Clinical trial NCT04279314
Conditions: Rett Syndrome
Sponsor: ACADIA Pharmaceuticals Inc.
Start date: 2020-01-29; Completion date: 2022-08-19
Enrollment: 154 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
To investigate the safety and tolerability of long-term treatment with oral trofinetide in girls and women with Rett syndrome

Detailed description:

Arms: arm 1: None
Interventions: intervention 1: Trofinetide
Intervention descriptions: intervention 1: Trofinetide solution of 30-60 mL based on subject's weight at Baseline, administered twice daily by mouth or gastrostomy tube (G-tube)

Protocol documents:
Prot_000.pdf: 
Official Title: A 40-Week, Open-label Extension Study of Trofinetide for the 
Treatment of Girls and Women with Rett Syndrome 
NCT Numbers: NCT04279314 
Document Date: 7 August 2020 


Study: ACP-2566-004 
 
Final Version: 1.0 
Clinical Study Protocol Amendment 2 
 
Date: 07 August 2020 
 
    Confidential and Proprietary Information of ACADIA Pharmaceuticals Inc. 
Page 1 of 76 
 
 
 
 
CLINICAL STUDY PROTOCOL 
 
A 40-Week, Open-label Extension Study of Trofinetide for the Treatment of 
Girls and Women with Rett Syndrome  
 
Protocol Number: ACP-2566-004 
Amendment 2 
 
Original Protocol Date: 02 April 2019 
Protocol Amendment 1 Date: 27 April 2020 
Protocol Amendment 2 Date: 07 August 2020 
 
 
 
 
Protocol Template Version: 1.0  
Confidentiality Statement 
This protocol is the confidential information of ACADIA Pharmaceuticals Inc. and is intended solely for the 
guidance of the clinical investigation. This protocol may not be disclosed to parties not associated with the 
clinical investigation or used for any purpose without the prior written consent of ACADIA Pharmaceuticals 
Inc. 


Study: ACP-2566-004 
 
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SPONSOR SIGNATURE PAGE 
Title: A 40-Week, Open-label Extension Study of Trofinetide for the Treatment of Girls and 
Women with Rett Syndrome 
 
ACADIA Head of Clinical Development: 
 
SVP, Head of Clinical Development 
ACADIA Pharmaceuticals Inc. 
  
 
 
 
 
 
 
 
 
see appended electronic signature page 
 
Signature 
 
Date 
 
 
ACADIA Project Lead: 
 
VP, Clinical Development 
ACADIA Pharmaceuticals Inc. 
 
 
 
 
 
 
 
see appended electronic signature page 
 
Signature 
 
Date 
 


Signature Page for ACP-2566-004 Protocol Amendment 2
Signature Page for ACP-2566-004 Protocol Amendment 2
Approve
13-Aug-2020 11:18:50 GMT+0000
Approve
20-Aug-2020 13:27:43 GMT+0000


Study: ACP-2566-004 
 
Final Version: 1.0 
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Date: 07 August 2020 
 
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DECLARATION OF INVESTIGATOR 
I confirm that I have read the above protocol. I understand it, and I will work according to the 
moral, ethical, and scientific principles governing clinical research as set out in the principles 
of Good Clinical Practice, as required by International Council for Harmonisation of 
Technical Requirements for Pharmaceuticals for Human Use (ICH) Guideline E6 and as 
described in the United States (US) Code of Federal Regulations (CFR) 21 CFR parts 50, 54, 
56, 312, and according to applicable local requirements. 
Confidentiality Statement 
The confidential information in this document is provided to you as a Principal Investigator 
or Consultant for review by you, your staff, and the applicable institutional review 
board/ethics committee. Your acceptance of this document constitutes agreement that you 
will not disclose the information contained herein to others without written authorization 
from the Sponsor. 
Principal Investigator 
 
 
Signature 
 
Date 
 
 
 
Name (printed) 
 
 
 
 


Study: ACP-2566-004 
 
Final Version: 1.0 
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Date: 07 August 2020 
 
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PROTOCOL SYNOPSIS1 
Protocol 
Number 
ACP-2566-004 
EudraCT 
Number 
Not applicable 
Protocol Title 
A 40-Week, Open-label Extension Study of Trofinetide for the Treatment 
of Girls and Women with Rett Syndrome 
Name of 
Investigational 
Product 
Trofinetide oral solution 
Indication 
Rett syndrome  
Phase of 
Development 
3 
Sponsor 
ACADIA Pharmaceuticals Inc. 
 
 
Primary Objective 
• To investigate the safety and 
tolerability of long-term treatment 
with oral trofinetide in girls and 
women with Rett syndrome 
 
Primary Endpoints 
• Treatment-emergent adverse events 
(TEAEs) 
• Serious adverse events (SAEs) 
• Withdrawals due to adverse events (AEs) 
• Potentially clinically important changes in 
other safety assessments 
Secondary Objectives 
• To investigate the efficacy of 
long-term treatment with oral 
trofinetide in girls and women with 
Rett syndrome 
 
Secondary Endpoints 
• Rett Syndrome Behaviour Questionnaire 
(RSBQ) total score – Change from 
Baseline to Week 40 
• Clinical Global Impression–Improvement 
(CGI-I) Score at Week 40 (assessing 
 
 
 
1 NOTE: In this protocol, as in FDA guidance documents, the use of the word ”should” means that something is 
suggested or recommended, but not required. If something “may be” done, the meaning is that something is 
allowed, but not required. The terms “must” and “will” mean that something is required. 


Study: ACP-2566-004 
 
Final Version: 1.0 
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Date: 07 August 2020 
 
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Page 6 of 76 
 
 
• To investigate the efficacy of 
long-term treatment with oral 
trofinetide in girls and women with 
Rett syndrome on overall ability to 
communicate  
• To investigate the benefit of long-term 
treatment with oral trofinetide on 
overall quality of life for girls and 
women with Rett syndrome 
• To investigate the efficacy of 
long-term treatment with oral 
trofinetide in girls and women with 
Rett syndrome on: 
o Hand function 
o Ambulation and other gross 
motor skills 
o Ability to communicate 
choices and preferences 
o Ability to communicate 
verbally 
• To investigate the efficacy of 
long-term treatment with oral 
trofinetide on a global assessment of 
the severity of illness in girls and 
women with Rett syndrome 
• To investigate the benefit of long-term 
treatment with oral trofinetide on the 
burden on caregivers of girls and 
women with Rett syndrome 
• To investigate the benefit of long-term 
treatment with oral trofinetide on the 
impact of the disability on the child’s 
and family’s everyday life 
improvement from Baseline of the present 
study) 
Change from Baseline to Week 40 in: 
• Communication and Symbolic Behavior 
Scales-Developmental Profile™ 
Infant-Toddler Checklist – Social 
Composite Score (CSBS-DP-IT Social) – 
Change from Baseline to Week 40 
• Overall Quality of Life Rating of the 
Impact of Childhood Neurologic Disability 
(ICND) Scale 
• Rett Syndrome Clinician Rating of Hand 
Function (RTT-HF) 
• Rett Syndrome Clinician Rating of 
Ambulation and Gross Motor Skills 
(RTT-AMB) 
• Rett Syndrome Clinician Rating of Ability 
to Communicate Choices (RTT-COMC) 
• Rett Syndrome Clinician Rating of Verbal 
Communication (RTT-VCOM) 
• Clinical Global Impression–Severity 
(CGI-S) 
• Rett Syndrome Caregiver Burden 
Inventory (RTT-CBI) Total Score (items 
1-24) 
• Impact of Childhood Neurologic Disability 
(ICND) Scale Total Score 
Pharmacokinetic Objectives 
• To characterize the pharmacokinetics 
(PK) of trofinetide in girls and women 
with Rett syndrome following 
Pharmacokinetic Endpoints 
• Whole blood concentration of trofinetide 
and possible metabolites 


Study: ACP-2566-004 
 
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long-term treatment with oral 
trofinetide 
• To assess the 
pharmacokinetic/pharmacodynamic 
(PK/PD) relationship using safety and 
efficacy endpoints in girls and women 
with Rett syndrome following 
long-term treatment with oral 
trofinetide 
• Trofinetide PK parameters using the 
population PK approach 
• PK/PD using appropriate PK/PD analysis 
methods  
Number of 
Study Sites 
Approximately 28 sites will participate in this study. 
Number of 
Subjects 
Planned 
Approximately 180 subjects are expected to be enrolled. 
Test Product, 
Dose, and 
Administration 
Subjects will receive an oral dose of trofinetide for up to 40 weeks. 
For subjects who remained on the originally assigned dose in the 
antecedent study, the assigned dose in this study will be based on the 
subject’s weight at the Baseline visit of the present study, as outlined 
below in Table S–1. Thus, for a particular subject whose weight at the 
Baseline of this study moves them into a different weight category 
according to Table S-1, the assigned dose given in this study may be 
different from the originally assigned dose given in the antecedent 
study. After Baseline the dose will not be changed based on a change in 
weight that places the subject into a different weight category.  
Subjects whose originally assigned dose in the antecedent study was 
decreased for tolerability reasons will remain on that same dose in this 
study. The dose should be increased at some point during the study, if 
tolerated, to the dose that would have been assigned based on the 
weight category at Baseline of the present study. 
Doses may be administered by gastrostomy (G) tube (doses 
administered via gastrojejunal [GJ] tubes must be administered through 
the G-port). 
Table S–1 
Dosing Schedule Based on Weight at Baseline of the 
Present Study 
Weight 
Dose  
Total Daily Dose 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 


Study: ACP-2566-004 
 
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Abbreviations: BID=twice daily 
If the subject cannot tolerate administration of the full assigned dose 
according to Table S-1 (for example, if the subject experiences 
diarrhea), the Investigator may instruct the caregiver to reduce study 
drug to a dose as low as half the assigned dose. If the subject’s assigned 
dose at the beginning of this study was decreased for tolerability in the 
antecedent Study ACP-2566-003, the dose in the present study may be 
reduced to a dose as low as half the originally assigned dose from Study 
ACP-2566-003. For example, a subject who was originally assigned a 
dose of 40 mL (8 g) BID in Study ACP-2566-003 cannot have their 
dose in this study reduced to less than 20 mL (4 g) BID. At no point 
may the subject be on a dose that is lower than half the assigned dose. 
In addition, up to 4 doses (in total, consecutive or non-consecutive) may 
be held for this reason.  
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 subject is again 
unable to tolerate that dose, the Investigator will continue treatment on 
the highest dose that the subject can tolerate which should be no lower 
than half the assigned dose. The final dose must be given BID, with the 
same dose given morning and evening. 
Coronavirus 
Disease 2019 
(COVID-19) 
Update 
As a result of the COVID-19 public health emergency, this protocol has 
incorporated revisions to study methodology in accordance with the 
FDA Guidance on Conduct of Clinical Trials of Medical Products 
during the COVID-19 Public Health Emergency. Specifically, 
alternative methods of performing safety and efficacy assessments 
include provisions for off-site assessments. The implementation of 
alternative processes were to be consistent with the protocol to the 
extent possible, and clinical investigators were to document the reason 
for any contingency measures implemented. 
Study Design 
This is a 40-week, multicenter, open-label extension (OLE) study to 
evaluate long-term safety and tolerability of trofinetide in girls and 
women with Rett syndrome. Subjects who complete the preceding 
double-blind study (ACP-2566-003) will be eligible to enroll in the 
OLE. Legally acceptable representatives (LARs)/subjects must be 
consented prior to the procedures being performed at the Week 12/end 
of treatment (EOT) visit of the antecedent study. The Week 12/EOT 
visit of the antecedent study will serve as the Baseline visit of the 
present study. The data gathered at the Week 12/EOT visit of the 
antecedent study serves as the baseline data of the present study.  
Because of the special circumstances surrounding the COVID-19 public 
health emergency in the spring of 2020, subjects who were discontinued 


Study: ACP-2566-004 
 
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from Study ACP-2566-003 before completion because of the 
COVID-19 public health emergency and subjects who completed Study 
ACP-2566-003, but were prevented from entering the present study 
because of the COVID-19 public health emergency, may be 
re-evaluated for eligibility at the Baseline visit of the present study. 
However, subjects in the future who are discontinued from Study 
ACP-2566-003, or who are not able to rollover to this study at the 
Week 12/EOT visit of the antecedent study, will not be able to enter the 
present study.   
The study will have two periods: 
• Treatment period: 40 weeks   
• Safety follow-up period: 30 days 
 
Open-label Treatment Period (40 Weeks) 
The Week 12/EOT visit of the antecedent study (ACP-2566-003) 
generally serves as the Baseline visit (Visit 1) of the present study.  
Those subjects for whom it is not possible to use the Week 12/EOT visit 
of the antecedent study (ACP-2566-003) as the Baseline visit (Visit 1) 
may be re-evaluated for study eligibility at the Baseline visit of the 
present study, with approval by the Sponsor or Medical Monitor. Those 
subjects will have blood drawn for Baseline values 3 to 14 days before 
the Baseline visit. These subjects include: 1) those who were 
discontinued from Study ACP-2566-003 before completion because of 
the COVID-19 public health emergency; and 2) those who completed 
Study ACP-2566-003, but were prevented from entering this study 
because of the COVID-19 public health emergency.  
All efficacy assessments (except the CGI-I) will be completed at the 
Baseline visit prior to administration of the first dose of study drug in 
the present study (Table S–2).  
The first dose of study drug for the present study is intended to be 
administered after all Baseline assessments are completed. Since in 
most cases, the subject will have taken the last dose of study drug from 
the antecedent study on the morning of the Baseline visit for this study, 
the first dose of study drug for this study will then be administered 
either as an evening dose after the Baseline visit has been completed or 
on the following day. A triplicate electrocardiogram (ECG) must be 
performed 2-3 hours after the first dose and a PK sample will be taken 
upon completion of the ECG.  
Study drug must be discontinued at any time in the study in the event 
that a post-enrollment QTcF duration of ≥500 ms or an increase of 
≥60 ms compared to the average QTcF interval at Baseline of the 
present study (before dosing) is observed. For visits at which more than 


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one ECG is completed, the average QTcF interval of all legible ECGs 
will be used to determine the QTcF interval for that visit. 
Dosing is twice a day, once in the morning and once in the evening.  
Investigational product will be shipped directly to the subject. 
Confirmation of delivery to the subject will be made by a visiting nurse. 
Study drug shipment, return, and accountability will be performed in 
accordance with the drug distribution plan. In addition, study drug will 
be dispensed at the site during the Baseline visit when the visit is 
conducted in the clinic.  
Study visits may be done off-site rather than in the clinic with the prior 
approval of the Sponsor or Medical Monitor. Efficacy assessments will 
be performed by qualified clinician raters and by caregivers at all the 
designated visits (Table S–2). 
Safety Follow-up Period (30 Days) 
Subjects who discontinue prematurely, or who complete but do not 
continue into the subsequent open-label study (ACP-2566-005) will 
receive a follow-up telephone call or telemedicine visit to assess safety 
30 days after the last dose of study drug. 
The study schematic is provided in Figure S–1. 
The schedule of assessments is provided in Table S–2. 
Study Duration 
The duration of participation for individual study subjects will be 
approximately 44 weeks, consisting of a treatment period of 40 weeks, 
and a safety follow-up period of 30 days. 
The study completion date is defined as the date the final subject, across 
all sites, completes their final protocol-defined assessment. 
Main Criteria 
for Inclusion and 
Exclusion 
To be eligible for this study, subjects must meet all of the inclusion 
criteria and none of the exclusion criteria. 
Inclusion Criteria: 
1. Informed consent prior to the conduct of any study procedures 
is required as follows: 
a. For subjects who are minors: written informed consent 
will be obtained from the legally acceptable 
representative (LAR). The subject should provide 
written or oral assent if deemed able by the Investigator. 
The process of obtaining informed consent will be 
conducted in accordance with institutional review board 
(IRB) or ethics committee (EC) policy and applicable 
local law. 


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b. For subjects who are not minors: written informed 
consent will be obtained from the LAR or the subject if 
deemed able by the Investigator. If the subject is 
deemed not able to provide consent, the subject should 
provide written or oral assent if deemed able by the 
Investigator. The process of obtaining informed consent 
will be conducted in accordance with IRB or EC policy 
and applicable local law. 
c. The subject’s caregiver must also provide written 
informed consent regarding their participation in the 
study prior to participating in any study procedures. 
2. Has completed the Week 12/EOT visit of the antecedent study, 
Study ACP-2566-003  
NOTE: An exception is made for subjects who either 
discontinued from or completed but did not directly rollover 
from the antecedent study due to the COVID-19 public health 
emergency. These subjects may enroll in this study within 
12 months of their withdrawal/completion from the antecedent 
study, with approval of the Sponsor or Medical Monitor.  
3. Met all entry criteria for the antecedent study  
4. May benefit from long-term treatment with open-label 
trofinetide in the judgment of the Investigator 
5. Can still swallow the study medication provided as a liquid 
solution or can take it by gastrostomy tube 
6. The subject’s caregiver is English-speaking and has sufficient 
language skills to complete the caregiver assessments  
Childbearing Potential 
7. Subjects of childbearing potential must abstain from sexual 
activity for the duration of the study and for at least 30 days 
thereafter. If a subject is sexually active or becomes sexually 
active during the study, she must use 2 clinically acceptable 
methods of contraception (e.g., oral, intrauterine device [IUD], 
diaphragm plus spermicide, injectable, transdermal or 
implantable contraception) for the duration of the study and for 
at least 30 days thereafter. Subject must not be pregnant or 
breastfeeding. 
Place of Residence 
8. Subject and caregiver(s) must reside at a location to which 
study drug can be delivered and have been at their present 
residence for at least 3 months prior to Baseline 
 


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Exclusion Criteria: 
1. Began treatment with growth hormone during the antecedent 
study  
2. Began treatment with IGF-1 during the antecedent study 
3. Began treatment with insulin during the antecedent study  
4. Has developed a clinically significant cardiovascular, endocrine 
(such as hypo- or hyperthyroidism, Type 1 diabetes mellitus, or 
uncontrolled Type 2 diabetes mellitus), renal, hepatic, 
respiratory, or gastrointestinal disease (such as celiac disease or 
inflammatory bowel disease) or has major surgery planned 
during the study 
5. Subject is judged by the Investigator or the Medical Monitor to 
be inappropriate for the study due to AEs, medical condition, or 
noncompliance with investigational product or study procedures 
in the antecedent study 
6. Has a clinically significant abnormality in vital signs at Baseline 
7. Has an average QTcF interval of >450 ms on the Baseline ECG 
performed before the first dose of trofinetide is given in the 
present study (i.e., the ECG performed at the Week 12/EOT 
visit of the antecedent study) 
8. Has developed a clinically significant ECG finding during the 
antecedent study 
9. Is judged by the Investigator or the Medical Monitor to be 
inappropriate for the study for any reason 
Pharmacokinetic 
Assessments 
PK blood samples will be collected for trofinetide concentration 
measurements at the Baseline visit (both before dosing and 
approximately 2-3 hours after dosing) and at Visit 2, Visit 3, Visit 4, and 
Visit 5.  
The second PK sample taken at the Baseline visit (Visit 1), taken 
approximately 2-3 hours after dosing, will take place as soon as 
possible after the postdose ECG is performed. 
PK samples taken at Visits 2, 3, 4, and 5 should be collected at one of 
the following time intervals: 
• 2-3 hours after dosing 
• 4-6 hours after dosing 
• 7-11 hours after dosing 
Every effort should be made to collect PK samples at discrete time 
intervals during Visits 2, 3, 4, and 5. However, if the interval is the same 
at each visit, then the collection time should vary within that interval. 


Study: ACP-2566-004 
 
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The following scenario is only for illustrative purposes, a number of 
other scenarios are possible.  
• For example, if the time interval of 4-6 hours after dosing is 
used for Visit 2, 3, 4, and 5, then every effort should be made to 
collect PK samples at 4, 4.5, 5, and 6 hours after dosing, 
respectively. 
Pharmacokinetic samples will also be collected, if possible, at any ET 
visit or the visit immediately following any SAE or following any AE 
leading to discontinuation. 
For all scheduled PK samples (and for unscheduled samples if 
possible), the dates and times of administration of the study drug, the 
dates, times and content of the meals, and the dates and times of the 
administration of concomitant medications over the 2 days prior to and 
on the morning of the PK sample draw, as well as the date and time of 
the sample draw, will be recorded. For samples collected from subjects 
who experience any SAE or experience an AE leading to 
discontinuation, the date and time of the last dose of study drug prior to 
the SAE or AE leading to discontinuation will also be recorded.  
Optional 
Biomarker 
Analysis 
Participation in the effort to identify biomarkers is an optional 
component of the study. Subjects for whom separate informed consent 
for the identification of biomarkers of response to trofinetide is 
provided will have blood samples taken at Baseline (before dosing) and 
at Visits 3, 4, and 5, or upon early termination. Blood samples will be 
used to investigate differences between responders and non-responders 
in trofinetide-treated in RNA transcripts (transcriptomics), proteins 
(proteomics), and metabolites (metabolomics). 
Sample Size 
Calculations 
Up to approximately 180 subjects will be enrolled. The sample size for 
this study is not based on statistical power, but will depend on the 
number of subjects who transition into this open-label extension study 
from the antecedent study.   
Statistical 
Methods 
The purpose of this study is to collect safety data from subjects exposed 
to trofinetide for up to 40 weeks in this study. Secondary objectives 
include assessment of efficacy outcome measures over time. No 
statistical testing is planned. 
Analysis Sets 
The Safety Analysis Set will include all subjects who received at least 
one dose of study drug in this study.  
Any other analysis sets, if necessary, will be defined in the statistical 
analysis plan (SAP). 


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General Statistical Approach 
All endpoints will be summarized for the Safety Analysis Set. 
Additional summaries by prior treatment may be included. 
Data from the Week 12/EOT visit procedures of Study ACP-2566-003 
will be carried over as Baseline information in the present study, as 
applicable (e.g., not for the CGI-I for which this is not applicable).   
Continuous measurement results will be reported using the number of 
subjects with data values, mean, standard error of the mean, median, 
standard deviation, minimum, and maximum. For each categorical 
outcome, the frequency and percentage of subjects in each category will 
be reported.  
Primary Analyses 
Adverse events will be classified into standard terminology using the 
Medical Dictionary for Regulatory Activities (MedDRA). All AEs will 
be listed and TEAEs will be summarized by system organ class and 
preferred term. A TEAE is defined as an AE that started after the first 
administration of study drug and no later than the last administration of 
study drug plus 30 days. Summaries by maximum severity and by 
relationship to study drug will also be provided. Serious TEAEs, fatal 
AEs, and TEAEs leading to discontinuation will also be summarized. 
Descriptive summary statistics for ECG, vital signs and body weight, 
and clinical laboratory parameters, including observed values and 
changes from Baseline, will be tabulated by timepoint. Additionally, 
categorical analyses will be conducted on the incidence of subjects with 
prolonged QTc intervals and changes in QTc intervals in accordance 
with International Council for Harmonisation (ICH) guidelines. 
Efficacy Analyses 
Descriptive summary statistics for RSBQ, CGI-I (observed value only, 
no change from Baseline), CGI-S, CSBS-DP-IT Social, each of the four 
Rett Syndrome Clinician Rating scales, RTT-CBI, and ICND (QoL 
score and total score) observed values and change from Baseline will be 
summarized by timepoint.  
Pharmacokinetic Analyses 
Pharmacokinetic (PK) and efficacy (PD) measures will be collected 
from all subjects at the Baseline visit before dosing, at the Baseline visit 
after dosing, and after dosing at Weeks 2, 12, 26 and 40/EOT.  
Whole blood concentration and possible metabolites data for trofinetide 
will be listed and summarized using descriptive statistics. If data allow, 
population PK analyses will be performed to further characterize the 
PK. The details of the PK analysis will be presented in a separate 


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population PK report and PK/PD report in accordance with a separate 
data analysis plan (DAP). 
Date 
07 August 2020 


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Figure S–1 
Schematic of Study Design for ACP-2566-004 
 
 
 
a 
Subjects who roll over into the subsequent open-label study (ACP-2566-005) will not have the safety follow-up telephone call or telemedicine visit. 


