the nervous 
system, skeletal system, renal or reproductive system were observed. 
x 
Pharmacodynamic studies in Tcf4+/- mice and wild-type, littermate controls found that 6 weeks of 
dosing at 100 mg/kg BID and 200 mg/kg BID were effective in reversing cognitive and 
neurobehavioral deficits analogous to the human PTHS phenotype.     
x 
The safety, tolerability, and PK of NNZ-2591 Oral Solution as determined by the First-in-Human 
(FIH) Phase 1 study. NNZ-2591 appeared to be safe and generally well-tolerated when 
administered orally twice daily for 7 days at doses up to 12 mg/kg BID. There were no SAEs. All 
AEs were mild or moderate. There were no NNZ-2591 related abnormal laboratory results, 
ECGs, or vital signs and no sentinel safety issues. This study is summarized in the IB. 
x 
The results of allometric scaling of exposure at steady state (AUCtau) in healthy, adult volunteers 
at 12 mg/kg indicate that exposures for the pediatric population at weights lower than 70 kg are 
estimated to be lower than those observed for the adult population in the Phase 1 study at 
12 mg/kg. Therefore, dosing all children at a maximum of 12 mg/kg BID will not put them at risk 
of exposures higher than that experienced by healthy adults. At the same time, the predicted 
exposures are expected to be in the range of what was identified as a potentially efficacious dose 
from the animal models so there is a prospect of benefit with dosing at the maximum proposed 
dose of 12 mg/kg BID. A summary of the allometric scaling analysis is presented in Appendix A.  


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x 
Data from a parallel PK study in the same background strain of mice utilized in the 
pharmacodynamic study was conducted to support comparison of exposure between mice and 
humans. This study, combined with PK data from the Phase 1 study, resulted in an estimate that a 
dose of approximately 10 mg/kg BID in adults should result in daily exposure equivalent to 
100 mg/kg BID in the mouse. The weight-based doses in the Phase 2 study are intended to result 
in a distribution of exposures equivalent to the 12 mg/kg dose in adults that will provide a 
reasonable opportunity for efficacy as well as being predicted to be safe and well-tolerated. 
In this Phase 2 study, NNZ-2591 Oral Solution, 50 mg/mL will be administered orally BID over the 
course of 13 weeks. Participants will be up-titrated according to the dosing schedule outlined in Section 
7.2. The dose titration schedule may be modified based on updated PK modelling including data from the 
study. 
6.2.1 
Rationale for Duration of Dosing 
Given the overall safety and tolerability profile of NNZ-2591 in preclinical studies to date, 13 weeks of 
treatment at this dose is expected to be safe and well-tolerated. NNZ-2591 has been shown to be well-
tolerated in animal studies for up to 13 weeks at up to 200 mg/kg/day for rats and 120 mg/kg/day for 
dogs. In addition, NNZ-2591 Oral Solution appears to exhibit generally linear kinetics with no time-
dependent effect on PK parameters and no evidence of accumulation in humans.  
A study duration of 13 weeks is expected to provide sufficient time for changes in synaptic plasticity to 
manifest in behavioral and symptom change. A number of functionally relevant symptoms in PTHS could 
show a more immediate response as a result of this change in synaptic function. Based on findings in a 
parallel program of another IGF-1 metabolite analog (trofinetide) in Rett syndrome, it has been observed 
that improvements in some symptoms can be seen at 4 or 6 weeks of treatment.1, 32 This includes 
improvements in symptoms clinicians considered clinically meaningful such as behavior, seizures, 
ambulation, social interaction, motor skills (hand use) and repetitive behaviors. As such, there is a 
prospect of benefit with dosing at the maximum proposed dose of 12 mg/kg BID for 7 weeks with a total 
duration of approximately 13 weeks of treatment in this Phase 2 study in PTHS.  
Other symptoms or skills such as communication may need longer than 4 to 6 weeks to manifest, and the 
treatment period of 13 weeks in this study may allow sufficient time for possible change in more complex 
symptoms to manifest. So, on the whole, within a 13-week period it is possible for there to be a range of 
symptoms that could improve and be beneficial to patients. In sum, this approach to dosing maintains a 
balance between risk and benefit for this first Phase 2 study in the PTHS population. 
6.2.2 
Rationale for Selection of Efficacy Outcomes 
Pitt-Hopkins syndrome is a severely limiting, lifelong disorder in which affected individuals cannot 
achieve the functional capacity to care for themselves, protect themselves from harm, form normal peer 
relationships, or achieve independent daily living skills. The constellation of symptoms in PTHS have 
significant impact on the functionality of affected individuals and their overall quality of life and well-
being. The debilitating impairments in cognition, language and social function hinder overall social, 
emotional, and academic development and independent execution of tasks of daily living. Adaptive 
behavior is severely impaired with individuals remaining at low developmental and functional levels even 
as they age.12 This greatly limits their ability to have any independence in executing activities of daily 
living rendering them dependent on support throughout their lives. Their severe disability puts them at 
further risk of adverse medical, developmental, psychosocial, and family outcomes.8, 33  


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The underlying neuropathology and severe neurological and neurodevelopmental impairments also lead 
to neurobehavioral dysfunction and dysregulation which substantially disrupt daily activities, put 
individuals at greater risk of harm, and can be potentially life-threatening. Hyperventilation and apnea 
result in a number of negative impacts on health including swollen abdomen, excessive burping, 
decreased oxygen saturation, changes in pallor, cyanosis, chronic hypoxia, and finger clubbing.6, 9, 10 
Seizures can be debilitating to cognitive and neurological development. Self-injurious behaviors put 
individuals with PTHS at risk of physical harm, potentially with long-term cumulative effects which 
decreases overall quality of life. There is currently no on-going natural history study of PTHS. Cohort 
studies, and a recent consensus statement paper on diagnosis and treatment3 have provided detailed 
information characterizing the clinical manifestations of the disorder and their functional impact. This has 
provided insights on the symptoms that are central to the disorder and are important to patient functioning 
and well-being. The Pitt Hopkins Research Foundation (PHRF) maintains a family registry as part of the 
Coordination of Rare Diseases (CoRDS) Registry at Sanford which collects information on clinical 
symptoms (https://pitthopkins.org/research/how-to-get-involved-in-research-today/family-registry/). The 
PHRF also did an assessment of the patient and family experience by conducting a trend analysis of data 
shared through social networks.34 In this analysis they identified the most concerning symptoms, most 
common methods of disease management, and unmet needs.  
Together, the output from these initiatives and expert clinical advice has provided guidance into the 
important domain areas to address with NNZ-2591 treatment that would be most relevant and potentially 
beneficial to patients with PTHS (see Section 9.5.9). The secondary outcome of efficacy in this study will 
be assessed by evaluating a range of symptom areas to determine if there is preliminary evidence of 
efficacy, but also importantly to gather information about the performance of outcome measures and the 
influence of baseline patient attributes on clinical response.  
6.3 
Study Objectives and Endpoints 
6.3.1 
Primary Objective 
To investigate the safety, tolerability, and PK of treatment with NNZ-2591 Oral Solution, 50 mg/mL in 
children and adolescents with PTHS.  
6.3.1.1 
Primary Endpoints 
Safety: Incidence of AEs and SAEs from commencement of IMP administration through to the time of 
the last study visit will be evaluated. Vital signs, laboratory measures, physical exam results 
(neurological, ophthalmologic, and general) urine output history, 
, and ECGs 
will also be evaluated from screening/baseline through the end of the study.  
Tolerability: Tolerability will be assessed based on caregiver reports and the caregiver diary, required 
dosing adjustments or discontinuations, dosing compliance and AEs related to participant experience. 
Pharmacokinetic:  
NNZ-2591 PK concentrations will be listed by participant and time point for each dose level with actual 
time they were acquired. The concentrations will be summarized by time, dose level and age group. For 
the concentrations measured at trough points dose relationship will be examined statistically with age 
group and/or body weight of participant as covariate. 
Overall exposure parameters for NNZ-2591 such as Cmax and AUCtau may be derived by sparse non-
compartmental analysis (NCA). Additionally, PK concentrations of NNZ-2591 measured in pediatric 


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participants will be used to refine a population PK model developed from adult NNZ-2591 PK data and 
extrapolated to pediatric population. After refinement, complete profiles for each participant will be 
simulated using a range of variability of model parameters and individual covariate effects. A standard 
NCA approach will be used for simulated profiles to calculate parameters Cmax, AUCtau, etc. for 
characterization of NNZ-2591 exposure in pediatric participants. 
6.3.2 
Secondary Objectives 
To investigate measures of efficacy during treatment with NNZ-2591 Oral Solution, 50 mg/mL in 
children and adolescents with PTHS. 
 
6.3.2.1 
Secondary Endpoints 
Within-participant changes will be assessed from the Baseline period to End of Treatment (EOT) using 
the following measures: 
Overall Improvement: 
x 
PTHS-specific Clinical Global Impression Scale–Improvement (CGI-I) Overall Improvement 
Score; clinician-assessed  
x 
PTHS-specific Clinical Global Impression Scale–Domain Improvement Scores; clinician-
assessed 
x 
Caregiver Global Impression of Change Scores; caregiver-assessed 
PTHS Symptom Severity: 
x 
Clinical Global Impression Scale–Severity (CGI-S)–Overall Severity Score; clinician-
assessed 
x 
Clinical Global Impression Scale–Severity (CGI-S)–Domain Scores; clinician-assessed  
x 
Caregiver Top 3 Concerns Likert Scale Scores; caregiver-assessed 
Communication 
x 
MacArthur-Bates Communicative Development Inventory (MB-CDI) Scores; caregiver-
assessed 
x 
Observer-Reported Communication Ability Measure (ORCA) Score; caregiver-assessed 
Maladaptive Behavior 
x 
Aberrant Behavior Checklist-2 (ABC-2) Scores; caregiver-assessed 
x 
Behavior Problem Inventory-Short Form (BPI-SF) Scores; caregiver-assessed 
Sleep: Child Sleep Habits Questionnaire (CSHQ) Scores; caregiver-assessed  
GI: Gastrointestinal Health Questionnaire (GIHQ) Scores; caregiver-assessed 
Adaptive Behavior: Vineland Adaptive Behavior Scales-3 Scores; clinician-assessed 
Motor 
o Bayley Scales of Infant Development 4 (BSID-4) motor scale, clinician-assessed (for 


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participants who are assessed on the BSID-4) 
o Modified Two-minute Walk Test scores; clinician-assessed 
Quality of Life (Child and Caregiver) 
x 
Quality of Life Inventory-Disability (QI-Disability) Scores; caregiver-assessed 
x 
Impact of Childhood Neurological Disability (ICND) Overall Quality of Life rating; 
caregiver-assessed  
6.3.2.2 
Exploratory Endpoints 
Blood and stool samples will be collected for an exploratory analysis of biomarkers and gut microbiome. 
The analysis will be detailed in a separate analysis plan but will not include DNA sequencing. Samples 
will be collected at Screening (Visit 1) and Baseline (Visit 3, before dosing) and at the EOT visit, or upon 
early termination. 
 
 


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7. STUDY DESIGN 
7.1 
Study Design Overview 
This is a Phase 2, open-label study of the safety and tolerability of NNZ-2591 Oral Solution, 50 mg/mL in 
male and female children and adolescents with Pitt-Hopkins syndrome. Approximately 10 to 20 male and 
female participants between the ages of 3 and 17 years will be enrolled and receive treatment of NNZ-
2591 Oral Solution, 50 mg/mL for a total of 13 weeks. Additional participants may be screened, enrolled, 
and treated to achieve approximately 20 study completers. 
There will be a total of 17 study visits, comprising 5 in-clinic visits, and 12 remote telemedicine/in-home 
nurse visits. The study will commence with an approximately 4 to 6 week Screening and Baseline period. 
During this Screening/Baseline period, participants will be assessed for study eligibility and data will be 
collected to establish the participant’s baseline characteristics and symptom severity using a variety of 
assessments (Section 9.2). Two in-clinic visits and one remote/in-home visit (Visits 1, 2, 3) comprise 
Screening and Baseline.  
 
Once eligibility is confirmed, participants will receive the starting dose of 4 mg/kg and then be up-titrated 
to the target dose (see Section 7.2). Participants will receive treatment for a total of 13 weeks. During the 
treatment period, there are three in-clinic visits: Week 2 (Visit 5), Week 6 (Visit 9) and Week 13 (Visit 16). 
There is a combined remote, telemedicine visit by the site investigator and an in-home visit by a visiting 
nurse for safety at Week 1 (Visit 4), Week 4 (Visit 7), Week 8 (Visit 11) and Week 10 (Visit 13). These 
combination remote/in-home visits will be conducted simultaneously whenever possible, or they will be 
conducted on the same day with the telemedicine visit occurring first. If it is not feasible to conduct these 
visits simultaneously or on the same day, the telemedicine portion of the visit and the nurse portion of the 
visit must be conducted on two consecutive days. In-home visits with a visiting nurse will be done at Weeks 
3 (Visit 6), 5 (Visit 8), 7 (Visit 10), 9 (Visit 12), 11 (Visit 14), and 12 (Visit 15). 
 
All participants will also have a remote post-treatment visit with the Investigator via telemedicine and an 
in-person home visit by a visiting nurse approximately 2 weeks after the end of treatment (Week 15, Visit 
17). 
 
For all visits designated as in-clinic, an in-person visit is preferred. Off-site assessments may be allowed 
due to extenuating circumstances, such as those related to the COVID-19 health emergency. All requests 
for off-site assessments must be approved in advance by the Sponsor or Medical Monitor. 
 
Participants will be divided into three groups by age:  
x 
13 to 17 years old (Group 1) 
x 
8 to 12 years old (Group 2) 
x 
3 to 7 years old (Group 3) 
 
Enrollment will commence with the oldest age group and will proceed as follows. After at least three 
participants in Group 1 have received two weeks of treatment at the starting dose, the Data Safety 
Monitoring Committee (DSMC) will review data on safety, and tolerability. If tolerability and safety for 
those participants during the specified period is deemed acceptable, enrollment for Group 2 will proceed 
and dosing will begin at the starting dose. When at least three participants in Group 2 have received the 
first two weeks of treatment, the DSMC will review data on safety, and tolerability. If tolerability and 
safety for Group 2 is deemed acceptable, enrollment for Group 3 will commence and dosing will begin at 
the starting dose. 


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7.1.1
Study Design 
Figure 1 
Neu-2591-PTHS-001 Study Design Diagram    
7.2
Dose-levels and Titration
The total administration period for NNZ-2591 Oral Solution, 50 mg/mL IMP will be 13 weeks. As 
outlined below in Table 4,the IMP will be administered open-label at a starting dose of 4 mg/kg BID for 
2 weeks. Following DSMC review and approval of each dose titration, participants will be up-titrated to 
8 mg/kg BID for 2 weeks, and then to the 12 mg/kg BID dose. The participant will then continue on the 
assigned dose (or highest dose tolerated) for the remainder of the 13 weeks. The process for DSMC 
review of dose escalation is described in Section 7.2.2. 
Table 4 
Dose Titration Schedule
Week 1 and
Week 2
Week 3
Week 4 
and
Week 5
Week 6
Week 7 
and 
Week 8
Week 9
Week 10 to 
Week 13
4 mg/kg 
BID 
(8 mg/kg 
daily)
4 mg/kg BID 
(8 mg/kg daily)1
8 mg/kg 
BID 
(16 mg/kg 
daily)
8 mg/kg BID 
(16 mg/kg 
daily)1
12 mg/kg 
BID 
(24 mg/kg 
daily)
12 mg/kg BID 
(24 mg/kg 
daily)
12 mg/kg 
BID 
(24 mg/kg 
daily)
DSMC 
Reviews Data 
Prior to Up-
Titration2 
DSMC 
Reviews Data 
Prior to Up-
Titration2 
DSMC 
Reviews Data 
After 2 weeks 
on 12 mg/kg 
to Confirm 
Safety2
BID = twice daily; DSMC = Data Safety Monitoring Committee
1Participants will stay on their current dose until the data is reviewed by the Data Safety Monitoring Committee (DSMC)
2Expected time for DSMC review will be one week.
The dose titration schedule may be modified based on updated PK modelling including data from the 
study.
7.2.1
Adjustment of the Dose for Tolerability
The dose for each participant is based on the participant’s weight at Baseline (Visit 3). The dose will not 
be increased or decreased if the participant’s weight at a postbaseline visit changes. Within the up-
titration period, if a participant is deemed not to be tolerating the current dose within the 2-week period 
for that dose, and they have not met any of the stopping criteria (Section 9.4.5), they can be down-titrated 
to the next lowest dose in the titration schedule. The Investigator must attempt to increase the dose as 
soon as it is possible based on the clinical situation. The aim is to return to the originally assigned dose. If 


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the originally assigned dose cannot be reached or the participant is again unable to tolerate that dose, the 
Investigator will continue treatment on the highest dose that the participant can tolerate. The process for 
DSMC review of dose escalation is described in section 7.2.2.  
 
7.2.2 
Dose Escalation Review by the DSMC 
Data from two weeks of dosing at each dose in the dose escalation will be reviewed by the DSMC before 
participants are allowed to be administered the next highest dose. Participants will stay on their current 
dose until the DSMC has reviewed the data and approved the participants to escalate to the next dose.  
 
After eligibility is confirmed, the participant will be administered NNZ-2591 IMP open label at a starting 
dose of 4 mg/kg BID for a total of approximately 3 weeks. After 2 weeks of dosing at 4 mg/kg BID, the 
DSMC will review the safety data. The participant will remain on the starting dose during the DSMC 
review. If the DSMC determines that tolerability and safety during the 2-week dosing period are 
acceptable, the participant will escalate to the next dose of 8 mg/kg BID and be dosed for a total of 
approximately 3 additional weeks at that dose. After 2 weeks of dosing at 8 mg/kg BID, the DSMC will 
review the safety data. If the DSMC determines that tolerability and safety during the 2-week dosing 
period on 8 mg/kg BID are acceptable, the participant will then escalate to the target dose of 12 mg/kg 
BID and be dosed for 2 weeks. After 2 weeks of dosing at 12 mg/kg BID, the DSMC will review the 
safety data to determine if the participant can continue dosing at 12 mg/kg BID for the remainder of the 
13 weeks.  
 
The DSMC will determine if dose escalation can proceed based on pre-specified stopping rules 
summarized for individual participants in Section 9.4.5. Note that early cessation of IMP treatment does 
not necessarily mean the participant is withdrawn from the study. With any early cessation, all 
evaluations and tests scheduled during the study must be conducted if possible. 
 
The site will inform the caregiver of the up-titration decisions as soon as possible after the site has 
received the official notification of the DSMC decision. The site will verify with the caregiver that 
instructions to up-titrate to 8 mg/kg BID have been followed at the Week 4 visit (Visit 7), and will verify 
with the caregiver that instructions to up-titrate to 12 mg/kg BID have been followed at the Week 7 visit 
(Visit 10). If the DSMC has not yet approved the participant to escalate to the next dose by the time of the 
relevant visit, the site should contact the caregiver within 24 hours of receiving the decision from the 
DSMC. If the participant is not approved to titrate to the next dose, the site will contact the caregiver to 
discuss appropriate follow-up as is clinically indicated. At each visit, the clinician or nurse should check 
the dose when reviewing the caregiver diary.  
 
7.3 
Treatment Assignment 
This is an open-label study in which all participants will be assigned to NNZ-2591 Oral Solution, 
50mg/mL and receive 13 weeks of treatment. All participants will be titrated to the dose of 12 mg/kg BID 
or the highest tolerated dose based on the Dosing Schedule in Section 7.2. The dose titration schedule 
may be modified based on updated PK modelling including data from the study. 
Participants who are unable to tolerate the study treatment may be discontinued from the study.  
If a participant meets all eligibility criteria and written informed consent has been provided by a legally 
authorized representative, the participant will be enrolled in the study.   


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7.4 
Study Timeline 
The study duration is 19 weeks from Screening to Follow-Up at Week 15 (+4 days).  
7.5 
COVID-19 
As a result of the Coronavirus disease (COVID-19) public health emergency, this protocol includes 
provision for alternative methods of performing safety and efficacy assessments in accordance with the 
FDA Guidance on Conduct of Clinical Trials of Medical Products during the COVID-19 Public Health 
Emergency.** Provision is made for off-site assessments where necessary and all efforts have been made 
to ensure the alternative processes are consistent as much as possible with in-clinic visits. For visits that 
are designated as in-clinic, an actual in-clinic visit is preferred. However, an off-site visit may be 
conducted for a scheduled in-clinic visit in extenuating circumstances due to the COVID-19 health crisis 
with the prior approval of the Sponsor and Medical Monitor. Clinical investigators are to document the 
reason for any contingency measures implemented. If at any time in the study, the participant or his or her 
caregiver tests SRVLWLYH IRU VHYHUH DFXWH UHVSLUDWRU\ V\QGURPH FRURQDYLUXV  6$56-CoV-2) or becomes 
ill with symptoms of COVID-19, the family should be instructed to contact the site as soon as possible. 
Once informed, the Investigator should contact the Medical Monitor. 
8. PARTICIPANT POPULATION  
Approximately 10 to 20 male and female participants between the ages of 3 and 17 years, inclusive, are 
planned to complete the study, balanced to the extent possible by age and the number of males and 
females. 
The participants will be categorized by age in three groups: 13 to 17-year-olds (Group 1), 8 to 12-year-
olds (Group 2), and 3 to 7-year-olds (Group 3). Enrollment will be balanced to the extent possible by sex 
and age with a minimum of 3 males and 3 females across all age groups, and a minimum of 3 participants 
in each age group.  
Enrollment will proceed by age group according to the following process. Enrollment will commence first 
in the oldest age group (Group 1). After at least three participants in Group 1 have received two weeks of 
treatment at the starting dose, safety, and tolerability data will be reviewed by the DSMC.  If tolerability 
and safety for those participants during that period is deemed acceptable, enrollment for Group 2 will 
proceed and dosing will commence at the starting dose. When at least three participants in Group 2 have 
received two weeks of treatment at the starting dose, safety and tolerability data will be reviewed by the 
DSMC. If tolerability and safety for Group 2 is deemed acceptable, enrollment for Group 3 will 
commence and dosing will begin at the starting dose. Participants in the younger age groups may be 
screened and enter the 4 to 6 week Screening period before DSMC review, but they will not be enrolled 
or dosed until the DSMC has reviewed and approved the initiation of dosing in the younger age group.  
 
** FDA Guidance on Conduct of Clinical Trials of Medical Products During the COVID-19 Public Health 
Emergency: Guidance for Industry, Investigators, and Institutional Review Boards (Center for Biologics 
Evaluation and Research; Center for Devices and Radiological Health; Center for Drug Evaluation and 
Research; Office of the Commissioner; Office of Clinical Policy and Programs; Office of Clinical Policy; 
Office of Good Clinical Practice) (2021). 


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8.1 
Inclusion Criteria 
To be eligible for this study, participants must meet all of the inclusion criteria and none of the exclusion 
criteria. Inclusion criteria include: 
1. Clinical diagnosis of PTHS with a documented disease-causing genetic etiology for the disorder.  
2. Males or females aged 3 to 17 years at Screening, inclusive. 
3. Body weight of 12 kg or higher at Screening. Weight measurements may be repeated during the 
Screening period in consultation with the Medical Monitor. 
4. Participants with a Clinical Global Impression – Severity (CGI-S) score of 4 or greater at the 
Screening visit. 
5. Not currently actively undergoing regression or loss of skills defined as no persistent loss of 
previously acquired developmental skills for a period within 3 months of the Screening visit. 
a. Previously acquired skills include any skill established and used on a daily basis for at 
least 3 months. 
b. Variations in use of skills that is part of a general pattern of ups and downs, as 
determined by the investigator, are not counted as regression. 
c. Skills may include language, non-verbal communication, fine motor skills, ambulation 
(including gait, coordination, independence of walking/standing), social skills, play, self-
help.  
6. Each participant must be able to swallow the IMP provided as a liquid solution.  
7. The participant’s caregiver(s) must have sufficient English language skills to complete caregiver 
assessments in English. 
 
8.2 
Exclusion Criteria 
To be eligible for this study, participants must meet all of the inclusion criteria and none of the exclusion 
criteria. Exclusion criteria include: 
     Laboratory and Screening Values 
1. 
Body weight < 12 kg at Screening. Weight may be repeated during the Screening period in 
consultation with the Medical Monitor. 
2. 
HbA1c values above 7% at Screening. 
3. 
Clinically significant abnormalities in safety laboratory tests (as determined by the Investigator) 
in safety laboratory values, vital signs, as measured at the Screening visit. Laboratory testing 
may be repeated during the Screening period in consultation with the Medical Monitor. 
4. 
Positive pregnancy test at the Screening visit. 
5. 
QTcF Exclusions (any of the following): 
a. Screening QTcF interval of > 450 ms assessed using Fridericia’s correction factor based on 
12-lead ECG obtained as a continuous ECG of at least 50 seconds. 
b. Hypokalemia (Serum potassium at the Screening visit < 3.0 mmol/L or mEq/L). Serum 
potassium may be repeated during the Screening period with the agreement of the Medical 
Monitor. 
c. QTcF prolongation that is currently controlled with medication, in which normal QTcF 
intervals can only be achieved with medication. 
6. 
Any other clinically significant finding (as determined by the Investigator) on ECG at the 
Screening visit that would increase the likelihood of a severe arrhythmia. 
 






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antibiotics, pain relievers, anti-diarrheals, and laxatives) whose doses and dosing regimen have 
not been stable for at least 4 weeks prior to the Screening visit. If the treatment was 
discontinued, the discontinuation must have occurred no fewer than 2 weeks prior to the 
Screening visit. 
a. This includes nutraceutical treatments and cannabinoids (tetrahydrocannabinol [THC] 
and cannabidiol [CBD]) which are allowable but also must meet the stability conditions 
above. 
b. Pain relievers and laxatives may be used as needed.  
c. Standard of care immunizations and vaccinations (including COVID-19) are allowed and 
are not subject to the above stability conditions. 
d. The Investigator should consult with the medical monitor if a participant is being treated 
with ocular toxic, hepatotoxic, nephrotoxic or antibiotic medications.  
e. If the Investigator is unsure if a medication is permitted, the Medical Monitor should 
confirm if the medication is allowed and will provide written documentation to support 
the use of the medication, if allowed. 
 
Regression of Skills 
 
37. 
Actively undergoing regression or loss of skills defined as a persistent loss of previously 
acquired developmental skills within 3 months of the Screening visit.  
a. Previously acquired skills include any skill established and used on a daily basis for at 
least 3 months. 
b. Variations in use of skills that is part of a general pattern of ups and downs, as 
determined by the investigator, are not counted as regression. 
c. Skills may include language, non-verbal communication, fine motor skills, ambulation 
(including gait, coordination, independence of walking/standing), social skills, play, self-
help.  
 
Seizures 
 
38. 
Seizure profile that is not stable 8 weeks prior to commencing IMP. The overall pattern of 
seizure activity for the participant should be consistent with their typical pattern of seizures and 
be stable within the 8-week period in terms of type, intensity, duration, and frequency as 
determined by the clinical Investigator.  
 
Medical Conditions 
39. 
Currently pregnant, lactating, or breastfeeding. 
40. 
Current clinically significant (as determined by the Investigator) cardiovascular, 
gastrointestinal, respiratory, or endocrine disease, or clinically significant organ impairment. 
The Investigator may consult with the Medical Monitor as needed if he/she is uncertain if 
symptoms are exclusionary. 
41. 
Current clinically significant (as determined by the Investigator) hypo- or hyperthyroidism. If 
the participant has hypo- or hyperthyroidism that is controlled with medication, the Investigator 
should consult with the Medical Monitor to confirm if the participant is eligible. 
42. 
Type 1 or Type 2 diabetes mellitus requiring insulin (whether well controlled or uncontrolled), 
or uncontrolled Type 1 or Type 2 diabetes.  
43. 
Has planned surgery during the study. 
44. 
History of, or current, cerebrovascular disease or brain trauma. 
45. 
History of, or current catatonia or catatonia-like symptoms. 
46. 
History of, or current, malignancy. 






