Conditions: Rett Syndrome
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 15
Healthy volunteers: no

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

Detailed description:

Arms:
arm 1: Oral dose of trofinetide

Interventions: intervention 1: Trofinetide
Intervention descriptions: intervention 1: Trofinetide solution of 10-30 mL based on subject's weight at Baseline, administered twice daily by mouth or gastrostomy tube (G-tube)

Protocol document:
Prot_000.pdf: 
Official Title: An Open-Label Study of Trofinetide for the Treatment of Girls Two to 
Five Years of Age who Have Rett Syndrome 
NCT Numbers: NCT04988867 
Document Date: 19 December 2022 


Study: ACP-2566-009 
Final Version: 1.0 
Clinical Study Protocol Amendment 2 
Date: 19 December 2022 
 
Confidential and Proprietary Information of Acadia Pharmaceuticals Inc. 
Page 1 of 77 
 
 
 
 
CLINICAL STUDY PROTOCOL 
 
An Open-Label Study of Trofinetide for the Treatment of Girls Two to Five 
Years of Age who Have Rett Syndrome 
 
Protocol Number: ACP-2566-009 
Amendment 2 
 
Original Protocol Date: 08 December 2020 
Protocol Amendment 1 Date: 30 June 2021 
Protocol Amendment 2 Date: 19 December 2022 
 
 
 
 
 
 
Protocol Template Version: 0.2 
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-2566-009 
 
Final Version: 1.0 
Clinical Study Protocol Amendment 2 
 
Date: 19 December 2022 
 
Confidential and Proprietary Information of Acadia Pharmaceuticals Inc. 
Page 2 of 77 
SPONSOR SIGNATURE PAGE 
Title: An Open-Label Study of Trofinetide for the Treatment of Girls Two to Five Years of 
Age who Have Rett Syndrome 
 
Acadia Head of Rare Disease and 
External Innovation: 
 
Senior Vice President 
Head of Rare Disease and External 
Innovation & Chief Scientific Officer 
Acadia Pharmaceuticals Inc. 
  
  
 
See appended electronic signature page 
 
Signature 
Date 
 
 
 
Acadia Study Lead: 
 
Vice President, Clinical Development 
Acadia Pharmaceuticals Inc. 
 
 
 
See appended electronic signature page 
 
Signature 
Date 
 
 
 
 
 
 


Signature Page for ACP-2566-009 Protocol Amendment 2
Signature Page for ACP-2566-009 Protocol Amendment 2
Approve
Approve


Study: ACP-2566-009 
 
Final Version: 1.0 
Clinical Study Protocol Amendment 2 
 
Date: 19 December 2022 
 
Confidential and Proprietary Information of Acadia Pharmaceuticals Inc. 
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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 an 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. 
Investigator 
 
 
Signature 
 
Date 
 
 
 
Name (printed) 
 
 
 
 


Study: ACP-2566-009 
 
Final Version: 1.0 
Clinical Study Protocol Amendment 2 
 
Date: 19 December 2022 
 
Confidential and Proprietary Information of Acadia Pharmaceuticals Inc. 
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PROTOCOL SYNOPSIS1 
Protocol 
Number 
ACP-2566-009 
EudraCT 
Number 
Not applicable 
Protocol Title 
An Open-Label Study of Trofinetide for the Treatment of Girls Two to 
Five Years of Age who Have Rett Syndrome 
Name of 
Investigational 
Product 
Trofinetide oral solution 
Indication 
Rett syndrome  
Phase of 
Development 
2/3 
Sponsor 
Acadia Pharmaceuticals Inc. 
 
 
Primary Objectives 
• To investigate the safety and 
tolerability of treatment with oral 
trofinetide in girls two to five 
years of age who have Rett 
syndrome 
• To characterize the 
pharmacokinetics of oral 
trofinetide in girls two to five 
years of age who have Rett 
syndrome 
Primary Endpoints 
The safety endpoints are as follows: 
• treatment-emergent adverse events (TEAEs) 
• serious adverse events (SAEs) 
• withdrawals due to adverse events (AEs) 
• potentially clinically important changes in other 
safety assessments 
The pharmacokinetic (PK) endpoints are as follows: 
• whole blood concentration of trofinetide 
• trofinetide PK parameters using the population 
PK approach 
 
 
 
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-2566-009 
 
Final Version: 1.0 
Clinical Study Protocol Amendment 2 
 
Date: 19 December 2022 
 
Confidential and Proprietary Information of Acadia Pharmaceuticals Inc. 
Page 6 of 77 
Exploratory Efficacy Objectives 
• To investigate the efficacy of 
treatment with oral trofinetide in 
girls two to five years of age who 
have Rett syndrome 
• To investigate the benefit of 
treatment with oral trofinetide on 
overall quality of life for girls 
two to five years of age who have 
Rett syndrome 
Exploratory Efficacy Endpoints 
• Clinical Global Impression–Improvement 
(CGI-I) score 
• Change from Baseline in Clinical Global 
Impression–Severity (CGI-S) score 
• Caregiver Global Impression–Improvement 
(CaGI-I) score 
• Change from Baseline in Overall Quality of Life 
Rating of the Impact of Childhood Neurologic 
Disability (ICND) Scale 
Number of Study 
Sites 
Approximately 10 sites will participate in this study. 
Number of 
Subjects Planned 
Approximately 10 to 15 subjects are expected to be enrolled, at least 
1 subject who weighs ≥9 to <11 kg, and at least 4 subjects who are 
less than 4 years of age at Screening, including at least one subject 
who is 2 years of age. 
Test Product, 
Dose, and 
Administration 
Note: Subjects who began treatment with trofinetide under the 
original protocol dated 08 December 2020 will continue to have 
trofinetide titration according to the dosing schedule in the original 
protocol.  
Subjects Who Began Treatment Under Original Protocol Dated 
08 December 2020 
Subjects will begin treatment with trofinetide 2 g (in 10 mL) twice 
daily (BID). The dose will be increased to 3 g (in 15 mL) BID at the 
Week 2 visit, 4 g (in 20 mL) BID at the Week 4 visit, and 5 g (in 
25 mL) BID at the Week 8 visit. In each case, the dose will be 
increased only if the Investigator judges that the subject is showing 
acceptable tolerability of the treatment. Doses will be administered 
orally or by gastrostomy (G) tube (G-tube) (doses administered via 
gastrojejunal [GJ] tubes must be administered through the G-port). 
At any point in the study, if the subject is not able to tolerate 
administration of the assigned dose, the Investigator may instruct the 
caregiver to reduce the dose of study drug to a dose as low as 1 g 
BID. In addition, up to four doses (in total, consecutive or 
non-consecutive) may be withheld within the first 6 weeks. After the 
subject is able to tolerate treatment, the Investigator will increase the 
dose as tolerated and will continue treatment on the highest dose the 
subject can tolerate (up to 5 g BID). This final assigned dose, i.e., the 
highest tolerated dose, must be given BID and the morning and 
evening doses must be identical. 