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Table S–2 
Schedule of Events and Assessments for Study ACP-2566-004 
Period 
 
Baseline 
Open-label Treatment Period 
Safety  
Follow-upk 
Visit Week 
0 
2 
12 
26 
40/EOT/ET 
EOT/ET 
+30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
±3 
±7 
±7 
+7 
+4 
Type of Visitl 
Clinic or Off-site 
Telephone or 
Telemedicine 
Informed consent 
Xa 
 
 
 
Xa 
 
Inclusion/exclusion criteria 
X 
 
 
 
 
 
Medical history update 
X 
 
 
 
 
 
Physical examinationl 
X 
X 
X 
X 
X 
 
Vital signsb 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
X 
 
Weightl 
Xl 
X 
X 
X 
X 
 
12-lead electrocardiogram (ECG) 
Xc 
X 
X 
X 
Xc 
 
Clinical laboratory tests (hematology, 
chemistry)m  
X 
X 
X 
X 
X 
 
Urinalysis 
X 
X 
X 
X 
X 
 
TSH, Free T3, Free T4 
X 
 
 
 
X 
 
Serum pregnancy testd 
X 
 
X 
X 
X 
 
Blood samples for pharmacokinetics 
Xe 
Xf 
Xf 
Xf 
Xf 
 
Blood sample for optional analysis 
for biomarkersg 
Xh 
 
X 
X 
X 
 
Rett Syndrome Behaviour 
Questionnaire (RSBQ) 
X 
X 
X 
X 
X 
 
Clinical Global Impression–
Improvement in the present study 
(CGI-I)i 
 
X 
X 
X 
X 
 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
X 
 
Communication and Symbolic 
Behavior Scales-Developmental 
Profile™ Infant-Toddler 
(CSBS-DP-IT) Checklist 
X 
X 
X 
X 
X 
 
Impact of Childhood Neurologic 
Disability (ICND) Scale 
X 
 
 
 
X 
 
Rett Syndrome Clinician Rating of 
Hand Function (RTT-HF) 
X 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Ambulation and Gross Motor Skills 
(RTT-AMB) 
X 
X 
X 
X 
X 
 
Table abbreviations and footnotes provided on next page 


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Table S–2 
Schedule of Events and Assessments for Study ACP-2566-004 
(Continued) 
Period 
Baseline 
Open-label Treatment Period 
Safety  
Follow-upk 
Visit Week 
0 
2 
12 
26 
40/EOT/ET 
EOT/ET 
+30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
±3 
±7 
±7 
+7 
+4 
Type of Visit l 
Clinic or Off-site 
Telephone or 
Telemedicine 
Rett Syndrome Clinician Rating of 
Ability to Communicate Choices 
(RTT-COMC)l 
X 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Verbal Communication (RTT-VCOM) 
X 
X 
X 
X 
X 
 
Rett Syndrome Caregiver Burden 
Inventory (RTT-CBI) 
X 
 
X 
 
X 
 
Dispensing and review of semi-
structured caregiver diary, including 
seizure diary 
X 
X 
X 
X 
X 
 
Concomitant medications 
X 
X 
X 
X 
X 
X 
Assessment of adverse events 
X 
X 
X 
X 
X 
X 
Study drug dispensedj 
X 
 
 
 
 
 
Authorization of study drug 
dispensationj 
X-------------------------------- X 
 
 
Study drug returnj 
 
X-----------------------------------X 
 
Study drug accountabilityj 
 
X 
X 
X 
X 
 
Abbreviations: EOT=end of treatment; ET=early termination 
a 
Unless subjects are enrolling in the present study under special circumstances due to the COVID-19 public health 
emergency (detailed in the inclusion/exclusion criteria), consent for the present study must be obtained prior to 
performance of the EOT procedures at the Week 12/EOT visit in the antecedent study. Data from the Week 12/EOT 
visit procedures of the antecedent study will be carried over as Baseline information in the present study, as applicable. 
Subjects who enter the present study under special circumstances due to the COVID-19 public health emergency will 
have consent obtained for the present study before any procedures are performed (e.g., the blood draw before the 
Baseline visit). For subjects who decide to continue into subsequent open-label extension (OLE) study (ACP-2566-
005), informed consent for ACP-2566-005 must be obtained prior to performing the Week 40/EOT procedures. 
b  
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure will be measured after the subject has been sitting for ≥3 minutes. 
c  
ECGs will be completed in triplicate at Visit 1 (Baseline) both before dosing and 2-3 hours after dosing and at Visit 5 
(Week 40/EOT/ET). For those subjects who completed the antecedent study, the ECG taken at the Week 12/EOT visit 
in the antecedent study is the predose Baseline ECG of the present study. A single ECG will be completed at Visit 2 
(Week 2), Visit 3 (Week 12), and Visit 4 (Week 26).  
d  
For subjects who have reached menarche and have not had surgical sterilization  
e 
The PK sample taken at the Week 12/EOT visit in the antecedent study is the predose PK blood sample for the present 
study. A postdose PK blood sample will be collected at the end of ECG assessment 2-3 hours after the first dose of 
study drug in the present study. 
f  
PK samples at Visits 2, 3, 4, and 5 will be collected at one of the following time intervals: 1) 2-3 hours after dosing OR 
2) 4-6 hours after dosing OR 3) 7-11 hours after dosing. Every effort should be made to collect the PK samples at 
discrete time intervals during Visits 2, 3, 4, and 5. However, if the interval is the same across these visits, then the 
collection time should vary within that interval.  


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g  
Participation in the effort to identify biomarkers is an optional component of the study requiring a separate informed 
consent. 
h  
The biomarker sample taken at the Week 12/EOT visit in the antecedent study is the Baseline sample for the present 
study. 
i  
The CGI-I assessments at Weeks 2, 12, 26, and 40/EOT will be a comparison with the baseline state of the illness in the 
present study (i.e., Visit 1 of ACP-2566-004). 
j 
Investigational product will be shipped directly to the subject. Confirmation of delivery to the subject will be made by a 
visiting nurse. Study drug shipment, return, and accountability will be performed in accordance with the drug 
distribution plan. In addition, study drug will be dispensed at the site during the Baseline visit when the visit is 
conducted in the clinic. 
k 
Subjects who roll over into the subsequent open-label study (ACP-2566-005) will not have the safety follow-up 
telephone call or telemedicine visit. 
l 
Study visits may be done off-site rather than in the clinic with the prior approval of the Sponsor or Medical Monitor. 
When a study visit takes place off-site, the physical examination will not be required. Weight should be measured 
whenever possible at off-site visits. Measurement of weight is necessary at Baseline in order to determine the starting 
dose. The RTT-COMC should be completed if possible, but it is not required. 
m 
Subjects for whom the Baseline visit of this study is not on the same day as the Week 12/EOT visit of the antecedent 
study will have blood drawn for Baseline values 3 to 14 days before the Baseline visit. 


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TABLE OF CONTENTS 
SPONSOR SIGNATURE PAGE ............................................................................................... 2 
DECLARATION OF INVESTIGATOR ................................................................................... 4 
PROTOCOL SYNOPSIS ........................................................................................................... 5 
TABLE OF CONTENTS ......................................................................................................... 20 
LIST OF TABLES ................................................................................................................... 24 
LIST OF FIGURES ................................................................................................................. 24 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................................. 25 
1 
INTRODUCTION ...................................................................................................... 27 
1.1 
Background Information ............................................................................................ 27 
1.2 
Investigational Product ............................................................................................... 28 
1.3 
Previous Clinical Experience ..................................................................................... 29 
1.4 
Study Rationale .......................................................................................................... 32 
2 
STUDY OBJECTIVES AND ENDPOINTS .............................................................. 33 
2.1 
Primary Objective....................................................................................................... 33 
2.1.1 
Primary Endpoints ...................................................................................................... 33 
2.2 
Secondary Objectives ................................................................................................. 33 
2.2.1 
Secondary Endpoints .................................................................................................. 34 
2.3 
Pharmacokinetic Objectives ....................................................................................... 34 
2.3.1 
Pharmacokinetic Endpoints ........................................................................................ 35 
3 
STUDY DESCRIPTION ............................................................................................ 35 
3.1 
Overview of Study Design ......................................................................................... 35 
3.1.1 
Open-label Treatment Period (40 Weeks) .................................................................. 36 
3.1.2 
Safety Follow-Up Period (30 Days) ........................................................................... 37 
4 
SUBJECT ELIGIBILITY AND WITHDRAWAL CRITERIA .................................. 37 
4.1 
Inclusion Criteria ........................................................................................................ 37 
4.2 
Exclusion Criteria ....................................................................................................... 38 
4.3 
Subject Withdrawal of Consent .................................................................................. 39 
4.4 
Subject or Study Discontinuation ............................................................................... 39 
4.4.1 
Post-Baseline QTcF Interval Stopping Criteria .......................................................... 40 
4.4.2 
Handling of Subject Discontinuation During the Treatment Period .......................... 40 
4.5 
Subject Lost to Follow-Up ......................................................................................... 41 


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4.6 
Concomitant Therapy ................................................................................................. 41 
4.6.1 
Permitted and Prohibited Medications ....................................................................... 41 
5 
INVESTIGATIONAL PRODUCT ............................................................................. 42 
5.1 
Investigational Product Description ........................................................................... 42 
5.1.1 
Formulation, Appearance, and Packaging .................................................................. 42 
5.1.2 
Product Storage and Stability ..................................................................................... 42 
5.1.3 
Dosing and Administration ......................................................................................... 42 
5.1.3.1 Dosing ........................................................................................................................ 42 
5.1.3.2 Administration of Study Drug .................................................................................... 44 
5.1.4 
Method of Assigning Subjects to Treatment Groups.................................................. 45 
5.1.5 
Blinding ...................................................................................................................... 45 
5.1.6 
Study Drug Compliance ............................................................................................. 45 
5.1.7 
Overdose ..................................................................................................................... 45 
5.2 
Investigational Product Accountability Procedures ................................................... 45 
6 
STUDY PROCEDURES ............................................................................................ 46 
6.1 
Efficacy Assessments ................................................................................................. 46 
6.1.1 
Rett Syndrome Behaviour Questionnaire (RSBQ) ..................................................... 46 
6.1.2 
Clinical Global Impression–Improvement (CGI-I) and Clinical Global Impression–
Severity (CGI-S)......................................................................................................... 47 
6.1.3 
Communication and Symbolic Behavior Scales Developmental Profile Infant-
Toddler (CSBS-DP-IT) Checklist .............................................................................. 48 
6.1.4 
Impact of Childhood Neurologic Disability (ICND) Scale ........................................ 49 
6.1.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF) .................................. 49 
6.1.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills (RTT-AMB)
 .................................................................................................................................... 49 
6.1.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices (RTT-COMC)
 .................................................................................................................................... 50 
6.1.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM) .............. 50 
6.1.9 
Rett Syndrome Caregiver Burden Inventory (RTT-CBI) ........................................... 50 
6.2 
Safety Assessments..................................................................................................... 51 
6.2.1 
Physical Examination ................................................................................................. 51 
6.2.2 
Vital Signs .................................................................................................................. 51 
6.2.3 
Height, Weight, and Body Mass Index ....................................................................... 51 
6.2.4 
Electrocardiograms ..................................................................................................... 51 
6.2.4.1 Post-Enrollment QTcF Interval Stopping Criteria ...................................................... 52 
6.2.5 
Laboratory Evaluations .............................................................................................. 52 
6.3 
Caregiver Diary .......................................................................................................... 54 


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6.4 
Pharmacokinetic Assessments .................................................................................... 54 
6.4.1 
Specimen Preparation, Handling, Storage, and Shipment.......................................... 55 
6.5 
Identification of Biomarkers of Response to Trofinetide in Rett Syndrome .............. 56 
7 
ADVERSE EVENTS ................................................................................................. 56 
7.1 
Specification of Safety Parameters............................................................................. 56 
7.1.1 
Definition of Adverse Event ....................................................................................... 56 
7.1.2 
Definition of Serious Adverse Event .......................................................................... 57 
7.2 
Classification of an Adverse Event ............................................................................ 59 
7.2.1 
Severity of Event ........................................................................................................ 59 
7.2.2 
Relationship to Study Drug ........................................................................................ 59 
7.2.2.1 Duration ...................................................................................................................... 59 
7.2.2.2 Frequency ................................................................................................................... 60 
7.2.2.3 Action Taken with Study Drug ................................................................................... 60 
7.2.2.4 Therapy ....................................................................................................................... 60 
7.2.2.5 Outcome ..................................................................................................................... 60 
7.2.2.6 Seriousness ................................................................................................................. 60 
7.2.3 
Definition of Unexpectedness .................................................................................... 60 
7.3 
Time Period and Frequency for Event Assessment and Follow-Up ........................... 60 
7.3.1 
Adverse Event Reporting ........................................................................................... 61 
7.3.2 
Serious Adverse Event Reporting............................................................................... 61 
7.3.3 
Reporting of Pregnancy .............................................................................................. 62 
7.3.4 
Reporting of Overdose ............................................................................................... 63 
8 
CLINICAL MONITORING ....................................................................................... 63 
9 
STATISTICAL METHODS AND DATA ANALYSIS .............................................. 63 
9.1 
Statistical and Analytical Plans .................................................................................. 63 
9.2 
Statistical Hypotheses................................................................................................. 63 
9.3 
Sample Size Determination ........................................................................................ 63 
9.4 
Subject Populations for Analysis ................................................................................ 64 
9.5 
Statistical Analyses ..................................................................................................... 64 
9.5.1 
General Approach ....................................................................................................... 64 
9.5.2 
Primary Analyses ........................................................................................................ 64 
9.5.3 
Secondary Analyses .................................................................................................... 65 
9.5.4 
Pharmacokinetic Analyses .......................................................................................... 65 
9.5.5 
Subgroup Analyses ..................................................................................................... 65 
9.6 
Interim Analyses ......................................................................................................... 65 


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9.7 
Measures to Minimize Bias ........................................................................................ 65 
9.8 
Breaking the Study Blind/Subject Code ..................................................................... 65 
10 
STUDY MANAGEMENT AND DATA COLLECTION .......................................... 65 
10.1 
Data Collection and Management Responsibilities.................................................... 65 
10.2 
Source Documents ...................................................................................................... 66 
10.3 
Case Report Forms ..................................................................................................... 66 
10.4 
Confidentiality ............................................................................................................ 66 
10.5 
Study Records Retention ............................................................................................ 66 
10.6 
Protocol Exceptions and Deviations........................................................................... 67 
10.7 
Protocol Amendments ................................................................................................ 67 
11 
STUDY MONITORING, AUDITING, AND INSPECTING .................................... 67 
11.1 
Quality Control and Quality Assurance...................................................................... 67 
11.2 
Risk Management ....................................................................................................... 68 
12 
ETHICAL CONSIDERATIONS ................................................................................ 69 
12.1 
Ethical Standard ......................................................................................................... 69 
12.2 
Institutional Review Board/Ethics Committee ........................................................... 69 
12.3 
Informed Consent Process .......................................................................................... 69 
12.3.1 Consent and Other Informational Documents Provided to Subjects .......................... 71 
12.3.2 Consent Procedures and Documentation .................................................................... 71 
13 
PUBLICATION PLAN .............................................................................................. 71 
14 
CONFLICT OF INTEREST POLICY ....................................................................... 71 
14.1 
Finance, Insurance, and Indemnity............................................................................. 71 
15 
LITERATURE REFERENCES .................................................................................. 72 
 


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LIST OF TABLES 
Table S–1 
Dosing Schedule Based on Weight at Baseline of the Present Study ................ 7 
Table S–2 
Schedule of Events and Assessments for Study ACP-2566-004 ..................... 17 
Table 1–1 
Change from Treatment Baseline (Day 14) to End of Treatment  (Day 54) 
in Core Efficacy Outcomes (mITT) (Neu-2566-RETT-002) ........................... 30 
Table 5–1 
Dosing Schedule Based on Weight at Baseline of the Present Study .............. 43 
Table 6-1 
PK Sampling Times ......................................................................................... 55 
 
LIST OF FIGURES 
Figure S–1 
Schematic of Study Design for ACP-2566-004 ............................................... 16 
Figure 1–1 
Comparison of Change from Treatment Baseline on the RSBQ at 
Follow-Up Assessments Between 200 mg/kg BID and Placebo BID 
Treatment Groups ............................................................................................ 31 
 


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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Term 
Definition 
AE 
adverse event(s) 
BID 
twice daily 
CGI-I 
Clinical Global Impression–Improvement 
CGI-S 
Clinical Global Impression–Severity 
CSBS-DP-IT 
Communication and Symbolic Behavior Scales-Developmental 
Profile™ Infant-Toddler 
EC 
ethics committee 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
EOT 
end of treatment 
ET 
early termination 
GCP 
Good Clinical Practice 
GJ 
gastrojejunal 
GPE 
glycine-proline-glutamate 
ICF 
informed consent form 
ICH 
International Council for Harmonisation  
ICND 
Impact of Childhood Neurologic Disability Scale 
IGF-1 
insulin-like growth factor 1 
IRB 
institutional review board 
IRT 
interactive response technology 
LAR 
legally acceptable representative 
MeCP2 
methyl-CpG binding protein 2  
MeCP2 
gene encoding methyl-CpG binding protein 2 (in animals) 
MECP2 
gene encoding methyl-CpG binding protein 2 (in humans) 
PK 
pharmacokinetic(s) 
PR interval 
PR interval of ECG 
QRS interval 
QRS interval of ECG 
QT interval 
QT interval for heart rate of ECG 
QTc 
corrected QT interval of ECG for heart rate 
QTcB 
corrected QT interval using Bazett’s correction method 
QTcF 
corrected QT interval using Fridericia’s correction method 
RSBQ 
Rett Syndrome Behaviour Questionnaire 
RTT 
Rett syndrome 
RTT-AMB 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor 
Skills 


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Term 
Definition 
RTT-CBI 
Rett Syndrome Caregiver Burden Inventory 
RTT-COMC 
Rett Syndrome Clinician Rating of Ability to Communicate 
Choices 
RTT-DSC 
RTT Domain Specific Visual Analog Scale 
RTT-HF 
Rett Syndrome Clinician Rating of Hand Function 
RTT-VCOM 
Rett Syndrome Clinician Rating of Verbal Communication 
SAE 
serious adverse event(s) 
TEAE 
treatment emergent adverse event(s) 
US 
United States 
 


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1 INTRODUCTION 
This document is a research protocol and the described study will be conducted in 
compliance with the protocol and the International Council for Harmonisation (ICH) 
Good Clinical Practice (GCP) Guideline.  
1.1 
Background Information 
Rett syndrome (RTT) is a seriously debilitating neurodevelopmental disorder for which there 
is currently no approved treatment. Its prevalence is reported as 1 in 10,000-15,000 female 
live births (Bienvenu et al. 2006; Neul et al. 2010). While the great majority of patients with 
RTT are females, males who meet the criteria for RTT have also been identified 
(Neul et al. 2019). In 96-98% of patients diagnosed with classic RTT, the disease is caused by 
mutations in the X-linked MECP2 gene (Percy et al. 2018). MECP2 encodes methyl-CpG 
binding protein 2 (MeCP2) which modulates gene expression by binding to methylated CpG 
dinucleotides, primarily by activating but also by repressing transcription (Ip et al. 2018; 
Neul et al. 2008; Kriaucionis and Bird 2003; Hite et al. 2009). The activity of the MeCP2 
protein is diminished in both neurons and astrocytes (Yasui et al. 2013). 
In patients with typical RTT, there is seemingly normal psychomotor development for the 
first 6 months of life, but soon thereafter the failure to reach normal developmental 
milestones is observed, followed by a period of developmental regression in which there is a 
loss of normal use of the hands and of spoken language (Samaco and Neul 2011). The period 
of developmental regression is accompanied by transient autistic features in many but not all 
individuals with RTT (Lee et al. 2013; Neul et al. 2014). Loss of social skills appears to 
stabilize or reverse after the regression period, and individuals with RTT demonstrate social 
intentions through eye contact (Young et al. 2008; Djukic and McDermott 2012; 
Urbanowicz et al. 2016). Nonetheless, social interaction and communication remain limited 
(Mount et al. 2003; Urbanowicz et al. 2015; Rose et al. 2013; Kaufmann et al. 2012; 
Woodyatt and Ozanne 1993). The intellectual disability in RTT appears to be profound; 
however, precise measurement of the extent of cognitive impairment is difficult because of 
the severe communication and motor deficits affecting most individuals 
(Byiers and Symons 2012; Clarkson et al. 2017). 
Seizures are common, although not diagnostic. Commonly observed symptoms include 
awake breathing disruptions, scoliosis, and interest in social interaction (intense eye 
communication) (Neul et al. 2010; Percy et al. 2010). Gastrointestinal symptoms, including 
constipation and chewing and swallowing difficulties, are observed in the majority of patients 
with RTT (Motil et al. 2012). Sudden changes in mood, screaming and inconsolable crying 
are common behaviors in children and adolescents with RTT (Mount et al. 2001, 2002; 
Robertson et al. 2006; Cianfaglione et al. 2015). These impairments can further exacerbate 


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other symptoms and disrupt activities of daily living including educational, recreational and 
treatment opportunities (Epstein et al. 2016; Perry et al. 1991; Thompson and Iwata 2001; 
Iemmi et al. 2016). Autonomic manifestations, which include abnormalities in cardiac and 
respiratory function, as well as in peripheral circulation, are considered to be predominantly 
of central nervous system origin. Rett syndrome is also characterized by impaired growth 
affecting the brain and other organ systems.  
Loss of purposeful hand use is a defining aspect of RTT during the onset of regression and at 
least 30% of individuals remain without any type of purposeful hand use (Downs et al. 2010). 
Individuals with some purposeful hand movements vary in their level of ability to reach for 
and grasp objects. The ability to self-feed is observed in 25-43% (Cass et al. 2003; 
Larsson et al. 2005; Downs et al. 2011). Poorer levels of hand function are associated with 
both age and severity of mobility impairment (Downs et al. 2011). 
In adulthood, a late motor deterioration stage occurs, characterized by worsening dystonia, 
rigidity, and in some cases deterioration in the ability to walk and parkinsonian symptoms. 
Affected individuals have a yearly death rate between 1% and 2%, with 25% of all deaths 
characterized as sudden and unexpected (Kerr et al. 1997; Samaco and Neul 2011). Women 
with RTT can survive into their fifties and occasionally longer (Samaco and Neul 2011). 
There are no medicines approved for the treatment of RTT. Treatment focuses on the 
management of each patient’s symptoms and, even in that regard, is often unsatisfactory and 
has only a limited effect on functional improvement. Accordingly, the burden on families and 
other caregivers is very great (Palacios-Ceña et al. 2018). 
1.2 
Investigational Product 
Trofinetide is a synthetic analog of glycine-proline-glutamate (also known as glypromate or 
GPE), a peptide that occurs naturally in the brain. GPE is the N-terminal tripeptide of the 
insulin-like growth factor 1 (IGF-1) protein. Trofinetide crosses the blood-brain barrier 
following oral administration. In the brain, it is believed to normalize decreased 
bioavailability of IGF-1 and GPE, as well as having an anti-inflammatory effect on 
pathologically activated glial cells. Both conditions contribute to deficits in synaptic 
development and functional maturation of synaptic plasticity that are fundamental to the 
wide-ranging effects of RTT. Tropea et al. (2009) observed that treatment with GPE reversed 
Rett-like symptoms in MeCP2 mutant mice. Therefore, the aim of treatment with trofinetide 
is to exert an effect on brain structure and function such as dendritic length and branching 
and long-term potentiation, which would be expected to lead to improvements across a wide 
range of symptoms of RTT. 


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1.3 
Previous Clinical Experience 
Trofinetide showed linear pharmacokinetics (PK) across the dose range tested in pediatric 
RTT patients. These PK results are in agreement with the data obtained previously in healthy 
subjects and in adult RTT patients. From a drug metabolism perspective, there was no 
accumulation, metabolic inhibition, or induction observed during treatment. For subjects 
treated with 50 mg/kg twice daily (BID), median maximum (peak) observed drug 
concentration (Cmax) was 17.7 μg/mL and median AUC0-12ss was 139.4 µg/mL·h. For subjects 
treated with 100 mg/kg BID, median Cmax was 52.6 μg/mL and median AUC0-12ss was 
338.6 µg/mL·h. For subjects treated with 200 mg/kg BID, median Cmax was 82.2 μg/mL and 
median AUC0-12ss was 505.1 µg/mL·h. The geometric mean of the apparent terminal 
elimination half-life (t1/2) varied from 5.3 h to 6.1 h across the three dosing groups. 
Trofinetide has exhibited a favorable safety and tolerability profile in two studies in subjects 
with Rett syndrome. Study Neu-2566-Rett-001 (NCT01703533) was a Phase 2 study of 
trofinetide in 56 adolescent and adult females with RTT (Glaze et al. 2017). Subjects were 
randomized 2:1 to 35 mg/kg trofinetide BID or placebo for 14 or 28 days (Cohorts 0 and 1, 
respectively) or 70 mg/kg trofinetide BID or placebo for 28 days (Cohort 2). Both dose levels 
were well-tolerated in this study, and no time- or dose-dependent adverse events (AEs) were 
apparent. The most common AE across all treatment groups was diarrhea (39% in the 
combined 35 mg/kg BID group, 11% in the 70 mg/kg BID group and 15% in the placebo 
group). The most common AE in the 70 mg/kg BID group was somnolence (17%; vs. 5% in 
the placebo group and 0% in the 35 mg/kg BID group). Four serious adverse events (SAEs) 
involving three subjects were reported during the study. In each of these instances, the AEs 
were deemed unrelated to the study medication. One subject withdrew from the study due to 
these events while the two subjects had already finished the study medication when their 
SAEs occurred. Each of these SAEs was deemed to be resolved by study conclusion. The 
results of this study also provided preliminary evidence of a treatment effect in the 70 mg/kg 
BID treatment group. 
A second Phase 2 study of oral trofinetide in pediatric and adolescent females with RTT, 
Study Neu-2566-RETT-002, assessed three dose levels of trofinetide (50 mg/kg BID, 
100 mg/kg BID, and 200 mg/kg BID vs. placebo) (Glaze et al. 2019). The study population 
consisted of female subjects between 5 and 15 years of age with a diagnosis of RTT and a 
proven mutation of the MECP2 gene. The primary analysis of efficacy was change from 
treatment Baseline (Visit 3, Day 14, end of placebo run-in) to End-of-Assessment (Visit 7, 
Day 54) between placebo and the 200 mg/kg BID treatment groups.  
At the time the study was initiated, there was no consensus upon the most appropriate and 
sensitive endpoint(s) for detecting beneficial drug effect in RTT, so the study examined 


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five unranked core efficacy outcome measures. Each of these five endpoints represented a 
different dimension or manifestation of the pathophysiology and clinical impairment directly 
associated with RTT. As shown below in Table 1–1, three of the five core endpoints showed a 
statistically significant difference from placebo at p<0.05 for the 200 mg/kg BID dose group: 
two clinician completed assessments (RTT Domain Specific Visual Analog Scale [RTT-DSC] 
and Clinical Global Impression–Improvement [CGI-I]) and one of the caregiver completed 
assessments (RSBQ). The remaining two primary endpoints were directionally positive. 
Table 1–1 
Change from Treatment Baseline (Day 14) to End of Treatment  
(Day 54) in Core Efficacy Outcomes (mITT) 
(Neu-2566-RETT-002) 
Outcome Measure 
Prespecified 
covariates 
p≤0.1 
Placebo 
(n=24) 
50 mg/kg 
(n=15) 
100 mg/kg 
(n=16) 
200 mg/kg 
(n=27) 
RSBQ Total 
D14 Treatment Baseline 
Change D14-D54 (LS mean) 
p-value vs. placeboa  
PR 
 
39.5 
-2.3 
 
 
44.7 
-3.0 
0.768 
 
40.3 
-1.5 
0.749 
 
42.2 
-6.7 
0.042 
RTT-DSC Total 
Exact Median Test 
D14 Treatment Baseline 
Change D14-D54 
p-value vs. placeboa 
UnAdj 
 
473.3 
-25.85 
 
 
450.0 
-32.50 
0.999 
 
445.3 
-12.10 
0.748 
 
516.6 
-76.00 
0.025 
CGI-I 
D14 Treatment Baseline 
Change D14-D54 (LS mean) 
p-value vs. placebob 
UnAdj 
 
(4) 
3.5 
 
 
(4) 
3.3 
0.391 
 
(4) 
3.4 
0.703 
 
(4) 
3.0 
0.029 
Top 3 Caregiver Concerns 
D14 Treatment Baseline 
Change D14-D54 (LS mean) 
p-value vs. placeboa 
UnAdj 
 
223.8 
-12.52 
 
 
237.6 
-16.56 
0.776 
 
211.5 
-2.09 
0.455 
 
245.9 
-18.54 
0.619 
MBA Total 
D14 Treatment Baseline 
Change D14-D54 (LS mean) 
p-value vs placeboa 
TBL, PR 
 
48.8 
-2.6 
 
 
46.6 
-2.8 
0.872 
 
48.6 
-2.4 
0.925 
 
46.6 
-2.9 
0.840 
Abbreviations: CGI-I=Clinical Global Impression–Improvement; LS =least squares; MBA=Motor Behavior 
Assessment; mITT=modified Intent-to-treat; PR=placebo response; RSBQ=Rett Syndrome Behaviour 
Questionaire; RTT-DSC=RTT Domain Specific Visual Analog Scale; TBL=treatment Baseline;  
UnAdj=unadjusted 
a 
Prespecified model covariates if p≤0.1: TBL and/or PR.  
b 
CGI-I has no pretreatment baseline value. The CGI-I values at Day 14 are the ratings of change from Day 0 
(the pretreatment Baseline) to Day 14 (end of placebo run-in). 
 