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the physical and neurological exam will be completed by the Investigator at the telemedicine visits based 
on the guidance documents provided to the Investigator. During the in-home component of the visit at 
Screening (Visit 2), the nurse will collect vital signs, weight, do a Targeted Physical Exam assessing 
general appearance, abdominal/costovertebral angel tenderness (CVA), and edema, and query oral intake. 
The nurse will review the caregiver diary during the visit and provide the caregiver with a new section of 
the diary. Post-enrollment, at the in-home component of the combined visit, the nurse will collect urine, 
vital signs, weight, and blood samples for a comprehensive metabolic panel (CMP), complete blood count 
(CBC) with differential, and coagulation (except Visit 1, when only CMP is collected).  The nurse will 
conduct a Targeted Physical Exam assessing: general appearance; the abdomen and the CVA region for 
tenderness; the extremities and peri-orbital region for edema. A suprapubic exam (bladder palpation) to 
assess for urinary retention may be performed. The visiting nurse will also review the caregiver diary, 
provide the caregiver with a new diary section, query oral intake, and a review tolerability. At the post-
treatment follow-up visit at Week 15, the visiting nurse will also collect a 12-lead ECG, in addition to the 
above assessments. 
 
In-home only visits with a visiting nurse will be done at Weeks 3 (Visit 6), 5 (Visit 8), 7 (Visit 10), 9 
(Visit 12), 11 (Visit 14), and 12 (Visit 15). At these visits, the nurse will collect urine, vital signs, weight, 
and a blood sample for the CMP. The nurse will also conduct a Targeted Physical Exam assessing general 
appearance, abdominal/CVA, and edema. A suprapubic exam (bladder palpation) to assess for urinary 
retention may be performed. The visiting nurse will review the caregiver diary, query oral intake, and 
review tolerability.  
 
A combined remote visit or nurse-only in-home visit may be conducted in-clinic by the study staff if 
agreed upon by the Investigator and the caregiver. The site should inform the nursing service of such a 
change. If an unscheduled nurse visit is needed for follow-up (e.g. repeat labs or other assessments), this 
can be requested by the site with the nursing service. The site should inform the Medical Monitor if they 
require an unscheduled nursing visit or will conduct a remote visit in the clinic. 
 
The nurse will send a report for all in-home nurse visits to the Investigator along with a copy of the 
completed Caregiver Diary. The Investigator is responsible for reviewing the Caregiver Diary and 
determining if any issues should be reported as an adverse event or require follow up. The visiting nurse 
will escalate any issues related to potential AE/SAEs, stopping criteria, major dosing issues or issues of 
potential safety concern to the Investigator the same day. Any changes noted will be provided to the 
Investigator who will be responsible for determining and reporting AE/SAEs.  
9.2 
Study Procedures by Visit 
9.2.1 
Visit 1 Screening/Baseline (in-clinic) – Week -4 (r 2 days) 
The following assessments will be conducted and data collected during Visit 1: 
x 
Eligibility 
o Informed consent (mandatory) 
o Consent for reuse of leftover Biomarker and PK samples (optional) 
o Inclusion/exclusion criteria 
o Medical history  
o Confirm documented PTHS diagnosis and genotype 
o PTHS history and exam 
o Confirm co-morbid psychiatric disorders by Diagnostic and Statistical Manual of Mental 
Disorders, Fifth Edition (DSM-5)  
o Autism Mental Status Exam 




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Remote/Telemedicine – Investigator and Appropriate Staff 
The following assessments will be conducted and data collected during Visit 2, conducted by the site via 
telemedicine or video facilities as appropriate. Caregiver assessments must be completed the day of the 
visit or no earlier than the day before the visit (except the caregiver diary which is completed daily) and 
reviewed by the Investigator. 
x 
Medical history (any updates since Visit 1) 
x 
Review of the Caregiver Diary (con-meds, seizures, voids, oral intake, breathing events, changes in 
symptoms)  
 
Caregiver Assessments 
The following assessments should be completed by the caregiver. Caregiver assessments must be 
completed the day of the visit or no earlier than the day before the visit. 
x 
Efficacy 
o Caregiver Top 3 Concerns  
o ABC-2 
o CSHQ 
o GIHQ 
 
In-home Nurse Visit 
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 2: 
 
x 
Vital Signs 
x 
Targeted Physical Exam: abdominal, CVA tenderness, general appearance, edema. Exam may include 
suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine output is noted. 
x 
Weight 
x 
Review of Caregiver Diary 
 
9.2.3 
Visit 3 Baseline (in-clinic) – Week 0 (r 2 days) 
The following assessments will be conducted and data collected during the Baseline visit (Visit 3). 
Caregiver assessments must be completed the day of the visit or no earlier than the day before the visit 
(except the caregiver diary which is completed daily) and reviewed by the Investigator. 
 
x 
Eligibility 
o Review inclusion/exclusion criteria 
o Medical history (updates from Screening visit) 
o Rapid SARS-CoV-2 test 
x 
Safety 
o Physical and neurological examination 
o Vital signs 
o Height/length 
o Weight 
o 12-lead ECG 
o CBC with differential 


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o Coagulation 
o CMP 
o Urinalysis 
x 
Review of the Caregiver Diary (con-meds, seizures, voids, oral intake, breathing events, changes 
in symptoms) 
x 
Biomarkers 
o Blood sample for biomarkers 
o Stool sample for microbiome 
x 
Efficacy 
o CGI-S 
o Vineland Adaptive Behavior Scales-3 
o MB-CDI 
o ORCA 
o Caregiver Top 3 Concerns  
o ABC-2 
o BPI-SF 
o ICND 
o QI-Disability 
o CSHQ 
o GIHQ 
o Modified two-minute walk test 
The Baseline visit (Visit 3) may be conducted over three consecutive days. It is preferrable that ECGs, 
safety labs and biomarker sample collection are conducted on the same day. All assessments must be 
completed within the two-day window for the visit (visit day ± 2 days).  For Visit 3, all assessments must 
be completed before administration of the IMP. 
 
x 
In-clinic administration of first dose 
o The first dose of IMP will be administered in the clinic after all Baseline assessments are 
completed, or, if the Investigator judges that it is too late in the day, on the following day. 
The participant should be monitored for at least three hours post the first dose.  
o If the first dose is administered the day following Baseline, vital signs (except height) 
should be collected before the first dose is administered. 
o The dose can be taken orally from a cup or from a dosing syringe. 
o Doses should be given on an empty stomach (2 hours after the previous meal or snack 
and 30 minutes prior to the next). 
o The IMP should not be mixed with water, liquid thickeners, or food for administration.   
o Day 1 of dosing is defined by the date when the first dose is taken. At this visit, the site 
will dispense an adequate supply of IMP for dispensing by the caregiver at home.  
9.2.4 
Visit 4 Treatment Period (telemedicine and in-home) – Week 1 (r 1 day) 
Visit 4 (Week 1) will be a remote telemedicine and in-home visit. The assessments to be conducted and 
data collected are divided accordingly below.  
Remote/Telemedicine – Investigator and Appropriate Site Staff 
The following assessments and data will be conducted or collected by the site via telemedicine or video 
facilities as appropriate during Visit 4. Caregiver-completed assessments should be reviewed by the 
Investigator.  


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x 
Recent Clinical History 
x 
AE/SAE and tolerability review 
x 
Review of the Caregiver Diary (dosing, con-meds, seizures, voids, oral intake, changes in symptoms)  
 
Caregiver Assessments 
The following assessments should be completed by the caregiver.  
x 
Caregiver diary 
 
In-home Nurse Visit 
 
The in-home visit will be conducted by a visiting nurse. The nurse will escalate any issues related to 
AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to the Investigator 
via telephone the same day. Any changes noted will be provided to the Investigator who will be 
responsible for determining and reporting AE/SAEs.  
 
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 4: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, and changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood for CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention may be performed 
if decreased urine output is noted. 
 
9.2.5 
Visit 5 Treatment Period (in-clinic) – Week 2 (r 2 days) 
Visit 5 (Week 2) will be an in-clinic visit. This visit may be conducted over three consecutive days. The 
following assessments should be conducted and data collected by site personnel during this visit. 
Caregiver assessments must be completed the day of the visit or no earlier than the day before the visit 
(except the Caregiver Diary which is completed daily) and reviewed by the Investigator. All assessments 
must be completed within the window for the visit (visit day ±2 days). 
x 
Safety 
o Recent clinical history 
o Physical and neurological examination 
o Vital signs 
o Weight 
o Review for AEs/SAEs 
o 12-lead ECG 
o CBC with differential 
o Coagulation 
o CMP  


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o Urinalysis 
x 
Caregiver Diary (con-meds, seizures, voids, oral intake, breathing events, changes in symptoms) 
x 
Review Dosing Compliance 
x 
Pharmacokinetics/Pharmacodynamics 
o Blood sampling for PK 
 
9.2.6 
Visit 6 Treatment Period (in-home nurse) – Week 3 (r1 day) 
The in-home visit at Visit 6 (Week 3) will be conducted by a visiting nurse. The nurse will escalate any 
issues related to AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to 
the Investigator via telephone the same day. Any changes noted will be provided to the Investigator who 
will be responsible for determining and reporting AE/SAEs.  
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 6: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood for CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted if decreased urine output is noted. 
 
9.2.7 
Visit 7 Treatment Period (telemedicine and in-home) – Week 4 (r1 day) 
Visit 7 (Week 4) will be a remote telemedicine and in-home visit. The assessments to be conducted and 
data collected are divided accordingly below.  
Remote/Telemedicine – Investigator and Appropriate Site Staff 
The following assessments and data will be conducted or collected by the site via telemedicine or video 
facilities as appropriate during Visit 7. Caregiver-completed assessments should be reviewed by the 
Investigator.  
x 
Recent Clinical History 
x 
Modified neurological exam 
x 
Modified physical exam 
x 
AE/SAE and tolerability review 
x 
Inform visiting nurse of dose titration, and confirm that dose titration instructions have been followed 
by the caregiver. 
x 
Review of the Caregiver Diary (con-meds, seizures, voids, oral intake, changes in symptoms)  
 
Caregiver Assessments 
The following assessments should be completed by the caregiver.  


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x 
Caregiver diary 
 
In-home Nurse Visit 
 
The in-home visit will be conducted by a visiting nurse. The nurse will escalate any issues related to 
AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to the Investigator 
via telephone the same day. Any changes noted will be provided to the Investigator who will be 
responsible for determining and reporting AE/SAEs.  
 
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 7: 
 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, and changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood labs 
o CBC with differential 
o Coagulation 
o CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 
9.2.8 
Visit 8 Treatment Period (in-home nurse) – Week 5 (r1 day) 
The in-home visit at Week 5 (Week 8) will be conducted by a visiting nurse. The nurse will escalate any 
issues related to AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to 
the Investigator via telephone the same day. Any changes noted will be provided to the Investigator who 
will be responsible for determining and reporting AE/SAEs.  
The following assessments should be conducted, and data collected by the visiting nurse at the 
participant’s home during Visit 8: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood for CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 


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9.2.9 
Visit 9 Treatment Period (in-clinic) – Week 6 (r 2 days) 
Visit 9 (Week 6) is an in-clinic visit. The following assessments will be conducted and data collected 
during Visit 9. This visit may be conducted over three consecutive days. Caregiver assessments must be 
completed the day of the visit or no earlier than the day before the visit (except the caregiver diary which 
is completed daily) and reviewed by the Investigator. All assessments must be completed within the 
window for the visit (visit day ±2 days). 
 
x 
Safety 
o Recent clinical history 
o Physical and neurological examination 
o Vital signs 
o Weight 
o Review for AEs/SAEs 
o 12-lead ECG 
o CBC with differential 
o Coagulation 
o CMP 
o Urinalysis 
o TSH, Free T3, Free T4 
o Serum Pregnancy Test (for female participants who have reached menarche) 
x 
Caregiver Diary (dosing, con-meds, seizures, voids, oral intake, breathing events, changes in 
symptoms) 
x 
Review Dosing Compliance 
x 
Pharmacokinetics/Pharmacodynamics 
o Blood sampling for PK 
x 
Efficacy 
o CGI-I 
o CGI-S 
o Caregiver Top 3 Concerns  
o ABC-2 
o CSHQ 
o GIHQ 
9.2.10 Visit 10 Treatment Period (in-home nurse) – Week 7 (r 1 day) 
The in-home visit at Week 7 (Visit 10) will be conducted by a visiting nurse. The nurse will escalate any 
issues related to AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to 
the Investigator via telephone the same day. Any changes noted will be provided to the Investigator who 
will be responsible for determining and reporting AE/SAEs.  
At Week 7, the site will also have a phone call with the caregiver to confirm the instructions for the dose 
titration have been followed. The site will also inform the visiting nurse so the dosing schedule can be 
reviewed with the family during the in-home visit.  
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 10: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, changes in symptoms 


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x 
Review Dosing Compliance 
x 
Collect blood for CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 
9.2.11 Visit 11 Treatment Period (telemedicine and in-home) – Week 8 (r 1 day) 
Visit 11 (Week 8) will be a remote telemedicine and in-home visit. The assessments to be conducted and 
data collected are divided accordingly below.  
Remote/Telemedicine – Investigator and Appropriate Site Staff 
The following assessments and data will be conducted or collected by the site via telemedicine or video 
facilities as appropriate during Visit 11. Caregiver-completed assessments should be reviewed by the 
Investigator.  
x 
Recent Clinical History 
x 
Modified neurological exam 
x 
Modified physical exam 
x 
AE/SAE and tolerability review 
x 
Review of the Caregiver Diary (dosing, con-meds, seizures, voids, oral intake, changes in symptoms)  
 
Caregiver Assessments 
The following assessments should be completed by the caregiver.  
x 
Caregiver diary 
 
In-home Nurse Visit 
 
The in-home visit will be conducted by a visiting nurse. The nurse will escalate any issues related to 
AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to the Investigator 
via telephone the same day. Any changes noted will be provided to the Investigator who will be 
responsible for determining and reporting AE/SAEs.  
 
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 11: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake and changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood labs 
o CBC with differential  
o Coagulation 
o CMP 


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x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 
 
9.2.12 Visit 12 Treatment Period (in-home nurse) – Week 9 (r 1 day) 
The in-home visit at Week 9 (Visit 12) will be conducted by a visiting nurse. The nurse will escalate any 
issues related to AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to 
the Investigator via telephone the same day. Any changes noted will be provided to the Investigator who 
will be responsible for determining and reporting AE/SAEs.  
 
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 12: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood for CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 
9.2.13 Visit 13 Treatment Period (telemedicine and in-home) – Week 10 (r1 day) 
Visit 13 (Week 10) will be a remote telemedicine and in-home visit. The assessments to be conducted and 
data collected are divided accordingly below.  
Remote/Telemedicine – Investigator and Appropriate Site Staff 
The following assessments and data will be conducted or collected by the site via telemedicine or video 
facilities as appropriate during Visit 13. Caregiver-completed assessments should be reviewed by the 
Investigator.  
x 
Recent Clinical History 
x 
Modified neurological exam 
x 
Modified physical exam 
x 
AE/SAE and tolerability review 
x 
Review of the Caregiver Diary (dosing, con-meds, seizures, voids, oral intake, changes in symptoms)  
 
Caregiver Assessments 
The following assessments should be completed by the caregiver.  


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x 
Caregiver diary 
 
In-home Nurse Visit 
 
The in-home visit will be conducted by a visiting nurse. The nurse will escalate any issues related to 
AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to the Investigator 
via telephone the same day. Any changes noted will be provided to the Investigator who will be 
responsible for determining and reporting AE/SAEs.  
 
The following assessments should be conducted, and data collected by the visiting nurse at the 
participant’s home during Visit 13: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, and changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood labs 
o CBC with differential  
o Coagulation 
o CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 
9.2.14 Visit 14 Treatment Period – (in-home nurse) Week 11 (r1 day) 
The in-home visit at Week 11 (Visit 14) will be conducted by a visiting nurse. The nurse will escalate any 
issues related to AE/SAEs, stopping criteria, major dosing issues or issues of potential safety concern to 
the Investigator via telephone the same day. Any changes noted will be provided to the Investigator who 
will be responsible for determining and reporting AE/SAEs.  
 
The following assessments should be conducted, and data collected by the visiting nurse in the 
participant’s home during Visit 14: 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: concomitant medications, voids, oral intake, changes in symptoms 
x 
Review Dosing Compliance 
x 
Collect blood for CMP 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 




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o HbA1c 
x 
Review Caregiver Diary (con-meds, seizures, breathing events, changes in symptoms) 
x 
Review Dosing Compliance 
x 
Pharmacokinetics/Pharmacodynamics 
o Blood sampling for PK 
x 
Biomarkers 
o Blood sample for biomarkers 
o Stool sample for microbiome 
x 
Efficacy 
o CGI-I 
o CGI-S 
o SB5 or Bayley-4 (Bayley is completed for participants who cannot achieve a basal on 
SB5) 
o Vineland Adaptive Behavior Scales-3 
o ASME 
o MB-CDI 
o ORCA 
o Caregiver Top 3 Concerns  
o ABC-2 
o BPI-SF 
o Caregiver Impression of Change 
o ICND 
o QI-Disability 
o CSHQ 
o GIHQ 
o Modified two-minute walk test 
9.2.17 Visit 17 Follow-up (telemedicine and in-home) – Week 15 (+ 4 days) 
Visit 17 (Week 15, Follow-up) will be a remote and in-home visit. The assessments to be conducted and 
data collected are divided accordingly below.  
Remote/Telemedicine – Investigator and Appropriate Site Staff 
The remote visit will be conducted by the site via telemedicine or video facilities as appropriate. 
Caregiver assessments must be completed the day of the visit or no earlier than the day before the visit 
(except the caregiver diary which is completed daily) and reviewed by the Investigator. 
The Investigator will also query changes in tolerability and note any changes. The Investigator is 
responsible for determining and reporting any changes that qualify as AEs. 
The following assessments will be conducted and data collected or reviewed by site personnel during 
Follow-up: 
x 
Safety 
o Recent clinical history 
o Physical and neurological examination: A modified version of the physical and 
neurological exam will be done based on the guidance document provided to the 
Investigator. 
o Review for AEs/SAEs 


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x 
Review Caregiver Diary (dosing, con-meds, seizures, voids, oral intake, breathing events, 
changes in symptoms) 
x 
Efficacy 
o CGI-I 
o CGI-S 
o Caregiver Top 3 Concerns  
o ABC-2 
o CSHQ 
o GIHQ 
x 
Study Exit Form 
 
In-home Nurse Visit 
 
The in-home visit will be conducted by a visiting nurse who will also query any changes in tolerability 
during the in-home visit. Any changes will be noted and provided to the Investigator.  
 
The following assessments will be conducted and data collected by the home nurse during Follow-up: 
 
x 
AE/SAE/tolerability query 
x 
Review caregiver diary: dosing, concomitant medications, voids, oral intake, changes in symptoms 
x 
Collect blood:  
o CMP 
o CBC with differential 
o Coagulation 
x 
Collect urine for urinalysis 
x 
Vital Signs 
x 
Weight 
x 
12-lead ECG 
x 
Targeted Physical Exam: general appearance, abdominal, CVA tenderness, check for edema. Exam 
may include a suprapubic exam (bladder palpation) to assess for urinary retention if decreased urine 
output is noted. 
 
9.3 
Screening and Baseline Procedures 
Screening and Baseline assessments are completed after informed consent during a period of 
approximately 4 to 6 weeks. During the Screening/Baseline period data will also be collected to establish 
the participant’s baseline characteristics and symptom severity based on assessments collected during the 
4 to 6 week period. There are two in-clinic visits and one remote/in-home visit for Screening and Baseline 
(Visits 1, 2, 3). Screening (Visit 1, in-clinic), Screening Visit 2, (combined remote/in-home nurse visit), 
Baseline (Visit 3, in-clinic). The second visit during the Screening period (Visit 2) should be conducted 2 
weeks ± 1 day after the Screening visit (Visit 1) . The participant will then remain in Screening for an 
additional 2 to 4 weeks. The caregiver diary must be completed daily during the entire Screening period 
whether it is 4 weeks or 6 weeks. 
 
During the Screening visit (Visit 1) informed consent to participate in the study will be obtained from the 
parent or legally authorized representative.  
 




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9.4 
Treatment of Participants 
9.4.1 
Investigational Medicinal Product 
Full information regarding the preparation, storage, and administration of NNZ-2591 Oral Solution, 50 
mg/mL is provided in the Pharmacy Manual. NNZ-2591 IMP is provided as a ready-to-use strawberry-
flavored liquid for oral administration. 
 
9.4.2 
Investigational Medicinal Product Administration 
NNZ-2591 Oral Solution, 50 mg/mL will be administered open-label at a target dose of 12 mg/kg BID. 
The total administration period for NNZ-2591 will be 13 weeks. Participants will be up-titrated to the 
target dose according to the dosing schedule outlined in Section 7.2. The dose titration schedule may be 
modified based on updated PK modelling including data from the study. 
NNZ-2591 Oral Solution, 50 mg/mL, is provided as a ready-to-use strawberry-flavored liquid for oral 
administration. The IMP should not be mixed with water, liquid thickeners, or food for administration.  
Each day there will be two dose administrations, one in morning and one in afternoon or evening. There 
should be at least 8 hours between doses. Doses should be given on an empty stomach (2 hours after the 
previous meal or snack and 30 minutes prior to the next). Doses may be taken over a 10-minute period 
with a follow up of 250 mL of water.  
For all participants, the total duration of NNZ-2591 treatment will be 91 days. Dosing at the end of 
Baseline counts as Day 1 whether one or two doses are given. Dosing may not exceed 91 days including 
Day 1, and all efficacy assessments for the EOT visit must be completed on or before Day 91 of 
treatment. 
The first dose of IMP will be administered in the clinic after all Baseline assessments are completed or, if 
the Investigator judges that it is too late in the day, on the following day. The participant should be 
monitored for at least three hours after the first dose. If the first dose is administered the day following 
Baseline, vital signs (except height) should be collected before the dose is administered. Day 1 of dosing 
is defined by the date when the first dose is taken. At this visit, the site will provide IMP for the caregiver 
to dispense at home. Enough IMP will be provided to cover the interval until the next in-clinic visit when 
the caregiver will be re-supplied from site.  
Within the up-titration period, if a participant is deemed not to be tolerating the current dose within the 
2-week period, and they have not met any of the stopping criteria below (see Section 9.4.5), they can be 
down-titrated to the next lower dose in the titration schedule (See Section 7.2).The Investigator must 
attempt to increase the dose as soon as it is possible based on the clinical situation. The aim is to return to 
the originally assigned dose. If the originally assigned dose cannot be reached, or the participant is again 
unable to tolerate that dose, the Investigator will continue treatment on the highest dose the participant 
can tolerate. Stopping rules for the discontinuation of the administration of IMP are described in 
Section 9.4.5. Investigators are to discontinue administration of IMP if the participant meets any of the 
criteria described in Section 9.4.5. 
Note that early cessation of IMP does not necessarily mean the participant is withdrawn from the study. 
With any early cessation, all evaluations and tests scheduled during the study must be conducted if 
possible. 
 






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x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Participants who have received IMP for longer than 2 weeks and discontinue prematurely any time after 
Visit 5 should return to the investigational site for final safety and efficacy assessments as scheduled for 
the EOT visit (Week 13, Visit 16) and complete the study exit form. 
Participants who discontinue from the trial prior to receiving any treatment or who discontinue after 
having received treatment only up to Visit 5 (Week 2 post-baseline) may be replaced.   
Participants with early treatment cessation due to SAEs must be followed-up and provided appropriate 
medical care until their signs and symptoms have remitted or stabilized, or for the remaining study period 
(until study end), whichever comes sooner.  “Study end” is when all participants have completed the 
study. 
9.4.6 
Concomitant Therapy 
Investigators are encouraged to follow current international and hospital guidelines and current best 
medical practice for the management of participants with PTHS. Medications should be used per current 
recommendations. Concomitant medications permitted in the study are outlined below and are described 
in the Exclusion Criteria (Section 8.2).  
Medications prohibited from the study are outlined below and in the Exclusion Criteria (Section 8.2).  
Participants should not have their existing medication withdrawn in order to enter the study. 
Excluded medications/treatments include:  
x 
Current treatment or treatment within the two weeks prior to Screening with MAO inhibitors, d-
cycloserine, oxytocin, carbetocin, tricyclic antidepressants, or bupropion. 
x 
Current treatment with more than 3 allowable psychotropic medications.   
o Includes medications used to treat problems with sleep onset and sleep continuity except 
melatonin. Melatonin for difficulties with sleep onset is permissible and is not included in 
the count of psychotropic medications.   
o The use of anti-epileptics for other indications such as the treatment of mood disorders 
counts towards the limit of permitted psychotropic medications.   
o Anti-epileptic medications used for treatment of seizures does not count towards the limit 
of psychotropic medications.  
x 
Current treatment with insulin or treatment within the last 12 weeks prior to commencement of 
IMP. 
x 
Current treatment with IGF-1 or treatment within 6 months of commencement of the IMP. 
x 
Current treatment with growth hormone or treatment within 12 months of commencement of the 
IMP. 
 


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The following medications are allowed but must be at stable doses before the start of the study as follows:  
 
x 
Non-pharmacologic somatic treatment (e.g., a ketogenic diet or vagal nerve stimulation): dosing 
regimen must be stable at least 4 weeks prior to Screening. If the treatment was discontinued, the 
discontinuation must have occurred no fewer than 2 weeks before Screening.  
x 
A new behavioral, educational, or cognitive therapy cannot be started during the period of the 
study or within 4 weeks prior to Screening. Changes to an ongoing treatment regimen that are due 
to school schedules or are otherwise seasonally related are not exclusionary as long as they are 
consistent with the typical type and pattern of behavioral treatment received by the participant.  
x 
Seizure medication: dosing regimen must be stable 8 weeks prior to commencing the IMP. 
Adjustments for changes in weight or age are allowable. If the treatment was discontinued, the 
discontinuation must have occurred no fewer than 2 weeks before Screening. 
x 
Psychotropics or any other medication used for a chronic illness (not including antibiotics, pain 
relievers, anti-diarrheals, and laxatives): dosing regimen must have been stable for at least 4 
weeks prior to Screening.  If the treatment was discontinued, the discontinuation must have 
occurred no fewer than 2 weeks before the commencement of IMP. 
o This includes nutraceutical treatments and cannabinoids (THC and CBD) which are 
allowable but also must meet the stability conditions above. 
o The Investigator should consult with the medical monitor if a participant is being treated 
with ocular toxic, hepatotoxic medications, nephrotoxic, or antibiotic medications.  
 
The following medications/treatments are permitted and may be adjusted as needed: 
x 
Procedural pain control medication. 
x 
Laxatives and anti-diarrheals.  
Standard of care immunizations and vaccinations (including COVID-19) are allowed and are not subject 
to the above stability conditions. 
 