Study: ACP-2566-009 
 
Final Version: 1.0 
Clinical Study Protocol Amendment 2 
 
Date: 19 December 2022 
 
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Subjects Enrolled Under Protocol Amendment 1 Dated 30 June 
2021  
Subjects participating under this amended protocol will begin 
treatment with trofinetide 2 g (in 10 mL) twice daily (BID). The dose 
will be increased to 4 g (in 20 mL) BID at the Week 2 visit. At the 
Week 4 visit, the dose will be increased to 5 g (in 25 mL) BID for 
subjects who weigh ≥9 to <12 kg (based on weight at Baseline), or 6 g 
(in 30 mL) BID for subjects who weigh 12 to <20 kg. In each case, 
the dose will be increased only if the Investigator judges that the 
subject is showing acceptable tolerability of the treatment. Doses will 
be administered orally or by gastrostomy (G) tube (G-tube) (doses 
administered via gastrojejunal [GJ] tubes must be administered 
through the G-port). 
At any point in the study, if the subject is not able to tolerate 
administration of the assigned dose, the Investigator may instruct the 
caregiver to reduce the dose of study drug to a dose as low as 1 g 
BID. In addition, up to four doses (in total, consecutive or 
non-consecutive) may be withheld within the first 6 weeks. After the 
subject is able to tolerate treatment, the Investigator will increase the 
dose as tolerated and will continue treatment on the highest dose the 
subject can tolerate (up to 5 g BID for subjects who weigh ≥9 to 
<12 kg, or up to 6 g BID for subjects who weigh 12 to <20 kg). This 
final assigned dose (i.e., the highest tolerated dose) must be given 
BID and the morning and evening doses must be identical. 
Study Design 
This is a multicenter, open-label study of trofinetide for the treatment 
of girls 2 to 5 years of age with RTT. Participating girls 2 to 4 years 
of age must weigh ≥9 kg and <20 kg. Girls 5 years of age who weigh 
≥9 kg and <12 kg can also be enrolled. 
The duration of the study will be 26 months and will have three main 
periods: 
• Screening period: up to 4 weeks 
• Treatment period 
• Period A: 12 weeks  
• Period B: up to approximately 21 months 
• Safety follow-up period: 30 days 
Screening Period (Up to 4 Weeks) 
During the Screening period, subjects will be assessed for study 
eligibility. Only those subjects who meet all inclusion and no 
exclusion criteria will be eligible for the study. 


Study: ACP-2566-009 
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Clinical Study Protocol Amendment 2 
Date: 19 December 2022 
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Page 8 of 77 
Subjects will be evaluated for the diagnosis of RTT. In addition, there 
must be verified documentation of a MECP2 (methyl-CpG–binding 
protein 2 gene) mutation. 
Investigators should not withdraw a subject’s prohibited medication 
for the purpose of enrolling them into the study. Medications should 
be discontinued only if it is deemed clinically appropriate to do so and 
in consultation with the treating physician. 
Caregivers will begin to keep a semi-structured caregiver diary 
during the Screening period. 
Treatment Period A (12 Weeks) 
Treatment Period A is designed for evaluation of the dosing, 
tolerability, pharmacokinetics, and exploratory efficacy of trofinetide 
in this population. The treatment period is approximately 12 weeks, 
the same length as the double-blind, placebo controlled study in girls 
and women 5 to 20 years of age (Table S–1). 
The Baseline visit (Visit 2) may occur after screening procedures are 
completed and have not ruled the subject out of eligibility for the 
study. 
Dosing is twice a day, once in the morning and once in the evening. 
The first dose of study drug will be administered after all Baseline 
assessments are completed, or, if the Investigator judges that it is too 
late in the day, on the following day. The day the first dose is taken 
will be considered Day 1. A triplicate ECG must be performed 
2 hours (±15 minutes) after the first dose. Two PK samples will also 
be collected after the first dose: the first, 2 hours (±15 minutes) after 
dosing, as close as possible to the postdose ECG, and the second, at 
least 1 hour after the first. Study drug must be discontinued in the 
event that a post-Baseline QTcF (QT interval corrected using 
Fridericia’s correction method) duration of ≥500 ms or an increase of 
≥60 ms compared to the average QTcF interval at Baseline (before 
dosing) is observed. 
After Treatment Period A (and the 30-day follow-up period, if 
applicable) is completed by all participants, an interim clinical study 
report of those data will be written. A final clinical study report will 
be written after the entire study is completed. 
Treatment Period B (Up to Approximately 21 Months) 
Treatment Period B is designed to assess the safety and exploratory 
efficacy of long-term treatment with trofinetide. Treatment Period B 
is similar in design and objectives to the open-label extension studies 
in girls and women 5 to 20 years of age. 