Among the three outcome measures that exhibited improvement compared to placebo for the 
200 mg/kg BID group, the median magnitude of effect (change) was 15% for RTT-DSC 
(vs. 5% for placebo) and the mean magnitude of effect (change) was 16% for RSBQ (vs. 6% 


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for placebo). For the CGI-I, the mean outcome was 3.0 for the 200 mg/kg BID group and was 
3.5 for placebo. For the CGI-I, more than 20% of subjects in the 200 mg/kg BID group were 
judged by clinicians to be “much improved”, compared with less than 5% of those on 
placebo. 
At the end of the dosing period, data indicate that clinical benefit (change from treatment 
Baseline) was continuing to accrue and that there was a divergent trend between the placebo 
and 200 mg/kg BID groups as highlighted below in Figure 1–1 for the RSBQ. Further, there 
is evidence of a significant diminution of effect following cessation of dosing, suggesting that 
the separation during the dosing period was due to beneficial treatment effect and not from 
other differences between the two arms that occurred simply by chance.  
Figure 1–1 
Comparison of Change from Treatment Baseline on the RSBQ at 
Follow-Up Assessments Between 200 mg/kg BID and Placebo BID 
Treatment Groups 
 
Abbreviations: BID=twice daily; LS=least squares; RSBQ=Rett Syndrome Behaviour Questionaire 
Note: End of treatment assessment is at Day 54. Treatment baseline is at Day 14, at the end of the placebo 
run-in. Day 66 is post-treatment. 
 
Trofinetide was generally safe and well tolerated. Most AEs reported during the double-blind 
treatment period were mild in intensity and the majority of events were deemed not related to 
study medication. The most common AEs reported from the start of the double-blind period 
across all treatment groups were diarrhea (27%), vomiting (15%), upper respiratory tract 
pyrexia (13%), and infection (12%). The most common AE from the start of the double-blind 
treatment period in the trofinetide treatment groups was diarrhea (27% in the 50 mg/kg BID 
group, 13% in the 100 mg/kg BID group, and 56% in the 200 mg/kg BID group compared 


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with 4% in the placebo group). The diarrhea associated with trofinetide may have been due to 
an improvement in gastrointestinal motility added to ongoing treatment with laxatives. Only 
three subjects experienced SAEs: one subject in the placebo BID group had an SAE of 
pneumonia, one subject in the 100 mg/kg BID group had an SAE of worsening of tonic 
cluster seizures, and one subject in the 200 mg/kg BID group had SAEs of streptococcal 
infection and pneumonia. In each of these instances, the SAEs were deemed not related to 
study medication and resolved by the end of the study. 
No changes in QTc interval attributable to treatment were seen in adolescent and adult 
subjects with RTT in study Neu-2566-RETT-001, nor in pediatric subjects in 
Neu-2566-Rett-002.   
Phase 2 studies of the oral formulation of trofinetide in adolescent and adult subjects with 
fragile X syndrome and of the intravenous formulation in adult subjects with moderate to 
severe traumatic brain injury (TBI) have also been completed and are described in the 
Investigator’s brochure. 
Always refer to the latest version of the trofinetide Investigator’s Brochure for the overall 
benefit/risk assessment and the most accurate and current information regarding drug 
metabolism, PK, efficacy, and safety. 
1.4 
Study Rationale 
As described above, Rett syndrome is a devastating disorder for which there is as yet no 
treatment beyond symptomatic care resulting in a great unmet medical need. In the two 
studies of trofinetide in girls and women with RTT, trofinetide has been well tolerated. The 
highest dose assessed, 200 mg/kg BID, was observed to provide benefit compared to placebo 
even in a relatively small study, as described above.  
The double-blind study ACP-2566-003, in which subjects are to be enrolled before being 
considered for entry into the present study, was designed to assess whether the effects of 
administration of trofinetide seen in the two Phase 2 studies, are confirmed in a larger 
population of children, adolescents, and young adults. The study compared the efficacy and 
safety of single trofinetide treatment group to treatment with placebo. Dosing was based on 
weight (Section 5.1.3).   
The present study is an open-label extension (OLE) that gives subjects who received placebo 
in the antecedent study a chance to receive treatment with trofinetide, allows subjects who 
received treatment with trofinetide in the antecedent study to continue treatment, and assesses 
the long-term safety and tolerability of trofinetide in this population. This study will also 
serve to provide data on the benefits of long-term treatment with trofinetide. 


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2 STUDY OBJECTIVES AND ENDPOINTS 
2.1 
Primary Objective 
The primary objective of this study is to investigate the safety and tolerability of long-term 
treatment with oral trofinetide in girls and women with Rett syndrome. 
2.1.1 
Primary Endpoints 
The primary endpoints are:  
• Treatment-emergent adverse events (TEAEs) 
• Serious adverse events (SAEs) 
• Withdrawals due to AEs 
• Potentially clinically important changes in other safety assessments 
2.2 
Secondary Objectives 
The secondary objectives are: 
• To investigate the efficacy of long-term treatment with oral trofinetide in girls and 
women with Rett syndrome 
• To investigate the efficacy of long-term treatment with oral trofinetide in girls and 
women with Rett syndrome on overall ability to communicate 
• To investigate the benefit of long-term treatment with oral trofinetide on overall 
quality of life for girls and women with Rett syndrome 
• To investigate the efficacy of long-term treatment with oral trofinetide in girls and 
women with Rett syndrome on: 
o Hand function  
o Ambulation and other gross motor skills  
o Ability to communicate choices and preferences 
o Ability to communicate verbally  
• To investigate the efficacy of long-term treatment with oral trofinetide on a global 
assessment of the severity of illness in girls and women with Rett syndrome 
• To investigate the benefit of long-term treatment with oral trofinetide on the burden 
on caregivers of girls and women with Rett syndrome 
• To investigate the benefit of long-term treatment with oral trofinetide on the impact of 
the disability on the child’s and family’s everyday life 


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2.2.1 
Secondary Endpoints 
The secondary endpoints for this study are: 
• Rett Syndrome Behaviour Questionnaire (RSBQ) total score – Change from Baseline 
to Week 40 
• Clinical Global Impression–Improvement (CGI-I) Score at Week 40 (assessing 
improvement from Baseline of the present study)  
Change from Baseline to Week 40 in: 
• Communication and Symbolic Behavior Scales-Developmental Profile™ 
Infant-Toddler Checklist – Social Composite Score (CSBS-DP-IT Social)  
• Overall Quality of Life Rating of the Impact of Childhood Neurologic Disability 
(ICND) Scale 
• Rett Syndrome Clinician Rating of Hand Function (RTT-HF) 
• Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills (RTT-AMB) 
• Rett Syndrome Clinician Rating of Ability to Communicate Choices (RTT-COMC) 
• Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM) 
• Clinical Global Impression–Severity (CGI-S) 
• Rett Syndrome Caregiver Burden Inventory (RTT-CBI) Total Score (items 1-24) 
• Impact of Childhood Neurologic Disability (ICND) Scale Total Score 
2.3 
Pharmacokinetic Objectives 
The pharmacokinetic objectives of this study are: 
• To characterize the pharmacokinetics (PK) of trofinetide in girls and women with 
Rett syndrome following long-term treatment with oral trofinetide 
• To assess the pharmacokinetic/pharmacodynamic (PK/PD) relationship using safety 
and efficacy endpoints in girls and women with Rett syndrome following long-term 
treatment with oral trofinetide 


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2.3.1 
Pharmacokinetic Endpoints 
The pharmacokinetic endpoints for this study are: 
• Whole blood concentration of trofinetide and possible metabolites 
• Trofinetide PK parameters using the population PK approach 
• PK/PD using appropriate PK/PD analysis methods 
 
3 STUDY DESCRIPTION 
3.1 
Overview of Study Design 
This is a 40-week, multicenter, open-label extension (OLE) study to evaluate long-term 
safety and tolerability of trofinetide in girls and women with Rett syndrome. Subjects who 
complete the preceding double-blind study (ACP-2566-003) will be eligible to enroll in the 
OLE. Legally acceptable representative (LARs)/subjects must be consented prior to the 
procedures being performed at the Week 12/EOT visit of the antecedent study. The Week 
12/EOT visit of the antecedent study will serve as the Baseline visit of the present study. The 
data gathered at the Week 12/EOT visit of the antecedent study serves as the baseline data of 
the present study.  
Because of the special circumstances surrounding the Coronavirus Disease 2019 
(COVID-19) public health emergency in the spring of 2020, subjects who were discontinued 
from Study ACP-2566-003 before completion because of the COVID-19 public health 
emergency and subjects who completed Study ACP-2566-003, but were prevented from 
entering the present study because of the COVID-19 public health emergency, may be 
re-evaluated for eligibility at the Baseline visit of the present study. 
However, subjects in the future who are discontinued from Study ACP-2566-003, or who are 
not able to rollover to this study at the Week 12/EOT visit of the antecedent study, will not be 
able to enter the present study.  
The duration of participation for individual study subjects will be approximately 44 weeks. 
Approximately 28 sites will participate in this study. 
The study will have two periods (Figure S–1): 
• Treatment period: 40 weeks  
• Safety follow-up period: 30 days 


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The schedule of events and assessments is provided in Table S–2. As shown in the schedule 
of events, study visits may be done off-site rather than in the clinic with the prior approval of 
the Sponsor or Medical Monitor. 
The study completion date is defined as the date the final subject, across all sites, completes 
their final protocol-defined assessment. An individual subject is considered to have 
completed the study on the date of her final protocol-defined assessment. Please note that the 
“final protocol-defined assessment” includes the follow-up visit or contact, whichever is 
later. Procedures for when a subject is lost to follow-up are provided in Section 4.5. 
3.1.1 
Open-label Treatment Period (40 Weeks) 
The Week 12/EOT visit of the antecedent study (ACP-2566-003) generally serves as the 
Baseline visit (Visit 1) of the present study.  
Those subjects for whom it is not possible to use the Week 12/EOT visit of the antecedent 
study (ACP-2566-003) as the Baseline visit (Visit 1) may be re-evaluated for study eligibility 
at the Baseline visit of the present study, with approval by the Sponsor or Medical Monitor. 
Those subjects will have blood drawn for Baseline values 3 to 14 days before the Baseline 
visit. These subjects include: 1) those who were discontinued from Study ACP-2566-003 
before completion because of the COVID-19 public health emergency; and 2) those who 
completed Study ACP-2566-003, but were prevented from entering this study because of the 
COVID-19 public health emergency.  
All efficacy assessments (except the CGI-I) will be completed at the Baseline visit prior to 
administration of the first dose of study drug in the present study (Table S–2). 
The first dose of study drug for the present study is intended to be administered after all 
Baseline assessments are completed. Since in most cases, the subject will have taken the last 
dose of study drug from the antecedent study on the morning of the Baseline visit for this 
study, the first dose of study drug for this study will then be administered either as an evening 
dose after the Baseline visit has been completed or on the following day. A triplicate 
electrocardiogram (ECG) must be performed 2-3 hours after the first dose and a PK sample 
will be taken upon completion of the ECG. 
Study drug must be discontinued at any time in the study in the event that a post-enrollment 
QTcF duration of ≥500 ms or an increase of ≥60 ms compared to the average QTcF interval 
at Baseline of the present study (before dosing) is observed. For visits at which more than 
one ECG is completed, the average QTcF interval of all legible ECGs will be used to 
determine the QTcF interval for that visit. 


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The dose will be administered according to Section 5.1.3. Dosing is twice a day, once in the 
morning and once in the evening.  
Investigational product will be shipped directly to the subject. Confirmation of delivery to the 
subject will be made by a visiting nurse. Study drug shipment, return, and accountability will 
be performed in accordance with the drug distribution plan. In addition, study drug will be 
dispensed at the site during the Baseline visit when the visit is conducted in the clinic.  
Study visits may be done off-site rather than in the clinic with the prior approval of the 
Sponsor or Medical Monitor. Efficacy assessments will be performed by qualified clinician 
raters and by caregivers at all the designated visits (Table S–2). 
For the 2 days before scheduled visits and the morning of the scheduled visit, the dates and 
times of trofinetide dosing, concomitant medication dosing, and meals will be recorded in the 
caregiver diary. Daily study drug dosing, including any dose modifications, missed or partial 
doses, will also be recorded in the caregiver diary. 
3.1.2 
Safety Follow-Up Period (30 Days) 
A 30-day safety follow-up telephone or telemedicine contact is to be completed for subjects 
who complete the treatment period of the study (and do not immediately enter any 
subsequent trofinetide study), as well as those who discontinue prematurely from the study. 
The telephone contact includes assessment of concomitant medications and treatments and 
assessment of AEs. 
4 SUBJECT ELIGIBILITY AND WITHDRAWAL CRITERIA 
To be eligible for this study, subjects must meet all of the inclusion criteria and none of the 
exclusion criteria. 
4.1 
Inclusion Criteria 
A subject must meet all of the following inclusion criteria to be eligible for participation in 
the study: 
1. Informed consent prior to the conduct of any study procedures is required as follows: 
a. For subjects who are minors: written informed consent will be obtained from 
the legally acceptable representative (LAR). The subject should provide 
written or oral assent if deemed able by the Investigator. The process of 
obtaining informed consent will be conducted in accordance with institutional 
review board (IRB) or ethics committee (EC) policy and applicable local law. 


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b. For subjects who are not minors: written informed consent will be obtained 
from the LAR or the subject if deemed able by the Investigator. If the subject 
is deemed not able to provide consent, the subject should provide written or 
oral assent if deemed able by the Investigator. The process of obtaining 
informed consent will be conducted in accordance with IRB or EC policy and 
applicable local law. 
c. The subject’s caregiver must also provide written informed consent regarding 
their participation in the study prior to participating in any study procedures. 
2. Has completed the Week 12/EOT visit of the antecedent study, Study ACP-2566-003  
NOTE: An exception is made for subjects who either discontinued from or completed 
but did not directly rollover from the antecedent study due to the COVID-19 public 
health emergency. These subjects may enroll in this study within 12 months of their 
withdrawal/completion from the antecedent study, with approval of the Sponsor or 
Medical Monitor. 
3. Met all entry criteria for the antecedent study 
4. May benefit from long-term treatment with open-label trofinetide in the judgment of 
the Investigator 
5. Can still swallow the study medication provided as a liquid solution or can take it by 
gastrostomy tube 
6. The subject’s caregiver is English-speaking and has sufficient language skills to 
complete the caregiver assessments 
Childbearing Potential 
7. Subjects of childbearing potential must abstain from sexual activity for the duration of 
the study and for at least 30 days thereafter. If a subject is sexually active or becomes 
sexually active during the study, she must use 2 clinically acceptable methods of 
contraception (e.g., oral, intrauterine device [IUD], diaphragm plus spermicide, 
injectable, transdermal or implantable contraception) for the duration of the study and 
for at least 30 days thereafter. Subject must not be pregnant or breastfeeding. 
Place of Residence 
8. Subject and caregiver(s) must reside at a location to which study drug can be 
delivered and have been at their present residence for at least 3 months prior to 
Baseline 
4.2 
Exclusion Criteria 
A subject must meet none of the following exclusion criteria to be eligible for the study: 
1. Began treatment with growth hormone during the antecedent study  


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2. Began treatment with IGF-1 during the antecedent study 
3. Began treatment with insulin during the antecedent study  
4. Has developed a clinically significant cardiovascular, endocrine (such as hypo- or 
hyperthyroidism, Type 1 diabetes mellitus, or uncontrolled Type 2 diabetes mellitus), 
renal, hepatic, respiratory, or gastrointestinal disease (such as celiac disease or 
inflammatory bowel disease) or has major surgery planned during the study 
5. Subject is judged by the Investigator or the Medical Monitor to be inappropriate for 
the study, due to AEs, medical condition, or noncompliance with investigational 
product or study procedures in the antecedent study 
6. Has a clinically significant abnormality in vital signs at Baseline 
7. Has an average QTcF interval of >450 ms on the Baseline ECG performed before the 
first dose of trofinetide is given in the present study (i.e., the ECG performed at the 
Week 12/EOT visit of the antecedent study) 
8. Has developed a clinically significant ECG finding during the antecedent study 
9. Is judged by the Investigator or the Medical Monitor to be inappropriate for the study 
for any reason 
4.3 
Subject Withdrawal of Consent 
In accordance with the Declaration of Helsinki and other applicable regulations, a subject and 
LAR consenting on behalf of a subject have the right to withdraw from the study at any time, 
and for any reason, without prejudice to her future medical care. 
Should a subject (or LAR) request or decide to withdraw consent, every reasonable effort will 
be made to complete and report observations as thoroughly as possible up to the date of 
withdrawal, including the evaluations specified at the ET or safety follow-up (whichever is 
applicable), as outlined in Table S–2. 
4.4 
Subject or Study Discontinuation 
Subjects may be discontinued from the study for a number of reasons, including, but not 
limited to, those listed below: 
• Adverse event 
• Death 
• Increase in post-enrollment QTcF interval (defined below and in Section 6.2.4.1) 
• Lack of efficacy 
• Lost to follow-up (Section 4.5) 


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• Non-compliance with study drug 
• Physician decision 
• Pregnancy 
• Protocol deviation 
• Study terminated by sponsor 
• Use of prohibited medication 
• Other 
The Sponsor reserves the right to discontinue the study at any time for any reason. Such 
reasons may be any of, but not limited to, the following: 
• Occurrence of AEs unknown to date in respect of their nature, severity, and duration 
or the unexpected incidence of known AEs 
• Medical, ethical or business reasons affecting the continued performance of the study 
Regulatory authorities also have the right to terminate the conduct of the study in their region 
for any reason. 
4.4.1 
Post-Baseline QTcF Interval Stopping Criteria 
Study drug must be discontinued in the event that a post-enrollment QTcF duration of 
≥500 ms or an increase of ≥60 ms compared to the average QTcF interval at Baseline (before 
dosing) is observed. For visits at which more than one ECG is completed, the average QTcF 
interval of all legible ECGs will be used to determine the QTcF interval for that visit. 
4.4.2 
Handling of Subject Discontinuation During the Treatment Period 
Unless the subject has withdrawn consent (or the LAR has withdrawn consent on behalf of 
the subject) to be contacted for this study, every reasonable effort should be made to 
complete Visit 5 and the safety follow-up (as outlined in Table S–2) if a subject discontinues 
prematurely for any reason. All information will be reported on the applicable pages of the 
electronic case report form (eCRF). 
If a subject is discontinued from the study because of an AE, every reasonable attempt should 
be made to follow the subject until the AE resolves or until the Investigator deems the AE to 
be chronic or stable. For subjects who continue to be followed for safety, SAEs should 
continue to be reported as described in Section 7.3.2. All SAEs will continue to be followed 
until such events have resolved or the Investigator deems them to be chronic or stable. 


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Pharmacokinetic samples will also be collected, if possible, at any ET visit or the visit 
immediately following any SAE or following any AE leading to discontinuation, even if it is 
an unscheduled visit. 
4.5 
Subject Lost to Follow-Up 
A subject will be considered lost to follow-up if they fail to attend a scheduled visit 
(excluding the safety follow-up telephone call) and the study site is unable to contact the 
subject or caregiver.  
Every reasonable effort should be made to contact the caregiver and will include a minimum 
of three documented telephone calls and, if necessary, a certified letter to the subject’s last 
known mailing address or local equivalent methods. All contact attempts are to be 
documented in the source documents. 
4.6 
Concomitant Therapy 
In order to ensure that appropriate concomitant therapy is administered, it is essential that 
caregivers be instructed not to administer any medication to the subject without prior 
consultation with the Investigator (unless the subject is receiving treatment for a medical 
emergency). 
The Investigator may prescribe appropriate medication to treat AEs. The Sponsor and 
Investigator or designee will confer to determine whether it is appropriate to continue such a 
subject in the trial if a prohibited medication is prescribed. 
In contrast to the requirements of the antecedent study, it is not necessary to make an effort to 
maintain stable regimens of concomitant medications and non-medicine based therapies 
throughout the course of the study. New medications and treatments may be initiated during 
the study, excluding the prohibited medications IGF-1, growth hormone, and insulin. 
4.6.1 
Permitted and Prohibited Medications 
Prohibited medications are IGF-1, growth hormone, and insulin. Prohibitions for concomitant 
medications will be followed between Visit 1 and Visit 5. Medications that can prolong 
QT interval are not prohibited but must be used with caution. Any use of medications that 
could interfere with study conduct must be discussed with the Medical Monitor. 
Use of medications that could interfere with study conduct or any questions regarding 
concomitant medications must be reviewed and/or discussed with the Medical Monitor. 
Subjects who require current treatment with a prohibited medication will be withdrawn from 
the study. 


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Subjects who have previously taken a prohibited medication during the study will be 
withdrawn from the study unless: 
• the prohibited medication has been discontinued AND 
• withdrawal from the study presents an unacceptable medical risk to the subject 
The justification to allow the subject to continue in the trial will be made by the 
Sponsor/Medical Monitor, with medical input from the Investigator, and will be documented. 
If a subject is allowed to remain in the trial, this will be reported as a major protocol 
deviation and not a waiver. 
5 INVESTIGATIONAL PRODUCT 
5.1 
Investigational Product Description 
The investigational product will be trofinetide oral solution. Dose will be based on weight as 
outlined in Table 5–1. Trofinetide will be provided in a ready-to-use aqueous solution for oral 
administration. Doses will be administered orally or by G tube (doses administered by 
gastrojejunal [GJ] tubes must be administered through the G-port). 
5.1.1 
Formulation, Appearance, and Packaging 
The Sponsor will supply trofinetide oral solution as an aqueous, ready-to-use, 
strawberry-flavored liquid in 500 mL (16 oz) round, high-density polyethylene (HDPE) 
plastic bottles with a child-resistant closure. 
Trofinetide oral solution is a clear, pink to red-colored liquid containing 1 gram of trofinetide 
in each 5 mL. The trofinetide oral solution also contains purified water, maltitol, strawberry 
flavor, sucralose, methylparaben sodium, propylparaben sodium, and FD&C Red #40 as 
inactive ingredients. 
Trofinetide is manufactured under current Good Manufacturing Practices. 
During the treatment period, study drug will be distributed in a quantity sufficient to ensure 
the subject has an adequate supply of study drug between study visits. 
5.1.2 
Product Storage and Stability 
Investigational product will be shipped refrigerated at a temperature between 2°C and 8°C 
(36°F and 46°F) and will be stored at this temperature. Do not freeze. 
5.1.3 
Dosing and Administration  
5.1.3.1 
Dosing 
The dose assigned to each subject in the antecedent double-blind study, ACP-2566-003, was 
based on the subject’s weight at Baseline of that study. In this study, the dose assigned to each 


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subject is based on the subject’s weight at Baseline of this study. The assigned dose is based 
on the range of weight in which the subject falls at Baseline (Table 5–1), unless the subject’s 
dose was set lower in the antecedent study for reasons of tolerability. In that case the subject’s 
assigned dose for this study will be the final dose from the antecedent study. 
The dose will not be increased or decreased if the subject’s weight at a postbaseline visit puts 
them in a new weight category. The dose may be decreased during the study for poor 
tolerability as discussed in Section 5.1.3.2. Study drug is administered twice a day, once in 
the morning and once in the afternoon or evening. There should be at least 8 hours between 
doses. 
Table 5–1 
Dosing Schedule Based on Weight at Baseline of the Present Study 
Weight 
Dose  
Total Daily Dose 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 
Abbreviation: BID=twice daily 
 
The first dose of study drug for the present study is intended to be administered after all 
Baseline assessments are completed. Since in most cases, the subject will have taken the last 
dose of study drug from the antecedent study on the morning of the Baseline visit for this 
study, the first dose of study drug for this study will then be administered either as an evening 
dose after the Baseline visit has been completed or on the following day. An ECG must be 
performed 2-3 hours after the first dose and a PK sample will be taken upon completion of 
the ECG. Off-site dosing at Baseline may only be done if it is possible to record an ECG 2-3 
hours after the first dose and draw blood for a PK sample upon completion of the ECG. The 
Investigator must review the ECG readings with special attention to the QTcF interval before 
the subject can receive another dose of study drug. Off-site dosing at Baseline must first be 
approved by the Sponsor or Medical Monitor. 
For subjects who remained on the originally assigned dose in the antecedent study, the 
assigned dose in this study will be based on the subject’s weight at the Baseline visit of the 
present study (see Table 5–1). Thus, for a particular subject whose weight at the Baseline of 
this study moves them into a different weight category according to Table 5–1, the assigned 
dose given in the present study may be different from the originally assigned dose given in 


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the antecedent study. After Baseline the dose will not be change based on a change in weight 
that places the subject into a different weight category.   
Subjects whose originally assigned dose in the antecedent study was decreased for 
tolerability reasons will remain on that same dose in this study. The dose should be increased 
at some point during the study, if tolerated, to the dose that would have been assigned based 
on the weight category at Baseline of the present study. 
The day the first dose is taken will be considered Day 1 of dosing.  
Investigational product will be shipped directly to the subject. Confirmation of any delivery 
to the subject will be made by a visiting nurse. Study drug shipment, return, and 
accountability will be performed in accordance with the drug distribution plan. In addition, 
study drug will be dispensed at the site during the Baseline visit when the visit is conducted 
in the clinic.  
5.1.3.2 
Administration of Study Drug 
Study drug is supplied in 500 mL (16 oz) bottles with a child-resistant closure. A press-in 
bottle adapter and syringe for accurately measuring the dose are supplied separately. 
The study drug must not be mixed with any food or liquid, including water.  
Doses may be taken orally or via gastrostomy tube.  
• When study drug is given orally, it may be administered directly from a syringe or 
transferred into another container to facilitate administration. 
• For gastrojejunal tubes, medication should be given via the gastric port. The tube 
must be flushed with water after study drug administration to clear the tube of study 
drug. No more than 250 mL of water should be used. Doses should be taken over a 
10-minute period. 
If the subject cannot tolerate administration of the full assigned dose according to Table 5–1 
(for example, if the subject experiences diarrhea), the Investigator may instruct the caregiver 
to reduce study drug to a dose as low as half the assigned dose. If the subject’s assigned dose 
at the beginning of this study was decreased for tolerability in the antecedent Study 
ACP-2566-003, the dose in the present study may be reduced to a dose as low as half the 
originally assigned dose from Study ACP-2566-003. For example, a subject who was 
originally assigned a dose of 40 mL (8 g) BID in Study ACP-2566-003 cannot have their 
dose in this study reduced to less than 20 mL (4 g) BID. At no point may the subject be on a 
dose that is lower than half the assigned dose. In addition, up to 4 doses (in total, consecutive 
or non-consecutive) may be held for this reason.  