If the Investigator is unsure if a medication is permitted, the Medical Monitor should confirm if the 
medication is allowed and will provide written documentation to support the medication’s use, if allowed. 
Concomitant medications will be monitored in a caregiver diary for the approximately 4 to 6 week 
Screening period to confirm there are no changes from the time of initial screening. 
Every effort should be made to maintain stable regimens of concomitant medications and allowed non-
medicine-based therapies throughout the course of the study, with the understanding that there will be 
some changes to non-medicinal treatment regimens that are due to school schedules or are otherwise 
seasonally related.  
Details of concomitant medications will be recorded in the participant notes and in the eCRF. 
9.5 
Assessments  
9.5.1 
Vital Signs  
Vital signs including weight, blood pressure (SBP and DBP in mmHg), heart rate (bpm), respiratory rate 
(rpm), and body temperature (qC) will be assessed at the Screening visits (Visits 1 and 2) and at the 
Baseline visit (Visit 3). Height will be measured at Screening (Visit 1), Baseline (Visit 3) and End of 
Treatment (Week 13, Visit 16). Blood pressure (SBP and DBP in mmHg), weight, heart rate (bpm), 
respiratory rate (rpm), and body temperature (qC) will also be assessed at all post-enrollment study visits. 
If the first dose is administered in clinic the day after the Baseline visit, blood pressure (SBP and DBP in 




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collecting the samples to reduce contamination. 
x Complete Blood Count with Differential (Visits 1, 3, 5, 7, 9, 11, 13, 16, 17): Hemoglobin (Hb), 
hematocrit (Hct), red cell count (RBC), white blood cell count (WBC), mean cell volume (MCV), 
mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), platelets (PLT), red cell 
distribution width (RDW-CV), and leukocyte count (neutrophils, lymphocytes, monocytes, 
eosinophils, and basophils). If abnormalities are seen, additional tests may be conducted as deemed 
necessary by the Investigator and Medical Monitor and as described below in the clinical management 
plan in Sections 9.5.2.1 and 9.5.2.3. 
x Coagulation (Visits 1, 3, 5, 7, 9, 11, 13, 16, 17): prothrombin time test, International normalized ratio 
(INR), activated partial thromboplastin (aPTT). If abnormalities are seen, additional tests may be 
conducted as deemed necessary by the Investigator and Medical Monitor and as described below in the 
clinical management plan in Sections 9.5.2.1 and 9.5.2.3. 
x Comprehensive Metabolic Profile (All Visits except Visit 2): amylase, sodium, glucose, potassium, 
blood urea nitrogen (BUN), enzymatic creatinine, BUN/creatinine ratio, estimated glomerular 
filtration rate (eGFR), bicarbonate, chloride, total calcium, phosphorus, magnesium, bilirubin (direct 
and indirect), creatine kinase, alkaline phosphatase (ALP), aspartate transaminase (AST), alanine 
amino transferase (ALT), gamma-glutamyl transferase (GGT), total protein, albumin, total cholesterol, 
and random triglycerides. If abnormalities are seen, additional tests may be conducted as deemed 
necessary by the Investigator and/or Medical Monitor and as described below in the clinical 
management plan in Sections 9.5.2.1 and 9.5.2.3. 
o eGFR is calculated using the modified Bedside Schwartz formula: 
eGFR=(0.413*height)/creatinine mg/dL 
o This formula will be used for all participants including those who may age up to 18 years 
old during the study 
x Urinalysis (All Visits except Visit 2): with microscopic analysis, glucose, bilirubin, ketones, specific 
gravity, blood, pH, protein, albumin, urobilinogen, nitrite, and leucocyte esterase. If abnormalities are 
seen on microscopy, additional tests for culture, sensitivity and protein analysis may be conducted as 
deemed necessary by the Investigator and/or Medical Monitor, and as described below in the clinical 
management plan in Section 9.5.2.2.  
x Urine will also be tested at Screening, for drugs of abuse, i.e., stimulants (e.g., amphetamine, 
methamphetamine, methylenedioxymethamphetamine [MDMA], cocaine), hallucinogens (e.g., 
phencyclidine), narcotics/analgesics/ opiates (e.g., codeine, morphine, methadone, propoxyphene), 
depressants/sedatives/hypnotics (e.g., barbiturates). 
x TSH, Free T3, Free T4 (Visits 1, 16): free triiodothyronine (T3), free thyroxine (T4), thyroid 
stimulating hormone (TSH). 
x Glycosylated hemoglobin (HbA1c) (Visits 1, 16) 
x Rapid test for SARS-CoV-2 (Visits, 1, 3, 16). The site may use any test with same day results 
approved for use for children and adolescents in the US.  












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submitted. The clinician will be queried to adjudicate which ECG should be considered the final read and 
entered into the database. 
ECGs will be sent via secure transmission and read by a qualified central reader. The results from the 
reports from the central reader and any alert flags should be reviewed by the Investigator who will 
determine if the values are clinically significant and sign the report. Results will be assessed against the 
ECG screening criteria for QTcF at the Screening visit and against the QTcF stopping criteria throughout 
the study.   
Investigational Medicinal Product must be discontinued at any time during the study in the event that a 
post-enrollment 47F) GXUDWLRQ RI   PV confirmed by a repeat continuous ECG, or a clinically 
significant LQFUHDVH RI   PV (confirmed by a repeat continuous ECG) compared to the average QTcF 
interval at Baseline (Visit 3, before dosing) is observed.  
9.5.9 
Battery of Pitt-Hopkins Syndrome Assessment Instruments 
The battery of PTHS specific measures will evaluate symptom change in clinically meaningful and 
functionally impactful symptom domains for individuals with PTHS including communication, cognition, 
motor function, maladaptive behavior, adaptive behaviors, sleep, seizures, breathing and GI issues (see 
Section 6.3.2). The selection of outcome measures for this study reflects the insights from the published 
literature, and expert clinical advice. 
All assessments will be administered in a standardized manner by trained practitioners. To the extent 
possible, all efforts should be made to maintain the same caregiver informant or investigator rater (as 
applicable) across visits for a single participant. If in extenuating circumstances (such as severe illness), 
the caregiver is unable to complete the rating for a visit, another appropriate rater can be designated to 
complete the assessment for that visit. This should be documented in the source and the EDC. The site 
staff should ensure the alternative rater has appropriate knowledge of the participant to complete the 
rating and is informed of the instructions for completing the assessments. 
Caregivers will complete most assessments via an online portal. Paper versions of assessments will also 
be used and may be made available for online versions in the case of difficulty accessing the electronic 
portal. These assessments should be completed the day of the visit or no earlier than the day before the 
visit. No assessments can be conducted until informed consent has been given by the caregiver or legally 
authorized representative. Also, training for the caregivers on all caregiver completed assessments must 
be completed before the caregiver rater may complete the assessments. 
Overall symptom severity and functional level will be assessed using the following tools: 
9.5.9.1 
Clinical Global Impression of Severity (CGI-S) 
Clinical Global Impression of Severity assessments will be done at Screening (Visit 1), Baseline (Visit 3), 
Week 6 (Visit 9), EOT (Week 13, Visit 16), and Follow-up (Week 15, Visit 17). The Clinical Global 
Impression - Severity scale (CGI-S)36 is a 7-point scale that requires the Investigator to rate the severity of 
the individual's overall illness at the time of assessment, relative to the Investigator's past experience with 
participants who have the same diagnosis.36 Considering total clinical experience, a participant is assessed 
on severity of illness at the time of rating: 1, normal, not at all impaired; 2, borderline, slightly impaired; 
3, mildly impaired; 4, moderately impaired; 5, markedly impaired; 6, severely impaired or 7, among the 
most extremely impaired ill. Following best practice for neurodevelopmental disorders, the CGI-S will be 
evaluated using PTHS-specific scoring anchors. The clinicians will provide an overall global severity 
rating and a severity rating for each of seven domains: language/communication, fine motor 
functioning/self-help, social initiation/social avoidance, challenging behavior, ambulation/gross motor, 


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gastrointestinal issues, and autonomic/breathing abnormalities. The clinician rater should assess the 
participant’s current state taking into consideration observations at the visit and symptoms within the last 
week. To ensure that interpretation of the scoring rubric is normalized among the raters, clinicians 
administering the CGI-S will be required to complete scoring calibration training. 
9.5.9.2 
Clinical Global Impression of Improvement (CGI-I) 
Clinical Global Impression of Improvement assessments will be made at Visit 9 (Week 6), EOT (Week 
13, Visit 16), and Follow-up (Week 15, Visit 17). The CGI-I scale36 requires the Investigator to rate how 
much the participant’s illness has improved or worsened relative to a baseline state. A 7-point scale is 
used with the following values: 1, very much improved; 2, much improved; 3, minimally improved; 4, no 
change; 5, minimally worse; 6, much worse; 7, very much worse.  The CGI-I will be evaluated using 
PTHS-specific scoring anchors. The clinicians will provide an overall global improvement rating and an 
improvement rating for each of seven domains: language/communication, social initiation/social 
avoidance, fine motor functioning/self-help challenging behavior, ambulation/gross motor, 
gastrointestinal issues, and autonomic/breathing abnormalities. The clinician should assess the 
participant’s current state taking into consideration observations at the visit and symptoms within the last 
week. The rating of change is compared with status at Baseline (Visit 3). To ensure that interpretation of 
the scoring rubric is normalized among the raters, clinicians administering the CGI-I will be required to 
complete scoring calibration training. 
9.5.9.3 
Bayley Scales of Infant Development v4 (BSID4) or Stanford-Binet Intelligence Scales 
v5 (SB5) 
The Stanford-Binet Intelligence Scales version 5 (SB5) assessment will be done at Screening (Visit 1) and 
EOT (Week 13, Visit 16). SB5 is validated for ages 2 to 85+ and measures both verbal and nonverbal 
intellectual and cognitive abilities through a set of 10 subtests.37 General cognitive ability is reported as a 
full-scale intellectual quotient score (FSIQ). Cognitive ability in five categories are reported in the index 
scores: fluid reasoning (FR), knowledge (KN), quantitative reasoning (QR), visual-spatial processing 
(VS), and working memory (WM). Non-verbal (NVIQ) and verbal intelligence quotients (VIQ) are also 
calculated. Scoring of index scores and IQ will be calculated using the z-deviation method for intellectual 
disability.38  
The Bayley Scales of Infant Development version 4 (BSID-4) is conducted for participants who cannot 
achieve a basal score on SB5or for whom the SB5 is developmentally inappropriate .39 Assessment will 
be done at Screening (Visit 1) and End of Treatment (Week 13, Visit 16). The Bayley is a norm-
referenced developmental assessment validated for ages 16 days to 42 months. It assesses cognitive 
ability, motor skills, and behavior and provides standardized scores in five domains: Cognitive, 
Language, Motor, Social-Emotional, and Adaptive Behavior. Scale scores, age equivalents and growth 
scale values (GSVs) can also be calculated for the scale subtests: cognition (CG), receptive 
communication (RC), expressive communication (EC), fine motor (FM), and gross motor (GM). Scaled 
scores and age equivalents can be calculated for the social emotional (SE) subscale. The Adaptive 
Behavior subscale will not be collected in this study. The Early Learning Composite score and scaled 
scores are calculated for children 42 months and younger. Raw scores, age equivalents, and growth scale 
values are calculated for all participants. 
If the SB-5 or the Bayley had previously been administered for the participant within 3 months of 
Screening, the scores from that administration may be used if it meets the following criteria: 1) 
assessment was administered by site personnel, or another study site; 2) the site has access to all the 
scores required to be entered in the EDC; 3) the site has permission to share the scores from the 
previously administered assessment to the study EDC. Assessments done by the school or other clinics 


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are not acceptable. Only scores from SB-5 and the Bayley are acceptable; cognitive scores from other 
assessments cannot be entered in the EDC in lieu of the SB-5 or the Bayley. 
9.5.9.4 
MacArthur-Bates Communicative Development Inventory (MB-CDI) 
The MacArthur-Bates Communicative Development Inventory (MB-CDI) will be completed at Baseline 
(Visit 3) and EOT (Week 13, Visit 16). This caregiver-completed instrument assesses language 
development through vocabulary comprehension and production, gestures, and grammar.40 Designed to 
assess early language development in infants and toddlers, it can also be used with older, developmentally 
delayed children to assess developmental progress in language development. The MB-CDI includes three 
modules based on chronological or developmental age: Words and Gestures (8- to 18- months or 
developmental equivalent), Words and Sentences (16- to 30-months or developmental equivalent) and the 
CDI-III, an extension of the MB-CDI used with children 30-37 months or developmental equivalent. The 
Word and Gestures module will be used for this study in line with the expected cognitive and language 
development of the study population. It assesses developing abilities in language including production of 
gestures, and vocabulary comprehension and production which are reported as number of instances and 
percentile scores. 
9.5.9.5 
ORCA (Observer-Reported Communication Ability Measure) 
The Observer-Reported Communication Ability Measure (ORCA) will be completed at Baseline (Visit 3) 
and EOT (Week 13, Visit 16). This caregiver-reported measure was originally developed and validated 
for Angelman syndrome,41 to assess the individual’s overall communicative ability and was designed to 
capture communicative abilities in low-verbal and non-verbal populations, so is relevant for PTHS. It was 
developed based on feedback from a patient-focused listening session with the advocacy community and 
the Food and Drug Administration following best practices for measure development including concept 
elicitation interviews with caregivers and communication experts, item generation, assessment of validity 
and psychometrics, and factor analysis confirming single overall communication ability score.41-43 The 
rating scale evaluates the caregiver’s observation of the individual’s communication ability over the past 
30 days.42 The measure consists of 84 total questions with 70 behavioral items. It assesses communication 
abilities of individuals across all modalities: gestures, AAC (augmentative and alternative 
communication) and speech so is not dependent solely on verbal abilities. Twenty-two communication 
concepts relevant to communicative ability in PTHS are assessed within the categories of expressive 
communication, receptive communication, and pragmatic communication rendering a total 
communication score.   
9.5.9.6 
 
Caregiver Top 3 Concerns Rating 
The Caregiver Top 3 Concerns Rating assessment is a syndrome-specific measure of 3 signs or symptoms 
of PTHS identified by caregivers at Screening/Baseline as being priority concerns in which they would 
like to see improvement as a result of treatment. The assessment will be completed at all Screening and 
Baseline visits (Visits 1, 2, 3), as well as Week 6 (Visit 9), EOT (Visit 16), and Follow-up (Visit 17). 
In this assessment, concerns are identified on an individual, per-participant basis. The clinician should 
review the assessment with the caregiver at the Screening visit, ideally as part of the overall medical and 
clinical history. Caregivers should be encouraged, in selecting the concerns, to identify distinct, 
observable symptoms rather than broad ones. And caregivers should be advised to consider symptoms 
that they feel are important and have an impact on their child’s daily functioning and well-being.  
Caregivers are asked to identify 3 concerns from among 11 clinically important symptom domains: 
Language/Communication, Social interest/avoidance, Repetitive Behaviors, Challenging Behaviors, 
Gross motor skills, Fine motor skills, Gastrointestinal problems, Breathing Disruptions, Sleep, Seizures, 


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and Self-care skills. Each individual concern identified should be from a different symptom domain, but 
caregivers can select any domain and choose any symptom of concern within that domain. The same three 
concerns identified at screening/baseline are carried forward and rated at the follow-up visits. The 
severity of each concern is scored by caregivers using a 10-point Likert scale or severity. The caregiver 
should give consideration to symptoms within the last two weeks.  
9.5.9.7 
 
Aberrant Behavior Checklist-2 (ABC-2) 
The ABC-2 will be completed at all Screening and Baseline visits (Visits 1, 2, 3), as well as Week 6 
(Visit 9), EOT (Week 13, Visit 16), and Follow-up (Visit 17). The Aberrant Behavior Checklist-2 (ABC-
2)44 is a caregiver-completed rating scale for assessing problem behaviors that has been validated in a 
variety of clinical populations and been used as an outcome measure in clinical trials of 
neurodevelopmental disorders (NDDs) across much of the lifespan.44 The rater considers the relative 
frequency of an individual’s behaviors and gives a rating of the severity of the problem it presents: 0, not 
at all a problem; 1, the behavior is a problem but slight in degree; 2, the problem is moderately serious; 3, 
the problem is severe in degree. The caregiver should give consideration to symptoms within the last two 
weeks.  
The ABC-2 includes 58 items providing five subscale scores: Irritability, Social Withdrawal, Stereotypic 
Behavior, Hyperactive/Noncompliance, and Inappropriate Speech. These subscales have been shown to 
have good consistency, reliability, and validity with normative data available for specific populations 
including children with intellectual disability and ASD.45 
9.5.9.8 
Behavior Problems Inventory -Short form (BPI-SF) 
The Behavior Problems Inventory Short Form (BPI-SF) will be completed at Baseline (Visit 3) and at the 
EOT (Week 13, Visit 16). The BPI-SF is a caregiver-completed rating scale for assessing self-injury, 
aggressive and stereotyped behavior.46, 47 Frequency is assessed for all three types of behavior on a five-
point scale from 0 (Never), 1 (Monthly), 2 (Weekly), 3 (Daily) and 4 (Hourly). For self-injury and 
aggression, severity is also assessed on a 3-point scale of mild, moderate, or severe. 
9.5.9.9 
Caregiver Impression of Change 
The Caregiver Impression of Change assessment will be done at the EOT (Week 13, Visit 16). The 
Caregiver is asked to rate the change in his or her child’s overall function and well-being since the 
Baseline visit (Visit 3), and the change in specific symptom domains. Ratings are made using a 7-point 
Likert scale:1-Very much improved, 2-Much improved, 3-Improved, 4-Unchanged, 5-Worse, 6-Much 
worse, 7-Very much worse. For the rating of overall function, the caregiver also identifies the one 
symptom area or feature that has most influenced his or her rating of the child’s overall function and 
provides a description of symptom changes and the impact of the changes. The assessment of change 
should be made since the Baseline visit (Visit 3). The caregiver should assess the participant’s current 
state at the visit taking into consideration observations at the visit and symptoms within the last week. 
9.5.9.10 
Quality of Life Inventory-Disability (QI-Disability) 
The Quality of Life Inventory-Disability (QI-Disability) will be completed at Baseline (Visit 3) and EOT 
(Week 13, Visit 16).48-50 The QI-Disability48 is a caregiver-report measure that assesses a child’s well-
being and quality of life over the past month. Designed to be relevant for children who have an 
intellectual disability, the QI-Disability includes 32 questions in 6 domains: Social Interaction, Physical 
Health, Independence, Positive Emotions, Leisure and the Outdoors, and Negative Emotions. The QI-
Disability has been validated in individuals ages 5 to 18 with Rett syndrome, Downs syndrome, CDKL5 


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Deficiency Disorder, Cerebral Palsy, and ASD. Items are rated on a 5-point Likert scale with a 0-100 
scaling such that the value of each rating is: never = 0, rarely =25, sometimes = 50, often = 75, and very 
often is 100. Domain scores are calculated by summing all the scores and dividing by the number of 
items. The total score is calculated by summing the domain scores and dividing by the number of 
domains.  
9.5.9.11 
Impact of Childhood Neurological Disability-Overall Quality of Life Rating (ICND) 
The Impact of Childhood Neurologic Disability Scale (ICND) will be administered at Baseline (Visit 3) 
and at End of Treatment (Week 13, Visit 16). The ICND was developed to evaluate the impact that a 
child’s condition has on the child’s and the family’s everyday life.51 The full scale assesses the effect of 
four conditions or health problems on the child’s or the family’s life, and evaluates the child’s overall 
quality of life. In this study, only the overall quality of life assessment will be completed. The caregiver 
will rate overall quality of life of the participant by responding to the following: “Please rate your child’s 
overall ‘Quality of Life’ on the scale below. Choose the number which you feel is best and circle it”. The 
choices range from 1 (“Poor”) to 6 (“Excellent”). The caregiver should consider the child’s status in the 
last month including their current condition. . 
9.5.9.12 
 Child Sleep Habits Questionnaire (CSHQ) 
The Child Sleep Habits Questionnaire (CSHQ) will be completed by the participant’s caregiver at all 
Screening and Baseline visits (Visits 1, 2, 3), as well as Week 6 (Visit 9), EOT (Week 13, Visit 16), and 
Follow-up (Visit 17). The CSHQ gathers information on bedtime and assesses sleep behavior, waking 
during the night, and morning waking/daytime sleepiness52 to assess both behavioral and medical sleep 
disorders. The CSHQ has been used across a range of NDDs including PTHS and has been used as an 
outcome measure for sleep intervention trials.53-55 It provides a total score and eight subscale scores: 
bedtime resistance, sleep onset delay, sleep duration, sleep anxiety, night awakenings, parasomnias, sleep 
disordered breathing, and daytime sleepiness. The caregiver should consider sleep behaviors within the 
last week in doing their ratings.  
9.5.9.13 
Vineland Adaptive Behavior Scales-3 
The Vineland Adaptive Behavior Scales Third Edition (Comprehensive Interview version, Vineland-3) 
will be completed at Baseline (Visit 3) and EOT (Week 13, Visit 16). This standardized measure assesses 
development and adaptive functioning which correspond to the domains of adaptive functioning used in 
the DSM-5: Communication, Daily Living Skills, and Socialization.56 The Vineland will be conducted as 
an interview by a trained site rater with the caregiver. The measure provides an overall adaptive 
functioning score, and standardized domain scores in Daily Living Skills, Communication, Socialization 
and Motor Skills. The Vineland also has a maladaptive behavior domain which will not be completed in 
this study. V-scale scores and growth scale values (GSV) can also be calculated for the subdomains. The 
GSV scores on the Vineland have been found to be more sensitive to change than standardized scores for 
developmentally delayed populations.57 Raw scores, standardized scores, v-scale scores, age equivalents, 
and GSV will be calculated for all participants. For participants 3 to 9 years of age, Motor Skills can be 
calculated; for those older than 9 years old, scores will not be calculated for Motor Skills. 
9.5.9.14 
GI Health Questionnaire (GIHQ) 
The Gastrointestinal Health Questionnaire (GIHQ) will be completed by the participant’s caregiver at all 
Screening and Baseline visits (Visits 1, 2, 3), as well as Week 6 (Visit 9), EOT (Week 13, Visit 16), and 
Follow-up (Visit 17). The measure was developed for use in NDDs and validated in individuals with Rett 
syndrome and includes GI symptoms that are relevant for PTHS.58 The GIHQ assesses the impact of GI 


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issues on the individual’s life over the last two weeks.58 Items are rated on a 5-point Likert scale as 
follows: 0= it is never a problem, 1=it is almost never a problem, 2=it is sometimes a problem, 3=it is 
often a problem, 4=it is almost always a problem. A total score and 9 subtest scores are calculated with 
the 9 subtests comprising issues in three domains: Gastrointestinal symptoms, Mood/Behavior, and Parent 
Concerns.  
9.5.9.15 
Modified 2-Minute Walk Test 
The modified 2-Minute Walk test (2MWT)59 is an assessment of how far an individual can walk during a 
2 minute period which assesses endurance and also has relevance for everyday functioning. The 2MWT 
will be done at Screening (Visit 1), Baseline (Visit 3) and at the EOT (Week 13, Visit 16).  For this 
assessment, the individual with PTHS walks back and forth between two cones on a 20-meter track for 
two minutes and the total distance covered is measured. The modified 2MWT requires two assessors and 
one walk assistant. The walk assistant knows the person with PTHS well (e.g., parent, caregiver, 
therapist) and provides the necessary physical support for the person to keep his/her balance and to 
maintain the highest possible gait speed.  
9.5.10 Caregiver Diary 
A Caregiver Diary will be completed daily by the caregiver and reviewed at all visits by the Investigator 
and by visiting nurse at remote visits. In the diary, the participant’s caregiver will record dosing 
compliance of IMP, any changes in concomitant medication or treatments, urine output measured by 
number of voids or wet diapers, any changes in symptoms including changes in oral intake, sleep activity, 
seizure activity, and breathing events. The diary also specifically queries changes in maladaptive behavior 
or self-injury which may suggest changes in the tendency to self-harm. At the Screening visit, the 
clinician will review the assessment with the caregiver and assist the caregiver to define the child’s 
typical seizure types, breathing abnormalities, sleep issues or other chronic conditions that are to be 
tracked in the diary. Any new, emergent types of seizures or breathing abnormalities, or any other new 
symptoms will also be documented if they occur.  
The Investigator must review the caregiver diary at or after each visit. At clinic visits, the Investigator 
will review the diary and the site will provide a new diary section to the caregiver. At remote visits, the 
nurse will review the diary in the home noting any potential issues in the nurse report and collect the diary 
to send to the Investigator along with notes from the visit. The nurse will provide the caregiver with a new 
diary section for the upcoming week. The nurse will not sign-off on the diary. The Investigator is 
responsible for reviewing the nurse notes, reviewing the diary, signing the diary, and determining if 
anything needs to be reported as an Adverse Event.  
 
9.6 
Pharmacokinetic Measurements 
Pharmacokinetic blood samples will be collected in accordance with the sampling schedule outlined in 
Table 2 for ages 7 to 17 years old and Table 3 for ages 3 to < 7 years old.  The date and actual time (hour 
and minute) that each sample is collected must be accurately recorded. The site must also ensure that the 
date and time of the previous evening’s dose and the morning and evening dose on the day the sample is 
collected is accurately collected in the Caregiver Diary.  
For the Week 2, 6, and 13 data collection, the previous day’s PM dose should be taken in the evening as 
late as possible, preferably before bedtime. The pre-dose sample should be taken 120 minutes to 
15 minutes before the morning dose is administered. For post-dose samples, sample collection can be 
± 15 minutes of the target time and must be no closer than 30 minutes from the previous sample (must be 


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at least 30 minutes from the previous sample). The actual collection time is recorded. The post-dose 
windows are determined from the first dose of the visit (morning dose) and all PK samples should be 
collected before the second dose (PM dose). Full details regarding the PK sampling collection, storage 
and shipping may be found in the Laboratory Manual for the study. 
PK blood samples may be collected from a cannula port or via venipuncture. Pre-prepared PK sampling 
tubes will be provided to each site within the lab visit kits for collection and storage of PK samples.  
Samples of whole blood will be collected, aliquoted, and will be stored at minus 20°C (or colder) no more 
than 2 hours following sample collection.  Samples will be shipped at designated times on dry ice and 
using temperature monitoring to the laboratory for storage and analysis. It is essential that accurate 
records are maintained regarding the date and time each PK sample is obtained, and the date and time 
each sample is placed in the 20°C freezer. The date and time of the most recent dose of IMP and most 
recent meal relative to that dose should be recorded in the Caregiver Diary.  
Minimum requirements for a participant to be considered evaluable within the PK population are 
administration of NNZ-2591 per protocol through to the morning dose at Visit 5 (Week 2) as a minimum, 
and collection of PK samples at the specified post-dose time point(s) through to Visit 5 (Week 2) as a 
minimum. 
 