Study: ACP-2566-009 
 
Final Version: 1.0 
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Date: 19 December 2022 
 
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During Treatment Period B, subjects will have assessments completed 
at 24 weeks and 52 weeks after the Baseline visit and then every 
26 weeks (6 months) thereafter until the study concludes or is 
terminated. Assessments may be completed in the clinic or off-site. 
Study visits may be done off-site at the discretion of the Principal 
Investigator and only with the prior approval of the Sponsor or 
Medical Monitor. 
Safety Follow-Up Period (30[+4] Days) 
A 30(+4)-day safety follow-up telephone or telemedicine contact is to 
be completed for subjects who complete the treatment period of the 
study or discontinue prematurely from the study (and do not begin to 
take marketed trofinetide within the 30 days after completing the 
treatment period of the study). 
The study schematic is provided in Figure S–1. 
The schedule of assessments is provided in Table S–1 (Screening, 
Baseline, and Treatment Period A) and Table S–2 (Treatment 
Period B, and Safety Follow-Up). 
Study Duration 
The duration of participation for individual study subjects will be 
approximately 26 months, consisting of a screening period of up to 
4 weeks, a 12-week initial treatment period, an approximately 
21-month extended treatment period, and a safety follow-up period of 
30 days. 
The study start date is defined as the date the first subject is enrolled, 
which is the baseline visit date for the first subject. 
The primary completion date is the last date that subject data was 
collected for the primary outcome measure. 
The study completion date is defined as the last date that subject data 
was collected for the primary outcome measure, secondary outcome 
measure(s), and adverse events, which includes the safety follow-up 
telephone call visit. 
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. Written informed consent will be obtained from the 
legally acceptable representative (LAR). 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. 


Study: ACP-2566-009 
 
Final Version: 1.0 
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Date: 19 December 2022 
 
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b. The subject’s caregiver must also provide written 
informed consent regarding their participation in the 
study prior to participating in any study procedures. 
2. Female subject  
a. 2 to 4 years of age and body weight ≥9 kg and <20 kg 
at Screening OR 
b. 5 years of age and body weight ≥9 kg and <12 kg at 
Screening 
3. Can swallow the study medication provided as a liquid 
solution or can take it by gastrostomy tube 
4. The subject’s caregiver is English-speaking and has sufficient 
language skills to complete the caregiver assessments 
Diagnosis 
5. Has classic/typical Rett syndrome (RTT) or possible RTT 
according to the Rett Syndrome Diagnostic Criteria 
(Appendix A) 
6. Has a documented disease-causing mutation in the MECP2 
gene 
7. Has a CGI-S score of ≥4 at Screening and Baseline 
Concomitant Treatment 
8. If the subject is taking or was taking an anticonvulsant or any 
other CNS-active medication (including cannabinoids): 
a. the treatment regimen has been stable for at least 
4 weeks before Baseline and there is no current plan 
to change the dose, OR 
b. if the medication was discontinued, the 
discontinuation has occurred no fewer than 2 weeks 
or 5 half-lives (whichever is greater) before Baseline 
9. If the subject is taking or was taking any other medication 
daily for a chronic illness (not including antibiotics, pain 
relievers, or laxatives): 
a. the treatment regimen of the medication has been 
stable for at least 2 weeks before Baseline and there is 
no current plan to change the dose, OR 
b. if the medication was discontinued, the 
discontinuation has occurred no fewer than 2 weeks 
or 5 half-lives (whichever is greater) before Baseline 
10. If the subject is receiving or was receiving a 
non-pharmacologic somatic treatment (e.g., a ketogenic diet 
or vagal nerve stimulation): 


Study: ACP-2566-009 
 
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a. the treatment regimen has been stable for at least 
2 weeks before Baseline and there is no current plan 
to change the treatment, OR 
b. if the treatment was discontinued, the discontinuation 
has occurred no fewer than 2 weeks before Baseline 
Seizures 
11. Has a stable pattern of seizures, or has had no seizures, within 
8 weeks prior to Screening 
Place of Residence 
12. 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 4 weeks prior to Screening 
 
Exclusion Criteria: 
Concomitant Treatment 
1. Has been treated with growth hormone within 12 weeks of 
Baseline 
2. Has been treated with IGF-1 within 12 weeks of Baseline 
3. Has been treated with insulin within 12 weeks of Baseline 
Medical Conditions Other Than RTT 
4. Has current 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. Has a history of, or current, cerebrovascular disease or brain 
trauma 
6. Has significant, uncorrected visual or uncorrected hearing 
impairment 
7. Has a history of, or current, malignancy 
Laboratory Studies, Vital Signs, and Electrocardiogram 
8. Has one or more clinical laboratory test value(s) outside the 
range specified below at Screening:  
a. hemoglobin below the normal range 
b. aspartate aminotransferase (AST) or alanine 
aminotransferase (ALT) value more than 1.5 × the 
upper limit of the normal range (ULN) for that 
subject’s age and gender 


Study: ACP-2566-009 
 
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c. total bilirubin value ≥1.5 × ULN 
d. serum creatinine ≥ULN  
e. serum potassium below the normal range; serum 
potassium may be repeated during the Screening 
period with the agreement of the Medical Monitor 
f. thyroid stimulating hormone (TSH) value outside the 
normal range 
9. Has a clinically significant abnormal laboratory value at 
Screening. Laboratory testing may be repeated during the 
Screening period with agreement of the Medical Monitor. 
10. Has clinically significant abnormality in vital signs at 
Screening or Baseline 
11. Has any of the following: 
a. QTcF interval of >450 ms at Screening or Baseline  
b. History of a risk factor for torsades de pointes 
(e.g., heart failure or family history of long QT 
syndrome) 
c. History of clinically significant QT prolongation that 
is deemed to put the subject at increased risk of 
clinically significant QT prolongation 
d. Other clinically significant finding on ECG at 
Screening or Baseline 
Other Criteria 
12. Has a significant sensitivity or allergic reaction to trofinetide 
or its excipients 
13. Has participated in another interventional clinical study within 
30 days prior to Screening  
14. 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 at each visit during Treatment 
Period A. At Baseline (Visit 2), a PK sample will be obtained before 
dosing. After study drug administration, two PK samples will be 
collected, the first one approximately 2 hours after dosing and the 
second approximately 1 hour later.  
In addition to the two PK samples taken at Baseline, two PK samples 
will be taken at each subsequent visit (or upon early termination [ET]) 
in Treatment Period A, for a total of ten postdose PK samples over the 
duration of the study. At Visit 3, Visit 4, Visit 5, and Visit 6, the two 
PK samples will be collected at one of the following time intervals: 
• 1-3 hours after dosing 
• 4-7 hours after dosing 