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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 subject is again unable to tolerate 
that dose, the Investigator will continue treatment on the highest dose the subject can tolerate 
which should be no lower than half the assigned dose. The final dose must be given BID, 
with the same dose given once in the morning and once in the evening.  
5.1.4 
Method of Assigning Subjects to Treatment Groups 
All eligible subjects will receive twice-daily doses of trofinetide over 40 weeks of treatment. 
Details of trofinetide dosing and administration are provided in Section 5.1.3. 
5.1.5 
Blinding 
This is an open-label study. 
5.1.6 
Study Drug Compliance 
If a subject misses one dose of study drug, she must not take an extra dose the next day. 
5.1.7 
Overdose 
An overdose is a deliberate or inadvertent administration of a treatment at a dose higher than 
the maximum recommended dose per protocol. It must be reported, irrespective of outcome, 
even if toxic effects were not observed (Section 7.3.4). All events of overdose are to be 
captured as protocol deviations. 
5.2 
Investigational Product Accountability Procedures 
The Investigator or designee will keep current and accurate records of the study drug product 
dispensed, used, and returned for each subject to assure the health authority and the Sponsor 
that the study drug is being handled appropriately. Caregivers must be instructed to return all 
packaging to the Investigator or designee at regularly scheduled clinic or home nursing visits 
as appropriate.  
At appropriate intervals during the study, study drug reconciliation will be performed by the 
Sponsor (or designee) who may return appropriate unused study drug and used and unused 
packaging to the Sponsor’s designee for destruction. 
At the conclusion of the study, final study drug reconciliation will be conducted at the site. 
Final study drug accountability documentation will be maintained at both the site and by the 
Sponsor or designee. Any remaining unused study drug and all used and unused packaging 
will be sent back to the Sponsor’s designee for destruction or destroyed at the site, as allowed 
by country-specific regulations. Documentation of study drug destruction will be recorded 
and maintained by both the Sponsor and the Sponsor’s designee. 


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The Investigator or designee is responsible for taking an inventory of each shipment of study 
drug received and comparing it with the accompanying material shipping form. The 
Investigator or designee will verify the accuracy of the information on the form, sign and date 
it, and provide a copy of it to the Sponsor or designee. Any study drug supplied is for use in 
this study only and must not be used for any other purpose. 
6 STUDY PROCEDURES 
Study-specific procedures are detailed below. All assessments will be completed according to 
the schedule described in Table S–2. Every effort should be made to complete the required 
procedures and evaluations at the designated visits and times. 
The assessments at the Week 12/EOT visit of the antecedent study will serve as the Baseline 
assessment of the present study as applicable (e.g., not for the CGI-I). 
6.1 
Efficacy Assessments 
All assessments will be administered in a standardized manner. Clinician completed measures 
will be completed by trained practitioners. Caregiver completed assessments will be reviewed 
by study personnel and caregivers will receive standardized training and guidance on how to 
complete the measures. To the extent possible, all efforts should be made to maintain the 
same caregiver (i.e., caregiver rater) and clinician rater (as applicable) across visits for a 
single subject. 
All efficacy assessments designated to be completed at Baseline are to be performed prior to 
administration of the first dose of study drug in the present study. 
6.1.1 
Rett Syndrome Behaviour Questionnaire (RSBQ) 
The Rett Syndrome Behaviour Questionnaire (RSBQ) is a 45-item caregiver-completed 
rating scale assessing a wide range of neurobehavioral symptoms known to be impaired in 
RTT (Mount et al. 2002). The RSBQ is a well-validated instrument that has been used in the 
Phase 2 study, Neu-2566-RETT-002, as well as in other observational and interventional 
studies in RTT (Glaze et al. 2019; Khwaja et al. 2014; O’Leary et al. 2018). The RSBQ has 
been correlated with functioning and quality of life and has been characterized and validated 
across a range of ages and genetic variations in RTT (Cianfaglione et al. 2015; 2016; 
Robertson et al. 2006; Barnes et al. 2015). The scale includes 45 items, including 8 subscales, 
whose ratings reflect the severity and frequency of symptoms. The caregiver rates items as 
“0” (not true), “1” (somewhat or sometimes true) or “2” (very true). The eight subscales 
include: 
1. General mood 
2. Breathing problems 


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3. Hand behavior 
4. Face movements 
5. Body rocking/expressionless face 
6. Nighttime behaviors 
7. Fear/anxiety 
8. Walking/standing 
As much as possible, caregiver raters will remain the same throughout the study. At the start 
of the study all caregiver raters will be required to complete a standardized training on how to 
complete the scale. 
The RSBQ assessment will be administered at Baseline and at all other designated visits.  
6.1.2 
Clinical Global Impression–Improvement (CGI-I) and Clinical Global 
Impression–Severity (CGI-S) 
The CGI-I scale will be administered at Visits 2, 3, 4, and 5. Completion of this scale requires 
the clinician to rate how much the subject’s illness has improved or worsened relative to a 
baseline state. A 7-point scale is used from 1=very much improved, 2=much improved, 
3=minimally improved, 4=no change, 5=minimally worse, 6=much worse, 7=very much 
worse. Since the CGI-I is a rating of improvement or worsening relative to a baseline state, 
by definition there cannot be a CGI-I rating at a baseline visit. 
The CGI-I assessed at the Week 12/EOT visit of the antecedent study (which serves as the 
Baseline visit of the present study) is the final CGI-I of the antecedent study and thus will be 
a comparison with the subject’s condition at the Baseline visit of the antecedent study.  
It is very important that clinicians are aware that the CGI-I assessments at all other 
designated visits in the present study will be a comparison with the subject’s condition at 
Baseline of the present study since they are an assessment of improvement in the present 
study. 
The CGI-S assessment will be made at Baseline and at Visits 2, 3, 4, and 5. The CGI-S is a 
7-point scale that requires the clinician to rate the severity of the subject’s illness at the time 
of assessment, relative to the clinician’s experience with subjects who have the same 
diagnosis. Considering total clinical experience, a subject is assessed on severity of illness at 
the time of rating: 1, normal, not at all ill; 2, borderline ill; 3, mildly ill; 4, moderately ill; 
5, markedly ill; 6, severely ill; or 7, extremely ill. 
In this study, the illness being assessed is Rett syndrome as a whole. 


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Following best practice, the CGI-S and CGI-I ratings for the study will be assessed using 
RTT-specific anchors across major symptom areas in the same manner as in the Phase 2 
studies (Neul et al. 2015; Busner and Targum 2007; Glaze et al. 2017; Glaze et al. 2019). 
6.1.3 
Communication and Symbolic Behavior Scales Developmental Profile Infant-
Toddler (CSBS-DP-IT) Checklist  
The Communication and Symbolic Behavior Scales-Developmental Profile™ (CSBS-DP) is 
standardized screening scale for assessing communication and pre-linguistic skills in young 
children 12-24 months (Wetherby et al. 2002) and can be used with older children  with 
developmental delay (Anagnostou et al. 2015; Urbanowicz et al. 2016). The CSBS-DP 
includes a suite of three separate measures: The Infant-Toddler Checklist, a follow-up 
Caregiver Questionnaire and a Behavior Sample. In this study only the Infant-Toddler 
(CSBS-DP-IT) Checklist will be used. 
Given the limited communication abilities of individuals with Rett Syndrome, the 
CSBS-DP-IT Checklist was assessed and a subset of items were found to be appropriate for 
assessing communication skills of individuals with Rett syndrome 8 to 19 years of age 
(Urbanowicz et al. 2016). The CSBS-DP-IT was assessed in a Phase 2 trial of mecasermin 
(recombinant human IGF-1) a compound related to trofinetide, in children with Rett 
syndrome 2 to 10 years of age (O’Leary et al. 2018). In that study, the first 16 items were 
completed, which allowed for calculation of the Social composite score. The CSBS-DP-IT 
demonstrated evidence of benefit in subjects in the active treatment group compared to those 
in the placebo treatment group (O’Leary et al. 2018). The CSBS-DP Social composite score 
has also shown evidence of sensitivity to change in behavioral intervention studies in other 
developmental disorders (e.g., Wetherby et al. 2014; Anagnostou et al. 2015). 
The CSBS-DP-IT Checklist is a 24-item rating scale completed by the caregiver. Each item is 
scored using a three-level rating of frequency: “not yet”, “sometimes” and “often”. Three 
composite scores assessing 7 skill areas can be calculated: 1) Social Composite (including 
Emotion and Eye Gaze, Communication Rate and Function, and Gestures); 2) Speech 
Composite (including Sounds and Words); 3) Symbolic Composite (including Understanding 
and Object Use). 
All 24 items on the Infant-Toddler Checklist are to be completed by the caregiver. The 
question after item 24 (“Do you have any concerns about your child’s development?”) is not 
to be completed. At each administration, the study staff will review the instructions and 
scoring rubric with the caregiver. The Social Composite raw score, comprised of items 1 to 
13, is used for the key secondary endpoint. 


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The CSBS-DP-IT Checklist will be administered at Baseline and at all other designated 
visits. 
6.1.4 
Impact of Childhood Neurologic Disability (ICND) Scale 
The Impact of Childhood Neurologic Disability (ICND) scale was developed to evaluate the 
impact that a child’s condition has on the child’s and the family’s everyday life at the present 
time and during the previous 3 months (Camfield et al. 2003). The parent or other caregiver 
evaluates the effect of a condition or health problem on 11 aspects of the child’s or the 
family’s life as “A lot”, “Some”, “A little”, “Not at all”, or “Does not apply”. The four 
conditions or health problems are 1) inattentiveness, impulsivity, or mood, 2) ability to think 
and remember, 3) neurologic or physical limitations, and 4) epilepsy. 
The caregiver then rates overall quality of life of the subject 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 assessment will be administered at Baseline and Visit 5.  
6.1.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF)  
The Rett Syndrome Clinician Rating of Hand Function is a clinician completed clinical 
assessment of the subject’s ability to use her hands for functional purposes (such as reaching 
for and grasping objects, self-feeding or drawing). The assessment is made on an 8-point 
Likert scale (0-7) with 0 denoting normal functioning and 7 the most severe impairment. This 
rating is a further development of the RTT-DSC Hand Use Rating used in Study 
Neu-2566-RETT-002. 
The assessment will be administered at Baseline and at all other designated visits. 
6.1.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
(RTT-AMB)  
The Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills is a clinician 
completed clinical assessment of the subject’s ability to sit, stand, and ambulate 
(e.g., walking, running, climbing stairs). The assessment is made on an 8-point Likert scale 
(0-7) with 0 denoting normal functioning and 7 the most severe impairment. This rating is a 
further development of the RTT-DSC Ambulation Rating used in Study Neu-2566-RETT-
002. 
The assessment will be administered at Baseline and at all other designated visits. 


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6.1.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
(RTT-COMC)  
The Rett Syndrome Clinician Rating of Ability to Communicate Choices is a clinician 
completed clinical assessment of the subject’s ability to communicate her choices or 
preferences, which can include the use of nonverbal means such as eye contact or gestures. 
The assessment is made on an 8-point Likert scale (0-7) with 0 denoting normal functioning 
and 7 the most severe impairment. This rating is a further development of the RTT-DSC 
Language/Communication Rating used in Study Neu-2566-RETT-002. 
The assessment will be administered at Baseline and at all other designated visits when the 
visit takes place in the clinic. When a study visit takes place off-site, the RTT-COMC should 
be completed if possible, but it is not required.   
6.1.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM)  
The Rett Syndrome Clinician Rating of Verbal Communication is a clinician completed 
clinical assessment of the subject’s ability to communicate verbally (e.g., words and phrases). 
The assessment is made on an 8-point Likert scale (0-7) with 0 denoting normal functioning 
and 7 the most severe impairment. This rating is a further development of the RTT-DSC 
Language/Communication Rating used in Study Neu-2566-RETT-002. 
The assessment will be administered at Baseline and at all other designated visits. 
6.1.9 
Rett Syndrome Caregiver Burden Inventory (RTT-CBI) 
The RTT-CBI is a syndrome-specific, caregiver-completed questionnaire that is based on the 
Caregiver Burden Inventory designed for Alzheimer’s disease (Lane et al. 2017; Novak and 
Guest 1989). The scale is intended to directly address caregiver burden and indirectly assess 
the significance of treatment effects on function in the context of activities of daily living. 
Caregivers rate how often a given statement describes their feeling or experience. Frequency 
ratings are on a 5-point Likert scale including: 0-never; 1-rarely; 2-sometimes; 3-frequently 
and 4-nearly always. As in the original Caregiver Burden Inventory, the RTT-CBI has 
24 negatively worded items (items 1 through 24) yielding a total score up to 96. The 
RTT-CBI also includes 2 positively worded items (items 25 and 26) that comprise the 
Optimism Index (Lane et al. 2017). In this study, as in the 2 previous studies of trofinetide in 
Rett syndrome, the total score is defined as the total Burden score (items 1-24). 
The RTT-CBI will be completed at Baseline, Visit 3, and Visit 5. 


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6.2 
Safety Assessments  
6.2.1 
Physical Examination 
A general physical examination will be conducted at Baseline and at all other designated 
visits. When a study visit takes place off-site, the physical examination will not be required. 
The physical exam procedures will include the following organ systems: 
• Neurological 
• Head, ears, eyes, nose, and throat 
• Skin 
• Cardiovascular 
• Respiratory 
• Abdomen 
• Genitourinary (optional) 
• Musculoskeletal 
6.2.2 
Vital Signs 
Vital signs will include body temperature, resting respiration rate, sitting systolic and 
diastolic blood pressure, and pulse rate. The sitting blood pressure will be measured after the 
subject has been sitting for ≥3 minutes. 
Vital signs to be measured at Baseline and at all other designated visits. 
6.2.3 
Height, Weight, and Body Mass Index 
Height will be measured at Baseline and Visit 5. 
Weight will be measured at Baseline, Visit 2, Visit 3, Visit 4, and Visit 5 when the visit takes 
place in the clinic. Measurement of weight is necessary at Baseline in order to determine the 
starting dose. When a study visit takes place off-site, weight should be measured whenever 
possible. 
Body mass index will be calculated using the following formula: 
Weight (kg) / [height (m)]2. 
6.2.4 
Electrocardiograms 
All 12-lead ECGs will be complete, standardized recordings, whenever possible. ECGs will 
be completed in triplicate at Visit 1 (Baseline) both before dosing and 2-3 hours after dosing 
and at Visit 5 (Week 40/EOT). For those subjects who completed the antecedent study, the 


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ECG taken at the Week 12/EOT visit in the antecedent study is the predose Baseline ECG of 
the present study. Subjects who were discontinued from the preceding study before 
completion because of the COVID-19 public health emergency and subjects who completed 
but were prevented from entering the present study because of the COVID-19 public health 
emergency, will have ECGs completed in triplicate at the Baseline visit which will be 
considered the predose Baseline ECG of the present study. A single ECG will be completed 
at Visit 2 (Week 2), Visit 3 (Week 12), and Visit 4 (Week 26). For visits at which more than 
one ECG is completed, the average QTcF interval of all legible ECGs will be used to 
determine the QTcF interval for that visit. 
The subject should rest for ≥5 minutes in a supine position before before the ECG is 
obtained. If it is impractical to rest in supine position due to the subject’s medical condition, 
the subject may remain in a partial supine or other position (e.g., upright in wheelchair) for 
≥5 minutes before the ECG is obtained and when the ECG is obtained. ECG tracings (paper 
or electronic) will be reviewed and interpreted by a qualified clinician for prolongation of the 
QTcF interval and for other cardiac irregularities. ECG tracings and results (ventricular rate, 
PR, QRS, QT, QTcF and QTcB intervals) will be included in the subject’s study records.  
At Baseline, the average QTcF interval of all legible ECGs will be used to determine 
eligibility. ECGs will also be read by a qualified central reader. The central reading will be 
the reading that is entered in the database. The results from the reports from the central reader 
will also be reviewed by the Investigator. 
6.2.4.1 
Post-Enrollment QTcF Interval Stopping Criteria 
In the event that a post-enrollment QTcF duration of ≥500 ms or an increase of ≥60 ms 
compared to the average QTcF interval at Baseline of the present study (before dosing) is 
observed, study drug administration is to be discontinued. For visits at which more than one 
ECG is completed, the average QTcF interval of all legible ECGs will be used to determine 
the QTcF interval for that visit. 
6.2.5 
Laboratory Evaluations 
Laboratory evaluations will be completed according to the schedule presented in Table S–2 
and procedures detailed in the laboratory manual. Additional safety testing may be performed 
at the discretion of the Investigator or designee. 
Clinical laboratory sample collection is not required to be completed under fasting 
conditions. The laboratory evaluations will include the following: 
• Clinical chemistry serum tests 


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o Sodium (Na), potassium (K), chloride (Cl), phosphorus (P), calcium (Ca), carbon 
dioxide (CO2), blood urea nitrogen (BUN), creatinine (CR), uric acid 
o Alanine aminotransferase (ALT), aspartate aminotransferase (AST), 
gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total 
bilirubin (TBIL), lactate dehydrogenase (LDH) 
o Glucose 
o Albumin (ALB), total protein 
o Thyroid stimulating hormone (TSH), free T3, and free T4  
 Thyroid function tests will be performed at Visit 1 (Baseline) and 
Visit 5 
• Pregnancy test 
o A serum pregnancy test will be performed at all designated visits (Table S–2) for 
subjects of childbearing potential 
• Hematology tests 
o Complete blood count (CBC) including: 
 White blood cell (WBC) count 
 Complete differential (relative and absolute) 
 Hematocrit (Hct), hemoglobin, red blood cells (RBC), platelets 
 Reticulocyte count 
• Urinalysis 
o Blood, RBCs, WBCs, protein, glucose, ketones, specific gravity, pH 
o Reasonable efforts will be made to collect a urine sample from all subjects. When 
collection of a urine sample proves impractical or impossible (e.g., because the 
subject is incontinent or unable to cooperate), failure to collect a urine sample will 
be recorded in the subject’s eCRF, and will not be considered a protocol deviation 
Subjects for whom the Baseline visit of this study is not on the same day as the Week 12/EOT 
visit of the antecedent study (e.g., subjects in the antecedent study who were prevented from 
entering this study by the COVID-19 public health emergency) will have blood drawn for 
Baseline values three to fourteen days before the Baseline visit. The investigator should have 
the results of the following standard laboratory studies available for review before enrolling 
the subject: the basic metabolic panel (Na, K, Cl, P, Ca, CO2, BUN, and CR), serum glucose, 


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the liver function tests (ALT, AST, GGT, ALP, TBIL and LDH), serum pregnancy test (if the 
subject is of childbearing potential), and CBC. 
6.3 
Caregiver Diary 
A semi-structured caregiver diary will be completed during treatment in which caregivers 
will record seizures and seizure-like spells if they are present.  
At the Baseline visit, the clinician will review subject’s seizure profile (if applicable) and will 
help to characterize the subject’s typical seizure types. These are recorded in the caregiver 
diary. 
For the 2 days before scheduled visits as well as the morning of the scheduled visit, the dates 
and times of trofinetide dosing, concomitant medication dosing, and meals will be recorded 
in the caregiver diary. Daily study drug dosing, including any dose modifications, missed or 
partial doses, will also be recorded in the caregiver diary. 
The caregiver diary will be completed and collected on an ongoing basis throughout the study 
from Baseline to Visit 5. The clinician will verbally ask about AEs (see Section 7.3.1), review 
the events recorded in the diary with the caregiver and will make a clinical evaluation, 
including an evaluation of whether an AE will be reported. 
6.4 
Pharmacokinetic Assessments 
Pharmacokinetic blood samples will be collected for measurement of whole blood 
concentrations of trofinetide and any relevant possible metabolites identified. 
Pharmacokinetic blood samples will be collected for trofinetide concentration measurements 
at the Baseline visit (both before dosing and after dosing) and at Visit 2, Visit 3, Visit 4, and 
Visit 5 in accordance with the sampling schedule outlined below (Table 6-1). 
The PK sample taken at the Week 12/EOT visit in the antecedent study is the predose 
PK blood sample for the present study. The second PK sample to be taken at the Baseline 
visit (Visit 1), approximately 2-3 hours after dosing, will take place as soon as possible after 
the postdose ECG is performed.  
PK samples at Visits 2, 3, 4, and 5 will be collected at one of the following time intervals: 
• 2-3 hours after dosing 
• 4-6 hours after dosing 
• 7-11 hours after dosing 
Every effort should be made to collect PK samples at discrete time intervals during Visits 2, 
3, 4, and 5. However, if the interval is the same at each visit, then the collection time should 


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vary within that interval. The following scenario is only for illustrative purposes, a number of 
other scenarios are possible.  
• For example, if the time interval of 4-6 hours after dosing is used for Visit 2, 3, 4, and 
5, then every effort should be made to collect PK samples at 4, 4.5, 5, and 6 hours 
after dosing, respectively. 
Pharmacokinetic samples will also be collected, if possible, at any ET visit or the visit 
immediately following any SAE or following any AE leading to discontinuation, even if it is 
an unscheduled visit. 
For all scheduled PK samples (and for unscheduled samples if possible), the dates and times 
of administration of the study drug, the dates, times and content of the meals, and the dates 
and times of the administration of concomitant medication over the 2 days prior to and on the 
morning of the PK sample draw, as well as the date and time of the sample draw, will be 
recorded. For samples collected from subjects who experience any SAE or experience an 
AE leading to discontinuation, the date and time of the last dose of study drug prior to the 
SAE or AE leading to discontinuation will also be recorded. 
Table 6-1 
PK Sampling Times 
Visit 
Timing of Sample  
(relative to start of dosing on the visit day) 
Week of Treatment 
1 (Baseline) 
Any time before dosing 
and 
2 – 3 hours after dosing (after ECG) 
Week 0 (Baseline) 
2, 3, 4, and 5 
2 – 3 hours after dosing  
or 
4 – 6 hours after dosing 
or 
7 – 11 hours after dosing 
Weeks 2, 12, 26, and 40/EOT 
Abbreviations: ECG=electrocardiogram; EOT=end of treatment 
6.4.1 
Specimen Preparation, Handling, Storage, and Shipment 
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. Blood samples will be processed for determination of trofinetide 
whole blood concentrations (and of concentrations of any relevant possible metabolites 
identified). At each timepoint, blood will be collected, processed, and samples will be 
shipped to the central laboratory for storage and to the bioanalytical laboratory for analysis. 
A laboratory manual will be provided for sample processing, storage, and shipping 
procedures. 