 
 


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10. ADVERSE EVENTS 
The Investigator and site staff are responsible for detection, recording and reporting of events that meet 
the criteria and definition of an AE or SAE, as described below. At in-home nurse visits, the nurse will do 
a query of any changes for potential AE/SAEs and document observations from the targeted physical 
exam. The nurse will escalate any issues related to AE/SAEs, stopping criteria, major dosing issues, or 
issues of potential safety concern to the Investigator the same day. Any changes noted will be provided to 
the Investigator who will be responsible for determining, recording, and reporting any events that qualify 
as AE/SAEs. 
10.1 Definition of Adverse Event 
An adverse event is any untoward medical occurrence associated with the use of a drug in humans, 
whether or not considered drug related. An AE can be any unfavorable and unintended sign (e.g., an 
abnormal laboratory finding), symptom or disease temporally associated with the use of a drug, without 
any judgment about causality.   
AEs can include the following: 
x 
Exacerbation of a chronic or intermittent pre-existing condition including either an increase in 
frequency and/or intensity of the condition. 
x 
Conditions detected or diagnosed after IMP administration even though it may have been present 
prior to the start of the study. 
x 
Signs, symptoms, or the clinical sequelae of a suspected interaction. 
x 
Signs, symptoms, or the clinical sequelae of a suspected overdose of either IMP or a concurrent 
medication. 
SAEs are a sub-category of AEs that are defined in Section 10.2, below. AEs/SAEs are recorded and 
reported from the time of first dose of IMP until Follow-up (Visit 17). 
Planned hospital admissions for an illness or a disease that existed prior to administration of IMP or 
enrollment in the clinical study are generally not to be considered AEs. Individuals who have planned 
surgical procedures that would occur during the study are to be excluded from the study (Exclusion 43).  
If the participant has a planned hospital admission (non-surgical), the Investigator should consult with the 
Medical Monitor to ensure this will not cause disruption of IP administration or would not qualify the 
participant for exclusion.  
Note that medical or surgical procedures are not considered AEs (e.g., endoscopy); however, the 
condition that leads to the procedure is an AE. 
10.2 Definition of Serious Adverse Event 
An adverse event or suspected 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, in-patient hospitalization or prolongation of existing hospitalization, a persistent or significant 
incapacity or substantial disruption of the ability to conduct normal life functions, or a congenital 
anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they may 
jeopardize the participant and may require medical or surgical intervention to prevent the outcomes listed 
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All AEs occurring after administration of the dose of IMP and on or before the final visit must be 
reported. These are referred to as treatment emergent adverse events (TEAEs).  All AEs must be recorded 
irrespective of whether they are considered drug related. 
At each visit/assessment in the period defined above, AEs will be evaluated by the Investigator and 
recorded. 
Any AEs already documented at a previous assessment and designated as ongoing, should be reviewed at 
subsequent visits. If these have resolved, this should be documented. 
10.7 Recording of AEs and SAEs 
When an AE/SAE occurs, it is the responsibility of the Investigator to review all documentation  
(e.g., hospital progress notes, laboratory, and diagnostics reports) relevant to the event. The Investigator 
should then record all relevant information regarding an AE/SAE on the eCRF. It is not acceptable for the 
Investigator to send photocopies of the participant’s medical records to the Sponsor in lieu of completion 
of the appropriate AE/SAE eCRF pages. However, there may be instances when the Sponsor requests 
copies of medical records for certain cases. In this instance, all participant identifiers will be removed on 
copies of the medical records prior to submission to the Sponsor. 
The Investigator will attempt to establish a diagnosis of the event based on signs, symptoms, and/or other 
clinical information. In such cases, the diagnosis should be documented as the AE/SAE and not the 
individual signs/symptoms.  
10.8 Evaluating AEs and SAEs 
10.8.1 Assessment of Intensity 
The Investigator will make an assessment of intensity for each AE and SAE reported during the study.  
The assessment will be based on the Investigator’s clinical judgment. The intensity of each AE and SAE 
recorded in the eCRF should be assigned to one of the following three categories: 
Mild:   
An event that is easily tolerated by the participant, causing minimal discomfort and not interfering with 
everyday activities. 
Moderate:   
An event that is sufficiently discomforting to interfere with normal everyday activities. 
Severe:   
An event that prevents normal everyday activities. 
An AE that is assessed as severe should not be confused with an SAE. Severity is a category utilized for 
rating the intensity of an event; and both AEs and SAEs can be assessed as severe. An event is defined as 
‘serious’ when it meets one of the pre-defined outcomes as described in Section 10.2 Definition of 
Serious Adverse Event.  
10.8.2 Assessment of Causality 
The Investigator is obligated to assess the relationship between IMP and the occurrence of each AE/SAE. 
The Investigator will use clinical judgment to determine the relationship. Alternative causes such as 
natural history of the underlying diseases, concomitant therapy, other risk factors, and the temporal 


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relationship of the event to the IMP will be considered and investigated. The Investigator will also consult 
the Investigator’s Brochure in the determination of his/her assessment.   
All AEs will be assessed by the Principal Investigator or qualified and authorized delegate as to whether 
there is a ‘reasonable possibility that the drug caused the event’ and on that basis be classified as either 
‘related’ or ‘not related’. 
There may be situations when a SAE has occurred and the Investigator has minimal information to 
include in the initial report to the Sponsor. However, it is very important that the Investigator always 
make an assessment of causality for every event prior to transmission of the SAE form to the Sponsor.  
The Investigator may change his/her opinion of causality in light of follow-up information, amending the 
SAE form accordingly.   
10.8.3 Follow-up of AEs and SAEs 
After the initial AE/SAE report, the Investigator is required to proactively follow each participant and 
provide further information to the Sponsor on the participant’s condition. 
All AEs and SAEs documented at a previous visit/contact and are designated as ongoing, will be 
reviewed at subsequent visits/contacts. 
All AEs will be followed until resolution, until the condition stabilizes, or until 30 days after the 
participant’s Visit 17 Follow Up, whichever occurs first. At which time, the appropriate AE/SAE eCRF 
page(s) will be updated. SAEs and renal AEs that are ongoing at the Follow-up visit (Visit 17) will be 
followed until resolution, stabilization of participant condition, or study end, whichever occurs first. In 
such cases, follow-up Serious Adverse Event Report (SAER) 
 forms will 
be completed at the Follow-up visit (Visit 17) as well as completed upon resolution, stabilization, or study 
end, whichever occurs first. “Study end” is when all participants have completed the study. 
The Investigator will ensure that follow-up includes any supplemental investigations as may be indicated 
to elucidate the nature and/or causality of the AE or SAE. This may include additional laboratory tests or 
investigations, histopathological examinations, or consultation with other health care professionals. 
The Sponsor may request that the Investigator perform or arrange for the conduct of supplemental 
measurements and/or evaluations to elucidate as fully as possible the nature and/or causality of the AE or 
SAE.  The Investigator is obligated to assist. If a participant dies during the study or during a recognized 
follow-up period, the Sponsor will be provided with a copy of the death certificate and any post-mortem 
findings, including histopathology. 
New or updated information will be recorded on the originally completed SAE 
 
with all changes signed and dated by the Investigator.  The updated SAE form should be re-sent to 
the Sponsor within the time frames outlined below in Section 10.8 Prompt Reporting of the SAEs to the 
Sponsor.  
Any clinical adverse event that is serious (as defined above in Section 10.2) 
 
  occurring during the course 
of the study, irrespective of the treatment received by the participant, must be reported to the Sponsor and 
Sponsor’s agent within 24 hrs (or sooner if possible) of the Investigator or designee becoming aware of 
the situation. 








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Pharmacokinetic (PK) Population 
 
The PK population will include all participants enrolled in this study who receive the IMP through to the 
morning dose of Visit 5 (Week 2) as a minimum, and who undergo PK sample collection at the specified 
post-dose time point(s) through to Visit 5 as a minimum.  
 
11.1 Sample Size Determination 
Sample sizes were estimated for this open-label study using within-participant change as the measure of 
improvement. Given the limited data from interventional trials in these patient populations, and that this is 
the first clinical study for NNZ-2591 IMP in this population, treatment change estimates were based on 
Clinical Global Impression Scale of Improvement (CGI-I) data from completed clinical trials in other 
rare, neurodevelopmental disorders.  
Based on the information provided in Glaze et al. 2019 (Double-blind, randomized, placebo-controlled 
study of trofinetide in pediatric Rett syndrome), the standard deviation of the CGI-I is approximately 0.7 
at a single timepoint.1 For a sample size of 10 participants, a two-sided significance level of 0.05, and a 
Wilcoxon signed-rank test, the study will be able to detect a difference of 0.87 units of change in the CGI-
I at a power of 0.90. For a power of 0.80, 0.85, and 0.90, the detectable difference would be 0.75, 0.80, 
and 0.87 or larger, respectively. The effect size for a power of 0.80, 0.85, and 0.90 is 1.07, 1.14, and 1.24, 
respectively. If the observed standard deviation is larger, the detectable differences would also be larger. 
For example, if the standard deviation is 1.5, the detectable difference for a power of 0.80, 0.85, and 0.90 
is 1.61, 1.71, and 1.86 or larger, respectively. For a sample size of 20 participants, a two-sided 
significance level of 0.05, and a Wilcoxon signed-rank test, the study will be able to detect a difference of 
0.55 units of change in the CGI-I at a power of 0.90. For a power of 0.80, 0.85, and 0.90, the detectable 
difference would be 0.48, 0.51, and 0.55 or larger, respectively. 
11.2 Safety Analyses 
Safety analyses will be conducted on the Safety Population and based on the safety endpoints listed in 
Section 6.3.1. Adverse events and other safety data will be summarized as frequencies and percentages, 
described in terms of severity and potential attribution. Objective safety assessments will be summarized 
over time on study; shift tables at time points of interest may be generated. The safety analysis details will 
be specified in the Statistical Analysis Plan (SAP). 
11.3 Efficacy Analysis 
Efficacy will be assessed by looking at within-participant changes from the Baseline period to the EOT 
using the efficacy endpoints listed in Section 6.3.2. 
 
The participant’s baseline characteristics and symptom severity will be established based on assessments 
collected during the 4 to 6 week Screening/Baseline period. Baseline values will be based on the average 
values of visits 1, 2, and 3 for assessments collected at more than one visit. A Wilcoxon signed rank test 
will be used to test for improvement and an exact significance level will be computed.  
 
Potentially influential factors will be considered and addressed with a linear regression model. This model 
may include covariates such as age, sex, nonverbal NVIQ or NVIQ equivalent, genotype, history of 
regression (yes/no), ASD diagnosis (yes/no) with no more than two covariates included in any model. 
Descriptive statistics and corresponding graphics will be used to present the results. The safety and 
efficacy analysis details will be specified in the SAP. 
 


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11.4 Pharmacokinetic Analysis 
The PK analysis will be based on the endpoints listed in Section 6.3.1. Pharmacokinetic (PK) measures 
will be collected from all participants at Visits 5 (Week 2), 9 (Week 6), and 16 (Week 13, EOT).   
NNZ-2591 PK concentrations will be listed by participant and time point for each dose level with actual 
time they were acquired. The concentrations will be summarized by time, dose level, and age group. For 
the concentrations measured at trough points dose relationship will be examined statistically with age 
group and/or body weight of participant as covariate. 
Overall exposure parameters for NNZ-2591 such as Cmax and AUCtau may be derived by sparse non-
compartmental analysis (NCA). Additionally, PK concentrations of NNZ-2591 measured in pediatric 
participants will be used to refine population PK model developed from adult NNZ-2591 PK data and 
extrapolated to pediatric population. After refinement, complete profiles for each participant will be 
simulated using range of variability of model parameters and individual covariate effects. A standard 
NCA approach will be used for simulated profiles to calculate parameters Cmax, AUCtau, etc. for 
characterization of NNZ-2591 exposure in pediatric participants. 
The PK analysis details will be specified in the PK SAP. 
11.5 Biomarkers and Microbiome 
Blood and stool samples will be collected for an exploratory analysis of biomarkers and gut microbiome. 
The analysis will be detailed in a separate analysis plan but will not include DNA sequencing. Samples 
will be collected at Screening (Visit 1) and Baseline (Visit 3, before dosing) and at the EOT visit (Week 
13, Visit 16), or upon early termination. 
12. STUDY DRUG MANAGEMENT  
12.0 Identity of Investigational Medicinal Product 
NNZ-2591 Oral Solution, 50 mg/mL IMP is supplied in 250 mL white PET PP28 neck bottles with white 
28 mm child resistant and tamper evident caps. NNZ-2591 Oral Solution, 50 mg/mL IMP is manufactured 
under GMP by Quay Pharmaceuticals Ltd. (28 Parkway, Deeside Ind Park, Flintshire CH5 2NS). 
• 
Name of Drug: NNZ-2591 
• 
INN: None 
• 
Chemical Name: Cyclo-L-*O\F\Oဨဨ$OO\O 3UROLQH F*ဨဨ$OO\O3 
• 
Molecular formula: C10H14N2O2 (net) 
• 
Chemical Abstracts Service (CAS) Registration Number: 847952-38-9 
• 
Physical appearance of formulation: Clear, pink to red, non-viscous, free from visible particulates 
• 
Odor and flavor of formulation:  Sweet strawberry odor and flavor 
NNZ-2591 Oral Solution, 50 mg/mL IMP is provided as a ready-to-use strawberry-flavored liquid for oral 
administration. The IMP is stored at 15-25°C in white 250 mL PET PP28 neck bottles with white 28 mm 


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child resistant and tamper evident white caps. The primary and secondary label text and design complies 
with U.S. FDA regulatory requirements and local codes of good manufacturing practice for medicinal 
products, as applicable, and will include the batch number, the manufacturing date of the IMP and the 
retest date of the product. 
12.1 Management of Clinical Supplies 
Investigational Medicinal Product, along with the dispensing cups and syringes, will be supplied to the 
site Pharmacist. The site Pharmacy contact is responsible for IMP management including receipt and 
correct storage/handling, dose calculation and IMP preparation, IMP accountability, and proper 
destruction of unused IMP and empty bottles. Full details regarding the preparation, storage, resupply, 
and stability of NNZ-2591 Oral Solution, 50 mg/mL may be found in the Pharmacy Manual. 
NNZ-2591 Oral Solution, 50 mg/mL bottles will be shipped at 15-25°C and should be stored at 15-25°C 
(temperature monitored), according to the procedures outlined in the Pharmacy Manual. NNZ-2591 Oral 
Solution, 50 mg/mL must be maintained in a secure area with access restricted to designated study 
personnel. Investigational site personnel should notify the Study Monitor immediately if IMP bottles are 
damaged or if temperature excursions occur during storage. 
12.1.0 Drug Accountability 
The Investigator or designees must maintain an inventory record of the IMP administered to ensure that 
the investigational drug has been stored as per specifications and administered only to enrolled 
participants, in accordance with this protocol. Refer to the Pharmacy Manual for accountability 
procedures. 
The IMP is to be prescribed only by the site Principal Investigator or named sub-Investigators and may 
not be used for any purpose other than that outlined in this protocol.  
The Pharmacist and clinical team (as applicable) have been delegated responsibility from the Principal 
Investigator to maintain accurate records detailing the quantity of IMP kits received, dispensed, used, 
destroyed, or returned. During the study, the following must be noted in the Treatment Log: 
x 
The study number and initials of the participant to whom IMP is dispensed. 
x 
The date(s) and quantity of IMP dispensed. 
Study participants will be instructed to return empty dosing bottles and any unused medication to the 
clinic. These returns will be accounted for in the clinic and the bottles and accountability form will be 
provided to the Pharmacy for reconciliation with dispensing and IP tracking logs.  
At the end of the study, all unused IMP will be returned to the Sponsor’s designee or destroyed by the site 
according to the Sponsor’s instructions, once it has been inventoried and the Study Monitor has reviewed 
the drug accountability records.  
12.2 Overdose 
Participants should not exceed the maximum daily doses or take any two doses within an 8-hour interval. 
If an administered dose is spat out, regurgitated, or vomited, the dose should NOT be re-administered, and 
this should be documented in the dosing diary or the source documents for the in-clinic dose. There is no 
specific antidote to NNZ-2591 IMP.  In the event of an overdose, appropriate supportive measures should 
be employed as clinically indicated and the Medical Monitor must be informed immediately.  


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13. QUALITY ASSURANCE AND DATA MANAGEMENT 
13.1 Data Quality Assurance 
The following measures will be implemented to ensure accuracy, consistency, completeness, and 
reliability of data: 
x Investigator discussions 
x Site initiation training 
x Early site visits following enrollment 
x Routine site management 
x Ongoing site communication and training 
x Periodic site monitoring  
x Data management quality control checks 
x Statistical quality control checks 
x Continuous data acquisition and cleaning 
x Internal review of data 
x Quality control check of the final study report 
13.2 Case Report Forms 
This study will utilize web-based eCRFs developed through a 21CFR Part 11 compliant platform. Prior to 
the initiation of the trial, each site will be contacted as to computer availability, hardware specifications, 
security infrastructure, internet connectivity, etc., to evaluate their capacity to use this type of data capture 
system. The Investigator and site personnel will be responsible for completing the eCRFs or ensuring the 
completion of forms to be completed by the caregivers and entered into the EDC via the secure portal for 
caregiver raters. The Investigator is responsible for the accuracy and reliability of all the information 
recorded on the eCRFs. All information requested on the eCRFs needs to be supplied, including 
participant identification data, visit date(s), assessment values, etc., and any omission or discrepancy will 
require explanation. All information on eCRFs must be traceable to source documentation at the site. 
During periodic monitoring visits, the site will make their computer and/or high-speed Internet access 
available to the Study Monitor so that he or she may verify the data entries with the source 
documentation.  
13.3 Monitoring 
A Sponsor’s Study Monitor will visit each site in accordance with the Clinical Monitoring Plan set forth 
for this trial as well as maintain regular telephone and written communication. Monitoring visits will be 
conducted by representatives of the Sponsor according to the ICH Guideline for Good Clinical Practice 
(GCP), and applicable regional regulations and guidelines. 
The Investigator will agree to the monitors making periodic site visits during the study. The timing of 
these visits will be agreed by the monitors and the study site personnel.  


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Monitoring will be on an ongoing basis by site. The assessments are to be captured on a specialized 
device/computer (e.g., 12-lead ECG) or in an online format or paper format (e.g., clinician and caregiver 
rating scales) and are to be obtained at the time of testing. It is imperative that the Investigator ensures 
that all tests are completed at each session, according to the participant’s capability. Data collected at the 
site will be entered into the eCRFs in centralized, password protected EDC system. Checking for 
completeness and rationality will be undertaken via the EDC at a central location. If discrepancies are 
noted, the site monitor will be alerted and site follow-up undertaken in order that problems may be 
resolved immediately. This may require retraining of investigators on the testing process and the 
importance of competing the testing correctly.  
13.4 Auditing 
The Sponsor (or Sponsor nominee) may conduct periodic audits of the study processes including, but not 
limited to, the clinical site and the bioanalytical laboratory. The Investigator must authorize access and 
make available all study related documents to authorized Sponsor’s representatives and regulatory 
authorities. 
The scope of a clinical site audit may include but will not be limited to drug accountability, regulatory 
documentation, the informed consent process, and comparison of eCRFs against source data. The 
Investigator agrees to cooperate with audits conducted at a reasonable time and in a reasonable manner. 
Regulatory agencies involved with the study may also audit the Investigator during or after the study. The 
Investigator should contact the Sponsor immediately if this occurs. 
13.5 Source Documents 
The Investigator will, within reason, prepare and maintain adequate and accurate source documents (e.g., 
medical records, ECGs, raw data collection forms, and electronic source documents) to record all 
observations and other pertinent data for each participant enrolled into the study. The Investigator will 
allow Sponsor representatives and authorized regulatory authorities to access documents pertaining to the 
study. 
13.6 Protocol Deviations 
This study will be conducted as described in this protocol, except during an emergency situation in which 
the protection, safety and well-being of the participant requires immediate intervention, based on the 
judgment of the Investigator (or appropriately qualified designee). In the event of a significant deviation 
from the protocol, the Investigator or designee must contact the Sponsor or the Medical Monitor at the 
earliest possible time by telephone. This will allow an early joint decision regarding the participant’s 
continuation in the study. The Investigator will document the outcome of the discussion in the source 
documents and the Sponsor likewise will retain a documentary record of the decision. The Sponsor will 
reserve the right to recommend suspension or discontinued involvement of the site if excessive protocol 
deviations occur. 
13.7 Records Retention 
The Investigator must maintain essential study documents (protocol and amendments, completed eCRFs, 
source documentation, signed informed consent forms, relevant correspondence and approvals, and all 
other supporting documentation) until at least 2 years after the last approval of a marketing application in 
an ICH region and until there are no pending or contemplated marketing applications in an ICH region or 
at least 2 years have elapsed since the formal discontinuation of clinical development of the 


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Investigational Medicinal Product. These documents should be retained for a longer period if required by 
the applicable regulatory requirements or the clinical site’s procedures. Participant identification codes 
(participant names and corresponding study numbers) will be retained for this same time period and 
stored separately.  Custody of the records may be transferred to another responsible party, acceptable to 
the Sponsor, which agrees to abide by the retention policies. Written notice of transfer must be submitted 
to the Sponsor. The Investigator must contact the Sponsors prior to disposing of any study records. 
Records retention will follow the Data Protection requirements in the participating country.


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14. ETHICS 
14.1 Informed Consent 
Written informed consent will be obtained from a parent or legally authorized representative of the 
participant before any study-related procedures are performed. The Investigator has an ethical and legal 
responsibility to ensure the parent or legally authorized representative of each participant being 
considered for inclusion in the study is given a full explanation of the study. The Investigator or designee 
shall inform the parent or legally authorized representative of each participant in writing of all aspects 
pertaining to the participant’s participation in the study including (but not limited to) aims, methods, 
anticipated benefits, and potential risks. The participant’s parent or legally authorized representative will 
have the opportunity to inquire about details of the study and to decide whether to participate.  
A participant’s parent or legally authorized representative should understand that they are free to refuse to 
allow the participant to participate in, and are free to withdraw the participant from, the study at any time 
without prejudice or loss of medical care to which they are otherwise entitled. They have the ability to 
withdraw the participant from the study at any time, for any reason. 
The parent or legally authorized representative of each participant must personally sign and date a study-
specific informed consent form on behalf of the participant to be a participant in the study. The 
participant’s caregiver who will complete the study assessments must also provide written informed 
consent regarding their participation in the study prior to participating in any study procedures. The form 
must be countersigned by the site Investigator who conducted the informed consent discussion (or an 
authorized designee). Authorized designees will be defined according to local center and ethics 
requirements. Their authorization will be documented on a written informed consent form, which, 
together with this protocol, will be approved by an IRB.  
A separate consent for potential re-use of leftover Biomarker and PK samples in later research, including 
potential genetic research, is optional. 
Each informed consent will include the elements required by U.S. 21 CFR 50 and ICH E6, Section 4.8. 
The Investigator agrees to obtain approval from the IRB and Sponsor for any written informed consent 
form proposed for use in the study prior to submission to the IRB. 
Each parent or legally authorized representative who provides written informed consent for a participant 
to participate in the study (by signing and dating of the form) will be given a copy of the signed informed 
consent form, and assent form if the minor is capable of signing an assent form or providing oral consent 
as required by local IRB regulations. The originals will be kept in the center’s regulatory file and a second 
copy filed in the participant’s medical record as permitted by the institution. The Investigator will inform 
participants and the parent or legally authorized representative of new information that may be relevant to 
the participant’s willingness to continue participation in the study according to local ethics requirements. 
It is important to obtain complete follow-up for all participants enrolled. Every attempt should be made to 
undertake all protocol specified assessments and complete the eCRF except for those participants who 
specifically withdraw consent for release of such information. 
14.2 Institutional Review Board Approval 
The Investigator will not begin the study until the protocol and informed consent form have been 
approved by the local Institutional Review Board (IRB) or specifically for the site by the central IRB as 
dictated by institutional requirements. Any amendments to the protocol must also be approved in writing 


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by the Sponsor, and the local IRB or central IRB for the site prior to implementation by the Investigator, 
except where necessary to eliminate an immediate hazard to study participants. 
Written IRB approval of the protocol and Study Information Forms/Informed Consent Forms will be 
typed on the Institutional letterhead and will refer to the Study Information Forms/Informed Consent 
Forms and to the study by title and protocol number given by Neuren Pharmaceuticals on the protocol.  A 
copy of the signed and dated letter of approval will be provided to Neuren Pharmaceuticals prior to study 
commencement. Any written information and/or advertisements to be used for volunteer recruitment will 
be approved by the IRB prior to use. 
All IRB correspondence including progress reports will be retained on file at the clinical site.  
14.3 Additional Protections for Minors 
The age-related inclusion criterion for the study protocol will allow enrollment of participants aged 3 to 
17 years. Participants aged younger than 18 are considered to be minors and, collectively, they constitute 
a vulnerable population. Additional regulations are in place for their protection (45 CFR Part 46 Subpart 
D).  Normally, minors cannot legally provide consent to participation in research on their own behalf.  
Consent must be provided by one or both parents, or a legally authorized representative, while the minor 
provides “assent” for participation. Assent is the minor’s affirmative agreement to participate in the 
research.   
Since this protocol involves greater than minimal risk but presents the prospect of direct benefit to all 
participants enrolled, consent shall be obtained from a guardian or at least one parent in accordance with 
local IRB requirements. Minors will be given the opportunity to assent to participation if and when they 
are considered capable of doing so by the Investigator and per local IRB requirements. 
 
 


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15. STUDY MANAGEMENT 
The Sponsor will appoint the project management team, and an independent DSMC. Project management 
may be delegated to a Contract Research Organization who will be overseen by the Sponsor. 
15.1 Principal Investigators 
A Principal Investigator will be appointed at each participating site to personally supervise all aspects of 
the conduct of the study at their site.  
15.2 Data Safety Monitoring Committee 
An independent DSMC will monitor the progress of the trial and ensure that the safety of trial participants 
is not compromised. The DSMC will provide independent affirmation that no safety or tolerability issues 
are arising in the study through their on-going review of the study data. Specifically, the DSMC will 
review the clinical safety data prior to any individual dose titration and prior to initiation of subsequent 
age groups. The DSMC will review safety data and make an adjudication on: 1) the ability for any 
individual participant to escalate to the next higher dose and 2) the ability for enrollment to begin in the 
younger age groups (Groups 2 and 3). 
The DSMC will minimally consist of a clinical chair, other physicians experienced in clinical trials with 
pediatric expertise (but not participating in this study), and a pediatric cardiologist. The DSMC Charter 
details the DSMC processes, duties, and responsibilities as well as the study-wide stopping criteria. 
15.3 Publications 
All publication rights are delineated in the Clinical Study Agreement and/or other separate agreements 
with the Investigator and/or Institution, as applicable. ICMJE uniform requirements will be respected, 
with regard to the composition, authorship and submission of manuscripts derived from this study. A 
publication committee will be composed that will include the principal investigators (or appropriate 
designees) as well as Sponsor representatives.  
 
 
 
 


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Appendix A. 
Allometric scaling to determine phase 2 exposures 
 
To determine the appropriate dose for pediatric participants, the Sponsor performed allometric scaling as 
discussed with the FDA in the Pre-IND Meeting. The findings from the allometric scaling analysis are 
summarized below. 
The allometric scaling analysis was performed using the pharmacokinetic data from the Phase 1 study of 
NNZ-2591 in healthy adults (PK Report Neu-2591-HV-001). The purpose of the allometric scaling is to 
predict exposure in younger and lighter participants based on observed exposures in adult healthy 
volunteers at steady-state from the completed clinical study (Neu-2591-HV-001) for the dose of 12 mg/kg 
(cohort M2). 
1. Methods for the Allometric Scaling 
Steady-state PK parameters were used in the modeling. Median and percentiles were estimated for steady-
state clearance CLss/F on Day 7 morning dose for the adult participants in the Neu-2591-HV-001 study to 
be used as input for allometric scaling. Median and 5th and 95th percentiles for the AUCtau at steady-state 
for the adult participants were used as a target exposure in children for comparison of the results.  
Fi value was already accounted for in the apparent steady state clearance CLss/F. The WTcentered value for 
the study was median body weight for the participants in M1 and M2 cohorts of the study Neu-2591-HV-
001 was equal to 73 kg.  
Median CLss/F derived for steady-state exposure for Day 7 morning dose was used as CLTV. Since the 
CLss/F was derived for body weight adjusted doses and hence was estimated as L/hr/kg for the 
simulations, the value was multiplied by the median weight of 73 kg to represent true value for the 
population of adult participants in the study Neu-2591-HV-001.  
For simulation of exposure for children 3-17 (the planned age group for the Phase 2 study), the Centers 
for Disease Control and Prevention (CDC) Growth Individual charts with 3rd and 97th percentiles (3rd, 
10th, 25th, 50th, 75th, 90th, and 97th) were downloaded from the CDC.gov website. The body weights 
for boys and girls from 36.5 months (3 years) to 17 years old (203.5 months) inclusive were combined for 
the input to Phoenix WinNonLin as a csv file. The file contained columns for 3rd, 5th, 10th, 25th, 50th, 
75th, 90th, 95th, and 97th percentiles for each month of age and gender individually. Column with gender 
was added for the combined dataset. Age groupings for the allometric scaling analysis were Group 1 (3 to 
< 6), Group 2 (6 to <12), and 12-17. 
The equation for allometric scaling was implemented in the Data Wizard function of Phoenix WinNonLin 
using weight for children of different age, gender, and percentile as direct input.  
Doses were varied in the range from 11 to 20 mg/kg for final estimate with 1 mg/kg increments. The dose 
levels 11, 12, 13, 14, … 20 mg/kg were used to calculate the related exposures for different body weight 
values and compare the resulting AUCtau values with adult results.  
Additionally, a 17.5-mg/kg dose was evaluated for the lowest body weights. Preliminary evaluation of 
suitable doses was done starting from 3 mg/kg dose levels. The parameters derived for this allometric 
scaling were AUCtau at steady state to compare with adult exposures. 
 


Neuren Pharmaceuticals Ltd.
Clinical Trial Protocol
Version 3.0
Confidential
Neu-2591-PTHS-001
October 30, 2023
Page 110 of 112
2. Allometric Scaling Results
Non-compartmental parameters for NNZ-2591 in adult participants from the study Neu-2591-HV-001 
were evaluated for dose proportionality of exposure after single, first doses and multiple steady-state 
doses of NNZ-2591 in healthy adult participants. Evaluation of dose proportionality supported the 
application of the published allometric scaling formula to steady state exposure parameter AUCtau to be 
matched to adult exposures using body weight normalized dose in mg/kg and apparent steady state 
clearance in healthy adult participants. The predicted exposures (steady-state AUCtau) were calculated 
from adult median CLss/F, estimated for morning and evening 12 mg/kg doses on Day 7 in the study 
Neu-2591-HV-001, for different dose values and body weights from CDC Growth Chart. 
The findings showed that overall the predicted pediatric exposures were lower than adult exposures for 
the 12mg/kg dose for comparisons of exposure for different dose values versus weight. Combining the 
dose into 3 age groups resulted in significant overlap of body weight and differences in the dose projected 
to match adult exposure for 12 mg/kg dose within the group. Hence, body weight-based dosing instead of 
age-related dosing was originally proposed for the dose selection for children. The combined plots of 
AUCtau vs weight are as shown in Figure A1. 
Figure A2 Predicted AUCtau of NNZ-2591 for different dose values vs weight all ages combined
Source: Figure 5 in Report Allometric scaling for PK of NNZ-2591 for Pediatric Population with PMS, AS, and 
PTHS 




Neuren Pharmaceuticals Ltd. 
Clinical Trial Protocol 
Version 3.0 
Confidential 
Neu-2591-PTHS-001 
October 30, 2023 
 
 
 
Page 112 of 112 
a Source: Neu-2591-HV-001 study adpc dataset, Phoenix 8.3 output for Project file Allometric 
Neuren.phxproj; CDC Growth chart reported in Figure 5 in the Allometric Scaling Report. 
b Report 1901515 and Report SOW6, 0621-20173NB_w_in-life_Bioanalytical and Pharmacokinetic 
Report_Mouse Whole Blood_NNZ-2591_27Aug2020 
This more conservative approach, which maintains the planned staggered enrollment of age groups, 
ensures that each nominal dose is administered to the older group before that dose is administered in the 
younger pediatric participants. As such the younger age groups receive only nominal doses that have been 
administered to the older group instead of exposure-equivalent weight-based doses at higher nominal 
doses. Given that it was estimated from allometric scaling that exposures would be lower for children < 
70 kg at 12 mg/kg than for adults at 12 mg/kg, dosing all children at a maximum of 12 mg/kg will not put 
them at risk of exposures higher than that experienced by healthy adults. So, this approach to the dosing 
maintains the balance between risk and benefit for this first Phase 2 study in the Angelman syndrome 
population.  
 