Study: ACP-2566-009 
 
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• 8-11 hours after dosing 
Every subject should provide at least two PK samples from each of 
the three specified time intervals over the course of Visits 3 through 6 
(1-3 hours after dosing, 4-7 hours after dosing, and 8-11 hours after 
dosing). The two PK samples taken within a time interval should be 
collected at least one hour apart. 
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, 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 
Caregiver 
Interview 
Participation of caregivers in an interview is an optional component of 
the study. Caregivers will be interviewed by a 3rd party central agency 
about the study subject’s and caregivers’ experience of treatment with 
trofinetide. Only caregivers who sign a separate informed consent will 
be interviewed. The results of the caregiver interviews will be 
summarized. The caregiver interview will be administered during 
Treatment Period B, at or before the final study visit (Week 104 
[EOT/ET]). 
Sample Size 
Calculations 
Approximately ten (10) to fifteen (15) subjects will be enrolled in this 
study, at least 1 subject who weighs ≥9 to <11 kg, and at least 
4 subjects who are less than 4 years of age at Screening, including at 
least one subject who is 2 years of age. The sample size is not based 
on statistical considerations and is believed to be adequate to 
characterize the PK of trofinetide in this population. 
Statistical 
Methods 
Analysis Sets 
The following populations will be defined and used in the analysis: 
Safety Analysis Set 
The Safety Analysis Set will consist of all enrolled subjects who 
received at least one dose of study medication. This analysis set will 
be used for all safety as well as any descriptive efficacy analyses.  
Pharmacokinetic (PK) Analysis Set 
The PK Analysis Set will consist of all subjects who receive at least 
1 dose of study drug and have sufficient blood concentration data to 


Study: ACP-2566-009 
 
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calculate at least one PK parameter. 
Safety Analyses 
Safety results will be summarized using descriptive statistics. Adverse 
events will be coded into standard terminology using the Medical 
Dictionary for Regulatory Activities (MedDRA). Treatment-emergent 
adverse events (TEAEs), TEAEs leading to discontinuation, TEAEs 
considered related to study drug, TEAEs by maximum severity, and 
serious adverse events (SAEs) reported after study medication start, 
will all be summarized. 
Descriptive statistics for ECG, vital signs and weight, and clinical 
laboratory parameters, including changes from Baseline, will be 
tabulated by timepoint. Additionally, categorical analyses will be 
conducted on the incidence of subjects with prolonged QTc (QT 
interval of ECG corrected for heart rate) intervals and changes in QTc 
intervals in accordance with International Council on Harmonisation 
(ICH) guidelines. 
Additional safety analysis details will be specified in the statistical 
analysis plan (SAP). 
Pharmacokinetic Analyses 
Pharmacokinetic (PK) and exploratory efficacy 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, 4, 8 and 12. 
Whole blood concentration data for trofinetide will be listed and 
summarized using descriptive statistics. Population PK analyses will 
be performed to characterize the PK in children 2 to 5 years of age. 
The details of the PK analysis will be presented in a separate 
population PK report in accordance with a separate data analysis plan 
(DAP). 
Efficacy Analyses 
Descriptive summary statistics for the CGI-I and CaGI-I (observed 
values only), and for the CGI-S and ICND QoL score (observed 
values and change from Baseline) will be summarized by visit using 
the Safety Analysis Set. 
Date 
19 December 2022 
 


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Figure S–1 
Schematic of Study Design for ACP-2566-009 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Baseline 
Visit 
 
Open-Label Treatment Period 
Treatment Period A (12 weeks) 
Treatment Period B (up to approximately 21 months) 
Treatment Period A 
Screening 
Visit 
Screening 
Period 
Up to 28 
Days 
Subjects 
N≈10-15 
Follow-Up Period 
30(+4 days) 
Treatment Period B 


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Table S–1 
Schedule of Events and Assessments for Study ACP-2566-009 
(Screening, Baseline, and Treatment Period A) 
Period 
Screening 
Baselinea 
Treatment Period A 
Visit  
 
 
Week 2b 
Week 4 
Week 8 
Week 12 
Visit Number 
1 
2 
3 
4 
5 
6 
Visit window (days) 
N/A 
N/A 
±3 
±3 
±3 
+3 
Type of Visitc 
Clinic or 
Off-site 
Clinic 
Clinic or Off-site 
Clinic 
Informed consent 
X 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
X 
 
 
 
 
Medical history and demographics 
X 
 
 
 
 
 
Confirm documented diagnosis of RTT 
or possible RTT 
X 
 
 
 
 
 
Confirm documented MECP2 
mutationd 
X 
 
 
 
 
 
Rett syndrome history 
X 
 
 
 
 
 
Rett Syndrome Clinical Severity Scale  
X 
 
 
 
 
 
Physical examinationc 
X 
X 
X 
X 
X 
X 
Vital signse 
X 
X 
X 
X 
X 
X 
Height 
X 
 
 
 
 
X 
Weightc 
X 
X 
X 
X 
X 
X 
Electrocardiogram (ECG)f 
Xf 
Xf 
X 
X 
X 
Xf 
Clinical laboratory tests 
X 
X 
X 
X 
X 
X 
Urinalysis 
X 
X 
X 
 
 
X 
TSH, free T3, free T4 
X 
 
 
 
 
 
Blood samples for pharmacokineticsg 
 
X 
X 
X 
X 
X 
Clinical Global Impression–
Improvement (CGI-I) 
 
 
X 
X 
X 
X 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
X 
X 
Caregiver Global Impression–
Improvement (CaGI-I) 
 
 
 
 
 
X 
Overall Quality of Life Rating of the 
Impact of Childhood Neurologic 
Disability (ICND) Scale 
 