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When possible, an additional PK sample will be collected from subjects who experience any 
SAE or experience an AE leading to discontinuation as soon as possible after the occurrence 
of that event. 
6.5 
Identification of Biomarkers of Response to Trofinetide in Rett Syndrome 
Subjects for whom separate informed consent for the identification of biomarkers of response 
to trofinetide is provided (where local regulations permit) will have blood drawn and stored 
for future investigations. Blood samples will be taken at Baseline (before dosing) and at 
Visit 3, Visit 4, and Visit 5, or upon early termination. The biomarker sample taken at the 
Week 12/EOT visit in the antecedent study is the Baseline sample for the present study. 
Participation in the effort to identify biomarkers is an optional component of the study 
requiring a separate informed consent, which may be obtained at any time during the study.  
Blood samples will be used to investigate differences between responders and non-responders 
in trofinetide-treated subjects in RNA transcripts (transcriptomics), proteins (proteomics), 
and metabolites (metabolomics). Unbiased analyses and targeted analyses will test candidate 
molecular pathways based on the available knowledge of RTT and trofinetide at the time of 
the investigation (Ehrhart et al. 2016; Shovlin and Tropea 2018; West et al. 2014; 
Buchovecky et al. 2013). The analysis of biomarkers does not include any DNA, genomic, or 
genetic testing or analysis.   
Stored samples and relevant clinical data will be made non-identifiable after the clinical 
study report has been issued. Any personal identifiers will be removed, and each study 
subject identifier will be replaced with a new number to limit the possibility of linking 
genetic data to a subject’s identity. 
7 ADVERSE EVENTS 
7.1 
Specification of Safety Parameters 
7.1.1 
Definition of Adverse Event 
An AE is defined as “any untoward medical occurrence in a patient or clinical study 
participant, temporally associated with the use of study drug, whether or not considered 
related to study drug”. 
An AE can therefore 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 or seriousness. An AE can arise from any use of the drug 
(e.g., off-label use, use in combination with another drug) and from any route of 
administration, formulation, or dose, including an overdose. 


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A suspected adverse reaction is any AE for which there is a reasonable possibility that the 
drug caused the AE. 
AEs do not include the following: 
• Stable or intermittent chronic conditions (such as myopia requiring eyeglasses) that 
are present prior to Baseline and do not worsen during the study 
• Medical or surgical procedures (e.g., surgery, endoscopy, tooth extraction, 
transfusion). The condition that leads to the procedure is an AE if not present at 
Baseline. 
• Overdose of concomitant medication without any signs or symptoms unless the 
subject is hospitalized for observation 
• Hospitalization for elective surgery planned prior to study (situation where an 
untoward medical occurrence has not occurred) 
• Pregnancy will not be considered an AE, but if it occurs, it will be reported on a 
pregnancy form 
 
7.1.2 
Definition of Serious Adverse Event 
In addition to the severity rating, each AE will be classified by the Investigator as “serious” 
or “not serious.” The seriousness of an event will be defined according to the applicable 
regulations and generally refers to the outcome of an event. An SAE is one that meets one or 
more of the following: 
• Is fatal 
• Is immediately life threatening 
• Results in disability or permanent damage 
• Requires hospitalization 
• Prolongs existing hospitalization 
• Is a congenital anomaly or birth defect (in an offspring) 
• Is medically significant 


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Definition of Life Threatening 
A life-threatening event places the subject at immediate risk of death from the event as it 
occurred. This does not include an AE, which, had it occurred in a more severe form, might 
have caused death. 
Definition of Hospitalization 
Hospitalization is defined by the Sponsor as a full admission to the hospital for diagnosis and 
treatment. This includes prolongation of an existing inpatient hospitalization.  
Examples of visits to a hospital facility that do not meet the serious criteria for 
hospitalization include: 
• Emergency room visits (that do not result in a full hospital admission) 
• Outpatient surgery 
• Preplanned or elective procedures 
• Protocol procedures 
• Social hospitalization, defined as admission to the hospital as a result of inadequate 
family support or care at the subject’s primary residence  
 
Definition of Disability or Permanent Damage 
Disability is defined as a persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions. 
Definition of Medically Significant 
Important medical events (medically significant events) that may not result in death, be life 
threatening, or require hospitalization may be considered to be an SAE when, based upon 
appropriate medical judgment, they may jeopardize the subject or may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. Examples of 
such events are intensive treatment in an emergency room or at home for allergic 
bronchospasm, blood dyscrasias, or convulsions that do not result in hospitalization or 
development of drug dependency or drug abuse. 
An SAE may also include any other event that the Investigator or Medical Monitor judges to 
be serious or that suggests a significant hazard, contraindication, side effect, or precaution. 


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7.2 
Classification of an Adverse Event 
7.2.1 
Severity of Event 
The severity of each AE will be graded on a 3-point scale and reported in detail as indicated 
on the eCRF: 
• Mild: awareness of sign or symptom but easily tolerated, causing minimal 
discomfort, and not interfering with normal everyday activities 
• Moderate: sufficiently discomforting to interfere with normal everyday activities 
• Severe: incapacitating and/or preventing normal everyday activities 
7.2.2 
Relationship to Study Drug 
The causality of each AE should be assessed and classified by the Investigator as “related” or 
“not related.” An event is considered related if there is a reasonable possibility that the event 
may have been caused by the product under investigation (i.e., there are facts, evidence, or 
arguments to suggest possible causation). 
Consider the following when assessing causality: 
• Temporal associations between the agent and the event 
• Response to cessation (de-challenge) or re-challenge 
• Compatibility with known class effect 
• Known effects of concomitant medications 
• Pre-existing risk factors 
• A plausible mechanism 
• Concurrent illnesses 
7.2.2.1 
Duration 
The start and stop dates for AEs will be recorded using the following criteria: 
• Start: Date of the first episode of the AE or date of significant sustained worsening in 
severity 
• Stop: Date when AE either ceased permanently or changed in severity 
For AEs of diarrhea, the start date and stop date of a decrease in severity will also be 
recorded. 


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7.2.2.2 
Frequency 
The frequency of the AE should be indicated according to the following definitions: 
• Single: Experienced once, without recurrence 
• Recurrent: More than one discrete episode with the same severity 
7.2.2.3 
Action Taken with Study Drug 
• Dose not changed: No change in study drug 
• Drug interrupted: Study drug temporarily stopped 
• Drug withdrawn: Study drug discontinued permanently 
• Dose decreased: Dose of study drug reduced 
7.2.2.4 
Therapy 
• None: No new treatment instituted 
• Medication: New treatment initiated as a direct result of AE 
• Other: Other action required 
7.2.2.5 
Outcome 
• Recovered/resolved: Recovered or resolved 
• Recovered/resolved with sequelae: Recovered or resolved with sequelae 
• Not recovered/not resolved: Not recovered or not resolved 
• Fatal: Death due to an AE 
• Unknown: Unknown 
7.2.2.6 
Seriousness 
• Not serious 
• Serious 
7.2.3 
Definition of Unexpectedness 
An AE, the nature or severity of which is not consistent with the information provided in the 
Reference Safety Information section of the current trofinetide Investigator’s Brochure. 
7.3 
Time Period and Frequency for Event Assessment and Follow-Up 
Adverse events with an onset before the first dose of open-label trofinetide in the present 
study will be recorded as an AE in the antecedent study. All ongoing AEs from Study 


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ACP-2566-003 will be carried over after informed consent has been obtained for the present 
study and recorded from Baseline for the present study until resolution or the follow-up 
safety assessment. An adverse event occurring after the first dose of open-label trofinetide in 
the present study will be recorded as an AE in the present study. Adverse events for the 
present study will be recorded from the time of first dose of open-label trofinetide through 
the safety follow-up period. 
All AEs must be either resolved or stable at the end of the safety follow-up period. If ongoing 
at the end of the safety follow-up period, the subject should be referred for appropriate 
treatment. 
In the event that a subject discontinues and has an ongoing AE at the time of discontinuation 
(Section 4.4.2) or is withdrawn from the study because of an AE, the subject should be 
followed and treated by the Investigator until the AE has resolved, stabilized, or a new 
chronic baseline has been established. 
7.3.1 
Adverse Event Reporting 
The Investigator must record all observed AEs and all reported AEs. At each visit, the 
Investigator should ask the subject a nonspecific question (e.g., “Have you noticed anything 
different since your last visit?”) to assess whether any AEs have been experienced since the 
last report or visit. 
Note that any use of medication (and specifically any newly prescribed medication) during 
the course of a study may indicate the occurrence of an AE that may need to be recorded on 
both the AE and the concomitant medication page. 
All AEs, serious and not serious, will be recorded on the AE eCRF page using appropriate 
medical terminology. Severity and relationship to study drug will be assessed by the 
Investigator. 
When possible, clinical AEs should be described by diagnosis and not by symptoms 
(e.g., “cold” or “seasonal allergies” instead of “runny nose”). 
All AEs, whether or not related to the study drug, must be fully and completely documented 
on the AE eCRF and in the subject’s notes. 
7.3.2 
Serious Adverse Event Reporting 
The reporting of SAEs by the Sponsor or designee to the regulatory authorities is a regulatory 
requirement. Each regulatory authority has established a timetable for reporting SAEs based 
upon established criteria. 


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Serious AEs must be reported within 24 hours of discovery to the Sponsor or its designee; use 
the appropriate form for initial and/or follow-up reporting.  
At a minimum, events identified by the Sponsor to require expedited reporting as serious, 
unexpected, and related to study drug must be brought to the attention of the responsible 
institutional review board/ethics committee (IRB/EC), as per applicable regulations. These 
will be provided by the Sponsor after their assessment. For European Union member states, 
the Sponsor or its designee will provide reports of suspected unexpected serious adverse 
reactions (SUSARs) directly to the ECs, as required by local legislation. In all other 
countries, it is the Investigator’s responsibility to provide these expedited reports to the 
responsible IRB/EC. It is also the Investigator’s responsibility to notify the responsible 
IRB/EC regarding any new and significant safety information. 
When an SAE occurs, Investigators will review all documentation related to the event and 
will complete the paper SAE form (for initial and/or follow-up information) and fax or email 
(within 24 hours of discovery) to the contact information provided on the SAE form. 
Subjects will be followed through the safety follow-up period for 30 days after last dose of 
study drug for any SAEs and/or other reportable information until such events have resolved 
or the Investigator, in conjunction with the Sponsor, deems them to be chronic or stable. 
In the event of any SAE (other than death), the study subject will be instructed to contact the 
Investigator (or designee) using the telephone number provided in the informed consent form 
(ICF). All subjects experiencing an SAE will be seen by the Investigator or designee as soon 
as is feasible following the report of the SAE. 
Serious AEs occurring after the study follow-up period (i.e., 30 days after last dose of study 
drug) should be reported if in the judgment of the Investigator there is “a reasonable 
possibility” that the event may have been caused by the product. 
SAEs should also be reported to the IRB/EC according to local regulations.  
7.3.3 
Reporting of Pregnancy 
Any subject who becomes pregnant during the study (with or without AEs) must be 
withdrawn from the study and the pregnancy must be reported on the Pregnancy form within 
24 hours of discovery to the Sponsor or its designee. Any subject who becomes pregnant 
during the study will be followed through the pregnancy outcome. 
Any AEs that are the consequence of pregnancy and which meet the criteria for serious 
should also be reported via the SAE form. 
7.3.3.1 Reporting Paternal Drug Exposure 


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Paternal drug exposure is defined as a father’s exposure to a medicinal product before or 
during his partner’s pregnancy. Any paternal drug exposure cases must be reported to the 
Sponsor within 24 hours of discovery via the Pregnancy form. Any AEs that are the 
consequence of paternal drug exposure and which meet the criteria for serious must also be 
reported to the Sponsor within 24 hours of discovery via the SAE form. Since no males are 
enrolling in this study, paternal drug exposure would occur only if a male who was not a 
study subject ingested study drug. 
7.3.4 
Reporting of Overdose 
An overdose is a deliberate or inadvertent administration of a treatment at a dose higher than 
the maximum recommended dose per protocol. It must be reported to the Sponsor or 
designee on the Overdose form within 24 hours of discovery. In addition, all events of 
overdose are to be captured as protocol deviations. 
8 CLINICAL MONITORING  
Routine monitoring of study sites is described in Section 11. 
Clinical site monitoring is conducted to ensure that the rights and well-being of human 
subjects are protected, that the reported study data are accurate, complete, and verifiable, and 
that the conduct of the study is in compliance with the currently approved protocol and 
amendment(s) as applicable, with GCP, and with applicable regulatory requirements. Details 
of the study site monitoring process are described in a separate clinical monitoring plan 
document. 
9 STATISTICAL METHODS AND DATA ANALYSIS 
9.1 
Statistical and Analytical Plans 
Statistical methods will be documented in detail in a statistical analysis plan (SAP) to be 
approved by the Sponsor prior to database lock. 
9.2 
Statistical Hypotheses 
No formal testing of hypotheses is planned. All outcomes will be summarized descriptively. 
9.3 
Sample Size Determination 
Up to approximately 180 subjects will be enrolled. The sample size for this study is not based 
on statistical power, but will depend on the number of subjects who transition into this 
open-label extension study from the antecedent study. 


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9.4 
Subject Populations for Analysis 
The Safety Analysis Set will consist of all subjects who have taken at least one dose of study 
drug. The Safety Analysis Set will be used for all analyses. 
Any other analysis sets, if necessary, will be defined in the statistical analysis plan (SAP). 
9.5 
Statistical Analyses 
9.5.1 
General Approach 
All endpoints will be summarized for the Safety Analysis Set. Additional summaries by prior 
treatment may be included. 
Data from the Week 12/EOT visit procedures of Study ACP-2566-003 will be carried over as 
Baseline information in the present study, as applicable (e.g., not for the CGI-I). An 
exception is made for those subjects for whom it was not possible to use the Week 12/EOT 
visit of the antecedent study (ACP-2566-003) as the Baseline visit (Visit 1) because of the 
COVID-19 public health emergency. For those subjects, data from the Week 12/EOT visit 
procedures of Study ACP-2566-003 will not be carried over as Baseline information in the 
present study. 
Continuous measurement results will be reported using the number of subjects with data 
values, mean, standard error of the mean, median, standard deviation, minimum, and 
maximum. For each categorical outcome, the frequency and percentage of subjects in each 
category will be reported. 
9.5.2 
Primary Analyses 
Adverse events will be classified into standard terminology using the Medical Dictionary for 
Regulatory Activities (MedDRA). All AEs will be listed and TEAEs will be summarized by 
system organ class and preferred term. A TEAE is defined as an AE that started after the first 
administration of study drug and no later than the last administration of study drug plus 
30 days. Summaries by maximum severity and by relationship to study drug will also be 
provided. Serious TEAEs, fatal AEs, and TEAEs leading to discontinuation will also be 
summarized. 
Descriptive summary statistics for ECG, vital signs and weight, and clinical laboratory 
parameters, including observed values and changes from Baseline, will be tabulated by 
timepoint. Additionally, categorical analyses will be conducted on the incidence of subjects 
with prolonged QTc intervals and changes in QTc intervals in accordance with International 
Council for Harmonisation (ICH) guidelines. 


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9.5.3 
Secondary Analyses 
Descriptive summary statistics for RSBQ, CGI-I (observed value only, no change from 
Baseline), CGI-S, CSBS-DP-IT Social, ICND (QoL score and total score), each of the four 
Rett Syndrome Clinician Rating scales, and the RTT-CBI observed values and change from 
Baseline will be summarized by timepoint. For the CGI-I there will be descriptive summary 
statistics of the observed value only. 
9.5.4 
Pharmacokinetic Analyses 
Pharmacokinetic (PK) and efficacy (PD) measures will be collected from all subjects at 
Baseline both before dosing and after dosing and after dosing at Weeks 2, 12, 26, and 
40/EOT.  
Whole blood concentration and possible metabolites data for trofinetide will be listed and 
summarized using descriptive statistics. If data allow, population PK analyses will be 
performed to further characterize the PK. The details of the PK analysis will be presented in a 
separate population PK report and PK/PD report in accordance with a separate data analysis 
plan (DAP). 
9.5.5 
Subgroup Analyses 
Selected analyses may be performed in subgroups. Details will be provided in the SAP. 
9.6 
Interim Analyses 
No interim analysis is planned in this study. 
9.7 
Measures to Minimize Bias 
Not applicable. The present study is open-label. 
9.8 
Breaking the Study Blind/Subject Code 
Not applicable. The present study is open-label. 
10 STUDY MANAGEMENT AND DATA COLLECTION 
10.1 Data Collection and Management Responsibilities 
All documents required for the conduct of the study as specified in the ICH GCP guidelines 
will be maintained by the Investigator in an orderly manner and made available for 
monitoring and/or auditing by the Sponsor and regulatory authorities. 
The Investigator and institution must permit authorized representatives of the Sponsor or 
designees (including monitors and auditors), regulatory authorities (including inspectors), 
and the IRB/EC direct access to source documents (such as original medical records). Direct 
access includes permission to examine, analyze, verify, and reproduce any records and 


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reports that are needed for the evaluation of the study. The Investigator must ensure the 
reliability and availability of source documents from which the information on the eCRF was 
derived. 
10.2 Source Documents 
All study specific information obtained at each study visit must be recorded in the subject’s 
record (source documentation), and then entered into a validated electronic data capture 
(EDC) database by trained site personnel. The source documentation may consist of source 
notes captured by site personnel and the caregiver diaries, as well as laboratory reports, 
ECG reports, and electronic source data. 
10.3 Case Report Forms 
Subject data required by this protocol are to be recorded in an EDC system on eCRFs. The 
Investigator and his or her site personnel will be responsible for completing the eCRFs. 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 subject 
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. 
10.4 Confidentiality 
The Investigator must ensure that each subject’s anonymity is maintained as described below. 
On the eCRFs or other documents submitted to the Sponsor or designees, subjects must be 
identified by a subject identification number only. Subject identifiers uniquely identify 
subjects within the study and do not identify any person specifically. Documents that are not 
for submission to the Sponsor or designees (e.g., signed ICFs) should be kept in strict 
confidence by the Investigator in compliance with Federal regulations or other applicable 
laws or ICH guidance on GCP. Data collection and handling should comply with the 
European Union General Data Protection Regulation (EU GDPR), where applicable. 
ACADIA has assigned a Data Protection Officer (DPO) as per the EU GDPR. 
10.5 Study Records Retention 
Investigators are required to maintain all essential study documentation as per ICH GCP 
guidelines. This includes, but is not limited to, copies of signed, dated and completed eCRFs, 
documentation of eCRF corrections, signed ICFs, subject-related source documentation, and 
adequate records for the receipt and disposition of all study drug. Investigators should 
maintain all essential study documentation, for a period of at least 2 years following the last 
approval of marketing application in an ICH region (US, Europe, and Japan), or until at least 


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2 years after the drug investigational program is discontinued, unless a longer period is 
required by applicable law or regulation. Only the Sponsor can notify an Investigator or 
vendor when any records may be discarded. Investigators should contact the Sponsor before 
destroying any files. 
10.6 Protocol Exceptions and Deviations 
No prospective entry criteria protocol deviations are allowed; all subjects must meet all 
eligibility criteria in order to participate in the study. Subjects with major eligibility protocol 
deviations identified in the antecedent study are not eligible for the present study even if the 
subject was allowed to continue in the antecedent study. 
Protocol waivers for eligibility will not be granted by the Sponsor under any circumstances. 
If, during the course of a subject’s post-enrollment participation in the trial it is discovered 
that the subject did not meet all eligibility criteria, he or she will be discontinued, unless the 
discontinuation presents an unacceptable medical risk. The justification to allow the subject 
to continue in the trial will be made by the Sponsor, with medical input from the Investigator, 
and will be documented. If allowed to remain in the trial, this will be reported as a major 
protocol deviation and not a waiver. All follow-up safety assessments must be completed and 
documented as outlined in the protocol (Section 3.1.2). The Investigator must report any 
protocol deviation to the Sponsor and, if required, to the IRB/EC in accordance with local 
regulations, within reasonable time. 
10.7 Protocol Amendments 
Changes to the protocol may be made only by the Sponsor (with or without consultation with 
the Investigator). All protocol modifications must be submitted to the site IRB/EC in 
accordance with local requirements and, if required, to regulatory authorities, as either an 
amendment or a notification. Approval for amendments must be awaited before any changes 
can be implemented, except for changes necessary to eliminate an immediate hazard to trial 
subjects, or when the changes involve only logistical or administrative aspects of the trial. 
No approval is required for notifications. 
11 STUDY MONITORING, AUDITING, AND INSPECTING 
11.1 Quality Control and Quality Assurance 
The Sponsor or designees and regulatory authority inspectors are responsible for contacting 
and visiting the Investigator for the purpose of inspecting the facilities and, upon request, 
inspecting the various records of the trial (e.g., eCRFs and other pertinent data) provided that 
subject confidentiality is respected. 


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The Sponsor’s or designee’s monitor is responsible for inspecting the eCRFs at regular 
intervals throughout the study to verify adherence to the protocol; completeness, accuracy, 
and consistency of the data; and adherence to local regulations on the conduct of clinical 
research. The monitor should have access to subject medical records and other study-related 
records needed to verify the entries on the eCRFs. 
The Investigator agrees to cooperate with the monitor to ensure that any problems detected in 
the course of these monitoring visits are resolved. 
In accordance with ICH guidance on GCP and the Sponsor’s audit plans, a certain percentage 
of sites participating in this study will be audited. These audits may include a review of site 
facilities (e.g., pharmacy, drug storage areas, and laboratories) and review of study-related 
records may occur in order to evaluate the trial conduct and compliance with the protocol, 
ICH guidance on GCP, and applicable regulatory requirements. 
The Sponsor’s or designee’s representatives, regulatory authority inspectors and IRB/EC 
representatives who obtain direct access to source documents should also respect subject 
confidentiality, taking all reasonable precautions in accordance with applicable regulatory 
requirements to maintain the confidentiality of subjects’ identities. 
11.2 Risk Management 
ACADIA utilizes the ICH E6 (GCP) Revision 2 risk management approach that includes 
methods to assure and control the quality of the trial proportionate to the risks inherent in the 
trial and the importance of the information collected. The intent is that all aspects of this trial 
are operationally feasible and that any unnecessary complexity, procedures, and data 
collection are avoided. The ACADIA risk management approach includes the following 
activities with a focus on critical processes and critical study data:  
• 
Risk Identification: risks to critical trial processes, governing systems, investigational 
product, trial design, data collection, and recording are identified. 
• Risk Evaluation: identified risks are evaluated by considering the following factors: 
(a) likelihood of occurrence, (b) impact on human subject protection and data 
integrity, and (c) detectability of errors. 
• Risk Control: risks that can be reduced (e.g., mitigating) or can be accepted are 
differentiated. Risk mitigation activities are incorporated in protocol design and 
implementation, study plans, training, processes, and other documents governing the 
oversight and execution of study activities. Where possible, predefined quality 
tolerance limits are to be defined to identify systematic issues that can impact subject 
safety or data integrity and deviations from the predefined quality tolerance limits will 


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trigger an evaluation and possibly an action. Contingency plans are developed for 
issues with a high risk factor that cannot be avoided. 
• Periodic risk review, communication, and escalation of risk management activities are 
ongoing during trial execution and risk outcomes are reported in the clinical study 
report (CSR). 
12 ETHICAL CONSIDERATIONS 
12.1 Ethical Standard 
The study will be conducted in compliance with the protocol, the Declaration of Helsinki, 
ICH GCP, and other applicable regulatory requirements (e.g., Serious Breach reporting, 
urgent safety measures, and EU GDPR). 
The study will be performed in accordance with the US Health Insurance Portability and 
Accountability Act (HIPAA) regulations, US FDA GCP Regulations (US Code of Federal 
Regulations [CFR] 21 parts 50, 54, 56, and 312), and ICH guidance on GCP (E6) and clinical 
safety data management (E2A). 
In accordance with Directive 75/318/EEC, as amended by Directive 91/507/EEC, the final 
clinical study report will be signed by an Investigator and/or Coordinating Investigator who 
will be designated prior to the writing of the clinical study report. 
12.2 Institutional Review Board/Ethics Committee 
The Investigator or designee will provide the IRB/EC with all requisite material, including a 
copy of the protocol, informed consent, and any subject information or advertising materials. 
The study will not be initiated until the IRB/EC provides written approval of the protocol and 
the informed consent and until approved documents have been obtained by the Investigator 
and copies received by the Sponsor. All amendments will be sent to the IRB/EC for 
information (minor amendment) or for submission (major amendment) before 
implementation. The Investigator will supply the IRB/EC and the Sponsor with appropriate 
reports on the progress of this study, including any necessary safety updates, in accordance 
with the applicable government regulations and in agreement with policy established by the 
Sponsor. 
12.3 Informed Consent Process 
In accord with the provisions of the US CFR 21 part 50, and since this study involves greater 
than minimal risk but presents the prospect of direct benefit to all subjects enrolled, consent 
shall be obtained from an LAR, typically 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 PI and per local IRB requirements. 


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Properly executed, informed consent/assent must be obtained from each LAR/subject prior to 
the final procedures being performed at the EOT visit in the antecedent study. Subjects who 
enter the present study under special circumstances due to the COVID-19 public health 
emergency will have consent obtained for the present study before any procedures are 
performed (e.g., such as the blood draw before the Baseline visit). The LAR is defined as 
“An individual or judicial or other body authorized under applicable law to consent on behalf 
of a prospective subject to the subject’s participation in the procedures involved in the 
research” (US CFR 21 part 50). 
For subjects who are minors, written informed consent will be obtained from the LAR. For 
subjects who are not minors, written informed consent will be obtained from the LAR or the 
subject if deemed able by the Investigator. When consent is being provided by an LAR, 
subject assent for participation should be documented, when possible. Assent is the 
affirmative agreement to participate in the research of a minor or of an adult who does not 
have the capacity to consent. If written assent is not possible, verbal assent is allowed and 
must be documented. If subject assent is not possible, then the site must document rationale 
for not being able to provide written or documented subject assent. 
If a subject’s 18th birthday takes place during the study and the subject is deemed able to 
consent by the Investigator, the subject should sign the informed consent. Reconsenting 
should take place if required by and in accordance with IRB or EC policy and applicable 
local law. 
The subject’s caregiver must also provide informed consent regarding their participation in 
the study prior to participating in any study procedures. 
The informed consent must, at a minimum, include the elements of consent described in the 
ICH guidance on GCP and the US CFR 21 part 50.25. A copy of the ICF planned for use will 
be reviewed by the Sponsor or designee for acceptability and must be submitted by the 
Investigator or designee together with the protocol, to the appropriate IRB/EC for review and 
approval prior to the start of the study at that investigational site. Consent forms must be in a 
language fully comprehensible to the LAR of the prospective subject. The Investigator must 
provide the Sponsor or designee with a copy of the IRB/EC letter approving the protocol and 
the ICF before the study drug supplies will be shipped and the study can be initiated. 
The consent form must be revised if new information becomes available during the study that 
may be relevant to the subject. Any revision must be submitted to the appropriate IRB/EC for 
review and approval in advance of use. 