SAP_003.pdf: 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
2 
 
This study will be conducted in compliance with the protocol, Good Clinical Practice 
and all other applicable regulatory requirements, including the archiving of 
essential documents. 
Confidential Information 
No use or disclosure outside Neuren Pharmaceuticals Ltd. is permitted without prior 
written authorization from Neuren Pharmaceuticals Ltd






Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
5 
 
1 Introduction 
This document describes additions and changes to the Final Version 1.0 of the Statistical 
Analysis Plan (SAP) for Protocol NEU-2591-PTHS-001. The section numbers following 
the introduction in this document correspond to those updated from the SAP Final 
Version 1.0; therefore, they are not sequential.  The reader is referred to SAP Final 
Version 1.0 for analysis descriptions for sections that are not included in this document. 
 
 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
6 
 
5.3 Definitions 
භ Number of missed doses will be derived based on the number of expected doses – 
the number of doses taken. If a subject took more doses than expected, the 
number of missed doses will be taken to be 0.  
The reader is referred to Section 5.3 of the SAP Final Version 1.0 for other analysis 
definitions. 
 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
7 
 
9.2.10 Behavior Problems Inventory-Short Form  
The BPI-SF will be completed at Visit 3 (Week 0) and at the End of Treatment (Week 13, 
Visit 16). The BPI-SF is a caregiver-completed rating scale for assessing self-injury, 
aggressive and stereotyped behavior. Each item is measured for frequency and items in 
the Self-Injurious Behavior and Aggressive/Destructive Behavior subscales are also 
assessed for severity. The frequency is scored on a 5-point scale, where 0=never/no 
problem; 1=monthly, 2=weekly, 3=daily, 4=hourly. The severity is scored on a 3-point 
scale, where 1=mild, 2=moderate, 3=severe. The items are grouped in three subscales: 
Self-Injurious Behavior (score ranges from 0 – 32 for frequency and 8 – 24 for severity), 
Aggressive/Destructive Behavior (score ranges from 0 – 40 for frequency and 10 – 30 for 
severity), and Stereotyped Behavior (score ranges from 0 – 48 for frequency). The score 
for each subscale is the sum of the items within that domain. The total frequency score is 
the sum of the frequency scores for the three domains. The total severity score is the sum 
of the severity scores for Aggressive/Destructive Behavior and Self-Injurious Behavior 
subscales. The total severity and frequency scores will only be calculated if all 
contributing domain scores are not missing. Missing items will be imputed as defined in 
Section 5.2.5. A subscale score will only be calculated if all items in that domain are 
answered or can be imputed. Lower scores indicate lesser frequency and severity in 
behavior problems. Within subject change is the difference between the score at EOT and 
the score at baseline. 
Summary statistics for the total score, subscale scores and their changes from baseline in 
the frequency and severity of BPI-SF scores will be displayed at End of Treatment. 
Within subject changes will be analyzed using a Wilcoxon signed rank test. The change 
from baseline in the total frequency and severity scores at EOT will be presented in plots 
as described in Section 9.2. The change from baseline in the subscale scores at EOT will 
be presented in a forest plot and waterfall plots. The subscale and total scores will be 
presented in line graphs at each visit. 
 
 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
8 
 
9.2.12 Gastrointestinal Health Questionnaire Scores 
The GIHQ will be completed at all Screening/Baseline period visits (Visits 1, 2, 3), as 
well as Week 6 (Visit 9), End of Treatment (Week 13, Visit 16), and Follow-up (Visit 
17).  Each item is scored on its frequency, relevancy, and importance to the caregiver. 
The Frequency is measured on a 5-point scale where 0=never; 1=almost never; 
2=sometimes; 3=often; 4=almost always. The surgery domain records occurrence as 
0=No and 1=Yes. The relevancy is scored on a scale from 1 to 4, where 1 is not relevant 
and 4 is very relevant. The importance is scored on a scale from 1 to 4, where 1 is not 
important and 4 is very important. Lower frequency scores indicate fewer gastrointestinal 
problems. For relevancy, higher scores indicate the question is more relevant and for 
importance, higher scores indicate the question is more important. The total frequency 
score is the sum of all item frequency scores. Similarly, the total relevancy and total 
importance scores are the sum of all item relevancy and importance scores, respectively. 
The items are grouped into 9 subscales. The subscale score is the sum of all items within 
the subscale, as shown on the CRF. Missing items will be imputed as defined in Section 
5.2.5. The subscale scores will only be calculated if all items within the domain are 
answered or can be imputed. The subscales are as follows: 
x General Health/Pain: Items 1 – 5, frequency score ranges from 0 – 20, relevancy 
score ranges from 5 – 20, importance score ranges from 5 – 20. 
x Eating, Chewing, and Swallowing: Items 1 – 9, frequency score ranges from 0 – 
36, relevancy score ranges from 9 – 36, importance score ranges from 9 – 36. 
x Reflux: Items 1 – 3, frequency score ranges from 0 – 12, relevancy score ranges 
from 3 – 12, importance score ranges from 3 – 12. 
x Gas and Bloating: Items 1 – 5, frequency score ranges from 0 – 20, relevancy 
score ranges from 5 – 20, importance score ranges from 5 – 20. 
x Diarrhea and Constipation: Items 1 – 6, frequency score ranges from 0 – 24, 
relevancy score ranges from 6 – 24, importance score ranges from 6 – 24. 
x Personality and Mood: Items 1 – 5, frequency score ranges from 0 – 20, 
Relevancy score ranges from 5 – 20, importance score ranges from 5 – 20. 
x Medications: Items 1 – 9, frequency score ranges from 0 – 36, relevancy score 
ranges from 9 – 36, importance score ranges from 9 – 36. 
x Surgery: Items 1 – 5, frequency score ranges from 0 – 5, relevancy score ranges 
from 5 – 20, importance score ranges from 5 – 20. 
x Parenting: Items 1 – 8, frequency score ranges from 0 – 24, relevancy score 
ranges from 8 – 24, importance score ranges from 8 – 24. 
Within subject change is the difference between the score at each relevant follow-up visit 
and the score at baseline. Summary statistics for the change from baseline in the total and 
each GIHQ domain score will be displayed at all post-baseline visits. Test-retest 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
9 
 
reliability will be assessed for all scores collected during the Screening period (Visits 1, 
2, and 3, pre-dosing) using ICC. Changes from baseline will be analyzed using a 
Wilcoxon signed rank test at Week 6 and EOT. The change from baseline in the total 
frequency score at EOT will be presented in plots as described in Section 9.2. The change 
from baseline in the domain frequency scores at EOT will be presented in a forest plot 
(with the exception of the Surgery domain) and waterfall plots. The domain and total 
frequency scores will be presented in line graphs at each visit, with the exception of the 
Surgery domain, which is scored differently from the other domains and is not 
comparable. 
 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
10 
 
9.2.16 Bayley Scales of Infant and Toddler Development version 4 
The BSID-4 will be performed at Screening (Visit 1) and End of Treatment (Week 13, 
Visit 16) on those subjects who cannot be assessed using the SB5. It assesses cognitive 
ability, motor skills, and behavior and provides standardized scores in five domains: 
Cognitive (COG), Language (LANG), Motor (MOT), Social-Emotional (SOEM), and 
Adaptive Behavior. The Adaptive Behavior scale was not collected for this study. 
Domain scores range from 40 – 160, where higher scores indicate greater ability in the 
domain area of development. Scaled scores, age equivalents and growth scale values 
(GSVs) can also be calculated for the scale subtests: cognition (CG), receptive 
communication (RC), expressive communication (EC), fine motor (FM), and gross motor 
(GM). Scaled scores and age equivalents can be calculated for the social emotional (SE) 
subtest. Scale subtest scores range from 1 – 19 with a mean of 10 and SD of 3. Age 
equivalent scores range from < 0:15 – >41:08, Growth scale scores range from 414   – 
559 with a mean of 500 and SD of 25. Higher scores indicate greater development in the 
scale subtest. Raw scores, age equivalent scores and GSV are collected for all subjects. 
Standard scores and scaled scores are only calculated for participants 42 months and 
younger.  
As part of the analysis, an overall developmental quotient, a verbal developmental 
quotient, and a non-verbal developmental quotient will also be calculated. These 
developmental quotients will only be calculated if the contributing scale scores are all 
non-missing. The developmental quotients will be derived as follows: 
Overall Developmental Quotient: 
(Mean of the age-equivalent scores in months of the cognition, fine motor, receptive 
communication, expressive communications scales/chronological age in months) x 100 
Verbal Developmental Quotient: 
(Mean of the age-equivalent scores in months of the expressive communication and 
receptive scales/chronological age in months) x 100 
 
 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
11 
 
Non-verbal Developmental Quotient: 
(Mean of the age-equivalent scores in months of the cognition and fine motor 
scales/chronological age in months) x 100 
Subtest/Subdomain Developmental Quotients: 
The developmental quotients for the BSID subtests will be derived. The developmental 
quotients for each subtest are calculated in a similar manner as the overall, non-verbal, 
and verbal developmental quotients. Subtest Developmental Quotient = (Age Equivalent 
Score in Months for the subtest / chronological age in months) x 100.  
Chronological age in months will be calculated as (date of assessment – date of birth + 
1)/30.4375. 
For age equivalent values < 2, the result is entered into EDC as 1.9, for analysis. For age 
equivalent values > X, where X is a numeric value, the result is entered into EDC as X.1, 
for analysis. For example, “>33” is entered in EDC as 33.1. The age equivalent score 
entered into EDC will be converted to months for analysis according to the following 
algorithm: 
x (a) Count the numeric portion of the result before the decimal as months. 
x (b) Count (the numeric portion of the result after the decimal multiplied by 10) as 
days and convert to months by dividing by 30.4375 
x Age equivalent score (months) = (a) + (b) 
The standard scores have a mean of 100 and a standard deviation of 15. The 
developmental quotient scores are a ratio of developmental age to chronological age and 
will have a range of 1 to 100. Higher scores indicate a higher IQ or better developmental 
progress. If any scores are missing, the subject will be excluded from corresponding 
summaries. 
Within subject change is the difference between the score at EOT and the score at 
baseline. If a score is missing, that subject will be excluded from the corresponding 
summaries. 
Summary statistics for the observed and change from baseline in the age equivalent, 
growth scale subtest scores, the standard and scale scores (for participants for whom the 
scores are calculated), and developmental quotients will be displayed at end of treatment. 
Changes from baseline will be analyzed using a Wilcoxon signed rank test. The change 
from baseline in the raw scores and GSV at EOT will be presented in plots as described 
in Section 9.2. The change from baseline in the raw and GSV domain scores will be 
presented in a forest plot, as described in Section 9.2. 
 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
12 
 
9.2.18 Stanford-Binet Intelligence Scales version 5 
The SB5 will be performed at Screening and Visit 16 (End of Treatment). Participants 
who cannot be assessed on the SB5 due to their developmental level will be assessed 
using the BSID-4 instead. General cognitive ability is reported as a full scale intellectual 
quotient score (FSIQ). Cognitive ability in five categories is reported in the index scores: 
fluid reasoning (FR), knowledge (KN), quantitative reasoning (QR), visual-spatial 
processing (VS), and working memory (WM). Non-verbal (NVIQ) and verbal 
intelligence quotients (VIQ) are also recorded. Scores range from 40 to 160, where higher 
scores indicate a higher IQ. For the analysis of index scores and IQ, the z-deviation 
method for intellectual disability will be used and z-deviation scores will be derived 
based on age at assessment according to the published algorithm (Sansone et al. 2014). 
Standard scores, change-sensitive scores, age equivalents and the raw scores are 
calculated and entered by the site rater.  
For age equivalent values < 2, the result is entered into EDC as 1.9, for analysis. For age 
equivalent values > x, where X is a numeric value, the result is entered into EDC as X.1, 
for analysis. For example, “>33” is entered in EDC as 33.1. 
Summary statistics on the observed and change from baseline in the z-deviation scores, 
change-sensitive scores and age equivalents for FSIQ, NVIQ, VIQ. Each category will be 
displayed at end of treatment. The change from baseline in FSIQ, NVIQ, and VIQ z-
deviation scores at EOT will be presented in plots as described in Section 9.2. The 
change from baseline in the domain z-deviation scores will be presented in a forest plot, 
as described in Section 9.2. 
 
 


Neuren Pharmaceuticals Ltd.  
Addendum to the Final Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
17MAY2024 
 
13 
 
9.3.2 Correlation of Efficacy Endpoints 
The potential covariates in this study are listed in Section 8.2. A 2x2 correlation analysis 
will be performed on all combinations to evaluate the relationship between the covariates. 
The Pearson Correlation Coefficient will be used to measure the association between age 
as a continuous variable and the NVIQ/DQ score as described in Section 9.2. If the 
coefficient is <.5, we will assume little to no association. The Point-Biserial Correlation 
Coefficient will also be used to measure the association between combinations of 
continuous and binary variables such as NVIQ/DQ and ASD diagnosis, respectively. The 
binary variables will be assigned a numeric value of 1 or 2. The Pearson Chi-Square test 
will be used to measure the association between combinations of categorical variables 
such as age group and genotype or sex and history of regression. A p-value < 0.10 would 
indicate association between the variables.  
The correlation analysis may inform the selection of covariates (or combinations thereof) 
to be used in a linear regression analysis on all pre-specified endpoints. Endpoints of 
interest for the linear regression analysis will be: 
x CGI-I Overall Score 
x Caregiver Impression of Change Overall Score 
x CGI-S Overall Score  
x ORCA t-score 
x BPI-SF Total frequency and severity scores 
x ABC-2 Total Score 
x CSHQ Total Score 
x GIHQ Total frequency, relevancy, and importance Scores 
x Vineland ABC score 
x Vineland GSV, raw, and standard domain scores 
x QI-Disability Overall Score 
x ICND Score  
A multiple linear regression model will be determined using the forward selection 
method where covariates meeting the entry criterion (p-value <0.2) enter into the model.  
The final model will include all parameters that have successfully been entered into the 
model. 
 
 




 
1 
 
Statistical Analysis Plan 
 
Protocol Title 
An Open-Label Study of the Safety, Tolerability, and 
Pharmacokinetics of Oral NNZ-2591 in Pitt Hopkins 
Syndrome 
Protocol Number: 
NEU-2591-PTHS-001 
Protocol Version: 
3.0 
SAP Version: 
1.0 
Date:  
30APR2024 
Sponsor:  
Neuren Pharmaceuticals Ltd. 
Suite 201, 697 Burke Road 
Camberwell, VIC 3124 
Australia 
 
Author: 
 
 
 
 
 
 
 
 
 
 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
2 
 
This study will be conducted in compliance with the protocol, Good Clinical Practice 
and all other applicable regulatory requirements, including the archiving of 
essential documents. 
Confidential Information 
No use or disclosure outside Neuren Pharmaceuticals Ltd. is permitted without prior 
written authorization from Neuren Pharmaceuticals Ltd


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
 
3 
 
 
Signatures / Approvals 
 
 
 
 
 
 
Chief Science Officer 
 
 
Name (Print or Type) 
Date 
 
 
 
 
Chief Medical Officer 
 
 
Name (Print or Type) 
Date 
 
 
 
 
Vice President, Clinical Development 
 
 
Name (Print or Type) 
Date 
 
 
 
 
Biostatistician  
. 
Name (Print or Type) 
Date 
 
 
 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
4 
 
Table of Contents 
1 
List of Abbreviations ................................................................................................... 6 
2 
Introduction ................................................................................................................. 9 
2.2.1 
Study Treatment .................................................................................. 22 
3 
Analysis Populations ................................................................................................. 23 
4 
Data Monitoring ........................................................................................................ 23 
5 
Statistical Methods .................................................................................................... 23 
5.2.1 
Imputation of Missing/Incomplete Dates............................................ 25 
5.2.2 
Imputation of Alphanumeric Data ...................................................... 26 
5.2.3 
Visit Windows .................................................................................... 26 
5.2.4 
Baseline Values ................................................................................... 27 
5.2.5 
Post-Baseline Values .......................................................................... 27 
6 
Subject Information ................................................................................................... 30 
7 
Safety Analysis .......................................................................................................... 33 
8 
Efficacy Analysis ....................................................................................................... 38 
9 
Efficacy Analysis Methods ........................................................................................ 40 
9.2.1 
Clinical Global Impression Scale - Overall Improvement Score ........ 41 
9.2.2 
Clinical Global Impression Scale - Domain Improvement Scores ..... 41 
9.2.3 
Caregiver Impression of Change Score .............................................. 42 
9.2.4 
Clinical Global Impression Scale - Severity - Overall Severity Score 43 
9.2.5 
Clinical Global Impression Scale - Severity - Domain Severity Scores
 
43 
9.2.6 
MacArthur-Bates Communicative Development Inventory Scores ... 44 
9.2.7 
Observer-Reported Communication Ability Score ............................. 45 
9.2.8 
Caregiver Top 3 Concerns Rating....................................................... 45 
9.2.9 
Aberrant Behavior Checklist-2 Scores ................................................ 46 
9.2.10 Behavior Problems Inventory-Short Form.......................................... 47 
9.2.11 Child Sleep Habits Questionnaire Scores ........................................... 48 
9.2.12 Gastrointestinal Health Questionnaire Scores .................................... 49 
9.2.13 Vineland Adaptive Behavior Scales-3 (VABS-3) .............................. 50 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
5 
 
9.2.14 Quality of Life Inventory - Disability (QI-Disability) Scores ............ 51 
9.2.15 Impact of Childhood Neurological Disability Overall quality of life 
rating 51 
9.2.16 Bayley Scales of Infant and Toddler Development version 4 ............ 52 
9.2.17 Modified two-minute walk test ........................................................... 53 
9.2.18 Stanford-Binet Intelligence Scales version 5 ...................................... 54 
9.3.1 
Biomarkers and gut microbiome ......................................................... 55 
9.3.2 
Correlation of Efficacy Endpoints ...................................................... 55 
9.3.3 
Additional exploratory analyses ......................................................... 56 
10 Tables, Listings, and Figures ..................................................................................... 56 
11 References ................................................................................................................. 57 
 
 
 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
6 
 
1 List of Abbreviations 
ABC-2  
 
Aberrant Behavior Checklist-2 
AE 
 
 
Adverse Event 
AMSE  
 
Autism Mental Status Exam 
APTT  
 
Activated Partial Thromboplastin Time 
ASD  
 
Autism Spectrum Disorder 
ATC  
 
Anatomical/Therapeutic/Chemical 
AUC  
 
Area under the plasma concentration-time curve 
BID 
 
 
Twice-a-day 
BPI-SF 
 
Behavior Problems Inventory - Short Form 
CFR   
 
Code of Federal Regulations 
CGI-I  
 
Clinical Global Impression - Improvement 
CGI-S  
 
Clinical Global Impression - Severity 
CRF 
 
 
Case Report Form 
CRO  
 
Contract Research Organization 
CSHQ  
 
Child Sleep Habits Questionnaire 
CSR 
 
 
Clinical Study Report 
COVID-19 
 
Coronavirus-19 
DSM5  
 
Diagnostic and Statistical Manual of Mental Disorders 
DSMC  
 
Data Safety Monitoring Committee 
ECG   
 
Electrocardiogram 
EDC  
 
Electronic Data Capture 
EGFR  
 
Estimated Glomerular Filtration Rate 
EOT 
 
 
End of Treatment 
ET 
 
 
Early Termination 
FDA  
 
Food and Drug Administration 
GCP  
 
Good Clinical Practice 
GIHQ  
 
Gastrointestinal Health Questionnaire 
GLP 
 
 
Good Laboratory Practice 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
7 
 
GMP  
 
Good Medical Practice 
IB 
 
 
Investigator’s Brochure 
ICF  
 
 
Informed Consent Form 
ICH 
 
 
International Conference on Harmonization 
ICND  
 
Impact of Childhood Neurological Disability 
IEC 
 
 
Independent Ethics Committee 
IMP 
 
 
Investigational Medicinal Product 
INR 
 
 
International Normalized Ratio 
IRB 
 
 
Institutional Review Board 
ITT 
 
 
Intent-to-Treat Population 
MB-CDI 
 
MacArthur-Bates Communicative Development Inventory 
MedDRA 
 
Medical Dictionary of Regulatory Affairs 
NVDQ  
 
BSID-4 nonverbal development quotient 
NVIQ  
 
SB5 nonverbal z-deviation IQ 
PT 
 
 
Prothrombin Time 
QI-Disability  
Quality of Life Inventory - Disability 
SAE 
 
 
Serious Adverse Event 
SAF 
 
 
Safety Population 
SAP 
 
 
Statistical Analysis Plan 
SB5 
 
 
Stanford-Binet Intelligence Scales version 5 
SD 
 
 
Standard Deviation 
SOC  
 
System Organ Class 
SOP 
 
 
Standard Operating Procedure 
ORCA  
 
Observer-Reported Communication Assessment 
PK 
 
 
Pharmacokinetics 
PMS  
 
Phelan McDermid Syndrome 
PMS-DSRS 
 
PMS Clinician Domain Specific Rating Scale 
PP 
 
 
Per-Protocol Population 
TEAE  
 
Treatment-Emergent Adverse Event 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
8 
 
TFLs  
 
Tables, Figures, and Listings 
TSH 
 
 
Thyroid-Stimulating Hormone 
UR 
 
 
Urine 
WHO  
 
World Health Organization 
 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
9 
 
2 Introduction 
Neuren Pharmaceuticals Ltd. is conducting a study to investigate a new treatment for Pitt-
Hopkins Syndrome (PTHS). The study background, design and subject assessments for 
the study are described in the study specific protocol.  
The statistical methods to be implemented during the analyses of data collected within 
the scope of this study will be outlined in this document. The purpose of this plan is to 
provide specific guidelines from which the statistical analysis will proceed. This 
statistical analysis plan (SAP) is to be interpreted in conjunction with the protocol. 
Should the SAP and protocol be inconsistent with respect to the planned analyses, the 
language of the SAP is governing. Any deviations from this plan will be documented in 
the clinical study report (CSR). 
The statistical principles applied in the design and planned analyses of this study are 
consistent with the International Conference on Harmonisation (ICH) guidelines E9 
(Statistical Principles for Clinical Trials). 
2.1 Study Objectives 
The primary objective of this study is to investigate the safety, tolerability, and 
pharmacokinetics of treatment with NNZ-2591 Oral Solution, 50mg/mL Investigational 
Medicinal Product (IMP) in children and adolescents with Pitt-Hopkins Syndrome.  
The secondary objective of this study is to investigate measures of efficacy during 
treatment with NNZ-2591 Oral Solution, 50mg/mL IMP in children and adolescents with 
Pitt-Hopkins Syndrome. 
2.2 Study Design 
This is a Phase 2, open-label study of the safety and tolerability of NNZ-2591 Oral 
Solution, 50mg/mL IMP in male and female children and adolescents with Pitt-Hopkins 
syndrome. Approximately 10-20 male and female subjects between the ages of 3 and 17 
years were planned to be enrolled and receive treatment of NNZ-2591 Oral Solution, 
50mg/mL IMP for a total of 13 weeks.  
There will be a total of 17 study visits, comprising 5 in-clinic visits and 12 remote 
telemedicine/in-home nurse visits. The study will commence with an approximately 4-6 
week Screening and Baseline period. During this Screening/Baseline period, subjects are 
assessed for study eligibility and data is collected to establish the subject’s baseline 
characteristics and symptom severity using a variety of assessments. Two in-clinic visits 
and one remote/in-home visit (Visits 1, 2, 3) comprise Screening and Baseline.  


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Protocol: NEU-2591-PTHS-001 
 
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Once eligibility is confirmed, subjects are dosed with the starting dose of 4 mg/kg and 
then be up-titrated to the target dose. Subjects receive IMP for a total of 13 weeks. 
During the treatment period, there are three in-clinic visits: Week 2 (Visit 5), Week 6 
(Visit 9) and Week 13 (Visit 16). There is a combined telemedicine/in-home visit by the 
site Investigator and an in-home visit by a visiting nurse for safety at Week 1 (Visit 4), 
Week 4 (Visit 7), Week 8 (Visit 11) and Week 10 (Visit 13). Ideally, these combination 
telemedicine/in-home visits will be conducted simultaneously whenever possible, or they 
will be conducted on the same day with the telemedicine visit occurring first. If this is not 
feasible the in-home and telemedicine components can be conducted over two 
consecutive days. In-home visits with a visiting nurse will be done at Weeks 3 (Visit 6), 5 
(Visit 8), 7 (Visit 10), 9 (Visit 12), 11 (Visit 14), and 12 (Visit 15).  
All subjects will also have a combined telemedicine/in-home follow-up visit with the 
Investigator via telemedicine and an in-person home visit by a visiting nurse 
approximately 2 weeks after the end of treatment (Week 15, Visit 17). 
For all visits designated as in-clinic, an in-person visit is preferred. Off-site assessments 
may be allowed due to extenuating circumstances due to the COVID-19 health 
emergency. All requests for off-site assessments must be approved in advance by the 
Sponsor or Medical Monitor. A combined remote visit or nurse-only in-home visit may 
be conducted in-clinic by the study staff if agreed upon by the Investigator and the 
caregiver. 
Subjects will be divided into three groups by age for enrollment: 
• 13 to 17 years old (Group 1) 
• 8 to 12 years old (Group 2) 
• 3 to 7 years old (Group 3) 
Enrollment commenced with the oldest age group and proceeded as follows. After at least 
three subjects in Group 1 have received two weeks of treatment at the starting dose, the 
Data Safety Monitoring Committee (DSMC) will review data on safety and tolerability. 
If tolerability and safety for those subjects during the specified period is deemed 
acceptable, enrollment for Group 2 will proceed and dosing will begin at the starting 
dose. When at least three subjects in Group 2 have received the first two weeks of 
treatment at the starting dose, the DSMC will review data on safety and tolerability. If 
tolerability and safety for Group 2 is deemed acceptable, enrollment for Group 3 will 
commence and dosing will begin at the starting dose. 
A summary of the schedule of events and assessments is provided in Table 1 below. 