X 
 
 
 
X 
Dispensing and review of semi-
structured caregiver diary 
X 
X 
X 
X 
X 
X 
Prior medications 
X 
 
 
 
 
 
Concomitant medications 
 
X 
X 
X 
X 
X 
Assessment of adverse events 
X 
X 
X 
X 
X 
X 
Authorization of study drug 
dispensationh 
 
X-------------------------------------------------------X 
Study drug returnh 
 
 
X----------------------------------------X 
Study drug accountabilityh 
 
 
X 
X 
X 
X 


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Abbreviations: AE=adverse event; EOT=end of treatment; MECP2=methyl-CpG–binding protein 2 gene; N/A=not 
applicable; PK=pharmacokinetic; RTT=Rett syndrome; SAE=serious adverse event; T3=triiodothyronine; 
T4=thyroxine; TSH=thyroid stimulating hormone 
a 
The first dose of study drug will be administered after all Baseline assessments are completed, or, if the Investigator 
judges that it is too late in the day, on the following day. In that case, the second day is still considered part of the 
Baseline visit. The day the first dose is taken will be considered Day 1. 
b 
Timing of post-Baseline visits will be calculated from the first day of dosing (Day 1) (i.e., the Week 2 visit will occur 
2 weeks [14±3 days] after the first day of dosing).  
c 
The Baseline and Week 12 visits must be done at the clinic. All other study visits may be done in the clinic or off-site. 
Study visits may be done off-site at the discretion of the Principal Investigator and only 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. 
d 
If documentation of the mutation is not adequate, genomic testing for a mutation in the MECP2 gene may be conducted 
as part of Screening. 
e 
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure should be measured after the subject has been sitting or supine for ≥3 minutes. 
f  
ECGs will be completed in triplicate at Visit 1 (Screening), at Visit 2 (Baseline), both predose and at 2 hours 
(±15 minutes) after dosing, and at Visit 6 (Week 12). A single ECG will be completed at all other designated visits. 
g 
A predose PK blood sample must be collected before administration of study drug at Baseline (Visit 2). After the first 
dose of study drug is administered, two PK samples will be collected: the first, 2 hours (±15 minutes) after dosing, as 
close as possible to the postdose ECG, and the second, at least 1 hour after the first.  
In addition to the two PK samples taken at Baseline, two PK samples will be taken at each subsequent visit (or upon 
early termination [ET]) in Treatment Period A, for a total of ten postdose PK samples over the duration of the study. At 
Visit 3, Visit 4, Visit 5, and Visit 6, the two PK samples will be collected at one of the following time intervals: 1-3 
hours after dosing, 4-7 hours after dosing, 8-11 hours after dosing. Every subject should provide at least two PK 
samples from each of the specified time intervals over the course of Visits 3 through 6 (1-3 hours after dosing, 4-7 
hours after dosing, and 8-11 hours after dosing). The two PK samples taken within a time interval should be collected at 
least one hour apart.  
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. 
h 
Study drug will be dispensed at the site during the Baseline visit when the visit is conducted in the clinic. For the 
remainder of the study, investigational product will be shipped directly to the subject. Study drug shipment, return, and 
accountability will be performed in accordance with the drug distribution plan.   
 
 


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Table S–2 
Schedule of Events and Assessments for Study ACP-2566-009 
(Treatment Period B, and Safety Follow-Up) 
Period 
Treatment Period B 
Safety 
Follow-Upg 
Visit  
Week 24  
Week 52 
(12 months) 
Week 78 
(18 months) 
Week 104 
EOT/ET 
(24 months) 
EOT/ET 
+30 days 
Visit Number 
7 
8 
9 
10 
 
Visit window (days) 
±7 
±7 
±7 
±7 
+4 
Type of Visita 
Clinic or Off-site 
Telephone or 
Telemedicine 
Physical examinationa 
X 
X 
X 
X 
 
Vital signsb 
X 
X 
X 
X 
 
Height 
 
 
 
X 
 
Weighta 
X 
X 
X 
X 
 
Electrocardiogram (ECG)a 
X 
X 
X 
X 
 
Clinical laboratory tests 
X 
X 
X 
X 
 
Urinalysis 
X 
X 
X 
X 
 
Blood samples for pharmacokineticsc 
 
 
 
Xc 
 
Clinical Global Impression–
Improvement (CGI-I) 
X 
X 
X 
X 
 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
 
Caregiver Global Impression–
Improvement (CaGI-I) 
X 
X 
X 
X 
 
Overall Quality of Life Rating of the 
Impact of Childhood Neurologic 
Disability (ICND) Scale 
X 
X 
X 
X 
 
Dispensing and review of semi-
structured caregiver diary 
X 
X 
X 
Xd 
 
Concomitant medications 
X 
X 
X 
X 
X 
Assessment of adverse events 
X 
X 
X 
X 
X 
Optional caregiver interviewe 
X 
 
Authorization of study drug 
dispensationf  
X------------------------------------------------------X 
 
Study drug returnf 
X------------------------------------------------------X 
 
Study drug accountabilityf  
X 
X 
X 
X 
 
Abbreviations: AE=adverse event; EOT=end of treatment; ET=early termination; SAE=serious adverse event 
a 
Study visits may be done in the clinic or off-site. Study visits may be done off-site at the discretion of the Principal 
Investigator and only with the prior approval of the Sponsor or Medical Monitor. The EOT visit should be done in the 
clinic whenever possible. 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. 
b 
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure should be measured after the subject has been sitting or supine for ≥3 minutes. 
c  
Pharmacokinetic samples will not be collected at the Week 104 visit or at an ET visit during Treatment Period B. 
Pharmacokinetic samples will be collected, if possible, at the visit immediately following any SAE or following any 
AE leading to discontinuation. 