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12.3.1 Consent and Other Informational Documents Provided to Subjects 
The subject/LAR must be given a copy of the signed informed consent and the original 
maintained in the designated location at the site. 
12.3.2 Consent Procedures and Documentation 
It is the Investigator or designee’s responsibility to obtain written informed consent from the 
subject/LAR after adequate explanation of the aims, methods, anticipated benefits, and 
potential hazards of the study. The LAR must be given ample time to decide about study 
participation and opportunity to inquire about details of the study. The IRB/EC-approved 
consent form must be personally signed and dated by the LAR with subject assent, if possible, 
and by the person who conducted the informed-consent discussion. The Investigator or 
appropriate site personnel must document the details of obtaining informed consent in the 
subject’s study documents. 
The subject’s caregiver must also indicate their understanding of the study and their role as a 
caregiver to the subject during the study. The subject’s caregiver must provide written 
consent prior to any study procedures being performed indicating their agreement to 
participate in the study in the caregiver role. 
Participation in the testing for identification of biomarkers is optional. Informed consent must 
be obtained, as appropriate, prior to blood draws for this procedure. 
Records related to a study subject’s participation will be maintained and processed according 
to local laws, and where applicable, the European Union General Data Protection Regulation 
(EU GDPR). The consent and study information documentation will include statements 
describing local and regional requirements concerning data privacy, and who to contact for 
questions. 
13 PUBLICATION PLAN 
All publication rights are delineated in the Clinical Study Agreement and/or other separate 
agreements with the Investigator and/or Institution, as applicable. 
14 CONFLICT OF INTEREST POLICY 
14.1 Finance, Insurance, and Indemnity 
Arrangements for finance, insurance, and indemnity are delineated in the Clinical Study 
Agreement and/or other separate agreements with the Investigator and/or Institution, as 
applicable. 
 
 


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15 LITERATURE REFERENCES 
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characteristics and assessment by anxiety scales. J Neurodev Disord. 2015:7(1):30. 
Bienvenu T, Philippe C, De Roux N, et al. The incidence of Rett syndrome in France. 
Pediatr Neurol. 2006;34(5):372-375. 
Buchovecky CM, Turley SD, Brown HM, et al. A suppressor screen in Mecp2 mutant mice 
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Busner J, Targum SD. The clinical global impressions scale: applying a research tool in 
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Byiers BJ, Symons FJ. Issues in estimating developmental level and cognitive function in 
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Downs J, Bebbington A, Jacoby P, et al. Level of purposeful hand function as a marker of 
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with Rett syndrome using qualitative methods. Am J Med Genet A. 2016;170(3):645-653. 
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Iemmi V, Knapp M, Brown FJ. Positive behavioural support for children and adolescents 
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Kerr AM, Armstrong DD, Prescott RJ, Doyle D, Kearney DL. Rett syndrome: analysis of 
deaths in the British survey. Eur Child Adolesc Psychiatry. 1997;6 Suppl 1:71-74. 
Khwaja O, Ho E, Barnes KV, et al. Safety, pharmacokinetics, and preliminary assessment of 
efficacy of mecasermin (recombinant human IGF-1) for the treatment of Rett syndrome. 
Proc Natl Acad Sci. U.S.A. 2014;111(12):4596-4601. 
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No 2:R221-227. 
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J Autism Dev Disord. 2017;47(4): 1102-1112. 


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Larsson G, Lindström B, Engerström IW. Rett syndrome from a family perspective: the 
Swedish Rett Center survey. Brain Dev. 2005;27 Suppl 1:S14-19. 
Lee JY, Leonard H, Piek JP, Downs J. Early development and regression in Rett syndrome. 
Clin Genet. 2013;84(6):572-576. 
Motil KJ, Caeg E, Barrish JO, et al. Gastrointestinal and nutritional problems occur 
frequently throughout life in girls and women with Rett syndrome. J Pediatr Gastroenterol 
Nutr. 2012;55(3):292-298. 
Mount RH, Charman T, Hastings RP, Reilly S, Cass H. Features of autism in Rett syndrome 
and severe mental retardation. J Autism Dev Disord. 2003;33(4):435-442. 
Mount RH, Charman T, Hastings RP, Reilly S, Cass H. The Rett Syndrome Behaviour 
Questionnaire (RSBQ): refining the behavioural phenotype of Rett syndrome. J Child 
Psychol Psychiatry. 2002;43(8):1099-1110. 
Mount RH, Hastings RP, Reilly S, Cass H, Charman T. Behavioural and emotional features in 
Rett syndrome. Disabil Rehabil. 2001;23(3-4):129-138. 
Neul JL, Benke TA, Marsh ED, et al. The array of clinical phenotypes of males with 
mutations in methyl-CpG binding protein 2. Am J Med Genet B Neuropsychiatr Genet. 
2019;180(1):55-67.  
Neul JL, Glaze D, Percy A, et al. Improving treatment trial outcomes for Rett syndrome: 
the development of Rett-specific anchors for the Clinical Global Impression scale. J Child 
Neurol. 2015;30(13):1743-1748. 
Neul JL, Lane JB, Lee HS, et al. Developmental delay in Rett syndrome: data from the 
natural history study. J Neurodevelop Dis. 2014;6:20. 
Neul JL, Kaufmann WE, Glaze DG, et al. Rett syndrome: revised diagnostic criteria and 
nomenclature. Ann Neurol. 2010;68(6):944-950. 
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Novak M, Guest C. Application of a multidimensional Caregiver Burden Inventory. 
Gerontologist. 1989;29(6):798-803. 
O’Leary HM, Kaufmann WE, Barnes KV, et al. Placebo controlled crossover efficacy 
assessment of mecasermin for the treatment of Rett syndrome. Ann Clin Transl Neurol 
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Palacios-Ceña D, Famoso-Pérez P, Salom-Moreno J, et al. “Living an obstacle course”: 
A qualitative study examining the experiences of caregivers of children with Rett syndrome. 
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Percy AK, Lane J, Annese F, Warren H, Skinner SA, Neul JL. When Rett syndrome is due to 
genes other than MECP2. Transl Sci Rare Dis. 2018;3(1):49-53. 
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28 girls with Rett syndrome. J Autism Dev Disord. 1991;21(4):551-556. 
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syndrome: an eye-tracking study of attention and recognition memory. Dev Med Child 
Neurol. 2013;55(4):364-371. 
Samaco RC, Neul JL. Complexities of Rett syndrome and MeCP2. J Neurosci. 
2011;31(22):7951-7959. 
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from different tissues. Orphanet J Rare Dis. 2018;13(1):113. 
Thompson RH, Iwata BA. A descriptive analysis of social consequences following problem 
behavior. J Appl Behav. Anal. 2001;34(2):169-178. 
Tropea D, Giacometti E, Wilson NR, et al. Partial reversal of Rett syndrome-like symptoms 
in MeCP2 mutant mice. Proc Natl Acad Sci U S A. 2009;106(6):2029-2034. 
Urbanowicz A, Downs J, Girdler S, Ciccone N, Leonard H. An exploration of the use of eye 
gaze and gestures in females with Rett syndrome. J Speech Lang Hear Res. 2016;59(6):1373-
1383. 
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abilities are influenced by MECP2 mutation type in girls with Rett syndrome. Am J Med 
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autism spectrum disorder in the blood plasma of children. PLoS One. 2014;9(11):e112445. 
Wetherby AM, Guthrie W, Woods J, et al. Parent-implemented social intervention for toddlers 
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communication and symbolic behavior scales developmental profile with very young 
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Yasui DH, Xu H, Dunaway KW, Lasalle JM, Jin LW, Maezawa I. MeCP2 modulates gene 
expression pathways in astrocytes. Mol Autism. 2013;4(1):3. 
Young DJ, Bebbington A, Anderson A, et al. The diagnosis of autism in a female: could it be 
Rett syndrome? Eur J Pediatr. 2008;167(6):661-669. 

SAP_001.pdf: 
Official Title: A 40-Week, Open-label Extension Study of Trofinetide for the 
Treatment of Girls and Women with Rett Syndrome 
NCT Numbers: NCT04279314 
Document Date: 20 Oct 2021 
 


 
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STATISTICAL ANALYSIS PLAN 
Protocol No.: 
ACP-2566-004 
Protocol Title: 
A 40-Week, Open-label Extension Study of Trofinetide 
for the Treatment of Girls and Women with Rett 
Syndrome 
Drug: 
trofinetide oral solution 
Sponsor:  
Acadia Pharmaceuticals Inc. 
Version No. and Date 
Version 3.0, 20 October 2021 
 
 
 


Protocol ACP-2566-004 
 
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Statistical Analysis Plan 
 
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SIGNATURE/APPROVAL PAGE 
AUTHORS 
 
____________________________________________                                    ___________       
                                                                                                        Date 
Associate Director, Biostatistics 
Acadia Pharmaceuticals Inc. 
 
 
APPROVERS 
 
 
_____________________________________________                               ____________       
 
                                                                                       Date 
VP, Clinical Development 
Acadia Pharmaceuticals Inc.                                                                                        
 
 
_____________________________________________                               ____________       
                                                                                                     Date 
Senior Director, Biostatistics 
Acadia Pharmaceuticals Inc. 
 
 
 
 
 


Signature Page for ACP-2566-004 Statistical Analysis Plan
Signature Page for ACP-2566-004 Statistical Analysis Plan
Approve
stics
22-Oct-2021 18:39:09 GMT+0000
Approve
22-Oct-2021 18:48:15 GMT+0000
Approve
atistics
22-Oct-2021 19:56:02 GMT+0000


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TABLE OF CONTENTS 
TABLE OF CONTENTS ............................................................................................................ 4 
LIST OF TABLES ...................................................................................................................... 7 
LIST OF FIGURES .................................................................................................................... 7 
ABBREVIATIONS .................................................................................................................... 8 
1. 
INTRODUCTION ........................................................................................................ 10 
2. 
OBJECTIVES ............................................................................................................... 11 
2.1 
Primary Objective ......................................................................................................... 11 
2.2 
Secondary Objectives ................................................................................................... 11 
2.3 
Pharmacokinetic Objectives ......................................................................................... 11 
3. 
STUDY DESIGN ......................................................................................................... 13 
3.1 
General Study Design ................................................................................................... 13 
3.2 
Schedule of Assessments .............................................................................................. 15 
3.3 
Randomization .............................................................................................................. 15 
3.4 
Blinding ........................................................................................................................ 15 
3.5 
Determination of Sample Size ...................................................................................... 15 
3.6 
Coronavirus Disease 2019 ............................................................................................ 15 
4. 
ANALYSIS SETS ........................................................................................................ 16 
5. 
DATA HANDLING CONVENTIONS ........................................................................ 17 
5.1 
General Data Reporting Conventions ........................................................................... 17 
5.2 
Derived Efficacy Variables .......................................................................................... 17 
5.2.1 
Rett Syndrome Behaviour Questionnaire (RSBQ) ................................................ 18 
5.2.2 
Clinical Global Impression–Improvement (CGI-I) ................................................ 20 
5.2.3 
Communication and Symbolic Behavior Scales Developmental Profile™ 
Infant-Toddler  Checklist – Social Composite Score (CSBS-DP-IT Social) ......... 20 
5.2.4 
Overall Quality of Life Rating of the Impact of Childhood Neurologic 
Disability (ICND) Scale ......................................................................................... 20 
5.2.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF) ............................. 21 
5.2.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
(RTT-AMB) ........................................................................................................... 21 
5.2.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
(RTT-COMC) ........................................................................................................ 21 
5.2.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM) ......... 21 
5.2.9 
Clinical Global Impression–Severity (CGI-S) ....................................................... 21 


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5.2.10 Rett Syndrome Caregiver Burden Inventory (RTT-CBI) ...................................... 21 
5.2.11 Impact of Childhood Neurologic Disability (ICND) Scale Total Score ................ 22 
5.3 
Study Day ..................................................................................................................... 22 
5.4 
Baseline Definition ....................................................................................................... 23 
5.5 
Analysis Visit Windows ............................................................................................... 23 
5.5.1 
Unscheduled Assessments ..................................................................................... 23 
5.5.2 
Multiple Measurements within Visit Windows ..................................................... 23 
5.6 
Missing or Incomplete Date for Last Dose of Study Drug ........................................... 24 
5.7 
Missing or Incomplete Dates for Prior or Concomitant Medications .......................... 24 
5.8 
Missing or Incomplete Date for Adverse Events .......................................................... 24 
5.9 
Missing Severity Assessment for Adverse Events ....................................................... 24 
5.10 
Missing Relationship to Study Drug for Adverse Events ............................................. 25 
5.11 
Character Values of Clinical Laboratory Variables ..................................................... 25 
6. 
SUBJECT DISPOSITION ............................................................................................ 26 
7. 
PROTOCOL DEVIATIONS ........................................................................................ 27 
8. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS ................................... 28 
9. 
MEDICAL HISTORY .................................................................................................. 29 
10. 
EXTENT OF EXPOSURE AND TREATMENT COMPLIANCE ............................. 30 
10.1 
Exposure to Study drug ................................................................................................ 30 
10.2 
Measurement of Treatment Compliance ...................................................................... 30 
11. 
PRIOR, CONCOMITANT, AND POST-TREATMENT MEDICATION .................. 32 
12. 
EFFICACY ANALYSES ............................................................................................. 33 
12.1 
Efficacy Variables ........................................................................................................ 33 
12.2 
Adjustment for Covariates ............................................................................................ 33 
12.3 
Handling of Missing Data ............................................................................................. 33 
12.4 
Multiple Comparisons / Multiplicity ............................................................................ 33 
12.5 
Examination of Subgroups ........................................................................................... 34 
12.5.1 MECP2 Mutation Groupings ................................................................................. 34 
13. 
METHODS OF EFFICACY ANALYSES ................................................................... 35 
13.1 
Analysis of Continuous Efficacy Endpoints ................................................................. 35 
14. 
SAFETY ANALYSES ................................................................................................. 36 
14.1 
Adverse Events ............................................................................................................. 36 
14.2 
Clinical Laboratory Variables ...................................................................................... 37 


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14.3 
Vital Signs .................................................................................................................... 41 
14.4 
Electrocardiogram (ECG) ............................................................................................. 42 
14.5 
Physical Examination ................................................................................................... 44 
14.6 
Other Safety Endpoints ................................................................................................. 44 
15. 
CLINICAL PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSES.. 45 
16. 
INTERIM ANALYSIS ................................................................................................. 46 
17. 
DATA MONITORING/REVIEW COMMITTEE ....................................................... 47 
18. 
COMPUTER METHODS ............................................................................................ 48 
19. 
CHANGES TO ANALYSES SPECIFIED IN PROTOCOL ....................................... 49 
20. 
REFERENCES ............................................................................................................. 50 
21. 
APPENDICES .............................................................................................................. 51 
21.1 
Schedule of Assessments .............................................................................................. 51 
21.2 
Summary of Version Changes ...................................................................................... 53 
 
 
 


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LIST OF TABLES 
Table 1 
Dosing Schedule Based on Weight at Baseline of the Present Study .................... 15 
Table 2 
Analysis Visit Windows......................................................................................... 23 
Table 3 
Dosing Schedule and Total Expected Volume Based on Weight at Baseline of 
the Present Study .................................................................................................... 30 
Table 4 
Conversion of Height of Remaining Liquid (cm) into Volume (mL). ................... 31 
Table 5 
Criteria for Potentially Clinically Important Laboratory Values – Hematology 
and Chemistry ........................................................................................................ 40 
Table 6 
Criteria for Potentially Clinically Important Laboratory Values - Urinalysis ....... 41 
Table 7 
Criteria for Potentially Clinically Important Vital Signs ....................................... 42 
Table 8  
Criteria for Potentially Clinically Important ECG Values ..................................... 43 
LIST OF FIGURES 
Figure 1 
Schematic of Study Design .................................................................................... 14 
 


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ABBREVIATIONS 
AE 
adverse event 
ATC 
Anatomical/Therapeutic/Chemical 
BMI 
body mass index 
CGI-I 
Clinical Global Impression – Improvement  
CGI-S 
Clinical Global Impression – Severity  
COVID-19 
coronavirus disease 2019 
DSMB 
Data and Safety Monitoring Board  
eCRF 
electronic case report form 
ECG 
Electrocardiogram 
EDC 
electronic data capture 
FDA 
Food and Drug Administration 
GJ 
Gastrojejunal 
GSD 
Guidance for Site Documentation and Data Management Querying of Data 
Impacted by COVID-19 
ICND 
Impact of Childhood Neurologic Disability Scale  
MedDRA 
Medical Dictionary for Regulatory Activities 
NDA 
new drug application 
PCI 
potentially clinically important 
PD 
pharmacodynamic(s) 
PHE 
public health emergency 
PK 
pharmacokinetic(s) 
QTcB 
QT Interval Corrected for Heart Rate using Bazett’s Formula 
QTcF 
QT Interval Corrected for Heart Rate using Fridericia’s Formula 
RSBQ 
Rett Syndrome Behaviour Questionnaire 
RTT 
Rett Syndrome 
RTT-AMB 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
RTT-CBI 
Rett Syndrome Caregiver Burden Inventory 
RTT-COMC 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
RTT-CSS 
Rett Syndrome Clinical Severity Scale 


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RTT-HF 
Rett Syndrome Clinician Rating of Hand Function 
RTT-VCOM 
Rett Syndrome Clinician Rating of Verbal Communication 
SAE 
serious adverse event 
SAP 
SD 
SE 
statistical analysis plan 
standard deviation 
standard error 
SOC 
system organ class 
TEAE   
treatment-emergent adverse event 
 
 
 


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1. 
INTRODUCTION 
This statistical analysis plan (SAP) provides a technical and detailed elaboration of the statistical 
analyses of efficacy and safety data as described in the protocol amendment 2, dated 07 August 
2020. 
Specifications for tables, figures, and listings are contained in a separate document. Statistical 
analyses for population pharmacokinetic (PK) and PK/pharmacodynamics (PD) modeling will be 
presented in a separate report and therefore will not be included in this SAP. 
This plan should be read in conjunction with the study protocol and the electronic case report 
forms (eCRFs). 


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2. 
OBJECTIVES 
2.1 
Primary Objective 
The primary objective of this study is to investigate the safety and tolerability of long-term 
treatment with oral trofinetide in girls and women with Rett syndrome. 
2.2 
Secondary Objectives 
The secondary objectives for this study are: 
• To investigate the efficacy of long-term treatment with oral trofinetide in girls and 
women with Rett syndrome 
• To investigate the efficacy of long-term treatment with oral trofinetide in girls and 
women with Rett syndrome on overall ability to communicate 
• To investigate the benefit of long-term treatment with oral trofinetide on overall quality 
of life for girls and women with Rett syndrome 
• To investigate the efficacy of long-term treatment with oral trofinetide in girls and 
women with Rett syndrome on: 
o Hand function 
o Ambulation and other gross motor skills 
o Ability to communicate choices and preferences 
o Ability to communicate verbally 
• To investigate the efficacy of long-term treatment with oral trofinetide on a global 
assessment of the severity of illness in girls and women with Rett syndrome 
• To investigate the benefit of long-term treatment with oral trofinetide on the burden on 
caregivers of girls and women with Rett syndrome 
• To investigate the benefit of long-term treatment with oral trofinetide on the impact of the 
disability on the child’s and family’s everyday life 
2.3 
Pharmacokinetic Objectives 
The pharmacokinetic objectives of this study are: 
• To characterize the pharmacokinetics (PK) of trofinetide in girls and women with Rett 
syndrome following long-term treatment with oral trofinetide 


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• To assess the pharmacokinetic/pharmacodynamic (PK/PD) relationship using safety and 
efficacy endpoints in girls and women with Rett syndrome following long-term treatment 
with oral trofinetide 


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3. 
STUDY DESIGN  
3.1 
General Study Design 
This is a 40-week, multicenter, open-label extension (OLE) study to evaluate long-term safety 
and tolerability of trofinetide in girls and women with Rett syndrome. Subjects who complete the 
preceding double-blind study (ACP-2566-003) will be eligible to enroll in the OLE. 
The Week 12/EOT visit of the antecedent study will serve as the Baseline visit of the present 
study. The data gathered at the Week 12/EOT visit of the antecedent study serves as the baseline 
data of the present study. 
Because of the special circumstances surrounding the Coronavirus Disease 2019 (COVID-19) 
public health emergency in the spring of 2020, subjects who were discontinued from Study 
ACP-2566-003 before completion because of the COVID-19 public health emergency and 
subjects who completed Study ACP-2566-003, but were prevented from entering the present 
study because of the COVID-19 public health emergency, may be re-evaluated for eligibility at 
the Baseline visit of the present study. 
However, subjects who are discontinued from Study ACP-2566-003 for other reasons other than 
COVID-19 pandemic, or who are not able to rollover to this study at the Week 12/EOT visit of 
the antecedent study, will not be able to enter the present study. 
The duration of participation for individual study subjects will be approximately 44 weeks. 
Approximately 28 sites will participate in this study. 
The study will have two periods (Figure 1): 
• Treatment period: 40 weeks 
• Safety follow-up period: 30 days 
The study design is summarized in Figure 1. 


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Figure 1 
Schematic of Study Design 
 
a    Subjects who roll over into the subsequent open-label study (ACP-2566-005) will not have the safety follow-up 
telephone call or telemedicine visit. 
 
The schedule of events and assessments is provided in the appendix (Section 21.1). As shown in 
the schedule of events, study visits may be done off-site rather than in the clinic with the prior 
approval of the Sponsor or Medical Monitor. 
For subjects who remained on the originally assigned dose in the antecedent study, the assigned 
dose in this study will be based on the subject’s weight at the Baseline visit of the present study, 
as outlined below in Table 1. Thus, for a particular subject whose weight at the Baseline of this 
study moves them into a different weight category according to Table 1, the assigned dose given 
in this study may be different from the originally assigned dose given in the antecedent study. 
After Baseline the dose will not be changed based on a change in weight that places the subject 
into a different weight category. 
Subjects whose originally assigned dose in the antecedent study was decreased for tolerability 
reasons will remain on that same dose in this study. The dose should be increased at some point 
during the study, if tolerated, to the dose that would have been assigned based on the weight 
category at Baseline of the present study. 
 
 


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Table 1 
Dosing Schedule Based on Weight at Baseline of the Present Study 
Weight 
Dose  
Total Daily Dose 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 
Abbreviations: BID=twice daily 
3.2 
Schedule of Assessments 
The schedule of assessments is in the Appendix (Section 21.1). 
3.3 
Randomization 
Not Applicable. This is not a randomized study. 
3.4 
Blinding  
This is an open-label extension study in which all patients will receive trofinetide. However, the 
investigators will not be unblinded of the antecedent study’s, ACP-2566-003, treatment 
assignment during the conduct of study ACP-2566-004. 
3.5 
Determination of Sample Size 
Up to approximately 180 subjects will be enrolled. The sample size for this study is not based on 
statistical considerations, but will depend on the number of subjects who transition into this 
open-label extension study from the antecedent study. 
3.6 
Coronavirus Disease 2019 
In March 2020, the emerging coronavirus disease 2019 (COVID-19) pandemic resulted in 
implementation of urgent safety measures designed to ensure subject safety. Mechanisms to 
record information on the potential impact of the COVID-19 pandemic on data itself, as well as 
data collection and integrity, were implemented (as detailed in the appendix of ACP-2566-004’s 
Data Management Plan titled “Guidance for Site Documentation and Data Management 
Querying of Data Impacted by COVID-19” [GSD] included in eTMF). 
Relationship to the public health emergency caused by the COVID-19 pandemic will be assessed 
for early terminations, protocol deviations, selected medications, and selected adverse events as 
detailed in the GSD. 


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4. 
ANALYSIS SETS 
Safety Analysis Set 
The Safety Analysis Set consists of all  subjects who received at least one dose of study drug in 
this study. 
Pharmacokinetics (PK) Analysis Set 
The PK Analysis Set consists of the subjects in Safety Analysis Set with at least one measurable 
trofinetide whole blood concentration. Subjects will be classified according to the actual 
treatment received. 