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Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
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The table of Events and Assessments is from version 2.2 which included ophthalmic 
exam at week 2 and week 6 (as did version 2.0 and 2.1). This was removed in version 
3.0. Since the majority of participants were enrolled on versions 2.0-2.2, and none were 
enrolled under version 3.0, the 2.2 version has been included in the SAP. 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
 
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Table 1: Schedule of Events and Assessments for Neu-2591-PTHS 
 
 
Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
Type of Visit 
Clinic 
Remote 
and In-
Home c,d 
Clinic 
Remote 
and In-
Home c,d 
Clinic 
In-
Homed 
Remote 
and In-
Homec,d 
In-
homed 
Clinic 
In-
Homed 
Remote 
and In-
Homec,d 
 
In-
Homed 
Remote 
and In-
Homec,d 
In-
Homed 
In-
homed 
Clinic 
Remote 
and In-
Home c,d 
Informed 
consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclu
sion criteria 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confirm 
documented 
PTHS 
diagnosis and 
genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
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Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
PTHS history 
and exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confirm co-
morbid 
psychiatric 
disorders by 
DSM-5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Rapid SARS-
CoV-2 test 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Autism Mental 
Status Exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Recent clinical 
history 
 
 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
X 
Physical 
examinationf 
X 
 
X 
 
X 
 
Xg 
 
X 
 
Xg 
 
Xg 
 
 
X 
Xg 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
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Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
Neurological 
examination 
X 
 
X 
 
X 
 
Xg 
 
X 
 
Xg 
 
Xg 
 
 
X 
Xg 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Abdominal/ 
CVA/General 
Appearancef 
 
N 
 
N 
 
N 
N 
N 
 
N 
N 
N 
N 
N 
N 
 
N 
Ophthalmic 
examinationh 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
X 
 
Vital signs 
X 
N 
X 
N 
X 
N 
N 
N 
X 
N 
N 
N 
N 
N 
N 
X 
N 
Height/length 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Weight 
X 
N 
X 
N 
X 
N 
N 
N 
X 
N 
N 
N 
N 
N 
N 
X 
N 
Review for 
AEs/SAEsi 
 
 
 
X,N 
X 
N 
X,N 
N 
X 
N 
X,N 
N 
X,N 
N 
N 
X 
X,N 


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Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
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Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
Caregiver 
Diary (dosing, 
conmeds, 
voids, seizures, 
changes in 
symptoms/ 
tolerability) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Review Dosing 
Compliance 
 
 
 
X,N 
X 
N 
X,N 
N 
X 
N 
X,N 
N 
X,N 
N 
N 
X 
X,N 
In-clinic 
administration 
of first dosej 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
12-lead 
electrocardiogr
am (ECG) 
X 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
X 
N 
CBC with 
differential 
X 
 
X 
 
X 
 
N 
 
X 
 
N 
 
N 
 
 
X 
N 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
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Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
Coagulation 
X 
 
X 
 
X 
 
N 
 
X 
 
N 
 
N 
 
 
X 
N 
Comprehensive 
Metabolic 
Panelk 
X 
 
X 
N 
X 
N 
N 
N 
X 
N 
N 
N 
N 
N 
N 
X 
N 
Urinalysisl 
X 
 
X 
N 
X 
N 
N 
N 
X 
N 
N 
N 
N 
N 
N 
X 
N 
Urinalysis-
Drugs of 
Abusel 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TSH, Free T3, 
Free T4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
HbA1c 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Serum 
pregnancy testm 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Blood samples 
for 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
X 
 


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Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
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Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
pharmacokineti
cs 
Blood sample 
for biomarkers 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Stool sample 
for microbiome 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
CGI-I 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
CGI-S 
X 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
Stanford-Binet 
Intelligence 
Scales version 
5 (SB5) or 
Bayley Scales 
of Infant 
Development 
ver 4n 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 


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Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
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Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
MB-CDI 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
ORCA 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Caregiver Top 
3 Concerns 
X 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
ABC-2 
X 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
Behavior 
Problems 
Inventory-Short 
Form (BPI-SF) 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Caregiver 
Impression of 
Change 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Quality of Life 
Inventory-
Disability (QI-
Disability) 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
Protocol: NEU-2591-PTHS-001 
 
30APR2024 
 
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Period 
Screening/ Baselinea 
 
4-6 weeks 
Baseline 
Treatment Period 
Follow-
upb 
Visit Week 
-4 to -6 
2 weeks 
from 
Visit 1 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13/EOTa 
15 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit window 
(days) 
±1a 
±1a 
±1a 
±1 
±2 
±1 
±1 
±1 
±2 
±1 
±1 
±1 
±1 
±1 
±1 
-3 
+4 
Impact of 
Childhood 
Neurological 
Disability- 
Overall quality 
of life rating 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
CSHQ 
X 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
Vineland 
Adaptive 
Behavior 
Scales-3 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
GIHQ 
X 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
Modified two-
minute walk 
test 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Study Exit 
Form 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xo 


Neuren Pharmaceuticals Ltd. 
 
Statistical Analysis Plan 
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X = Conducted by site personnel or caregiver as applicable 
N = Conducted by visiting nurse 
Abbreviations: ABC-2, Aberrant Behavior Checklist-2; AE, adverse event; Behavior Problems Inventory-Short Form; BPI-SF; CBC: Complete Blood Count; CGI-I, Clinical Global Impression Scale-Improvement; 
CGI-S, Clinical Global Impression Scale-Severity; CMP: Comprehensive Metabolic Panel; CSHQ, Child Sleep Habits Questionnaire; DSM-5, Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition; 
EOT, end of treatment; GIHQ, GI Health Questionnaire; ICND, Impact of Childhood Neurological Disability; IMP, Investigational Medicinal Product; MB-CDI, MacArthur-Bates Communicative Development 
Inventory; ORCA, Observer-Reported Communication Assessment; PTHS, Pitt-Hopkins syndrome; QI-Disability, Quality of Life Inventory- Disability; SAE, serious adverse events; Stanford Binet Intelligence 
Scales ver 5 
 
a The Screening/Baseline period will be conducted over 4-6 weeks. The second visit during the Screening period (Visit 2) should be conducted 2 weeks after the Screening visit (Visit 1) ± 1 day. The subject will then 
remain in Screening for an additional 2-4 weeks. The caregiver diary must be completed daily during the entire Screening period whether it is 4 weeks or 6 weeks. The Screening visit (Visit 1) and Baseline visit 
(Visit 3) may be conducted over two days. It is preferrable that ECGs, safety labs and biomarker sample collection are conducted on the same day. No assessments can be conducted until after informed consent has 
been given by the caregiver or legal guardian. Also, training for the caregivers on all caregiver completed assessments must be done before the caregiver rater may complete the assessments. All assessments must be 
done within the two-day window for the visit (visit day + or – 1 day). For Visit 3, all assessments must be done before administration of the IMP. 
 
b Also for early termination. Subjects who have received IMP for longer than 2 weeks and discontinue prematurely any time after Visit 5 should return to the investigational site for final safety and efficacy 
assessments as scheduled for the End of Treatment visit (Visit 16) and complete the study exit form. 
 
c This is a combined remote and in-person nurse visit. The remote visit is conducted by the site via telemedicine or video facilities as appropriate. Any caregiver-completed assessments should be completed the same 
day of the visit or no earlier than the day before the visit (except the Caregiver Diary which is completed daily) and reviewed by the Investigator. A modified version of the physical and neurological exam will be 
done at the remote visits based on the guidance document provided to the Investigator. At Week 4, the site will inform the caregiver of the dose titration. See Section 9.2. 
 
d In-home visits will be conducted by a visiting nurse. At every nurse visit, the nurse will collect urine, vital signs including weight, and blood samples for a comprehensive metabolic panel (CMP). They will also 
conduct an exam assessing general appearance, abdominal/costovertebral angle tenderness, and edema. A suprapubic exam for bladder palpation may be done if decreased urine output is reported. The visiting nurse 
will also review the dosing diary, query oral intake, and do a review of tolerability. Blood samples for CBC with differential and coagulation will be collected at Weeks 4, 8, 10 and Week 15. At the post-treatment 
follow-up visit at week 15, the visiting nurse will also collect ECG, in addition to the above assessments. At Week 7, the site will also have a phone call with the caregiver to inform him/her of the dose titration. The 
site will also inform the visiting nurse so the dosing schedule can be reviewed with the family during the in-home visit. See Section 9.1 and Section 9.2. 
 
e A clinic visit is preferred but an off-site visit may be conducted for a scheduled in-clinic visit in extenuating circumstances due to the COVID-19 health crisis. All requests for off-site visit assessments due to 
extenuating circumstances must be approved in advance by the Sponsor or Medical Monitor. 
 
f The in-clinic physical exam will include an abdominal exam and an assessment of costovertebral angle tenderness (CVA). A suprapubic exam for bladder palpation may be done if decreased urine output is reported. 
General awareness will be captured as part of the neurological exam. For in-home nursing visits, the nurse will do an exam assessing general appearance (e.g., alertness), an abdominal/CVA, and edema. A 
suprapubic exam for bladder palpation may be done if decreased urine output is reported. 
 
g A modified version of the neurological and physical exam will be done for the remote visit based on the guidance document provided to the Investigator. 
 
h A complete ophthalmological exam will be conducted by an ophthalmologist at Screening (Visit 1) and End of Treatment (Week 13, Visit 16). Abrief exam (undilated fundoscopy exam) will be done by the 
ophthalmologist at Week 2 and Week 6. See section 9.5.7). 
 
i The visiting nurse will query any changes in tolerability during the in-home visit. Any changes will be noted and provided to the Investigator. The Investigator will also query changes in tolerability and note any 
changes in the remote assessment. The Investigator is responsible for determining and reporting any changes that qualify as Adverse Events. 
 
j The first dose of IMP will be administered in the clinic after all Baseline assessments are completed, or, if the Investigator judges that it is too late in the day, on the following day. The subject should be monitored 
for at least three hours post first dose. Day 1 of dosing is defined by the date when the first dose is taken. At the visit, the site will dispense an adequate supply of IMP for dispensing by the caregiver at home. 
 
k A Comprehensive Metabolic Panel will be assessed every post-enrollment visit (see Section 9.5.2). If abnormal findings are observed on renal or liver function tests, additional tests as is clinically indicated may be 
conducted as described in Sections 9.5.2.1 and 9.5.2.3). 
 


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Statistical Analysis Plan 
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l Drugs of abuse will be evaluated at Screening (See Section 9.5.2). 
 
m For female subjects who have reached menarche. 
 
n The Bayley is done for subjects who cannot obtain a basal score on SB5. 
 
o The study exit form should also be completed if the subject terminates the study early. 
 
 


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2.2.1 Study Treatment 
The total administration period for NNZ-2591 Oral Solution, 50 mg/mL Investigational 
Medicinal Product (IMP) is 13 weeks. Following up-titration, the IMP is administered 
open-label at the target dose of 12 mg/kg BID.  
The DSMC reviews the clinical safety data prior to any individual dose titration and prior 
to initiation of subsequent age groups. For dose titration, after a subject has had two 
weeks of dosing at the dose level, the data is reviewed by the DSMC during which time 
the subject stays on their current dose.  As outlined below in Table 2, the IMP is 
administered at a starting dose of 4 mg/kg BID for up to 3 weeks. Following DSMC 
review and approval of each dose titration, subjects are up-titrated to 8 mg/kg BID for up 
to 3 weeks, and then to the 12 mg/kg BID dose. The subject then continues on the 
assigned dose (or highest dose tolerated) for the remainder of the 13 weeks. 
Table 2: Dose Titration Schedule 
Week 1 and 
Week 2 
Week 3 
Week 4 and 
Week 5 
Week 6 
Week 7 and 
Week 8  
Week 9 
Week 10 to 
Week 13 
4 mg/kg BID  
(8 mg/kg daily) 
4 mg/kg BID 
(8mg/kg 
daily)1 
8 mg/kg BID  
(16 mg/kg 
daily) 
8 mg/kg BID  
(16 mg/kg 
daily)1 
12 mg/kg BID  
(24 mg/kg 
daily) 
12 mg/kg BID  
(24 mg/kg 
daily) 
12 mg/kg BID  
(24 mg/kg 
daily) 
DSMC 
Review Data 
Prior to Up-
Titration2 
DSMC 
Review Data 
Prior to Up-
Titration2 
DSMC 
Reviews Data 
After 2 weeks 
on 12 mg/kg 
to Confirm 
Safety2 
1Subjects will stay on their current dose until the data is reviewed by the Data Safety Monitoring Committee (DSMC) 
2Expected time for DSMC review will be one week. 
Subjects who are unable to tolerate the study treatment may be discontinued from the 
study. 
 
 


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Statistical Analysis Plan 
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3 Analysis Populations 
Intent to Treat Population (ITT): The Intent to Treat population will consist of all 
subjects deemed eligible and enrolled into the study. 
Safety Population (SAF): The Safety population will consist of all subjects in the ITT 
population who received at least one dose of IMP. 
Modified Intent to Treat Population (mITT): The modified Intent to Treat population 
will consist of all subjects in the ITT population who completed treatment and completed 
the EOT visit. 
The pharmacokinetic analyses will be described in a separate analysis plan and 
summarized separately.  
4 Data Monitoring 
An independent DSMC will monitor the progress of the trial and ensure that the safety of 
trial subjects is not compromised. The DSMC will review the clinical safety data prior to 
any individual dose titration and prior to initiation of subsequent age groups. The DSMC 
will review safety data and make an adjudication on: 1) the ability for any individual 
subject to escalate to the next higher dose and 2) the ability for enrollment to begin in the 
younger age groups (Groups 2 and 3). The DSMC consists of a clinical chair (pediatric 
cardiologist), and two physicians experienced in clinical trials with pediatric expertise 
(but not participating in this study). The DSMC Charter details the DSMC processes, 
duties, and responsibilities as well as the study-wide stopping criteria. 
Data are provided to the DSMC as raw data listings for individual titration and age group 
reviews, as specified in the DSMC Charter. Additional summary analyses may be 
requested.  For quarterly data reviews safety listings are provided to the DSMC. There is 
no separate Statistical Analysis Plan for the DSMC; the DSMC received as subset of the 
safety TFLs as required for the quarterly data reviews. 
5 Statistical Methods  
The statistical analyses will be reported using summary tables, listings, and figures 
(TLFs). Numbering for TLFs will be based on the recommended numbering convention 
provided by the International Conference on Harmonization (ICH).  
The analyses described in this plan are considered a priori, in that they have been defined 
prior to database lock. Any analysis added after the database lock will be considered 


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Statistical Analysis Plan 
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30APR2024 
 
24 
 
exploratory. Post hoc analyses will be labeled as such on the output and identified in the 
CSR.  
Unless noted otherwise, all statistical tests will be two-sided with a significance level of α 
= 0.05 and descriptive in nature. Continuous variables will be summarized with means, 
standard deviations, medians, minimums, and maximums. Categorical variables will be 
summarized by counts and percent of subjects in corresponding categories. Missing 
values are not considered for percent calculations, unless stated otherwise. Footnotes may 
be used to specify the percent calculation as appropriate. Baseline for all analyses is 
defined in Section 5.2.3.  
Individual subject data obtained via the electronic data capture (EDC) system and from 
external vendors (excluding PK data) will be presented in listings. 
All analyses and tabulations will be performed using SAS version 9.4 or higher. Tables, 
listings, and figures will be presented in portable document format (PDF) and in Rich 
Text Format (RTF). All analyses will be subject to formal verification procedures. 
Specifically, results will be verified by independent programming. All documents will be 
reviewed by the lead statistician to ensure accuracy and consistency of analyses.  The 
validation process will be used to confirm that statistically valid methods have been 
implemented and that all data manipulations and calculations are accurate. Checks will be 
made to ensure accuracy, consistency with this plan, consistency within tables and 
consistency between tables and corresponding data listings. 
5.1 Determination of Sample Size 
Sample sizes were estimated for this open-label study using within-subject change as the 
measure of improvement. Given the limited data from interventional trials in these patient 
populations and that this is the first clinical study of NNZ-2591 IMP in this population, 
treatment change estimates were based on Clinical Global Impression Scale of 
Improvement (CGI-I) data from completed clinical trials in other rare, 
neurodevelopmental disorders. Based on the information provided in Glaze et al. 2019 
(Double-blind, randomized, placebo-controlled study of trofinetide in pediatric Rett 
syndrome), the standard deviation of the CGI-I is approximately 0.7 at a single timepoint. 
For a sample size of 10 subjects, a two-sided significance level of 0.05, and a Wilcoxon 
signed-rank test, the study will be able to detect a difference of 0.87 units of change in 
the CGI-I at a power of 0.90. For a power of 0.80, 0.85, and 0.90, the detectable 
difference would be 0.75, 0.80, and 0.87 or larger, respectively. The effect size for a 
power of 0.80, 0.85, and 0.90 is 1.07, 1.14, and 1.24, respectively. If the observed 
standard deviation is larger, the detectable difference would also be larger. For example, 
if the standard deviation is 1.5, the detectable difference for a power of 0.80, 0.85, and 


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0.90 is 1.61, 1.71, and 1.86 or larger, respectively. For a sample size of 20 subjects, a 
two-sided significance level of 0.05, and a Wilcoxon signed-rank test, the study will be 
able to detect a difference of 0.55 units of change in the CGI-I at a power of 0.90. For a 
power of 0.80, 0.85, and 0.90, the detectable difference would be 0.48, 0.51, and 0.55 or 
larger, respectively. 
5.2 Handling of Data 
5.2.1 Imputation of Missing/Incomplete Dates 
An incomplete date is any date for which either the day, month or year is unknown, but 
all three fields are not unknown. An incomplete date occurs when the exact date an event 
occurred or ended cannot be obtained from a patient. For many of the analyses, a 
complete date is necessary to determine if the event should be included in the analysis 
(i.e., if the event is treatment-emergent or a medication is concomitant). In such cases, 
incomplete or missing dates will be imputed.  
For incomplete or missing onset dates related to adverse events and concomitant 
medications, the dates will be imputed as follows:  
• For an incomplete start date with missing day but month and year are present: 
If the reported month and year are the same as Day 1 month and year, then the 
date will be imputed as the date of Day 1. Otherwise, the start day will be 
imputed to the first day of the month. 
• For an incomplete start date with missing day and month but year is present: If 
the reported year is the same as Day 1 year, then the date will be imputed as 
the date of Day 1. Otherwise, the start day will be imputed to January 1. 
• If the reported date is completely missing, it will be imputed as the date of 
Day 1. 
For concomitant medications, end dates will be imputed as follows: 
• For an incomplete end date with missing day but month and year are present: 
The reported date will be imputed as the last day of the month. 
• For an incomplete end date with missing day and month but year is present: If 
the reported year is the same as the study completion year, then the date will 
be imputed as the date of study completion. Otherwise, the end day will be 
imputed to December 31. 
• If the date is completely missing, the event will be imputed as the date of 
study completion. 


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If any imputed date causes the end date to occur prior to the start date of the event, the 
start date of the event will be used for the imputation of the end date. If the imputed date 
is later than the date of study withdrawal, then the date of study withdrawal will be 
imputed for the date.  
If the date of last dose is missing, then it will be imputed to the subject participation end 
date. 
Dates related to efficacy measures will not be imputed. 
In subject data listings, start and stop date of events will be displayed as reported on the 
eCRF (i.e., imputed values will not be listed). 
5.2.2 Imputation of Alphanumeric Data 
Should there be instances where a clinical laboratory parameter is reported with 
embedded non-numeric characters, for example, “<0.1” or “>10”, the data will be 
imputed for quantitative summaries. The actual values as reported in the database will be 
presented in data listings.  
For incorporation in quantitative summaries, the following imputation rule will be 
employed: 
• For values reported as less than a lower limit of detection, the value will be 
imputed as half of the numeric result preceded by the ‘<’ symbol. For example, 
“<0.1” will be imputed to 0.05. 
• For values reported as greater than an upper limit of detection, the value will be 
imputed as the numeric result preceded by the ‘>’ symbol. For example, “>10” 
will be imputed as 10. 
5.2.3 Visit Windows 
Unscheduled assessments that occur on the same day as a scheduled visit will be 
considered with the corresponding scheduled visit for analysis. Otherwise, unscheduled 
assessments will be considered with the most recently completed scheduled visit for 
analysis. If multiple assessments are windowed to the same visit for analysis, the results 
from the latest assessment will be used, as repeats may have been done to confirm 
abnormalities, per the protocol. Any unscheduled visits occurring during the Screening 
period will not be mapped to a scheduled visit but will be considered in the derivation of 
the Baseline value. 


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5.2.4 Baseline Values 
Baseline values that are missing will not be imputed for safety or efficacy measures. For 
efficacy assessments scheduled to be collected at more than one visit during the 
Screening/Baseline period, the baseline value will be based on the average of all non-
missing values from assessments conducted prior to the first dose of study drug. For 
assessments scheduled to be collected once during the Screening/Baseline period, the 
value from the most recent non-missing assessment prior to the first dose will be used. 
For safety and other assessments, the value from the most recent non-missing assessment 
prior to the first dose will be used. 
5.2.5 Post-Baseline Values 
For subjects who discontinue the study, data will be analyzed up to the last observation 
for safety and efficacy measures. Missing data for safety assessments will not be 
imputed. For efficacy rating scale measures other than the exceptions listed below, if an 
assessment has been completed but has missing items, missing values shall be imputed as 
follows: 
• If 3 or fewer items are missing, the last observation for that particular data 
element (e.g., item response) will be carried forward. 
• The scoring algorithms will be applied including the observed values and last 
observation carried forward (LOCF) for the missing items 
These rules will only apply to individual items. Total scores, subdomain and subscale 
scores will not be carried forward. If the assessment is completely missing, items will not 
be imputed.  
These imputation rules do not apply to the CGI-I, Clinical Global Impression Scale – 
Severity (CGI-S) or Caregiver Impression of Change assessments. If a particular domain 
is missing for these assessments, the participant will be excluded from the corresponding 
analysis. These imputation rules also do not apply to the Caregiver Top 3 Concerns 
assessment. If a concern is missing for this assessment, the average severity will still be 
calculated from all non-missing concern severity scores. 
5.3 Definitions 
● Age will be derived as the integer value of (informed consent/assent date - date of 
birth + 1)/365.25. 
● The age at time of assessment will be derived as the integer value of (date of 
assessment – date of birth + 1)/365.25. 


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● Body Mass Index (BMI) will be derived as weight in kilograms divided by height 
in meters squared. 
● The change from Baseline measurement for a variable at a particular visit will be 
derived as the measurement taken at the specified visit minus the baseline 
measurement. For the Clinical Global Impression Scale – Improvement and 
Caregiver Impression of Change, change from Baseline is the value at the visit. 
● Treatment duration in days will be derived as follows: Date of Last Dose – Date 
of First Dose + 1. 
● The number of expected doses will be derived as (2*treatment duration in days)-
1, as subjects are expected to receive only their morning dose on the last day of 
treatment 
● The number of doses taken will be derived as the number of doses taken as 
indicated by the Administration of First Dose CRF and by the Daily Dosing Log 
CRF for remaining doses, based on completed date and dose fields 
● Number of missed doses will be derived based on the number of expected doses – 
the number of doses taken 
● Study Day will be derived as Date of Event – Date of First Dose (+1 if the date of 
event ≥ date of first dose) 
● Screening/baseline duration in days will be derived as follows: Date of First Dose 
– Date of Screening 
● Study duration will be defined as the total number of days in the study (date of 
last visit – date of informed consent + 1). 
● History of developmental regression will be determined from the PTHS diagnosis 
page of the CRF question: “Has the child undergone a regression of skills in the 
past?” If coded as “yes” the subject is considered as having had a past 
developmental regression. 
● Autism Spectrum Disorder (ASD) diagnosis will be derived from the PTHS 
diagnosis page of the CRF. If ASD is checked under “confirmation of psychiatric 
co-morbidities”, the subject is considered as having the disorder. 
● Bipolar Disorder will be derived from the PTHS diagnosis page of the CRF. If 
“Bipolar disorders” is checked under “confirmation of psychiatric co-
morbidities”, the subject is considered as having the disorder. 


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● Attention Deficit Disorder will be derived from the PTHS diagnosis page of the 
CRF. If “Attention Deficit Disorder” is checked under “confirmation of 
psychiatric co-morbidities”, the subject is considered as having the disorder. 
● Obsessive-compulsive Disorder will be derived from PTHS diagnosis page of the 
CRF. If “Obsessive-compulsive disorder” is checked under “confirmation of 
psychiatric co-morbidities”, the subject is considered as having the disorder. 
● There will be three age group definitions use for the analysis and applied to all 
analyses requiring an analysis by age group: 
o Enrollment Age Group 
▪ 13 to 17 years old 
▪ 8 to 12 years old  
▪ 3 to 7 years old  
o Child/Adolescent Age Group 
▪ 13 to 17 years old  
▪ 3 to 12 years old  
o Divided Age Group 
▪ 16 to 17 years old  
▪ 13 to 15 years old 
▪ 8 to 12 years old  
▪ 3 to 7 years old 
● Dose modification is defined as dose increased, dose reduced, or dose interrupted 
responses for the action taken with study treatment on the AE CRF. 
● TEAEs are defined as those adverse events that occurred after the administration 
of first dose of study drug. 
● 
 
 
 
 
 
● Improvement in the Top 3 concerns will be derived as a decrease in severity 
(change from baseline < 0). This is calculated for the average score and for 
individual concerns by the symptom domains as defined in Section 9.2.8. 


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● The baseline seizure frequency will be determined based on the number of 
seizures recorded in the diary during the 4–6-week screening period. The baseline 
monthly seizure frequency will be derived by dividing the total number of 
seizures during the screening/baseline period by the screening/baseline duration in 
months (screening/baseline duration divided by 30.4375). During the treatment 
period (Visits 4 – 6), the seizure frequency will be derived by summing the 
number of seizures recorded in the diary over treatment. The monthly seizure 
frequency will be derived by dividing the total number of seizures during the 
treatment period by the treatment duration in months (treatment duration divided 
by 30.3475). 
● The baseline major breathing episode frequency will be determined based on the 
number of major breathing episodes recorded in the Major Respiratory Events log 
of the CRF during the 4-6 week screening period. The baseline monthly major 
breathing episode frequency will be derived by dividing the total number of major 
breathing episodes during the screening/baseline period by the screening/baseline 
duration in months (Screening/baseline duration divided by 30.4375). During the 
treatment period (Visits 4 – 16), the major breathing episode frequency will be 
derived by summing the number of major breathing episodes recorded in the diary 
over treatment. The monthly major breathing episode frequency will be derived 
by dividing the total number of major breathing episodes during the treatment 
period by the treatment duration in months (treatment duration divided by 
30.3475). 
● Prior medications are those medications started prior to and stopped before the 
first dose of study drug. Concomitant medications include: 1) medications started 
prior to, but still ongoing as of the first dose of study drug, 2) medications with a 
start date on or after first dose date.  
● The number of concomitant medications will be summed for each subject for all 
unique medications that are taken while on treatment. Medications taken with a 
frequency of PRN or “as needed” will be excluded from this count. 
6 Subject Information 
6.1 Subject Disposition 
Information regarding subject disposition will be summarized for all subjects by age 
groups as defined in Section 5.3 and overall.  
Summaries will include: number of subjects enrolled, number of subjects who screen 
failed, number of subjects in the ITT, Safety, and mITT populations, number of subjects 


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who complete the study, and number of subjects who discontinue the study early.  For 
those who discontinue early, the primary reason for discontinuation will be summarized. 
The primary reason for discontinuation from the CRF will be mapped to the 
Completion/Reason for Non-Completion SDTM Terminology for presentation in the 
table. A data listing by subject will also be generated. 
6.2 Protocol Deviations 
Protocol deviations will be collected throughout the duration of the study. Protocol 
deviations will be assigned a sponsor-defined category type. Each deviation will be 
defined as important or not important. In addition, a by-subject listing of all protocol 
deviations (important and not important) will be produced.  
6.3 Demographic and Baseline Characteristics 
Demographics variables will include: age, age groups, sex, ethnicity, race, height at 
baseline, weight at baseline, and BMI at baseline. Age and BMI will be derived as 
specified in Section 5.3. 
Baseline characteristics will include: PTHS Variant Types (as recorded on the CRF), 
history of developmental regression, Autism Mental Status Exam, seizure types at 
screening (as recorded on the CRF), presence of co-morbid psychiatric disorders (e.g. 
Autism Spectrum Disorder (ASD), bipolar disorders, attention deficit disorder, obsessive-
compulsive disorder), and Stanford-Binet Intelligence Scales version 5 (SB5) NVIQ 
standard and z-deviation score or the Bayley Scales of Infant Development – Version 4 
(BSID-4) NVDQ for subjects who cannot basal on the SB5 or for whom the SB5 is not 
developmentally appropriate. If the SB5 was attempted but the child was unable to basal 
on the assessment, the BSID-4 should be administered. If partial scores for the SB5 are 
entered in the EDC in addition to the BSID-4, only the BSID-4 scores will be 
summarized. If the child has complete sets of scores on the SB5 and the BSID-4, the 
scores for the assessment that was captured at both Screening and EOT will be 
summarized.  If a participant has a complete set of scores for the SB5 and BSID-4 at both 
Screening and EOT, these cases will be adjudicated by the Sponsor before database lock.  
Demographics and baseline characteristics will be summarized for all analysis 
populations (ITT Population, Safety Population, mITT Population).  
Subjects will be evaluated for PTHS at the Screening Visit. The diagnosis and genotype 
will be documented. PTHS genotypes will be summarized and listed by subject. 
History of developmental regression will be determined from the question as reported on 
the PTHS diagnosis page in the CRF: “Has the child undergone a regression of skills in 


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the past?” If coded as “yes” the subject is considered as having had a past developmental 
regression. 
The Autism Mental Status Exam (AMSE) will be performed at the Screening Visit. The 
AMSE is an 8-item observational assessment used to document communicative and 
behavioral functioning in subjects with ASD. Each question is scored as 0, 1, or 2. The 
range of the AMSE score is 0 – 14. Higher scores indicate greater severity of autistic 
symptoms. A question assessing overall impairment is also completed evaluated on a 4- 
point scale: No impairment, Mild, Moderate and Severe. 
The scoring algorithm is below: 
• Question 1-4, Eye Contact, Interest in Others, Pointing Skills, Language: each 
response is assigned a score of 0, 1, or 2 in the order they appear on the CRF. 
• Question 5 Pragmatics of Language: Not Impaired or Not Applicable=0, 
Reported=1, Observed=2 
• Question 6 Repetitive Behaviors/Stereotypy: None=0, Insists on routines=1, any 
other response=2 
• Question 7-8 Unusual or Encompassing Preoccupations, Unusual Sensitivities: 
None=0, Reported=1, Observed=2 
The AMSE score is the sum of the individual question scores above and will only be 
calculated if no items are missing. The AMSE score and the overall impairment will be 
summarized. 
Seizure type (“Clinical Type” in Seizures and Spells Log form on the CRF recorded at 
Baseline) and characteristics of seizures (“Description in Seizure Types at Screening” 
form on the CRF: change in awareness, loss of urine or bowel control, loss of ability to 
communicate, automatic repeated movements, rhythmic jerking muscle, falls if standing 
or sitting, head drops, warning before seizure occurred, common triggers, muscle 
stiffness, muscle twitch, other) are recorded at the Screening Visit for subjects. These 
descriptive characteristics will be summarized using frequencies and percentages. 
The presence of other co-morbid psychiatric disorders will be recorded on the PTHS 
Diagnosis page of the CRF. If the disorder is checked on the CRF, it will be considered 
as present in the subject. 
6.4 Medical History 
Medical history will be coded using Medical Dictionary for Regulatory Activities 
(MedDRA) version 24.0 and listed by System Organ Class (SOC), Preferred Term (PT), 
and verbatim term. A complete medical history will be recorded at all Screening and 


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Baseline visits (Visits 1, 2, 3). Medical history will be summarized using frequencies and 
percentages according to the SOC and PT.  
7 Safety Analysis 
The primary objective of the study is to investigate the safety, tolerability, and 
pharmacokinetics of treatment with NNZ-2591 in children and adolescents with PTHS. 
All safety analyses will be based on the Safety Population.  
Safety will be assessed based on incidence of treatment emergent adverse events 
(TEAEs), serious adverse events (SAEs), changes in vital signs, changes in laboratory 
measures, neurological, ophthalmological, and physical exam results, 
 
and changes in ECG’s. 
Tolerability will be assessed based on daily dosing logs, requiring dosing adjustments or 
discontinuations, dosing compliance, and AEs related to subject experience.  
Pharmacokinetics will be summarized separately outside of this SAP.  
7.1 Dosing Summary 
Treatment duration and study duration will be derived as stated in Section 5.3 and 
summarized using descriptive statistics. 
The maximum dose level (8 mg/kg BID, or 12 mg/kg BID) reached by each subject will 
be summarized according to the Dosing Compliance CRF as a frequency distribution. 
The number of missed doses as defined in Section 5.3 will be summarized. The number 
of missed doses will only be defined prior to discontinuation of treatment. 
7.2 Adverse Events 
Adverse events will be recorded from the time of first dose of IMP until Follow-up (Visit 
17). Adverse events summaries will only consider treatment emergent adverse events 
(TEAEs). TEAEs are defined as those adverse events that occurred after the 
administration of the first dose of study drug.  If it cannot be determined whether the 
adverse event is treatment emergent due to missing or incomplete (partial) onset dates, 
dates will be imputed for the purposes of determining treatment emergence as described 
in Section 5.2.1.  
Verbatim terms entered into the clinical database via the EDC system will be mapped to 
preferred terms and system organ classes using version 24.0 of MedDRA. 