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d  
At the Week 104 EOT/ET visit, the caregiver diary will be returned and reviewed, but no diary will be dispensed. 
e  
Caregiver interviews will be conducted remotely via telephone during Treatment Period B, at or before the final study 
visit (Week 104 [EOT/ET]). Participation in the caregiver interview is an optional component of the study requiring a 
separate informed consent. 
f  
Study drug will be dispensed at the site during the Baseline visit when the visit is conducted in the clinic. For the 
remainder of the study, investigational product will be shipped directly to the subject. Study drug shipment, return, and 
accountability will be performed in accordance with the drug distribution plan.  
g 
A 30(+4)-day safety follow-up telephone or telemedicine contact is to be completed for subjects who complete the 
treatment period of the study or discontinue prematurely from the study (and do not begin to take marketed trofinetide 
within the 30 days after completing the treatment period of the study). 
 


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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 ................................................................................................................... 25 
LIST OF FIGURES ................................................................................................................. 25 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................................. 26 
1 
INTRODUCTION ...................................................................................................... 28 
1.1 
Background Information ............................................................................................ 28 
1.2 
Investigational Product ............................................................................................... 28 
1.3 
Previous Clinical Experience ..................................................................................... 29 
1.3.1 
Clinical Pharmacology ............................................................................................... 29 
1.3.1.1 Linearity and Dose Proportionality ............................................................................ 29 
1.3.1.2 Comparison of Single-Dose and Multiple-Dose Pharmacokinetics ........................... 29 
1.3.1.3 Absorption .................................................................................................................. 29 
1.3.1.4 Distribution ................................................................................................................. 29 
1.3.1.5 Metabolism ................................................................................................................. 29 
1.3.1.6 Elimination ................................................................................................................. 30 
1.3.1.7 Intrinsic and Extrinsic Factors .................................................................................... 30 
1.3.1.8 Overview of Clinical Pharmacokinetic/Pharmacodynamic Relationships ................. 31 
1.3.2 
Phase 2 Studies ........................................................................................................... 31 
1.3.3 
Phase 3 Studies ........................................................................................................... 32 
1.4 
Study Rationale .......................................................................................................... 33 
1.4.1 
Rationale for Study Design ........................................................................................ 33 
1.4.2 
Rationale for Dose Selection ...................................................................................... 34 
1.5 
Benefit/Risk Assessment ............................................................................................ 34 
1.5.1 
Known Potential Risks ............................................................................................... 34 
1.5.2 
Known Potential Benefits ........................................................................................... 36 
2 
STUDY OBJECTIVES AND ENDPOINTS .............................................................. 36 
2.1 
Primary Objectives ..................................................................................................... 36 
2.1.1 
Primary Endpoints ...................................................................................................... 37 
2.2 
Exploratory Efficacy Objectives ................................................................................ 37 


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2.2.1 
Exploratory Efficacy Endpoints ................................................................................. 37 
3 
STUDY DESCRIPTION ............................................................................................ 37 
3.1 
Overview of Study Design ......................................................................................... 37 
3.1.1 
Screening Period (Up to 4 Weeks) ............................................................................. 38 
3.1.2 
Treatment Period A (12 Weeks) .................................................................................. 39 
3.1.3 
Treatment Period B (Up to Approximately 21 Months) ............................................. 40 
3.1.4 
Safety Follow-Up Period (30[+4] Days) .................................................................... 40 
4 
SUBJECT ELIGIBILITY AND WITHDRAWAL CRITERIA .................................. 40 
4.1 
Inclusion Criteria ........................................................................................................ 40 
4.2 
Exclusion Criteria ....................................................................................................... 42 
4.3 
Subject Withdrawal of Consent .................................................................................. 43 
4.4 
Subject or Study Discontinuation ............................................................................... 44 
4.4.1 
Post-Baseline QTcF Interval Stopping Criteria .......................................................... 44 
4.4.2 
Handling of Subject Discontinuation During the Treatment Period .......................... 45 
4.5 
Subject Lost to Follow-Up ......................................................................................... 45 
4.6 
Prior and Concomitant Therapy ................................................................................. 45 
4.6.1 
Prior Medication ......................................................................................................... 45 
4.6.2 
Concomitant Medication ............................................................................................ 45 
4.6.2.1 Permitted and Prohibited Medications ....................................................................... 46 
5 
INVESTIGATIONAL PRODUCT ............................................................................. 46 
5.1 
Investigational Product Description ........................................................................... 46 
5.1.1 
Formulation, Appearance, and Packaging .................................................................. 47 
5.1.2 
Product Storage and Stability ..................................................................................... 47 
5.1.3 
Dosing and Administration ......................................................................................... 47 
5.1.3.1 Dosing ........................................................................................................................ 47 
5.1.3.2 Administration of Study Drug .................................................................................... 49 
5.1.4 
Method of Assigning Subjects to Treatment Groups.................................................. 50 
5.1.5 
Blinding ...................................................................................................................... 50 
5.1.6 
Study Drug Compliance ............................................................................................. 50 
5.1.7 
Overdose ..................................................................................................................... 50 
5.2 
Investigational Product Accountability Procedures ................................................... 50 
6 
STUDY PROCEDURES ............................................................................................ 51 
6.1 
Screening Assessments ............................................................................................... 51 
6.1.1 
Confirm Diagnosis of RTT and MECP2 Mutation .................................................... 51 
6.1.2 
Medical History, Including RTT History, and Demographics .................................... 51 