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5. 
DATA HANDLING CONVENTIONS 
All data collected in the study will be listed. 
5.1 
General Data Reporting Conventions 
Continuous variables will be summarized using the following descriptive statistics: number of 
subjects, mean, standard error (SE), standard deviation (SD), median, minimum, and maximum. 
For descriptive summaries of efficacy, the 95% confidence interval (CI), calculated as 
mean ± 1.96*SE, will be presented. Unless specified otherwise, means, medians, and CIs will be 
presented to one more decimal place than the raw data, and the SDs and SEs will be presented to 
two more decimal places than the raw data. In general, the maximum number of decimal places 
is 4 and values will be truncated to 4 decimal places in situations where there are more than 
4 decimal places. Wherever possible data will be decimal aligned. 
Height, weight and BMI will be presented with a maximum of one decimal place. 
Categorical variables will be summarized by the number of subjects and the percentage of 
subjects in each category; the number of subjects and the percentage of subjects with missing 
data will be summarized for demographic and baseline characteristics (if applicable). Categories 
with zero counts will not have zero percentages displayed. For demographic summaries, 
percentages will be calculated by using the total number of subjects in the given treatment group 
of the antecedent study as the denominator. Percentages will be presented with one decimal 
place. 
Duration in months will be calculated as ([the number of days / 365.25] *12). 
Lab values, not including PK concentration data, that are collected with “<” or “>” signs will 
generally be analyzed as the numerical value without the sign in tables and figures. In listings, 
these data will be reported as collected with the sign. 
For each continuous measure in safety and efficacy analyses, change from baseline results will 
be presented by treatment group of the antecedent study and overall in two ways: 
1. Main analysis: using the baseline of this study, as defined in Section 5.5, to report the 
changes across the timepoints of this open-label Study ACP-2566-004. 
2. Exploratory analysis: using the baseline from the antecedent study, ACP-2566-003, to 
report the changes across the timepoints of this open-label Study ACP-2566-004. 
5.2 
Derived Efficacy Variables  
In general, assessment total scores and subscores will be derived within the analysis datasets. In 
the event that total scores and/or subscores are also collected on the electronic case report from 


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(eCRF), the derived values will be used for all analyses. Both the raw and derived scores will be 
presented in listings. 
5.2.1 
Rett Syndrome Behaviour Questionnaire (RSBQ)  
The RSBQ is a 45-item caregiver-completed rating scale in which the caregiver rates items as 
“0” (Not True), “1” (somewhat or sometimes true) or “2” (very true). The RSBQ includes 8 
subscales:1) general mood, 2) breathing problems, 3) hand behavior, 4) face movements, 5) body 
rocking/expressionless face, 6) night-time behaviors, 7) fear/anxiety, and 8) walking/standing. 
The total score ranging from 0 to 90 will be calculated as the sum of the scores for all 45 items. 
Score for item 31 (Uses eye gaze to convey feelings, needs and wishes.) will be reversed 
(2-observed item score) in the calculation of total score and subscores.  Higher total scores 
indicate greater frequency of symptoms. If there are 9 or less missing item scores, the total score 
will be calculated by the arithmetic mean of non-missing items multiplied by 45, rounded to the 
nearest integer. The RSBQ total score will be considered missing if there are missing scores for 
10 or more items. 
For the calculation of RSBQ subscores, the items under each RSBQ subscore will be summed. If 
an item is missing it will not be imputed and the subscore will be considered missing. 
RSBQ Subscores 
1) General mood consists of: 
a. Abrupt changes in mood (Item 14) 
b. Spells of screaming for no apparent reason during the day (Item 2) 
c. There are times when she appears miserable for no apparent reason (Item 16) 
d. Spells of inconsolable crying for no apparent reason during the day (Item 30) 
e. There are times when she is irritable for no apparent reason (Item 29) 
f. Screams hysterically for long periods of time and cannot be consoled (Item 22) 
g. There are certain days/periods where she performs worse than usual (Item 15) 
h. Vocalises for no apparent reason (Item 36) 
2) Breathing problems consists of: 
a. There are times when breath is held (Item 5) 
b. Swallows air (Item 19) 
c. Abdomen fills with air and sometimes feels hard (Item 25) 
d. There are times when breathing is deep and fast (hyperventilation) (Item 1) 
e. Air or saliva expelled from mouth with force (Item 6) 
3) Hand behaviors consists of: 
a. Does not use hands for purposeful grasping (Item 18) 
b. Restricted repertoire of hand movement (Item 24) 


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c. Hand movements uniform and monotonous (Item 20) 
d. The amount of time spent looking at an object is longer than time spent holding or 
manipulating them (Item 43) 
e. Has difficulty in breaking/stopping hand stereotypies (Item 35) 
f. Has frequent naps during the day (Item 21) 
4) Repetitive face movements consists of: 
a. Makes mouth grimaces (Item 28) 
b. Makes repetitive tongue movements (Item 32) 
c. Makes grimacing expressions with face (Item 34) 
d. Makes repetitive movements involving fingers around tongue (Item 4) 
5) Body rocking and expressionless face consists of: 
a. Rocks body repeatedly (Item 41) 
b. Expressionless face (Item 12) 
c. Rocks self when hands are prevented from moving (Item 33) 
d. Seems to look through people in to the distance (Item 17) 
e. Uses eye gaze to convey feelings, needs and wishes (reversed) (Item 31) 
f. Tendency to bring hands together in front of chin or chest (Item 40) 
6) Night-time behaviours consists of: 
a. Spells of screaming for no apparent reason during the night (Item 13) 
b. Spells of inconsolable crying for no apparent reason during the night (Item 42) 
c. Spells of laughter for no apparent reason during the night (Item 37) 
7) Fear/Anxiety consists of: 
a. Spells of apparent panic (Item 38) 
b. Spells of apparent anxiety/fear in unfamiliar situations (Item 7) 
c. Seems frightened when sudden changes in own body position (Item 9) 
d. There are times when parts of the body are held rigid (Item 10) 
8) Walking/standing consists of: 
a. Walks with stiff legs (Item 39) 
b. Although can stand independently tends to lean on objects or people (Item 23) 
Items not included in Subscores 
a. Makes repetitive hand movements with hands apart (Item 3) 
b. Grinds teeth (Item 8) 
c. Shifts gaze with a slow horizontal turn of head (Item 11) 
d. Spells of laughter for no apparent reason during the day (Item 26) 
e. Has wounds on hands as a result of repetitive hand movements (Item 27) 
f. Appears isolated (Item 44) 
g. Vacant ‘staring’ spells (Item 45) 


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5.2.2 
Clinical Global Impression–Improvement (CGI-I) 
The CGI-I scale is used by the clinician to rate how much the subject’s illness has improved or 
worsened relative to a baseline state. A 7-point scale is used from 1=very much improved, 
2=much improved, 3=minimally improved, 4=no change, 5=minimally worse, 6=much worse, 
7=very much worse. 
Higher CGI-I scores denote more severe illness and less improvement in the illness respectively. 
5.2.3 
Communication and Symbolic Behavior Scales Developmental Profile™ Infant-
Toddler  Checklist – Social Composite Score (CSBS-DP-IT Social)  
The Checklist consists of 24 questions that range from 0 to 4 points within each of 7 Clusters. 
Credit of 0 points is given for items checked “Not Yet”, 1 point for items checked “Sometimes”, 
or 2 points for items checked “Often”. For items that describe a series of numbers or ranges, 
credit of 0 points is given for items checked “None” and 1 to 4 points for items containing 
numbered choices. 
Three composite scores assessing 7 skill areas can be calculated. The Social Composite score is 
comprised of 13 items in the skill areas “Emotion and Eye Gaze” (items 1 to 4), 
“Communication” (items 5 to 8), and “Gestures” (items 9 to 13). The Speech Composite score, 
including the skill areas “Sounds” and “Words” is based on items 14 to 18. The Symbolic 
Composite score, including the skill areas “Understanding” and “Object Use”, is based on items 
19 to 24. 
The Social Composite raw score (comprised of items 1 to 13) ranging from 0 to 26 will be 
calculated as the sum of the scores for all 13 items. Higher Social Composite raw scores indicate 
better social communication development. If there are 2 or less missing item scores, the Social 
Composite raw score will be calculated by the arithmetic mean of the non-missing item scores 
multiplied by 13, rounded to the nearest integer. The Social Composite raw score will be 
considered as missing if there are missing scores for 3 or more items. 
5.2.4 
Overall Quality of Life Rating of the Impact of Childhood Neurologic Disability 
(ICND) Scale 
The overall quality of life score rating of the ICND will be analyzed as a separate endpoint. The 
numeric scale of the child’s overall quality of life ranges from 1 (“Poor”) to 6 (“Excellent”); 
lower overall quality of life scores indicate lower quality of life. 


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5.2.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF) 
The RTT-HF is a clinician completed clinical assessment of the subject’s ability to use her hands 
for functional purposes. The assessment is made on an 8-point Likert scale (0-7) with 0 denoting 
normal functioning and 7 the most severe impairment. 
5.2.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
(RTT-AMB) 
The RTT-AMB is a clinician completed clinical assessment of the subject’s ability to sit, stand, 
and ambulate. The assessment is made on an 8-point Likert scale (0-7) with 0 denoting normal 
functioning and 7 the most severe impairment. 
5.2.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
(RTT-COMC) 
The RTT-COMC is a clinician completed clinical assessment of the subject’s ability to 
communicate her choices or preferences, which can include the use of nonverbal means such as 
eye contact or gestures. The assessment is made on an 8-point Likert scale (0-7) with 0 denoting 
normal functioning and 7 the most severe impairment.  
5.2.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM) 
The RTT-VCOM is a clinician completed clinical assessment of the subject’s ability to 
communicate verbally. The assessment is made on an 8-point Likert scale (0-7) with 0 denoting 
normal functioning and 7 the most severe impairment.  
5.2.9 
Clinical Global Impression–Severity (CGI-S) 
The CGI-S is a 7-point scale that requires the clinician to rate the severity of the subject’s illness 
at the time of assessment, relative to the clinician’s experience with subjects who have the same 
diagnosis. Considering total clinical experience, a subject is assessed on severity of illness at the 
time of rating: 1=normal, not at all ill; 2=borderline ill; 3=mildly ill; 4= moderately ill; 
5=markedly ill; 6=severely ill; or 7=extremely ill. 
Higher CGI-S scores denote more severe illness and less improvement in the illness respectively. 
5.2.10 
Rett Syndrome Caregiver Burden Inventory (RTT-CBI) 
The RTT-CBI consists of 24 negatively worded items (Items 1 through 24). Frequency ratings 
are on a 5-point Likert scale including: 0-never; 1-rarely; 2-sometimes; 3-frequently and 4-nearly 
always. The RTT-CBI also includes 2 positively worded items (items 25 and 26) that comprise 
the Optimism Index; this index will not be used for analysis. The total score ranging from 0 to 96 


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will be calculated as the sum of the scores for Items 1-24. If there are 4 or fewer missing item 
scores, the RTT-CBI total score will be calculated by  the arithmetic mean of the non-missing 
item scores multiplied by 24, rounded to the nearest integer. The RTT-CBI total score will be 
considered as missing if there are missing scores for 5 or more items.  
5.2.11 
Impact of Childhood Neurologic Disability (ICND) Scale Total Score 
The ICND scale evaluates the effect of four health problems on 11 aspects of the child’s or the 
family’s life which are scored 0 (“Not at all”), 1 (“A little”), 2 (“Some”), 3 (“A lot”), or “Does 
not apply”. The four health problems are 1) inattentiveness, impulsivity, or mood, 2) ability to 
think and remember, 3) neurologic or physical limitations, and 4) epilepsy. The ICND total score 
ranging from 0 to 132 will be calculated as follows: 
1. For each health problem, the score will be calculated as the sum of the 11 item scores. 
The health problem score will be considered missing 
a.  if more than 5 item scores are either missing or “Does not apply” 
b. or if more than 3 item scores are missing for reasons other than the response of 
“Does not apply”. 
2. The ICND total score will be calculated as the sum of the average of each problem score 
multiplied by 11.  
𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆= ෍𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝_𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠_𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑒𝑒𝑘𝑘∗11
4
𝑘𝑘=1
 
where k indexes the four health problems. 
The ICND total score will be considered missing if at least 1 health problem score is 
missing. Imputed ICND total scores will be rounded to the nearest integer. 
Higher ICND total scores indicate worse health problems. The ICND total score does not include 
the Overall Quality of Life Rating. 
5.3 
Study Day 
If the date of assessment occurs on or after the first dose date, then study day will be calculated 
as (date of assessment – date of first dose) + 1. If the date of assessment occurs prior to the first 
dose date, then study day will be calculated as (date of assessment – date of first dose). There is 
no study day 0. 


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5.4 
Baseline Definition 
Data from the latest predose value of (Study ACP-2566-003 Week 12/EOT visit, unscheduled 
visit within Week 12 window, visit within 2 weeks of last double-blind dose, or Study ACP-
2566-004 Baseline Visit) will be carried over as Baseline information in the present study, as 
applicable (e.g., not for the CGI-I for which this is not applicable). 
Subjects for whom the Baseline visit of this study is not on the same day as the Week 12/EOT visit 
of the antecedent study (e.g., subjects in the antecedent study who were prevented from entering this 
study by the COVID-19 public health emergency) will have blood drawn for Baseline values three to 
fourteen days before the Baseline visit. 
5.5 
Analysis Visit Windows  
Efficacy, safety, and PK assessments will be summarized by analysis visit as presented in 
Table 2 below. 
Table 2 
Analysis Visit Windows 
Analysis Visit  
Target Study Day 
Study Day Interval 
Baseline (Day 1) 
1 
< 1 
Day 1 Post First Dose* 
1 
1 
Week 2 
15 
2 – 49 
Week 12 
85 
50 – 133 
Week 26 
183 
134 – 231 
Week 40 
281 
232 – 310 
*For ECG and PK assessments only; other assessments post-first dose on Day 1 will go to the 
Week 2 analysis visit window. 
5.5.1 
Unscheduled Assessments 
Both Scheduled and Unscheduled assessments, including the assessments at early termination 
visits, will be included for planned timepoint analyses based on the above analysis visit 
windowing rules. All assessments will be presented in data listings. 
5.5.2 
Multiple Measurements within Visit Windows 
In the event that more than one assessment falls within a given window, the assessment closest to 
the target study day will be selected for the by-visit analysis. If two assessments are equidistant 
from the target study day, then the chronologically last assessment will be used. Exceptions may 
be made for incomplete assessments, in which case, more complete assessments may be given 
priority. Details are provided in a separate programming specifications document. 
For safety analyses where the extreme values should be selected (e.g., overall post-Baseline 
minimum, overall post-Baseline maximum, and potentially clinically important values), all 


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non-missing post-Baseline values should be considered, regardless of whether the value is 
selected for the by-visit summaries. All assessments will be presented in data listings. 
5.6 
Missing or Incomplete Date for Last Dose of Study Drug  
For subjects with completely missing last dose date, the last dose date will be imputed by the last 
expected dosing date, defined as the earliest of the following dates: last drug kit dispense date + 
scheduled dosing interval per protocol, EOT/ET date and the return date of the last dispensed 
drug kit.  
For subjects with partial missing last dose date, the imputation will be compared against the last 
expected dosing date as defined above. Detailed algorithms will be documented in a separate 
programming specifications document. The missing or incomplete dates will be displayed in the 
data listings as reported on the eCRF rather than the imputed dates.  
5.7 
Missing or Incomplete Dates for Prior or Concomitant Medications  
Missing or incomplete medication start or stop dates will be imputed for the purpose of 
determining whether the medication is taken concomitantly or not (see Section 11 for definition). 
When the chronological order of medication use relative to the study drug treatment period is 
unclear due to missing or incomplete date(s), the medication will be considered as concomitant. 
The imputation algorithms will be detailed in the analysis dataset specification document. The 
missing or incomplete dates as captured on the eCRF will be displayed in the data listings.   
5.8 
Missing or Incomplete Date for Adverse Events  
Missing or incomplete adverse event (AE) start dates will be imputed for the purpose of 
determining whether the AEs are treatment-emergent or not (see Section 12.2 for definition). 
When the chronological order of an AE onset relative to the study drug treatment period is 
unclear due to missing or incomplete date(s), the AE will be considered as treatment-emergent. 
The imputation algorithms will be detailed in the analysis dataset specification document. The 
missing or incomplete dates captured on the eCRF will be displayed in the data listings. 
5.9 
Missing Severity Assessment for Adverse Events 
If the severity is missing for an AE starting on or after the date of the first dose of study drug, 
then a severity of “Severe” will be assigned. The imputed values for severity assessment will be 
used for incidence summaries, while the actual values will be used in data listings. 


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5.10 
Missing Relationship to Study Drug for Adverse Events 
If the relationship to study drug is missing for an AE starting on or after the date of the first dose 
of study drug, a causality of “Related” will be assigned. The imputed values for relationship to 
study drug will be used for incidence summaries, while the actual values will be presented in 
data listings.  
5.11 
Character Values of Clinical Laboratory Variables  
If the reported value of a clinical laboratory variable cannot be used in a statistical analysis due 
to, for example, a character string reported for a numeric variable, an appropriately determined 
coded value may be used in the statistical analysis. The coding algorithms will be detailed in the 
analysis dataset specification document. The actual values as reported in the database will be 
presented in data listings. 


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6. 
SUBJECT DISPOSITION 
The number of sites that screened at least 1 subject, number of sites that enrolled at least 
1 subject, and number of subjects enrolled will be tabulated. In addition, the number of subjects 
enrolled at each site will also be tabulated by Analysis Set. 
The number and percentage of subjects who completed the study, discontinued early (all 
discontinued and by discontinuation reasons including reason due to the COVID-19 public health 
emergency (PHE), and the reason for discontinuation will be summarized using the Safety 
Analysis Sets. All subjects excluded from the Safety Analysis Set and the reason(s) for exclusion 
will be listed. 


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7. 
PROTOCOL DEVIATIONS 
Protocol deviations will be reviewed periodically over the course of the study. The review 
process, definition of the deviation categories, and the classification of a deviation as major or 
minor are detailed in the Protocol Deviation Management Plan. Protocol deviations will also be 
assessed with respect to relationship to the COVID-19 PHE.  
A summary of the number and percentage of subjects with major protocol deviations for each 
deviation category will be presented for the Safety Analysis Set in three ways: all protocol 
deviations, COVID-19-PHE related protocol deviations, and non-COVID-19-PHE related 
protocol deviations.  
Three data listings of all protocol deviations, COVID-19-PHE related protocol deviations, and 
non-COVID-19-PHE related protocol deviations will be provided. 


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8. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS 
Demographics and baseline characteristics will be summarized for the Safety Analysis Set using 
descriptive statistics. Variables include, but are not limited to, age, age group (5-10 years old, 
11-15 years old, and 16-20 years old), race, ethnicity, height, weight, BMI, Baseline RSBQ total 
score, Baseline RSBQ severity (< 35 total score and ≥ 35 total score), and Baseline CGI-S score. 
Race will also be categorized by White vs. Non-White. The reported age reflects a subject’s age 
at the informed consent date. Baseline characteristics for subjects in ACP-2566-004 will also be 
summarized using the baseline of the antecedent study separately. 


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9. 
MEDICAL HISTORY 
Medical history reported terms will be coded with Medical Dictionary for Regulatory Activities 
(MedDRA), version 23.0 or newer. The subject incidence will be summarized for each system 
organ class (SOC) and preferred term for the Safety Analysis Set. A subject will be counted only 
once per SOC or per preferred term for the summary. 
A listing of the SOC, preferred term, body system, verbatim for the medical history 
condition/event, start and stop dates (when available), and an indicator for whether or not the 
condition is ongoing will be provided. 
Rett Syndrome History 
The age at Rett syndrome diagnosis, the age at  first symptoms noticed, the number and 
percentage of subjects by MECP2 gene mutation, and the number and percentage of subjects 
with more than one MECP2 mutation (Yes, No) collected on the Rett History eCRF page will be 
summarized. 
Rett syndrome history will be listed. 


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10. 
EXTENT OF EXPOSURE AND TREATMENT COMPLIANCE 
Extent of exposure and treatment compliance will be summarized as both continuous variables 
and categorical variables for Safety Analysis set.  
10.1 
Exposure to Study drug  
Duration of exposure to study drug will be calculated for each subject as (last dose date – first 
dose date + 1). The number and percentage of subjects within each of the following exposure 
levels in terms of duration of exposure will also be tabulated: <2 weeks (1 to 13 days), 2 to 
<12 weeks (14 to 83 days), 12 to <26 weeks (84 to 181 days), 26 to <40 weeks (182 to 
279 days), and >=40 weeks (280 days or longer). 
10.2 
Measurement of Treatment Compliance 
The study drug is provided in liquid form supplied in a 500 mL bottle. Study drug compliance 
will be calculated based on the drug accountability and dose modification data as collected on 
the eCRF. The study drug compliance will be calculated as (the total volume of drug actually 
taken (in mL) divided by the total volume of drug expected to be taken)*100. 
The total volume of drug expected to be taken will be based on the duration of exposure and 
dosing schedule as in Table 3. However, if there is any dose modification prescribed by the 
investigator due to intolerance, the total drug expected to be taken will be adjusted accordingly to 
account for the modified prescribed dose schedule as recorded in the EDC. 
The total volume of drug actually taken will be calculated as (total drug dispensed – total drug 
returned (height in cm of drug remaining converted into volume in mL based on the following 
conversion Table 4). 
Treatment compliance will be summarized as a categorical variable. The number and percentage 
of subjects within each of the following compliance levels will be tabulated: <80%, 80 to 120% 
and >120%.  
Details of treatment compliance calculation are provided in a separate programming 
specifications document. 
Table 3 
Dosing Schedule and Total Expected Volume Based on Weight at Baseline of the 
Present Study 
Weight  
Dose 
Total Daily Dose 
Total Expected Volume 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
60 mL x Number of Days Expected to be Dosed  
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
80 mL x Number of Days Expected to be Dosed  
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
100 mL x Number of Days Expected to be Dosed  
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 
120 mL x Number of Days Expected to be Dosed  
BID = twice daily 


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Table 4 
Conversion of Height of Remaining Liquid (cm) into Volume (mL). 
Height of Remaining 
Liquid (cm) 
Estimated Volume (ml)  
Height of Remaining 
Liquid (cm) 
Estimated Volume (ml)  
0.5 
18 
7.0 
248 
1.0 
35 
7.5 
266 
1.5 
53 
8.0 
283 
2.0 
71 
8.5 
301 
2.5 
89 
9.0 
319 
3.0 
106 
9.5 
336 
3.5 
124 
10.0 
354 
4.0 
142 
10.5 
372 
4.5 
159 
11.0 
389 
5.0 
177 
11.5 
407 
5.5 
195 
12.0 
425 
6.0 
212 
12.5 
443 
6.5 
230 
13.0 
460 
 
 
 


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11. 
PRIOR, CONCOMITANT, AND POST-TREATMENT MEDICATION  
Prior medication is defined as any medication with stop dates prior to the date of the first dose of 
double-blind study drug from study ACP-2566-003. Concomitant medication is defined as any 
medications that are ongoing at the first dose of study drug or with a start date between the dates 
of the first and last doses of study drug, inclusive. Post-treatment medication is defined as any 
medication with a start date after the date of the last dose of study drug. Medications will be 
coded using WHO Drug Global Dictionary March 2020 or newer version. 
The number and percentage of subjects taking prior, concomitant and post-treatment medications 
will be tabulated separately by each drug class (ATC Level 3) and medication preferred term, 
treatment group of the antecedent study and overall for Safety Analysis Set. Multiple medication 
usage by a subject in the same category will be counted only once. Listings of the prior, 
concomitant and post-treatment medications will also be provided. 
COVID-19 Infection Related Medications 
Concomitant medication analyses described above will also be tabulated and listed by 
relationship to COVID-19 Infection (Not related to COVID-19 Infection vs. Related to COVID-
19 Infection). 


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12. 
EFFICACY ANALYSES 
Unless otherwise specified, all efficacy analyses will be performed using the Safety Analysis Set. 
No hypothesis testing is planned. Descriptive summaries of all efficacy endpoints will be 
presented. 
12.1 
Efficacy Variables 
Secondary Efficacy Endpoints 
• RSBQ total score – change from Baseline to Weeks 2, 12, 26, and 40 
• CGI-I score at Weeks 2, 12, 26, and  40 (assessing improvement from the Baseline of the 
present study) 
Change from Baseline to Weeks 2, 12, 26, and 40 in: 
• CSBS-DP-IT Social 
• Overall Quality of Life Rating of the ICND  
• RTT-HF score 
• RTT-AMB score 
• RTT-COMC score 
• RTT-VCOM score 
• CGI-S score 
• RTT-CBI total score (items 1-24) 
• ICND total score 
Response rate based on  CGI-I at Weeks 2, 12, 26, and 40 
12.2 
Adjustment for Covariates 
Not applicable. 
12.3 
Handling of Missing Data 
Missing data imputation rules are described in Section 5.2. 
12.4 
Multiple Comparisons / Multiplicity 
No hypothesis testing is planned. 


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12.5 
Examination of Subgroups 
Subgroup analysis by MECP2 mutation groupings may be performed for RSBQ and CGI-I. 
12.5.1 
MECP2 Mutation Groupings 
The number and percentage of subjects will be examined in the following MECP2 mutation 
groupings. 
Mutations 
Code in EDC 
Category 
Severity 
R106W 
1 
R106W 
Severe 
R133C 
2 
R133C 
Mild 
T158M 
3 
T158M 
Moderate 
R168X 
4 
R168X 
Severe 
R255X 
5 
R255X 
Severe 
R270X 
6 
R270X 
Severe 
R294X 
7 
R294X 
Mild 
R306C 
8 
R306C 
Mild 
C298G (L100V) 
9 
Other mutations 
Mild 
G317A (R106Q 
10 
Other mutations 
Mild 
C421G (Y141X) 
11 
Other mutations 
Unknown 
C455G (P152R) 
12 
Other mutations 
Severe 
C302G (P101R) 
13 
Other mutations 
Severe 
C401G (S134C) 
14 
Other mutations 
Severe 
C423G (Y141X) 
15 
Other mutations 
Unknown 
C468G (D156E) 
16 
Other mutations 
Mild 
C674G (P225R) 
17 
Other mutations 
Mild 
C965T (P322L) 
18 
Other mutations 
Mild 
710del1 
19 
Other mutations 
Severe 
806del1 
20 
Other mutations 
Severe 
807del1 
21 
Other mutations 
Severe 
1157del41 
22 
C-terminal Truncations 
Mild 
1157del44 
23 
C-terminal Truncations 
Mild 
1163del26 
24 
C-terminal Truncations 
Mild 
1163del35 
25 
C-terminal Truncations 
Mild 
1164del44 
26 
C-terminal Truncations 
Mild 
1168del6 
27 
C-terminal Truncations 
Mild 
Exon 1+2 
28 
Large Deletions 
Severe 
Exon 3 
29 
Large Deletions 
Severe 
Exon 3+4 
30 
Large Deletions 
Severe 
Exon 4 
31 
Large Deletions 
Severe 
Exons 1-4 
32 
Large Deletions 
Severe 
All Others 
33 
Other mutations 
Derived from CRF 
Missing and unknown severity will not be imputed. 


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13. 
METHODS OF EFFICACY ANALYSES 
13.1 
Analysis of Continuous Efficacy Endpoints 
Descriptive statistics for all efficacy endpoints listed in Section 12.1 will be tabulated by 
treatment group of the antecedent study. The summaries of the change from baseline results will 
be presented in two ways (main analysis and exploratory analysis) as specified in Section 5.1. 