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Summaries that are displayed by SOC and PT will be ordered by descending order of 
incidence of SOC and PT within each SOC. Adverse event summaries of the following 
types will be presented for the Safety Population: 
• Overall summary of the TEAEs which contain an overview of each item below. 
• Subject count and incidence rate of TEAEs and total number of unique TEAEs by 
MedDRA system organ class and preferred term. 
• Subject count and incidence rate of TEAEs by MedDRA preferred term and 
greatest intensity. Intensity is defined by three categories: “Mild”, “Moderate”, 
and “Severe.” At each level of subject summarization, a subject is classified 
according to the greatest intensity if the subject reported one or more events. 
Adverse events with missing intensity will be considered severe for this summary.  
• Subject count and incidence rate of TEAEs by MedDRA preferred term and 
closest relationship to study drug (Related/Not Related). At each level of subject 
summarization, a subject is classified according to the closest relationship if the 
subject reported one or more events. Adverse events with missing relationship 
will be considered related for this summary. 
• Subject count and incidence rate of Serious TEAEs by MedDRA SOC and PT. 
• Subject count and incidence rate of TEAEs by dose level, SOC, and PT. 
• Subject count and incidence rate of related TEAEs by dose level, SOC, and PT. 
• Subject count and incidence rate of TEAEs leading to withdrawal of study 
medication by MedDRA SOC and PT. 
• Subject count and incidence rate of TEAEs leading to dose modification (defined 
as dose increased, dose reduced, or dose interrupted on the CRF) by MedDRA 
SOC and PT. 
• Subject count and incidence of TEAEs with an outcome of death by MedDRA 
SOC and PT. 
• 
 
 
 
 
A separate listing of related TEAEs will be generated. 
7.3 Clinical Laboratory Evaluations 
Blood will be collected at Visit 1 (Screening), Visit 3 (Week 0), and Visit 13 (End of 
Treatment). Urine samples will be collected at all visits. Laboratory parameters 
(chemistry, hematology, urinalysis) will be presented in data listings. Abnormal values 
will be flagged as high or low relative to normal ranges, where applicable, by the 
laboratory vendor.  


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Change from baseline in quantitative laboratory parameter values will be summarized 
using descriptive statistics at all visits. 
For those laboratory tests in which normal ranges are available, shifts from baseline will 
be presented. Categories for the shift tables will be high, normal, and low. 
Laboratory parameters will be classified according to the categories given by the 
laboratory vendor. The laboratory parameters from the vendor listed below will be 
assigned to the following categories for data summaries. 
Chemistry 
Hematology 
Urinalysis 
Bun/Creatinine Ratio 
EGFR (Bedside Schwartz) 
Hemoglobin A1C 
Pregnancy, qualitative 
Thyroxine, Free 
Triiodothyronine, Free 
TSH Ultrasensitive 
Urine Drug Screen w/ Con 
PT/INR/APTT 
Microalbumin, UR 
Random 
Urine Chemistry 
7.4 Physical and Neurological Examination 
 
The physical examination will be conducted at all in-clinic visits.  The exam will note 
Normal and Abnormal readings. For each abnormal reading, clinical significance will be 
determined. The physical exam procedure will include the following organ systems: 
Head, ears, eyes, nose, and throat; Skin; Cardiovascular; Respiratory; Gastrointestinal; 
Genitourinary (as appropriate or indicated); Musculoskeletal; and Allergies. The exam 
will include an assessment for sign of bladder or kidney pathology including an exam of 
the abdomen and both flanks. Assessment of edema will also be done. Exam may include 
suprapubic exam (bladder palpation) if decreased urine output is noted.  
A neurological examination will be conducted at all in-clinic visits.  A standard 
neurological exam will be conducted to include an evaluation of mental status, cranial 
nerves, motor exam, sensory exam, cerebellar (coordination, gait), and reflexes.  


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A modified version of the physical and neurological exams will be done by a physician 
via telemedicine at the remote visits. As part of the in-home visit, the nurse will conduct a 
targeted physical exam including assessment of general appearance, an abdominal and a 
costovertebral angle tenderness exam, assessment for signs of cystitis, and an assessment 
of edema. Exam may include suprapubic exam (bladder palpation) if decreased urine 
output is noted. 
 
  
All examination results will be reported as normal or abnormal. Clinical significance will 
be determined for abnormal results.  
The results of all examinations will be presented in data listings. 
7.5 Ophthalmology Assessment 
A comprehensive ophthalmological exam will be undertaken at Screening (Visit 1) and 
End of Treatment (Week 13, Visit 16). Routine visual inspection of each eye was 
undertaken at in-clinic Visit 5 (Week 2) and Visit 9 (Week 6) for subjects enrolled under 
protocol version 2.0, 2.1 and 2.2.  At these interim visit exams, an exam with dilation 
may be done if abnormalities are present or the assessment is indeterminate, or as deemed 
necessary by the ophthalmologist, Medical Monitor or Principal Investigator. The 
complete exam conducted at Visit 1 and Visit 16 could include up to 2 attempts at 
assessments of estimated visual acuity, dilated refractive error measurement, dilated slit 
lamp examination of the lens, and a dilated fundus examination. 
The results will be presented in data listings.  
7.6 Vital Signs 
Weight, blood pressure (systolic and diastolic in mmHg), heart rate (bpm), respiratory 
rate (rpm), and body temperature (°C) will be collected at the Screening visits (Visit 1 
and 2) and all post-baseline study visits. Ideally, vital signs will be taken after the subject 
has been seated for 5 minutes. The initial blood pressure reading will be summarized in 
the table. Height will be taken at the Screening visit (Visit 1), Visit 3 (Week 0), and End 
of Treatment (Week 13, Visit 16).   
Summary statistics for each measurement and the change from baseline will be presented 
at each post-baseline visit. All vital sign measurements will be presented in data listings. 
A separate table and listing will be provided for blood pressure. 


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7.7 Electrocardiogram (ECG) 
The ECG assessment will measure PR Interval (msec), QRS Duration (msec), QT 
Interval (msec), QTcF Interval (msec), Heart Rate (bpm), and RR Interval (msec). The 
assessment will be done at the Screening visit (Visit 1), Visit 3 (Week 0), Visit 5 (Week 
2), Visit 9 (Week 6), End of Treatment (Week 13, Visit 16) and Follow-Up (Visit 17).  
The interpretation of the results will be assessed as Normal or Abnormal, and any 
Abnormal results will be assessed for clinical significance. Ideally, the subject should be 
supine for at least 5 minutes before the first ECG is taken. If this cannot be done, this will 
be documented in the CRF. The ECG will be obtained as a continuous ECG of at least 50 
ms. All readable ECGs of at least 10 ms will be interpreted by the central reader and 
reported.  
Summary statistics for each ECG parameter and the change from baseline will be 
presented at each post-baseline visit. Additionally, summary statistics for the shifts in 
interpretation from baseline will be presented at each post-baseline visit. All ECG 
measurements will be presented in data listings. 
7.8 Prior and Concomitant Medications 
Prior and concomitant medication verbatim terms captured via the EDC system will be 
mapped to Anatomical/Therapeutic/Chemical (ATC) class and Preferred Names using the 
March 1, 2023 version of the World Health Organization (WHO) Drug Dictionary 
Enhanced available. 
Prior medications are those medications started prior to and stopped before the first dose 
of study drug. Concomitant medications include: 1) medications started prior to, but still 
ongoing as of the first dose of study drug, 2) medications with a start date on or after first 
dose date. Prior and concomitant medications will be summarized separately by WHO 
ATC class and preferred name. These summaries will present the number and percent of 
subjects using each medication. Subjects may have more than one medication per ATC 
class and preferred name. At each level of subject summarization, a subject is counted 
once if one or more medications at that level is reported for the subject. The drug 
classification will be the ATC classification 4. If this classification is not present, the next 
available classification will be utilized. Medications taken with a frequency of PRN or 
“as needed” will be excluded from the tabular summaries. Each summary will be ordered 
by descending order of incidence of ATC class and preferred term. 
 
 


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7.9 Monthly Seizure Episodes 
Monthly seizure frequency will be derived as defined in Section 5.3. Seizure frequency 
will be assessed from a seizure log that is part of the Caregiver Diary which is completed 
daily by the caregiver. 
Summary statistics on the observed and the change from baseline in monthly seizures 
will be displayed at baseline and end of treatment. Within subject changes will be 
assessed using a Wilcoxon Signed Rank Test. 
7.10 Monthly Major Breathing Episodes 
Monthly frequency of major breathing events will be derived as defined in Section 5.3. 
Breathing event frequency will be assessed from a Major Respiratory Events log that is 
part of the Caregiver Diary which is completed daily by the caregiver. 
Summary statistics on the observed and the change from baseline in monthly breathing 
episodes will be displayed at baseline and end of treatment. Within subject changes will 
be assessed using a Wilcoxon Signed Rank Test. 
8 Efficacy Analysis 
The secondary objective is to investigate measures of efficacy during treatment with 
NNZ-2591 Oral Solution in children and adolescents with PTHS.  
All efficacy analyses will be conducted on the ITT and mITT populations. 
8.1 Efficacy Variables 
There are no primary efficacy variables. 
Secondary variables for efficacy to be considered include:  
• PTHS-specific Clinical Global Impression Scale–Overall Improvement (CGI-
I) Score  
• PTHS-specific Clinical Global Impression Scale–Domain Improvement 
Scores  
• Clinical Global Impression Scale - Severity (CGI-S) - Overall Severity Score  
• Clinical Global Impression Scale - Severity (CGI-S) - Domain Severity Scores  
• MacArthur-Bates Communicative Development Inventory (MB-CDI) Scores  
• Observer-Reported Communication Ability (ORCA) Score  
• Caregiver Top 3 Concerns Likert Scale Scores  


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• Aberrant Behavior Checklist-2 (ABC-2) Scores  
• Behavior Problems Inventory-Short Form (BPI-SF) Scores  
• Child Sleep Habits Questionnaire (CSHQ) Scores  
• Gastrointestinal Health Questionnaire (GIHQ) Scores  
• Caregiver Impression of Change Scores 
• Vineland Adaptive Behavior Scales-3 Scores  
• Quality of Life Inventory-Disability (QI-Disability) Scores  
• Impact of Childhood Neurological Disability (ICND) Overall quality of life 
rating  
• Modified Two-minute walk test (2MWT) total distance 
• SB5 raw, z-deviation, age-equivalent, and Change Sensitive scores or BSID-4 
scores (raw, GSV, age-equivalent; standard and scaled scores may be assessed 
for participants for whom the standard scores can be calculated) 
Biomarker variables and gut microbiome analyses will be described in a separate analysis 
plan. 
8.2 Adjustments for Covariates 
A list of potential covariates to be used in descriptive summaries or analyses are provided 
below. 
• Age at Baseline  
• Sex (Male, Female) 
• SB5 NVIQ or BSID-4 NVDQ at Baseline 
• History of developmental regression (yes, no) 
• ASD diagnosis (yes, no) 
• CGI-S Overall Score at Baseline 
8.3 Examination of Subgroups 
No subgroup analyses are planned. 
8.4 Multicenter Studies 
All sites will be pooled for this analysis. 
8.5 Multiplicity Comparison/Multiplicity 
No formal statistical analysis is planned; therefore, there will be no adjustments for 
multiplicity. 


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9 Efficacy Analysis Methods 
9.1 Primary Efficacy Analysis 
There is no primary efficacy endpoint. 
9.2 Secondary Efficacy Analysis 
The efficacy endpoints will be analyzed using a Wilcoxon signed rank test on the paired 
subject data from baseline to end of treatment (Visit 16) and Visit 9 (Week 6) when 
applicable. Descriptive statistics will be used to summarize the data. For total scores (and 
certain subscale score as defined below) waterfall plots and histograms or bar charts (as 
appropriate) will also be created at the End of Treatment (EOT) visit with plots for all 
subjects and separate plots indicating each of the following: age groups, NVIQ/DQ score 
(>= 50, < 50), sex (Male, Female) and number of concomitant medications (> 6, ≤ 6). 
The number of concomitant medications will be determined as defined in Section 5.3. 
Line graphs plotting scores by visit will also be generated for the mean group data and 
showing individual participants.  The NVIQ/DQ score is the SB5 Non-Verbal 
Intelligence Quotient (z-deviation), or the BSID-4 Non-verbal Developmental Quotient 
depending on which assessment the subject took. The number and percentage of subjects 
who had an IQ/DQ score >= 50 and < 50 at Baseline and End of Treatment will be 
presented in a table for NVIQ/DQ, VIQ/DQ, and FSIQ/Overall DQ. For assessments that 
use discrete scores, the histograms will be changed to bar charts. If the change from 
baseline of the discrete scores is presented, then the change from baseline value will be 
rounded using conventional rounding standards for ease of presentation and 
understanding in the bar chart. For scales with domain scores, forest plots displaying the 
mean change from baseline and 95% CI for each domain score at EOT will be generated. 
All figures will be generated on the ITT and mITT populations. 
Exploratory analyses on some endpoints using linear regression may be performed to 
assess the relationship between treatment and specified covariates or combinations of 
covariates. The empirical cumulative distribution function will be plotted for certain 
variables. As appropriate, collective response analysis may be done to assess data across 
subjects. 
A correlation analysis of the efficacy measures will be done using Pearson correlation 
coefficients at Baseline and EOT. The correlation of each total score will be measured 
against the others and presented in a table and a heat map/matrix. CGI-I and CGI-S 
domain scores will also be included in the correlation analysis. Test-retest reliability will 
be assessed for continuous efficacy assessments collected more than once during the 
Screening period (Visits 1, 2, and 3, pre-dosing) using Intraclass Correlation Coefficients 


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(ICC). The ICC will be based on covariance parameter estimates from a Restricted 
Maximum Likelihood Mixed Model including results from assessments conducted during 
the Screening/Baseline period with a random effect for subject. The ICC will be derived 
as: ICC = Within-subject covariance/Total covariance. ICC values range from 0 to 1, 
with values closer to 1 indicating greater test-retest reliability. 
A treatment policy estimand will be used for all endpoints. The variables of interest are 
numeric scores associated with subject responses. Summaries will be performed on the 
observed data. There will be no imputations for completely missing scores or intercurrent 
events. Handling of missing responses items that contribute to total and subscale scores 
are defined in Section 5.2.5. 
Results for each efficacy variable will also be listed by subject. 
9.2.1 Clinical Global Impression Scale - Overall Improvement Score 
CGI-I assessments are completed at Visit 9 (Week 6), End of Treatment (Week 13, Visit 
16), and Follow-up (Visit 17). The clinician rates how much the subject’s illness has 
improved or worsened relative to Visit 3 (Week 0). The Overall CGI-I score is rated on a 
7-point scale, where 1=’very much improved’; 2=’much improved’; 3=’minimally 
improved’; 4=’no change’; 5=’minimally worse’; 6=’much worse’; 7=’very much 
worse’. Scores of 1, 2, and 3 indicate improvement. Within subject change is indicated by 
the score at each relevant follow-up visit (Week 6, Visit 9 and Week 13, EOT, Visit 16). 
If an overall score is missing, that subject will be excluded from analysis at that visit. 
Summary statistics for the CGI-I Overall score will be displayed at all post-baseline 
visits. Within subject changes will be analyzed using a Wilcoxon signed rank test at EOT 
and Week 6. The overall score will be presented in figures as described in Section 9.2. As 
this score is categorical, the waterfall plots will be adjusted so a score of “7 – Very Much 
Worse” will be presented at the bottom of the graph and a score of “1 – Very Much 
Improved” will be presented at the top of the graph. The overall score will also be 
presented as a line graph at post-baseline visits. 
9.2.2 Clinical Global Impression Scale - Domain Improvement Scores 
CGI-I assessments are completed at Visit 9 (Week 6), End of Treatment (Week 13, Visit 
16), and Follow-up (Visit 17). The clinician rates how much the subject’s illness has 
improved or worsened relative to Visit 3 (Week 0). The seven domain improvement 
scores are: Language/Communication, Social Interaction, Ambulation/Gross Motor, Fine 
Motor Functioning/Self-Help, Gastrointestinal, Autonomic/Breathing Abnormalities, and 
Challenging Behaviors: Repetitive Behavior/Self-Injury/Aggression. The individual 
domain CGI-I scores are rated on a 7-point scale, where 1=’very much improved’; 


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2=’much improved’; 3=’minimally improved’; 4=’no change’; 5=’minimally worse’; 
6=’much worse’; 7=’very much worse’. Scores of 1, 2, and 3 indicate improvement. If a 
domain score is missing, that subject will be excluded from analysis for that domain. 
Within subject change is indicated by the score at each relevant follow-up visit (Week 6, 
Visit 9 and Week 13, EOT, Visit 16). If a domain score is missing, that subject will be 
excluded from analysis for that domain at the relevant visit. The domain scores will be 
presented in figures as described in Section 9.2. Domain scores at EOT will be presented 
in a forest plot. Domain scores will be presented as line graphs at post-baseline visits. 
Domain scores as reported on the CRF will be summarized at each post-baseline visit, in 
the same manner as the CGI-I Overall Score. 
9.2.3 Caregiver Impression of Change Score 
The Caregiver Impression of Change assessment will be completed at the end of 
treatment visit (Week 13, Visit 16).  
The caregiver is asked to rate the change in his or her child’s overall function and well-
being since Visit 3 (Week 0), and the change in specific symptoms. This is rated on a 7-
point Likert scale: 1-Very much improved, 2-Much improved, 3-Improved, 4-
Unchanged, 5-Worse, 6-Much worse, 7-Very much worse. Scores of 1, 2 or 3 indicate 
improvement in overall function and symptoms. 
The assessment of change should be made relative to Visit 3 (Week 0). The caregiver 
should assess the subject’s current state at the visit taking into consideration observations 
at the visit and symptoms within the last week. 
For the rating of overall function, the caregiver also identifies the one symptom area or 
feature that has most influenced his or her rating of the child’s overall function and 
provides a description of symptom changes and impact of the changes.  
The symptom domains rated are: communication, social interaction, behavior, motor 
abilities, seizures, cognition and learning, self-care, gastrointestinal (GI) problems, and 
breathing problems.  Within subject change is indicated by the score at EOT. If a score is 
missing for a domain, that subject will be excluded from the corresponding domain 
summaries.  
Summary statistics on the Caregiver Impression of Change overall and domain scores 
will be displayed at End of Treatment. The overall score will be presented in waterfall 
plots, and histograms or bar charts (as appropriate), and the domain scores will be 
presented in a forest plot, as described in Section 9.2. 


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9.2.4 Clinical Global Impression Scale - Severity - Overall Severity Score 
Clinical Global Impression of Severity assessments will be made at the Screening visit 
(Visit 1), Visit 3 (Week 0), Visit 9 (Week 6), End of Treatment (Week 13, Visit 16), and 
Follow-up (Week 15, Visit 17).  The overall severity score is scored on a 7-point scale, 
where 1=’typical for age, not at all impaired’; 2=’borderline, slightly impaired’; 
3=’mildly impaired’; 4=’moderately impaired’; 5=’markedly impaired’; 6=’severely 
impaired’; 7=’among the most severely impaired’. Lower scores reflect less impairment. 
Within subject change is the difference between the score at each relevant follow-up visit 
and the score at baseline. If a score is missing, the subject will be excluded from the 
corresponding summaries. 
The overall score and change from baseline at each post-baseline visit will be 
summarized using descriptive statistics. Within subject changes will be analyzed using a 
Wilcoxon signed rank test at Week 6 and EOT. Test-retest reliability will be assessed for 
all scores collected during the Screening period (Visits 1 and 3) using ICC. The change 
from baseline in the overall score at EOT will be presented in figures as described in 
Section 9.2. The overall score will also be presented as a line graph at post-baseline 
visits. 
9.2.5 Clinical Global Impression Scale - Severity - Domain Severity Scores 
The CGI-S assessments will be made at the Screening visit (Visit 1), Visit 3 (Week 0), 
Visit 9 (Week 6), End of Treatment (Week 13, Visit 16), and Follow-up (Week 15, Visit 
17). The seven domain severity scores are: Language/Communication, Social Interaction, 
Ambulation/Gross Motor, Fine Motor/Self-Help, Gastrointestinal, Autonomic/Breathing 
Abnormalities, and Challenging Behaviors: Repetitive Behavior/Self-Injury/Aggression. 
Each domain score is scored on a 7-point scale, where 1=’typical for age, not at all 
impaired’; 2=’borderline, slightly impaired’; 3=’mildly impaired’; 4=’moderately 
impaired’; 5=’markedly impaired’; 6=’severely impaired’; 7=’among the most severely 
impaired’. Lower scores reflect less impairment. Within subject change is the difference 
between the total score at each relevant follow-up visit and the score at baseline. If a 
score is missing, the subject will be excluded from the corresponding summaries. 
The change from baseline in CGI-S domain scores will be analyzed in the same manner 
as the CGI-S overall score. The change from baseline in domain scores at EOT will be 
presented in forest plots as described in Section 9.2. Domain scores will be presented as 
line graphs at post-baseline visits. 