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6.1.3 
The Rett Syndrome Clinical Severity Scale (RTT-CSS) ............................................ 52 
6.2 
Exploratory Efficacy Assessments ............................................................................. 52 
6.2.1 
Clinical Global Impression–Improvement (CGI-I) and Clinical Global Impression–
Severity (CGI-S)......................................................................................................... 52 
6.2.2 
Caregiver Global Impression–Improvement (CaGI-I) ............................................... 53 
6.2.3 
Overall Quality of Life Rating of the Impact of Childhood Neurologic Disability 
(ICND) Scale .............................................................................................................. 53 
6.3 
Safety Assessments..................................................................................................... 54 
6.3.1 
Physical Examination ................................................................................................. 54 
6.3.2 
Vital Signs .................................................................................................................. 54 
6.3.3 
Height, Weight, and Body Mass Index ....................................................................... 54 
6.3.4 
Electrocardiograms ..................................................................................................... 54 
6.3.4.1 Post-Baseline QTcF Interval Stopping Criteria .......................................................... 55 
6.3.5 
Laboratory Evaluations .............................................................................................. 55 
6.4 
Caregiver Diary .......................................................................................................... 56 
6.5 
Caregiver Interview .................................................................................................... 57 
6.6 
Pharmacokinetic Assessments .................................................................................... 57 
6.6.1 
Blood Sampling .......................................................................................................... 58 
6.6.2 
Specimen Preparation, Handling, Storage, and Shipment.......................................... 60 
7 
ADVERSE EVENTS ................................................................................................. 60 
7.1 
Specification of Safety Parameters............................................................................. 60 
7.1.1 
Definition of Adverse Event ....................................................................................... 60 
7.1.2 
Definition of Serious Adverse Event .......................................................................... 61 
7.2 
Classification of an Adverse Event ............................................................................ 62 
7.2.1 
Severity of Event ........................................................................................................ 62 
7.2.2 
Relationship to Study Drug ........................................................................................ 63 
7.2.3 
Duration ...................................................................................................................... 63 
7.2.4 
Frequency ................................................................................................................... 63 
7.2.5 
Action Taken with Study Drug ................................................................................... 63 
7.2.6 
Therapy ....................................................................................................................... 64 
7.2.7 
Outcome ..................................................................................................................... 64 
7.2.8 
Seriousness ................................................................................................................. 64 
7.2.9 
Definition of Unexpectedness .................................................................................... 64 
7.3 
Time Period and Frequency for Event Assessment and Follow-up ........................... 64 
7.4 
Reporting Procedures ................................................................................................. 65 
7.4.1 
Adverse Event Reporting ........................................................................................... 65 
7.4.2 
Serious Adverse Event Reporting............................................................................... 65 


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7.4.3 
Reporting of Overdose ............................................................................................... 66 
8 
CLINICAL MONITORING ....................................................................................... 66 
9 
STATISTICAL METHODS AND DATA ANALYSIS .............................................. 66 
9.1 
Statistical and Analytical Plans .................................................................................. 66 
9.2 
Statistical Hypotheses................................................................................................. 67 
9.3 
Sample Size Determination ........................................................................................ 67 
9.4 
Subject Populations for Analysis ................................................................................ 67 
9.5 
Statistical Analyses ..................................................................................................... 67 
9.5.1 
General Approach ....................................................................................................... 67 
9.5.2 
Safety Analyses .......................................................................................................... 67 
9.5.3 
Pharmacokinetic Analyses .......................................................................................... 68 
9.5.4 
Efficacy Analyses ....................................................................................................... 68 
9.6 
Interim Analyses ......................................................................................................... 68 
9.7 
Measures to Minimize Bias ........................................................................................ 68 
9.8 
Breaking the Study Blind/Subject Code ..................................................................... 68 
10 
STUDY MANAGEMENT AND DATA COLLECTION .......................................... 68 
10.1 
Data Collection and Management Responsibilities.................................................... 68 
10.2 
Source Documents ...................................................................................................... 69 
10.3 
Case Report Forms ..................................................................................................... 69 
10.4 
Confidentiality ............................................................................................................ 69 
10.5 
Study Records Retention ............................................................................................ 69 
10.6 
Protocol Exceptions and Deviations........................................................................... 70 
10.7 
Protocol Amendments ................................................................................................ 70 
11 
QUALITY MANAGEMENT .................................................................................... 70 
11.1 
Risk Management ....................................................................................................... 70 
11.2 
Quality Control and Quality Assurance...................................................................... 71 
12 
ETHICAL CONSIDERATIONS ................................................................................ 72 
12.1 
Ethical Standard ......................................................................................................... 72 
12.2 
Institutional Review Board/Ethics Committee ........................................................... 72 
12.3 
Informed Consent Process .......................................................................................... 72 
12.3.1 Consent and Other Informational Documents Provided to Subjects .......................... 73 
12.3.2 Consent Procedures and Other Informational Documents Provided to Subjects ....... 73 
13 
PUBLICATION PLAN .............................................................................................. 74 


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14 
CONFLICT OF INTEREST POLICY ....................................................................... 74 
14.1 
Finance, Insurance, and Indemnity............................................................................. 74 
15 
LITERATURE REFERENCES .................................................................................. 75 
16 
APPENDICES ............................................................................................................ 76 
Appendix A Rett Syndrome Diagnostic Criteria ................................................................... 76 
 


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LIST OF TABLES 
Table S–1 
Schedule of Events and Assessments for Study ACP-2566-009 
(Screening, Baseline, and Treatment Period A) ............................................... 16 
Table S–2 
Schedule of Events and Assessments for Study ACP-2566-009 (Treatment 
Period B, and Safety Follow-Up) .................................................................... 18 
Table 1–1  
Gastrointestinal TEAEs Reported in ≥2 Trofinetide-Treated Subjects 
During or Following Double-Blind Treatment: Study Neu-2566-RETT-
002 – ITT Population ....................................................................................... 35 
Table 5–1 
Dosing Schedule for Trofinetide for Subjects Who Began Treatment 
Under Original protocol Dated 08 December 2020 ......................................... 48 
Table 5–2 
Dosing Schedule for Trofinetide, Based on Weight at Baseline, for 
Subjects Who Enrolled Under Protocol Amendment 1 Dated 30 June 2021 .. 49 
Table 6–1 
Blood Collection Volume Limits in Affected Children ................................... 59 
Table 6–2 
Blood Collection Volumes for MECP2 Mutation Testing, Safety Labs and 
Pharmacokinetic Samples ................................................................................ 59 
 
LIST OF FIGURES 
Figure S–1 
Schematic of Study Design for ACP-2566-009 ............................................... 15 
 