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14. 
SAFETY ANALYSES 
All safety analyses will be performed using the Safety Analysis Set. Safety summaries will be 
summarized using descriptive statistics by treatment group of the antecedent study and overall. 
For each continuous measure in clinical laboratory variables, vital signs, and electrocardiogram, 
change from baseline results will be presented in two ways as specified in Section 5.1. 
The primary safety endpoints are: 
• Treatment-emergent adverse events (TEAEs) 
• Serious adverse events (SAEs) 
• Withdrawals due to adverse events (AEs) 
• Potentially clinically important changes in other safety assessments 
14.1 
Adverse Events  
Adverse events will be coded using MedDRA dictionary, Version 23.0 or newer. 
An AE (classified by preferred term) will be considered a treatment-emergent AE (TEAE) if 
started after first dose administration and no later than last dose date + 30 days. AEs reported on 
Day 1 based on Baseline (pre-dose) findings (e.g., clinically significantly abnormal vital signs, 
laboratory test results, or electrocardiogram parameters) will not be considered as TEAEs. 
The event counts, the number, and percentage of subjects reporting TEAEs in the treatment 
group of the antecedent study and overall will be tabulated by system organ class (SOC) and 
preferred term; by SOC, preferred term, and maximum severity; and by SOC, preferred term, and 
relationship to study drug. If more than one TEAE occurs with the same preferred term for the 
same subject, then the subject will be counted only once for that preferred term using the most 
severe and most related occurrence for the summarization by severity and by relationship to 
study drug. The display in these tables will be sorted alphabetically by SOC and then by 
descending subject frequency for the preferred terms (combined counts from overall group) 
within each SOC. 
The event counts, the number and percentage of subjects with any TEAEs will also be tabulated 
by preferred term without SOCs. This table will be sorted by descending subject frequency. 
The incidence of most frequently reported (preferred terms reported by ≥ 5% of subjects overall) 
TEAEs, SAEs reported after treatment start, TEAEs leading to drug withdrawn, and TEAEs 
related to study drug will be summarized by SOC and preferred term. The tables will be sorted 
alphabetically by SOC and then by descending frequency within each SOC in overall group. In 
addition, the incidence of fatal treatment-emergent AEs (i.e., events that cause death) will be 
summarized separately by SOC and preferred term.  


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These summary tables except for the most frequently reported TEAEs tables will also be 
presented by relationship to COVID-19 Infection (Not related to COVID-19 Infection vs. 
Related to COVID-19 Infection). 
An AE listing by subject will display all events, including those which are not treatment-
emergent, and will include the verbatim term in addition to the MedDRA SOC and preferred 
term. This listing will also include all relevant eCRF data associated with the event: date of 
onset, date resolved, date of last dose, severity, frequency, outcome, relationship to study drug, 
and action taken with study drug. Separate listings will be presented for subjects with treatment-
emergent SAEs, related TEAEs, TEAEs leading to drug withdrawn, fatal TEAEs (if any), and 
TEAEs Related to COVID-19 Infection. 
14.2 
Clinical Laboratory Variables 
Due to COVID-19 PHE related disruptions, it is possible that some test results may be collected 
from a local laboratory. Local laboratory results and the associated normal ranges will be 
converted to SI units; local laboratory results, in SI units, will then be normalized to central lab 
ranges to be  included in summary data analysis together with the central laboratory results. The 
normalization will be performed using the following scale transformation equation: 
𝑠𝑠= 𝐿𝐿𝑠𝑠+ (𝑥𝑥−𝐿𝐿𝑥𝑥) 𝑈𝑈𝑠𝑠−𝐿𝐿𝑠𝑠
𝑈𝑈𝑥𝑥−𝐿𝐿𝑥𝑥
 
where s is the normalized individual laboratory value to be used for summary; x is the original 
value from the local lab; Lx and Ux are the lower and upper limits from the local lab; Ls and Us 
are the lower and upper limits from the central lab.  
For labs with only a single upper (or lower) limit, the following scale transformation equation 
will be used: 
𝑠𝑠= 𝑥𝑥𝑈𝑈𝑠𝑠
𝑈𝑈𝑥𝑥
 
where s is the normalized individual laboratory value to be used for summary; x is the original 
value from the local lab; Us is the upper (or lower) limit from the central lab; Ux is the upper 
(or lower) limit from the local laboratory. Local laboratory results and normalized results will be 
included in data listings. Only central lab and normalized local lab results will be used for 
summary of change from baseline, shift, and potentially clinically important (PCI) analyses. 
Clinical laboratory assessments are performed at Baseline, Week 2, Week 12, Week 26 and 
Week 40/EOT.  
• Clinical chemistry serum tests include the following: 


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o Sodium (Na), potassium (K), chloride (Cl), phosphorus (P), calcium (Ca), carbon 
dioxide (CO2), blood urea nitrogen (BUN), creatinine (CR), uric acid 
o Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-
glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total bilirubin 
(TBIL), lactate dehydrogenase (LDH) 
o Glucose 
o Albumin (ALB), total protein 
o Thyroid stimulating hormone (TSH), free T3, and free T4 
 Thyroid Function tests will be performed at Visit 1 (Baseline) and Visit 5 
• Pregnancy test 
o A serum pregnancy test will be performed at all designated visits for subjects of 
childbearing potential 
• Hematology tests include the following: 
o Complete blood count (CBC) including: 
- White blood cell (WBC) count 
- Complete differential (relative and absolute) 
- Hematocrit (Hct), hemoglobin, red blood cells (RBC), platelets 
- Reticulocyte count 
• Urinalysis tests include the following: 
o Blood, RBCs, WBCs, protein, glucose, ketones, specific gravity, pH 
Clinical laboratory values (in Système International [SI] units) and the change from Baseline 
values will be summarized at each post-Baseline visit using descriptive statistics. The overall 
minimum, maximum as well as the last post-Baseline observed and change from Baseline values 
will also be summarized. For urinalysis with categorical results, the number and percentage of 
subjects will be tabulated by category at Baseline and each post-Baseline visit, and the 
denominator is the number of subjects with non-missing values for the given parameter and visit.  
Laboratory values will also be summarized in shift tables to determine the number and 
percentage of subjects with values classified as below, within, and above normal ranges at each 
post-Baseline visit relative to the same classification at the Baseline visit. For the by-visit shift 
summary, the denominator is the number of subjects with non-missing values at Baseline and the 
given visit for the given parameter. For the shift to the overall post-Baseline minimum or 


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maximum, all post-Baseline values will be considered, including unscheduled and out of window 
values and the denominator is the number of subjects with non-missing Baseline value and at 
least 1 post-Baseline value for the given parameter.  
Clinical laboratory values are potentially clinically important (PCI) if they meet either the low or 
high PCI criteria listed in Tables 5 and 6. The number and percentage of subjects with post-
Baseline PCI values for each of the categories in Table 5 and 6 will be summarized for selected 
parameters. For the overall post-Baseline summaries of PCI values, all post-Baseline values will 
be considered, including unscheduled and out of window values. Subjects with multiple PCI 
values for a given parameter will be counted only once for that parameter. For the overall 
post-Baseline summary, the numerator of the percentage is the number of subjects with at least 
1 post-Baseline PCI laboratory value for the given parameter, and the denominator is the number 
of subjects with at least 1 post-Baseline laboratory value for the given parameter.  
 
 


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Table 5 
Criteria for Potentially Clinically Important Laboratory Values – Hematology 
and Chemistry 
Analyte 
Conventional 
Unit 
Low PCI 
Criteria 
High PCI 
Criteria 
 
SI Unit 
Low PCI 
Criteria 
High PCI 
Criteria 
Hematology (whole 
blood) 
 
 
 
 
 
 
Hemoglobin (female) 
g/dL 
<10 
>17 
g/L 
<100 
>170 
Hematocrit (female) 
% 
<30 
>50  
L/L 
<0.3 
>0.5  
Leukocyte (White 
Blood Cell Count) 
x 103/uL 
≤2.8 
≥15 
x 109/L 
≤2.8 
≥15 
Neutrophils 
x 103/uL 
≤1.5 
No upper 
limit 
x 109/L 
≤1.5 
No upper 
limit 
Platelet Count 
x 103/uL 
≤75 
≥700 
x 109/L 
≤75 
≥700 
Chemistry (serum 
or plasma) 
 
 
 
 
 
 
ALT 
U/L 
No lower 
limit  
≥3 X ULN 
U/L 
No lower 
limit  
≥3 X ULN 
AST 
U/L 
No lower 
limit  
≥3 X ULN 
U/L 
No lower 
limit  
≥3 X ULN 
Total Bilirubin 
mg/dL 
No lower 
limit 
≥1.5 ULN 
umol/L 
No lower 
limit 
≥1.5 ULN 
BUN 
mg/dL 
No lower 
limit 
≥30.0 
mmol/L 
No lower 
limit 
≥10.71 
Sodium 
mEq/L 
≤125 
≥155 
mmol/L 
≤125 
≥155 
Potassium 
mEq/L 
≤3.0 
≥5.5 
mmol/L 
≤3.0 
≥5.5 
Calcium, total 
mg/dL 
<8.0 
>11.0 
mmol/L 
<2.0 
>2.75 
Lactate 
Dehydrogenase 
(LDH) 
U/L 
No lower 
limit 
≥3 X ULN 
U/L 
No lower 
limit 
≥3 X ULN 
Alkaline Phosphatase 
U/L 
No lower 
limit 
≥3 X ULN 
U/L 
No lower 
limit 
≥3 X ULN 
Uric acid (female) 
mg/dL 
No lower 
limit 
≥8.5 
umol/L 
No lower 
limit 
≥505.75 
Albumin 
g/dL 
≤2.6 
≥6.0 
g/L 
≤26 
≥60 
Total Protein 
g/dL 
≤5.0 
≥10.0 
g/L 
≤50 
≥100 
Chloride 
mEq/L 
≤85 
≥120 
mmol/L 
≤85 
≥120 
Glucose (random) 
mg/dL 
≤45.1 
≥200.0 
mmol/L 
≤2.48 
≥11 
Serum Creatinine 
mg/dL 
Not 
Applicable 
>1.5 ULN 
umol/L 
Not 
Applicable 
>1.5 ULN 
Gamma-Glutamyl 
Transferase (GGT) 
U/L 
Not 
Applicable 
≥3 ULN 
U/L 
Not 
Applicable 
≥3 ULN 


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Table 6 
Criteria for Potentially Clinically Important Laboratory Values - Urinalysis 
Urinalysis (qualitative dipstick) 
Low PCI Criteria 
High PCI Criteria 
Blood (occult blood) 
Not Applicable 
≥ Moderate 
Protein 
Not Applicable 
≥ 100 mg/dL 
Glucose 
Not Applicable 
≥ 500 mg/dL 
 
Clinical laboratory data will be displayed in data listings with date and study day of collection. 
All units will be displayed according to SI conventions for units. Out of range values will be 
flagged in the data listings (i.e., ‘L’ or ‘H’). A separate listing will be provided for a subset of the 
chemistry, hematology, and urinalysis analytes with values classified as PCI. 
At the initiation of the study, the main reference ranges (the reference ranges provided by the 
central laboratory validated for use for the general population) were used for the purpose of 
flagging abnormal laboratory values for the investigator to assess clinical significance. During 
the study, at the request of a leading investigator an alternative set of reference ranges, more 
specific to patients with Rett syndrome rather than to the general population, was used for the 
purpose of flagging abnormal lab values for the investigator to assess clinical significance.  
All analysis will be done using the main reference ranges, not the alternative ranges. During the 
course of the study the main reference ranges were updated by the central laboratory. The latest 
version of the reference ranges provided by the central laboratory will be used for all analysis; 
PCI status and categorical labs (Low, Normal, High) and shifts will be derived from the latest 
version of the main reference range. Both of the reference ranges (main and alternative) and the 
corresponding categorical assessment will be listed. 
The pregnancy results (positive or negative) will be presented in a listing. 
14.3 
Vital Signs 
Vital signs will be collected throughout the study; height will be measured at Baseline and Week 
40/EOT; weight will be measured at Baseline, Week 2, Week 12, Week 26 and Week 40/EOT. 
Observed vital signs including weight and BMI and the changes from Baseline at each post-
Baseline visit will be summarized using descriptive statistics. 
Vital sign values will be considered PCI if they meet the criteria listed in Table 7. The number 
and percentage of subjects with post-Baseline vital signs that are PCI will be summarized at each 
post-Baseline visit and for overall post-Baseline. For the overall post-Baseline summaries, all 
post-Baseline values will be considered, including unscheduled and out of window values. 
Subjects with multiple PCI values for a given parameter will be counted only once for that 
parameter. For the by-visit summary, the numerator for the percentage is the number of subjects 


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with a post-Baseline PCI vital sign for the given parameter and visit, and the denominator is the 
number of subjects with non-missing values for the given parameter and visit. For the overall 
post-Baseline summary, the numerator for the percentage is the number of subjects with at least 
1 post-Baseline PCI vital sign for the given parameter, and the denominator is the number of 
subjects with at least 1 post-Baseline vital sign for the given parameter. A listing of overall and 
of subjects with any PCI vital sign values will be provided.  
Table 7 
Criteria for Potentially Clinically Important Vital Signs 
Vital Sign 
Parameter 
 
Unit 
Criteria 
Observed Value 
And/Or 
Change Relative 
to Baseline 
Change from Supine 
to Standing 
Systolic blood 
pressure 
(supine or sitting) 
mmHg 
≥180 
And 
Increase of ≥20 
- 
≤90 
And 
Decrease of ≥20 
- 
Diastolic blood 
pressure (supine or 
sitting) 
mmHg 
≥105 
And 
Increase of ≥15 
- 
≤50 
And 
Decrease of ≥15 
- 
Pulse (supine or 
sitting) 
bpm 
≥120 
And 
Increase of ≥15 
- 
≤50 
And 
Decrease of ≥15 
- 
Weight  
kg 
Not Applicable 
Increase of ≥7% 
- 
Decrease of ≥7% 
- 
14.4 
Electrocardiogram (ECG) 
12-lead ECGs are collected throughout the study at every visit.  
When ECG is collected multiple times (e.g. at Screening visit, Baseline visit both before dosing 
and 2-3 hours after dosing, and Week 40/EOT/ET visit), the average of all available values on 
the same day will be considered as one assessment for the summaries, except for Day 1 where 
averages will be by pre-dose and post-dose averages. The averages will be rounded to integer 
values. 
Observed (average) values of ECG variables (e.g., heart rate, PR interval, QRS interval, QT 
interval, QTcB and QTcF interval) and the changes from Baseline at each assessment time point 
will be summarized by treatment group.  
Observed (average) values of QTcB and QTcF will also be categorized into the following 
categories (msec) and the number and percentage of subjects in each category will be 
summarized by treatment group at each visit and for the overall post-Baseline maximum: 
• Observed: ≤450, 451 - ≤480, 481 - ≤500, and >500; >450; >480. 


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• Change from Baseline: ≤10, 11 – 30, 31 – 60, and >60; >30. 
For cardiologist’s interpretations, the number and percentage of subjects with ECG results that 
are determined as normal or abnormal will be summarized at scheduled visits. The overall post-
baseline worst interpretation will also be summarized (i.e. if a subject has one or more post-
baseline ECG results that is/are considered as abnormal, this subject will be counted in the 
abnormal category). Cardiologist’s interpretations will also be summarized in a shift table. The 
shifts from Baseline to overall post-Baseline worst interpretation will also be presented. For the 
by-visit shift summary, the denominator is the number of subjects with non-missing 
cardiologist’s interpretation at Baseline and the given visit for the given treatment group. For the 
summaries of shift from Baseline to the overall post-Baseline worst interpretation, the 
denominator is the number of subjects with non-missing Baseline and at least 1 post-Baseline 
cardiologist’s interpretation for the given treatment group. 
Electrocardiogram variable average values will be considered PCI if they meet the criteria listed 
in Table 8. The number and percentage of subjects with post-baseline PCI values will be 
summarized by treatment group at each post-Baseline visit and for overall post-Baseline. For the 
by-visit summary, the numerator for the percentage is the number of subjects with a 
post-Baseline PCI ECG for the given parameter, visit and treatment group, and the denominator 
is the number of subjects with non-missing values for the given parameter, visit and treatment 
group. For the overall post-Baseline summary, the numerator for the percentage is the number of 
subjects with at least 1 post-Baseline PCI ECG for the given parameter and treatment group, and 
the denominator is the number of subjects with at least 1 post-Baseline ECG value for the given 
parameter and treatment group. A listing of overall and of all subjects with any PCI ECG values 
will be provided.  
Table 8  
Criteria for Potentially Clinically Important ECG Values 
ECG Parameter 
Unit 
High PCI Criteria 
QRS Interval 
msec 
≥120 
PR Interval 
msec 
≥220 
QTcB 
msec 
>500 
QTcF 
msec 
>500 
QTcB: change from baseline  
>60 msec  
QTcF: change from baseline  
>60 msec  


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14.5 
Physical Examination 
Physical examinations are performed throughout the study at every visit in the clinic. Physical 
examination results (normal, abnormal, and not done) will be summarized in a frequency table 
by body system and visit. A listing of physical examination data will be listed. 
14.6 
Other Safety Endpoints 
There are no other safety endpoints in this study. 


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15. 
CLINICAL PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSES 
For trofinetide-treated subjects, whole blood concentration for trofinetide will be listed. Whole 
blood concentration data for trofinetide will be summarized for the PK Analysis Set at each visit 
using descriptive statistics. Concentrations that are below the limit of quantification (BLQ) will 
be displayed as “BLQ” in the data listings and imputed as 0 for computing summary statistics. 
If data allow, population PK and PK/PD analyses will be performed to further characterize the 
PK profile and exposure response relationship of trofinetide using measures of safety and 
efficacy parameters. The results of population PK and PK/PD modeling will be presented in a 
separate report. Trofinetide whole blood concentration data will remain blinded until the 
unblinding of the clinical database at the end of the study. 


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16. 
INTERIM ANALYSIS 
No interim analysis is planned in this study, If a new drug application (NDA) submission 
happens before the completion of this study, an interim data cut will be scheduled for that 
purpose. 


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17. 
DATA MONITORING/REVIEW COMMITTEE 
Data from this study will be reviewed by the Data and Safety Monitoring Board (DSMB) for 
study ACP-2566-003.  


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18. 
COMPUTER METHODS  
Statistical analyses will be performed using Version 9.4 (or newer) of SAS on a qualified and 
validated environment. 
Validation and quality control of the tables, listings and figures containing the results of the 
statistical analyses will follow appropriate standard operating procedures. 


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19. 
CHANGES TO ANALYSES SPECIFIED IN PROTOCOL 
No changes are made to the analyses specified in the protocol.  


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20. 
REFERENCES 
EMA (2009). Guideline on Missing Data in Confirmatory Clinical Trials, European Medicines 
Agency, London, UK. 
FDA Guidance on Conduct of Clinical Trials of Medical Products during COVID-19 Pandemic 
Guidance for Industry, Investigators, and Institutional Review Boards March 2020 
 
Points to consider on implications of Coronavirus disease 4 (COVID-19) on methodological 
aspects of ongoing clinical 5 trials, March 25 2020. 
 


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21. 
APPENDICES 
21.1 
Schedule of Assessments 
Period 
Baseline 
Open-label Treatment Period 
Safety 
Follow-upk 
Visit Week 
0 
2 
12 
26 
40/EOT/ET 
EOT/ET+ 
30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
±3 
±7 
±7 
+7 
+4 
Type of Visitl 
Clinic or Off-site 
Telephone or 
Telemedicine 
Informed consent 
Xa 
 
 
 
Xa 
 
Inclusion/exclusion criteria 
X 
 
 
 
 
 
Medical history update 
X 
 
 
 
 
 
Physical examinationl 
X 
X 
X 
X 
X 
 
Vital signsb 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
X 
 
Weightl 
Xl 
X 
X 
X 
X 
 
12-lead electrocardiogram (ECG)e 
Xc 
X 
X 
X 
Xc 
 
Clinical laboratory tests (hematology, 
chemistry)m 
X 
X 
X 
X 
X 
 
Urinalysis 
X 
X 
X 
X 
X 
 
TSH, Free T3, Free T4 
X 
 
 
 
X 
 
Serum pregnancy testd 
X 
 
X 
X 
X 
 
Blood samples for pharmacokinetics 
Xe 
Xf 
Xf 
Xf 
Xf 
 
Blood sample for optional analysis for 
biomarkersg 
Xh 
 
X 
X 
X 
 
Rett Syndrome Behaviour 
Questionnaire (RSBQ) 
X 
X 
X 
X 
X 
 
Clinical Global Impression–
Improvement (CGI-I)i 
 
X 
X 
X 
X 
 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
X 
 
Communication and Symbolic 
Behavior Scales-Developmental 
Profile™ Infant-Toddler (CSBS-DP-
IT) Checklist  
X 
X 
X 
X 
X 
 
 


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21.1 
Schedule of Assessments (Continued) 
Period 
Baseline 
Open-label Treatment Period 
Safety 
Follow-upk 
Visit Week 
0 
2 
12 
26 
40/EOT/ET 
EOT/ET+ 
30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
±3 
±7 
±7 
+7 
+4 
Type of Visitl 
Clinic or Off-site 
Telephone or 
Telemedicine 
Impact of Childhood Neurologic 
Disability Scale (ICND) 
X 
 
 
 
X 
 
Rett Syndrome Clinician Rating of 
Hand Function (RTT-HF) 
X 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Ambulation and Gross Motor Skills 
(RTT-AMB) 
X 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Ability to Communicate Choices 
(RTT-COMC)l 
X 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Verbal Communication (RTT-VCOM) 
X 
X 
X 
X 
X 
 
Rett Syndrome Caregiver Burden 
Inventory (RTT-CBI) 
X 
 
X 
 
X 
 
Dispensing and review of semi-
structured caregiver diary 
X 
X 
X 
X 
X 
 
Concomitant medications 
X 
X 
X 
X 
X 
X 
Assessment of adverse events 
X 
X 
X 
X 
X 
X 
Study drug dispensedj 
X 
 
 
 
 
 
Authorization of study drug 
dispensationj 
X--------------------------------------X 
 
 
Study drug returnj 
 
X---------------------------------------X 
 
Study drug accountabilityj 
 
X 
X 
X 
X 
 
Abbreviations: EOT=end of treatment; ET=early termination 
 
a 
Unless subjects are enrolling in the present study under special circumstances due to the COVID-19 public health 
emergency (detailed in the inclusion/exclusion criteria), consent for the present study must be obtained prior to 
performance of the EOT procedures at the Week 12/EOT visit in the antecedent study. Data from the Week 12/EOT 
visit procedures of the antecedent study will be carried over as Baseline information in the present study, as applicable. 
Subjects who enter the present study under special circumstances due to the COVID-19 public health emergency will 
have consent obtained for the present study before any procedures are performed (e.g., the blood draw before the 


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Baseline visit). For subjects who decide to continue into subsequent open-label extension (OLE) study (ACP-2566-
005), informed consent for ACP-2566-005 must be obtained prior to performing the Week 40/EOT procedures.  
b 
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure will be measured after the subject has been sitting for ≥3 minutes.  
c 
ECGs will be completed in triplicate at Visit 1 (Baseline) both before dosing and 2-3 hours after dosing and at Visit 5 
(Week 40/EOT/ET). For those subjects who completed the antecedent study, the ECG taken at the Week 12/EOT visit 
in the antecedent study is the predose Baseline ECG of the present study. A single ECG will be completed at Visit 2 
(Week 2), Visit 3 (Week 12), and Visit 4 (Week 26).  
d 
For subjects who have reached menarche and have not had surgical sterilization  
e 
The PK sample taken at the Week 12/EOT visit in the antecedent study is the predose PK blood sample for the present 
study. A postdose PK blood sample will be collected at the end of ECG assessment 2-3 hours after the first dose of 
study drug in the present study.  
f 
PK samples at Visits 2, 3, 4, and 5 will be collected at one of the following time intervals: 1) 2-3 hours after dosing OR 
2) 4-6 hours after dosing OR 3) 7-11 hours after dosing. Every effort should be made to collect the PK samples at 
discrete time intervals during Visits 2, 3, 4, and 5. However, if the interval is the same across these visits, then the 
collection time should vary within that interval.  
g 
Participation in the effort to identify biomarkers is an optional component of the study requiring a separate informed 
consent.  
h 
The biomarker sample taken at the Week 12/EOT visit in the antecedent study is the Baseline sample for the present 
study.  
i 
The CGI-I assessments at Weeks 2, 12, 26, and 40/EOT will be a comparison with the baseline state of the illness in the 
present study (i.e., Visit 1 of ACP-2566-004).  
j 
Investigational product will be shipped directly to the subject. Confirmation of delivery to the subject will be made by a 
visiting nurse. Study drug shipment, return, and accountability will be performed in accordance with the drug 
distribution plan. In addition, study drug will be dispensed at the site during the Baseline visit when the visit is 
conducted in the clinic.  
k 
Subjects who roll over into the subsequent open-label study (ACP-2566-005) will not have the safety follow-up 
telephone call or telemedicine visit.  
l 
Study visits may be done off-site rather than in the clinic with the prior approval of the Sponsor or Medical Monitor. 
When a study visit takes place off-site, the physical examination will not be required. Weight should be measured 
whenever possible at off-site visits. Measurement of weight is necessary at Baseline in order to determine the starting 
dose. The RTT-COMC should be completed if possible, but it is not required.  
m 
Subjects for whom the Baseline visit of this study is not on the same day as the Week 12/EOT visit of the antecedent 
study will have blood drawn for Baseline values 3 to 14 days before the Baseline visit. 
21.2 
Summary of Version Changes  
Version No: 
Document History 
Description of Update 
Author(s) 
Version Date 
1.0 
Original version 
 
09 December 
2020 
2.0 
Added MECP2 Groupings; added 
definitions of RSBQ subscores; 
clarified analysis visit windowing 
 
21 April 2021 
3.0 
Clarified text throughout. 
 
20 October 2021