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9.2.6 MacArthur-Bates Communicative Development Inventory Scores 
The MB-CDI will be completed at Visit 3 (Week 0) and End of Treatment (Week 13, 
Visit 16). This caregiver-completed instrument assesses language development in 
vocabulary comprehension and production, gestures, and grammar. Designed to assess 
early language development in infants and toddlers, it can also be used with older, 
developmentally delayed children to assess developmental progress in language 
development. The MB-CDI includes three modules based on chronological or 
developmental age: Words and Gestures (8 to 18 months or developmental equivalent), 
Words and Sentences (16 to 30 months or developmental equivalent) and the CDI-III, an 
extension of the MB-CDI used with children 30 to 37 months (or developmental 
equivalent). Only the Words and Gestures module will be used for this study in line with 
the cognitive and language development of the patient population. 
The number of “yes” answers in “First signs of understanding” and “sometimes” and 
“often” in “Starting to Talk” will be summarized. The number of phrases understood, the 
number of words understood, number of words produced, number of early gestures, 
number of later gestures, and number of gestures will be summarized. For these scores, 
scores calculated by the site rater and entered in the EDC will be used for the analysis. 
Scores may also be derived if the EDC score calculations were not completed. The 
calculations are as follows: 
• Phrases understood: total count of phrases selected in “Phrases” section (total 
phrases 28) 
• Words Understood: total count of words selected as either “understands” or 
“understands and says” in the “Vocabulary Checklist”. If for any given word both 
“understands” and “understands and says” are selected, the word is counted only 
once. Blanks are not counted. Total words 396. 
• Words Produced: total count of words selected as “understands and says”. If for 
any given word both “understands” and “understands and says” are selected, the 
word is counted only once. Blanks are not counted. Total words 396. 
• Early Gestures: total count of “sometimes” and “often” responses in “First 
Communicative Gestures” and “yes” answers in “Games and Routines”. 18 
maximum. 
• Later Gestures: total count of “yes” answers in “Actions with Objects,” 
“Pretending to be a Parent,” and “Imitating Other Adult Actions”. 45 maximum. 
• Total Gestures: Total of Early Gestures and Later Gesture scores. 63 maximum. 
Change from baseline for the scores will be analyzed using a Wilcoxon signed rank test. 
The change from baseline in the “words understood” score and the “words produced 
score” at EOT will be presented in plots as described in Section 9.2. Exemplars of new 


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words may be listed for individual subjects who show increases in vocabulary from 
baseline based on an increase in number of words produced.  
9.2.7 Observer-Reported Communication Ability Score 
The ORCA measure will be completed at Visit 3 (Week 0) and End of Treatment (Week 
13, Visit 16). This caregiver-reported measure was developed to assess communicative 
abilities in verbal and low-verbal/non-verbal populations. The caregiver evaluates the 
individual’s observed communication ability over the past 30 days. The ORCA measure 
will be scored using the R program provided by the ORCA developers (Center for Health 
Measurement, Duke University). The macro will produce an ORCA t-score as long as at 
least one of the 20 concepts or items has a score. A concept score will be given only if all 
items within the concept are answered. Higher scores reflect greater communication 
ability. The ORCA t-score range is 25.8 to 83.8. 
Alterative scoring algorithms are in the process of being developed by the ORCA 
developers based on feedback from users in the research community. These alternative 
algorithms would provide scoring alternatives to provide credit for emerging skills in 
addition to mastery of skills. These algorithms are in development and are not yet 
available from the ORCA developers. In line with these approaches, a modified t-score 
will also be calculated as an exploratory measure. The SAS macro used to calculate the 
total score will be edited to give credit for both “always” and “sometimes” responses (to 
assess emerging skills). This modification to the analysis of the items will be used to 
calculate the modified t-score. 
The t-score and modified t-score will be summarized using descriptive statistics. Change 
from baseline will be analyzed using a Wilcoxon signed rank test. The change from 
baseline in the t-score and modified t-score at EOT will be presented in plots as described 
in Section 9.2. 
9.2.8 Caregiver Top 3 Concerns Rating 
The Caregiver Top 3 Concerns Rating assessment will be completed at all 
Screening/Baseline period visits (Visits 1, 2, 3), as well as Week 6 (Visit 9), End of 
Treatment (Week 13, Visit 16), and Follow-up (Visit 17). The three concerns identified at 
Screening/Baseline are carried forward and rated at the follow-up visits.  
Caregivers are asked to identify 3 concerns from among 11 clinically important symptom 
domains: Language/Communication, Social interest/Avoidance, Repetitive Behaviors, 
Challenging Behaviors, Gross motor skills, Fine motor skills, Gastrointestinal problems, 
Breathing Disruptions, Sleep, Seizures, and Self-care skills. Each individual concern 


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identified should be from a different symptom domain, but caregivers can select any 
domain and choose any symptom of concern within that domain. 
The severity of each concern is scored using a 10-point Likert scale of severity. Average 
Top 3 concern severity is defined as the average of severity scores for the three concerns. 
Lower scores indicate lesser severity. The average score (i.e., average of the severity 
scores for the Top 3 concerns) will have a range of 0 to 10. Improvement in concern 
average severity is defined as a change from baseline score < 0. 
If the caregiver does not list the same 3 concerns on the Top 3 Concerns Follow-Up CRF 
that were recorded on the Top 3 Concerns Screening CRF (completed at Visit 1), then 
their responses will be reviewed and may not be included in analysis. A review of all 
concerns will be done prior to database lock, and visits with incorrect concerns will be 
flagged as unusable for analysis. 
The frequency of any symptom being chosen for all concerns combined will be 
summarized. Summary statistics for the change from baseline in the average severity and 
by symptom domain will be displayed at all post-baseline visits. Change from baseline 
will be analyzed using a Wilcoxon signed rank test at Week 6 and EOT. The frequency of 
subjects with improvement in the concern severity will be summarized by average 
concern and symptom domain. Test-retest reliability will be assessed for all scores 
collected during the Screening period (Visits 1, 2, and 3, pre-dosing) using ICC. The 
change from baseline in the average concern severity at EOT will be presented in plots, 
as described in Section 9.2. The change from baseline in domain severity scores at EOT 
will be presented in a forest plot. The domain and average concern severity will be 
presented in line graphs at each visit. 
9.2.9 Aberrant Behavior Checklist-2 Scores 
The ABC-2 will be completed at all Screening/Baseline period visits (Visits 1, 2, 3), as 
well as Week 6 (Visit 9), End of Treatment (Week 13, Visit 16), and Follow-up (Visit 
17).  Each item is scored on a 4-point scale, where 0= Not at all a problem; 1= The 
behavior is a problem but slight in degree; 2= The problem is moderately serious; 3= The 
problem is severe in degree. The items are grouped into 5 subscales: Irritability (Subscale 
I, score ranges from 0 – 45), Social Withdrawal (Subscale II, score ranges from 0 – 48), 
Stereotypic Behavior (Subscale III, score ranges from 0 – 21), 
Hyperactivity/Noncompliance (Subscale IV, score ranges from 0 – 48), and Inappropriate 
Speech (Subscale V, score ranges from 0 – 12). The total score has a range from 0 – 174. 
Lower scores indicate fewer behavior issues. The subscale scores are calculated via a 
scoring algorithm in the EDC. These calculated scores will be used for the analysis. If an 
item is missing and as such a subscale and total score was not calculated in the EDC, the 


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missing items will be imputed and subscale or total scores calculated from answered and 
imputed items. Missing items will be imputed as defined in section 5.2.5. The total score 
will only be calculated if all items are answered or can be imputed. If a total score is 
missing after applying the imputation algorithm, then that subject will be excluded from 
the corresponding summaries. The subscales and their corresponding items are listed 
below. 
• Subscale I (Irritability): Items 2, 4, 8, 10, 14, 19, 25, 29, 34, 36, 41, 47, 50, 52, 57. 
• Subscale II (Social Withdrawal): Items 3, 5, 12, 16, 20, 23, 26, 30, 32, 37, 40, 42, 
43, 53, 55, 58 
• Subscale III (Stereotypic Behavior): Items 6, 11, 17, 27, 35, 45, 49 
• Subscale IV (Hyperactivity/Noncompliance): Items 1, 7, 13, 15, 18, 21, 24, 28, 
31, 38, 39, 44, 48, 51, 54, 56 
• Subscale V (Inappropriate Speech): Items 9, 22, 33, 46 
The subscale scores are calculated as the sum of item scores within the subscale. The 
total score is calculated as the sum of all items across all subscales. 
Within subject change is the difference between the score at each relevant follow-up visit 
and the score at baseline. Summary statistics for the change from baseline in total and 
each subscale ABC-2 score will be displayed at all post-baseline visits. Change from 
baseline in the total and subscale scores will be analyzed using the Wilcoxon signed rank 
test at Week 6 and EOT. Test-retest reliability will be assessed for all scores collected 
during the Screening period (visits 1, 2, and 3, pre-dosing) using ICC. The change from 
baseline in the total score at EOT will be presented in plots as described in Section 9.2. 
The change from baseline in subscale scores at EOT will be presented in a forest plot. 
The total and subscale scores will be present in line graphs at each visit. 
9.2.10 Behavior Problems Inventory-Short Form  
The BPI-SF will be completed at Visit 3 (Week 0) and at the End of Treatment (Week 13, 
Visit 16). The BPI-SF is a caregiver-completed rating scale for assessing self-injury, 
aggressive and stereotyped behavior. Each item is measured for frequency and items in 
the Self-Injurious Behavior and Aggressive/Destructive Behavior subscales are also 
assessed for severity. The frequency is scored on a 5-point scale, where 0=never/no 
problem; 1=monthly, 2=weekly, 3=daily, 4=hourly. The severity is scored on a 3-point 
scale, where 1=mild, 2=moderate, 3=severe. The items are grouped in three subscales: 
Self-Injurious Behavior (score ranges from 0 – 32 for frequency and 8 – 24 for severity), 
Aggressive/Destructive Behavior (score ranges from 0 – 40 for frequency and 10 – 30 for 
severity), and Stereotyped Behavior (score ranges from 0 – 48 for frequency). The score 
for each subscale is the sum of the items within that domain. The total frequency score is 


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the sum of the frequency scores for the three domains. The total severity score is the sum 
of the severity scores for Aggressive/Destructive Behavior and Self-Injurious Behavior 
subscales. Missing items will be imputed as defined in Section 5.2.5. A subscale score 
will only be calculated if all items in that domain are answered or can be imputed. Lower 
scores indicate lesser frequency and severity in behavior problems. Within subject change 
is the difference between the score at EOT and the score at baseline. 
Summary statistics for the total score, subscale scores and their changes from baseline in 
the frequency and severity of BPI-SF scores will be displayed at End of Treatment. 
Within subject changes will be analyzed using a Wilcoxon signed rank test. The change 
from baseline in the total frequency and severity scores at EOT will be presented in plots 
as described in Section 9.2. The change from baseline in the subscale scores at EOT will 
be presented in a forest plot and waterfall plots. The subscale and total scores will be 
presented in line graphs at each visit. 
9.2.11 Child Sleep Habits Questionnaire Scores 
The CSHQ will be completed at all Screening/Baseline period visits (Visits 1, 2, 3), as 
well as Week 6 (Visit 9), End of Treatment (Week 13, Visit 16), and Follow-up (Visit 
17). Each item is scored on a 3-point scale, where 1=rarely; 2=sometimes; 3=usually. 
Items 32 and 33 are scores as 1 = not sleepy, 2 = very sleepy and 3 = falls asleep. A 
higher score reflects more disturbed sleep behavior. The items are grouped into 4 
domains and 8 subscale scores are derived from the items in these domains. The subscale 
scores are a sum of the items within that subscale, except for a few items which are 
reversed for scoring.  Missing items will be imputed as defined in Section 5.2.5. The 
subscale scores will only be calculated if all items in the subscale are answered or can be 
imputed. The subscales and their corresponding items are listed below. 
• Bedtime Resistance: Items 1, 3, 4, 5, 6, 8, where items 1 and 3 are reversed. 
Subscale score ranges from 6 – 18. 
• Sleep Onset Delay: Item 2 where item 2 is reversed. Subscale score ranges from 1 
– 3. 
• Sleep Duration: Items 9, 10, 11, where items 10 and 11 are reversed. Subscale 
score ranges from 3 – 9.  
• Sleep Anxiety: Items 5, 7, 8, 21. Subscale score ranges from 4-12. 
• Night Wakings: Items 16, 24, 25. Subscale score ranges from 3-9.  
• Parasomnias: Items 12, 13, 14, 15, 17, 22, 23. Subscale score ranges from 7-21. 
• Sleep Disordered Breathing: Items 18, 19, 20. Subscale score ranges from 3-9. 
• Daytime Sleepiness: Items 26, 27, 28, 29, 30, 31, 32, 33, where item 26 is 
reversed. Subscale score ranges from 8-24.  


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The total score will be calculated as a sum of all 33 items, with items 1 – 3, 10 – 11, and 
26 reversed. The total score will range from 33 – 99 and will only be calculated if all 
items are answered or can be imputed. Missing items will be imputed as defined in 
Section 5.2.5. Within subject change is the difference between the score at each relevant 
follow-up visit and the score at baseline. 
Summary statistics for the scores and their changes from baseline in the total score and 
subscale CSHQ scores will be displayed at all post-baseline visits. Test-retest reliability 
will be assessed for all scores collected during the Screening period (Visit 1, 2, and 3, 
pre-dosing) using Intraclass Correlation Coefficients (ICC). Changes from baseline will 
be analyzed using a Wilcoxon signed rank test at Week 6 and EOT. The change from 
baseline in the total score at EOT will be presented in plots as described in Section 9.2. 
The change from baseline in the subscale scores at EOT will be presented in a forest plot, 
as described in Section 9.2. The subscale and total scores will be presented in line graphs 
at each visit. 
9.2.12 Gastrointestinal Health Questionnaire Scores 
The GIHQ will be completed at all Screening/Baseline period visits (Visits 1, 2, 3), as 
well as Week 6 (Visit 9), End of Treatment (Week 13, Visit 16), and Follow-up (Visit 
17).  Each item is scored on its frequency, relevancy, and importance to the caregiver. 
The Frequency is measured on a 5-point scale where 0=never; 1=almost never; 
2=sometimes; 3=often; 4=almost always. The surgery domain records occurrence as 
0=No and 1=Yes. The relevancy is scored on a scale from 1 to 4, where 1 is not relevant 
and 4 is very relevant. The importance is scored on a scale from 1 to 4, where 1 is not 
important and 4 is very important. Lower frequency scores indicate fewer gastrointestinal 
problems. For relevancy, higher scores indicate the question is more relevant and for 
importance, higher scores indicate the question is more important. The total frequency 
score is the sum of all item frequency scores. Similarly, the total relevancy and total 
importance scores are the sum of all item relevancy and importance scores, respectively. 
The items are grouped into 9 subscales. The subscale score is the sum of all items within 
the subscale, as shown on the CRF. Missing items will be imputed as defined in Section 
5.2.5. The subscale scores will only be calculated if all items within the domain are 
answered or can be imputed. The subscales are as follows: 
• General Health/Pain: Items 1 – 5, frequency score ranges from 0 – 20, relevancy 
score ranges from 5 – 20, importance score ranges from 5 – 20. 
• Eating, Chewing, and Swallowing: Items 1 – 9, frequency score ranges from 0 – 
36, relevancy score ranges from 9 – 36, importance score ranges from 9 – 36. 
• Reflux: Items 1 – 3, frequency score ranges from 0 – 12, relevancy score ranges 
from 3 – 12, importance score ranges from 3 – 12. 


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• Gas and Bloating: Items 1 – 5, frequency score ranges from 0 – 20, relevancy 
score ranges from 5 – 20, importance score ranges from 5 – 20. 
• Diarrhea and Constipation: Items 1 – 6, frequency score ranges from 0 – 24, 
relevancy score ranges from 6 – 24, importance score ranges from 6 – 24. 
• Personality and Mood: Items 1 – 5, frequency score ranges from 0 – 20, 
Relevancy score ranges from 5 – 20, importance score ranges from 5 – 20. 
• Medications: Items 1 – 9, frequency score ranges from 0 – 36, relevancy score 
ranges from 9 – 36, importance score ranges from 9 – 36. 
• Surgery: Items 1 – 5, frequency score ranges from 0 – 5, relevancy score ranges 
from 5 – 20, importance score ranges from 5 – 20. 
• Parenting: Items 1 – 8, frequency score ranges from 0 – 24, relevancy score 
ranges from 8 – 24, importance score ranges from 8 – 24. 
Within subject change is the difference between the score at each relevant follow-up visit 
and the score at baseline. Summary statistics for the change from baseline in the total and 
each GIHQ domain score will be displayed at all post-baseline visits. Test-retest 
reliability will be assessed for all scores collected during the Screening period (Visits 1, 
2, and 3, pre-dosing) using ICC. Changes from baseline will be analyzed using a 
Wilcoxon signed rank test at Week 6 and EOT. The change from baseline in the total 
frequency score at EOT will be presented in plots as described in Section 9.2. The change 
from baseline in the domain frequency scores at EOT will be presented in a forest plot 
and waterfall plots. The domain and total frequency scores will be presented in line 
graphs at each visit, with the exception of the Surgery domain, which is scored differently 
from the other domains and is not comparable. 
9.2.13 Vineland Adaptive Behavior Scales-3 (VABS-3) 
The VABS-3 (Comprehensive Interview version) will be completed at Visit 3 (Week 0) 
and End of Treatment (Week 13, Visit 16) as a clinical interview. Scores are calculated 
by the site rater. An Adaptive Behavior Composite (ABC) score will be calculated, along 
with a standard score for each of the 4 domains: Communication, Daily Living Skills, 
Socialization, Motor Skills. For the motor skills domain, standard scores can only be 
calculated for participants ages 3-9. These range from 20 to 140.  
In each of the 11 subdomains, the raw scale (range 0 to 116), v-scale (range 1 to 24), age 
equivalent (range 0:0 to 22:0+), and growth scale (range 10 to 197) scores are recorded. 
Lower scores reflect less functional abilities. If a score is missing, that subject will be 
excluded from corresponding summaries. Within subject change is the difference 
between the score at EOT and the score at baseline. 


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Summary statistics for the change from baseline in the Vineland Adaptive Behavior 
Scales-3 composite and domain standard scores, and subdomain raw, v-scale, and growth 
scale scores will be displayed at end of treatment. Changes from baseline will be 
analyzed using a Wilcoxon signed rank test. The change from baseline in the composite 
standard score, raw scores and GSV at EOT will be presented in plots as described in 
Section 9.2. The change from baseline in the domain standard scores and subdomains at 
EOT will be presented in a forest plot. The domain standard scores and subdomain scores 
will be presented in line graphs at each visit. 
9.2.14 Quality of Life Inventory - Disability (QI-Disability) Scores 
The QI-Disability Score will be completed at Visit 3 (Week 0) and at the End of 
Treatment (Week 13, Visit 16). Each item is scored on a 5-point scale, where 1=never; 
2=rarely; 3=sometimes; 4=often; 5=very often. The items are grouped into six domains 
for analysis: physical health (items 1-4), positive emotions (items 5-8), negative emotions 
(items 9-15), social interaction (items 16-22), leisure and the outdoors (items 23-27), and 
independence (items 28-32). The domain scores are calculated by first transforming the 
5-point scale, so that 1=0, 2=25, 3=50, 4=75, and 5=100. Items 9-15 are reversed in 
scoring, so that 1=100, 2=75, 3=50, 4=25, and 5=0. The domain score is the average of 
the transformed score of the items within that domain. Missing items will be imputed as 
defined in Section 5.2.5. A domain score will be calculated as long as at least one of the 
items within the domain is answered. The overall score is the average of all domain 
scores and will be calculated as long as at least one of the domain scores can be 
calculated. Higher scores indicate a better quality of life. Within subject change is the 
difference between the score at EOT and the score at baseline. 
Summary statistics for the change from baseline in the QI-Disability overall and domain 
scores will be displayed at end of treatment. Changes from baseline will be analyzed 
using a Wilcoxon signed rank test. The change from baseline in the overall score at EOT 
will be presented in plots as described in Section 9.2. The change from baseline in the 
domain scores at EOT will be presented in a forest plot. The domain and overall scores 
will be presented in line graphs at each visit. 
9.2.15 Impact of Childhood Neurological Disability Overall quality of life rating  
The ICND Scale will be administered at Visit 3 (Week 0) and at End of Treatment (Week 
13, Visit 16). The ICND is scored on a scale from 1 to 6, where 1=poor and 6=excellent. 
Higher scores indicate better quality of life. Within subject change is the difference 
between the score at EOT and the score at baseline. If a score is missing, that subject will 
be excluded from corresponding summaries. If in the EDC, the numeric score is missing 
and only the descriptor is present, the numeric equivalent score will be used for analysis.  


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Summary statistics for the observed and change from baseline in the ICND score will be 
displayed at end of treatment. Change from baseline will be analyzed using a Wilcoxon 
signed rank test. The change from baseline in the ICND score at EOT will be presented in 
plots, as described in Section 9.2. 
9.2.16 Bayley Scales of Infant and Toddler Development version 4 
The BSID-4 will be performed at Screening (Visit 1) and End of Treatment (Week 13, 
Visit 16) on those subjects who cannot be assessed using the SB5. It assesses cognitive 
ability, motor skills, and behavior and provides standardized scores in five domains: 
Cognitive (COG), Language (LANG), Motor (MOT), Social-Emotional (SOEM), and 
Adaptive Behavior. The Adaptive Behavior scale was not collected for this study. 
Domain scores range from 40 – 160, where higher scores indicate greater ability in the 
domain area of development. Scaled scores, age equivalents and growth scale values 
(GSVs) can also be calculated for the scale subtests: cognition (CG), receptive 
communication (RC), expressive communication (EC), fine motor (FM), and gross motor 
(GM). Scaled scores and age equivalents can be calculated for the social emotional (SE) 
subtest. Scale subtest scores range from 1 – 19 with a mean of 10 and SD of 3. Age 
equivalent scores range from < 0:15 – >41:08, Growth scale scores range from 414   – 
559 with a mean of 500 and SD of 25. Higher scores indicate greater development in the 
scale subtest. Raw scores, age equivalent scores and GSV are collected for all subjects. 
Standard scores and scaled scores are only calculated for participants 42 months and 
younger.  
As part of the analysis, an overall developmental quotient, a verbal developmental 
quotient, and a non-verbal developmental quotient will also be calculated. These 
developmental quotients will only be calculated if the contributing scale scores are all 
non-missing. The developmental quotients will be derived as follows: 
Overall Developmental Quotient: 
(Mean of the age-equivalent scores in months of the cognition, fine motor, receptive 
communication, expressive communications scales/chronological age in months) x 100 
Verbal Developmental Quotient: 
(Mean of the age-equivalent scores in months of the expressive communication and 
receptive scales/chronological age in months) x 100 
 
 


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Non-verbal Developmental Quotient: 
(Mean of the age-equivalent scores in months of the cognition and fine motor 
scales/chronological age in months) x 100 
Subtest/Subdomain Developmental Quotients: 
The developmental quotients for the BSID subtests may also be derived if EDC 
calculations were not completed. The developmental quotients for each subtest are 
calculated in a similar manner as the overall, non-verbal, and verbal developmental 
quotients. Subtest Developmental Quotient = (Age Equivalent Score in Months for the 
subtest / chronological age in months) x 100.  
Chronological age in months will be calculated as (date of assessment – date of birth + 
1)/30.4375. 
For age equivalent values < 2, the result is entered into EDC as 1.9, for analysis. For age 
equivalent values > X, where X is a numeric value, the result is entered into EDC as X.1, 
for analysis. For example, “>33” is entered in EDC as 33.1. 
The standard scores have a mean of 100 and a standard deviation of 15. The 
developmental quotient scores are a ratio of developmental age to chronological age and 
will have a range of 1 to 100. Higher scores indicate a higher IQ or better developmental 
progress. If any scores are missing, the subject will be excluded from corresponding 
summaries. 
Within subject change is the difference between the score at EOT and the score at 
baseline. If a score is missing, that subject will be excluded from the corresponding 
summaries. 
Summary statistics for the observed and change from baseline in the age equivalent, 
growth scale subtest scores, the standard and scale scores (for participants for whom the 
scores are calculated), and developmental quotients will be displayed at end of treatment. 
Changes from baseline will be analyzed using a Wilcoxon signed rank test. The change 
from baseline in the raw scores and GSV at EOT will be presented in plots as described 
in Section 9.2. The change from baseline in the raw and GSV domain scores will be 
presented in a forest plot, as described in Section 9.2. 
9.2.17 Modified two-minute walk test  
The modified 2MWT be administered at Screening (Visit 1), Visit 3 (Week 0) and at End 
of Treatment (Week 13, Visit 16). The walk test measures how far an individual can walk 
during a 2-minute period and assesses endurance which has relevance for everyday 


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functioning. The distance is measured in meters and greater total distance indicates 
greater physical ability. Within subject change is the difference between the score at EOT 
and the score at baseline. If a score is missing, that subject will be excluded from 
corresponding summaries. 
Summary statistics for the observed and change from baseline in the total distance 
measured during the walk test will be displayed at end of treatment. Change from 
baseline will be analyzed using a Wilcoxon signed rank test. Test-retest reliability will be 
assessed for all scores collected during Screening period (Visits 1 and 3) using ICC. The 
change from baseline in total distance at EOT will be presented in plots, as described in 
Section 9.2. 
9.2.18 Stanford-Binet Intelligence Scales version 5  
The SB5 will be performed at Screening and Visit 16 (End of Treatment). Participants 
who cannot be assessed on the SB5 due to their developmental level will be assessed 
using the BSID-4 instead. General cognitive ability is reported as a full scale intellectual 
quotient score (FSIQ). Cognitive ability in five categories is reported in the index scores: 
fluid reasoning (FR), knowledge (KN), quantitative reasoning (QR), visual-spatial 
processing (VS), and working memory (WM). Non-verbal (NVIQ) and verbal 
intelligence quotients (VIQ) are also recorded. Scores range from 40 to 160, where higher 
scores indicate a higher IQ. For the analysis of index scores and IQ, the z-deviation 
method for intellectual disability will be used and z-deviation scores will be derived 
based on age at assessment according to the published algorithm (Sansone et al. 2014). 
Standard scores, change-sensitive scores, age equivalents and the raw scores are 
calculated and entered by the site rater.  
For age equivalent values < 2, the result is entered into EDC as 1.9, for analysis. For age 
equivalent values > x, where X is a numeric value, the result is entered into EDC as X.1, 
for analysis. For example, “>33” is entered in EDC as 33.1. 
Summary statistics on the observed and change from baseline in the z-deviation scores, 
change-sensitive scores and age equivalents for FSIQ, NVIQ, VIQ, and the domain 
scores: FR, KN, QR, VS, WM. Each category will be displayed at end of treatment. The 
change from baseline in FSIQ, NVIQ, and VIQ z-deviation scores at EOT will be 
presented in plots as described in Section 9.2. The change from baseline in the domain z-
deviation scores will be presented in a forest plot, as described in Section 9.2. 
 
 


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9.3 Exploratory Efficacy Analysis  
9.3.1 Biomarkers and gut microbiome 
Blood and stool samples will be collected at Screening (Visit 1), Week 0 (Visit 3, before 
dosing), and at End of Treatment (Visit 8), or upon early termination. Biomarker and gut 
microbiome will be analyzed separately outside of this analysis plan. 
9.3.2 Correlation of Efficacy Endpoints 
The potential covariates in this study are listed in Section 8.2. A 2x2 correlation analysis 
will be performed on all combinations to evaluate the relationship between the covariates. 
The Pearson Correlation Coefficient will be used to measure the association between age 
as a continuous variable and the SB5 NVIQ or BSID-4 NVDQ as described in Section 
9.2. If the coefficient is <.5, we will assume little to no association. The Point-Biserial 
Correlation Coefficient will also be used to measure the association between 
combinations of continuous and binary variables such as SB5 NVIQ/BSID-4 NVDQ and 
ASD diagnosis, respectively. The binary variables will be assigned a numeric value of 1 
or 2. The Pearson Chi-Square test will be used to measure the association between 
combinations of categorical variables such as age group and genotype or sex and history 
of regression. A p-value < 0.10 would indicate association between the variables.  
The correlation analysis will support the selection of covariates (or combinations thereof) 
to be used in a linear regression analysis on all pre-specified endpoints. Endpoints of 
interest will be: 
• CGI-I Overall Score 
• CGI-S Overall Score  
• ORCA t-score 
• BPI-SF Total score 
• ABC-2 Total Score 
• CSHQ Total Score 
• GIHQ Total Score 
• Vineland ABC score 
• Vineland GSV, raw, and standard domain scores 
• QI-Disability Overall Score 
• ICND Score  


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9.3.3 Additional exploratory analyses 
Additional analyses will be performed to examine the empirical cumulative distribution 
function of the change from baseline at EOT for the ORCA t-score, CGI-I, CGI-S,  ABC-
2 total score, CSHQ total score, GIHQ total frequency score, the BPI-SF total score, each 
BPI-SF domain score, VABS-3 composite score, VABS-3 GSV scores, VABS-3 raw 
scores, QL-Disability overall score, and ICND score will be plotted. 
Test-retest reliability will be assessed for assessments collected more than once during 
the Screening period (Visits 1, 2, and 3, pre-dosing) using ICC. A correlation analysis of 
the efficacy measures will be done using Pearson correlation coefficients. The correlation 
of each total or overall score and CGI-I and CGI-S domain scores at EOT will be 
measured against the others and presented in a table and a heat map/matrix.  
Waterfall plots may also be created for each post-baseline visit with the plots cross-
hatched to indicate genotype, History of Regression (yes, no), ASD diagnosis (yes no), 
depending on the number of subjects in each subgroup. 
10 Tables, Listings, and Figures 
A list of the tables, listings, and figures will be maintained outside of this document and 
may be amended as needed. 
Data listings will be sorted by subject number, then chronologically (if appropriate) as 
indicated by visit, date, and time. All data recorded on the CRFs will be included in the 
listings as appropriate as well as some derived information. Data on other treatments (e.g. 
behavioral treatments, supplements and special diets) were originally collected on three 
separate CRFs in the EDC: “Other Treatments”, “Supplements and Complementary 
Treatments” and “Behavioral/Educational Treatments”. Since the CRFs were duplicative 
the “Supplements and Complementary Treatments” and “Behavioral/Educational 
Treatments” were closed and only the “Other Treatments” CRF page was used. For the 
listing, the data from these three CRF pages will be combined and included in a single 
listing “Other Treatments.” The “New Symptoms Log” was closed so the listing will only 
include data up until the time the log was closed in the EDC.  
Listings of Subject Disposition, Protocol Deviations, and Inclusion/Exclusion Criteria 
Not Met will display results for All Subjects. Other listings will display results for the 
Safety or Intent to Treat Populations as appropriate. 
Rounding for all variables will occur only as the last step, immediately prior to 
presentation in listings, tables, and figures.  No intermediate rounding will be performed 


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on derived variables.  The standard rounding practice of rounding numbers ending in 0-4 
down and numbers ending in 5-9 up will be employed.   
Tables will generally be presented with the parameter in the first column followed by the 
summary column(s). The presentation of numerical values will adhere to the following 
guidelines:  
• Raw measurements will be reported to the number of significant digits as captured 
electronically or on the CRFs.  
• Rounding: 
o For derived parameters or raw results with more than 2 decimals:  
▪ Standard deviations will be reported to four decimal places 
▪ Means and medians will be reported to three decimal places  
▪ Minimums and maximums will be reported to two decimal places 
o For other parameters: 
▪ Standard deviations will be reported to two decimal places beyond 
the number of decimal places the original parameter is presented. 
▪ Means and medians will be reported to one decimal place beyond 
the number of decimal places the original parameter is presented.  
▪ Minimums and maximums will be reported to the same number of 
significant digits as the parameter.  
• Calculated percentages will be reported with no decimals. 
P-values will be presented to four decimal places. If a p-value is less than 0.0001 then it 
will be presented as “<0.0001”.  
11 References 
Sansone SM, Schneider A, Bickel E, Berry-Kravis E, Prescott C, Hessl D. Improving IQ 
measurement in intellectual disabilities using true deviation from population norms. J 
Neurodev Disord. 2014;6(1):16. 
ORCA Measure Scoring Manual. 2023. https://pattern.health/wp-
content/uploads/2023/04/ORCA-Measure-Scoring-Manual_Final_2023_04_18.pdf