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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Term 
Definition 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
BCRP 
breast cancer resistance protein 
BID 
bis in die; twice daily 
CaGI-I 
Caregiver’s Global Impression–Improvement 
CFR 
Code of Federal Regulations 
CGI-I 
Clinical Global Impression–Improvement 
CGI-S 
Clinical Global Impression–Severity 
EC 
ethics committee 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
EOT 
end of treatment 
ET 
early termination 
EU GDPR 
European Union General Data Protection Regulation 
FXS 
fragile X syndrome 
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  
IGF-1 
insulin-like growth factor 1 
IRB 
institutional review board 
LAR 
legally acceptable representative 
MATE2-K 
multidrug and toxin extrusion protein 2-K 
MECP2 
gene encoding methyl-CpG binding protein 2 (in humans) 
PD 
pharmacodynamic 
P-gp 
P-glycoprotein 
PK 
pharmacokinetic(s) 
QT interval 
QT interval for heart rate of ECG 
QTc 
QT interval of ECG corrected for heart rate 
QTcB 
QT interval corrected using Bazett’s correction method 
QTcF 
QT interval corrected using Fridericia’s correction method 


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Term 
Definition 
RSBQ 
Rett Syndrome Behaviour Questionnaire 
RTT 
Rett syndrome 
RTT-CBI 
Rett Syndrome Caregiver Burden Inventory 
RTT-CSS 
Rett Syndrome Clinical Severity Scale 
RTT-DSC 
RTT Domain Specific Visual Analog Scale 
SAE 
serious adverse event 
TBI 
traumatic brain injury 
TEAE 
treatment emergent adverse event 
ULN 
upper limit of normal 
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 and applicable regulatory requirements. 
1.1 
Background Information 
Rett syndrome (RTT) is a rare, seriously debilitating neurodevelopmental disorder which 
manifests in early childhood and affects primarily females. The main clinical features include 
a loss of acquired hand skills and spoken language, characteristic repetitive hand stereotypies, 
and either gait problems or an absence of gait. Patients with RTT cannot feed themselves and 
walk only with assistance. RTT patients require lifelong 24-hour care. There are no medicines 
currently approved for the treatment of RTT. 
RTT is characterized by a distinctive pattern of disease onset and progression: (1) relatively 
normal initial development; (2) regression of acquired skills; (3) a plateau or 
“pseudostationary” period; and (4) a late motor decline (Neul and Chang 2020). Psychomotor 
development may appear normal for the first six months of life, followed by failure to reach 
normal developmental milestones. Developmental regression, especially loss of purposeful 
hand movements and spoken language, occurs between 18 and 30 months. After regression, 
people with RTT enter a plateau or “pseudostationary” period in which skills are no longer 
lost. Social interaction improves but the characteristic hand stereotypies become prominent 
(Neul and Chang 2020). 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 (Samaco and Neul 2011). 
1.2 
Investigational Product 
Trofinetide is a novel synthetic analog of the tripeptide glycine-proline-glutamate (GPE), a 
product of the naturally occurring cleavage of insulin-like growth factor 1 (IGF-1), intended 
to treat the core symptoms of RTT by reducing neuroinflammation and supporting synaptic 
function. In the central nervous system (CNS), IGF-1 is produced by neurons and microglia. 
IGF-1 in the brain is critical for both normal development and for response to injury and 
disease. Trofinetide crosses the blood-brain barrier following oral administration and is 
thought to normalize insufficient bioavailability of IGF-1. Furthermore, it is believed that 
trofinetide inhibits the production of inflammatory cytokines as well as the overactivation of 
microglia and astrocytes. Both the inflammatory cytokines and pathologically activated 
microglia contribute to deficits in synaptic development and functional maturation of 
synaptic plasticity that are fundamental to the wide-ranging effects of RTT. 


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In the US, trofinetide for the treatment of RTT was granted Fast Track designation on 
04 June 2013, Orphan Drug designation on 11 February 2015, and Rare Pediatric Disease 
designation on 02 March 2020. 
1.3 
Previous Clinical Experience 
1.3.1 
Clinical Pharmacology 
The pharmacokinetics (PK) of trofinetide was assessed in healthy subjects, subjects with 
RTT, fragile X syndrome (FXS), and traumatic brain injury (TBI). 
1.3.1.1 
Linearity and Dose Proportionality 
Trofinetide exhibits linear kinetics with no time- or dose-dependent effect on PK parameters. 
Systemic exposure to trofinetide was dose-proportional across the studied dose range, with 
no metabolic auto-inhibition or auto-induction. 
1.3.1.2 
Comparison of Single-Dose and Multiple-Dose Pharmacokinetics 
Following oral administration of trofinetide, decline from peak concentration occurred in a 
biphasic manner. Minimal to no accumulation was observed following multiple-dose 
administration, and as such the single-dose PK profile is considered representative of the 
steady-state profile. 
1.3.1.3 
Absorption 
Following oral administration, trofinetide was rapidly absorbed (Tmax [time to maximum drug 
concentration], approximately 2 hours). There is no clinically relevant food effect and no 
difference in PK following morning or evening dosing. 
1.3.1.4 
Distribution 
In vitro, trofinetide has shown low protein binding in human plasma (<6%). The volume of 
distribution at steady state was approximately 60 L, which suggests that trofinetide reaches 
beyond the extracellular fluid, with limited distribution into tissues (intracellularly). 
Nonclinical data demonstrated increased penetration of trofinetide into rat brain in the 
absence of injury. 
1.3.1.5 
Metabolism 
In contrast to GPE, trofinetide displayed resistance to proteolytic degradation in human or rat 
plasma, and in human Caco-2 cells. Nonclinical data indicated that metabolism is not likely 
to be an important route of trofinetide disposition. 


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1.3.1.6 
Elimination 
Trofinetide is primarily excreted unchanged in urine, and hepatic metabolism is not likely to 
be an important route of trofinetide disposition. Trofinetide has an initial short half-life of 
approximately 1.2 hours, followed by a slower terminal elimination phase (half-life of 
approximately 13 hours). The initial half-life is considered the effective half-life for 
trofinetide. 
1.3.1.7 
Intrinsic and Extrinsic Factors 
Intrinsic Factors 
Population PK analysis in pediatric patients identified body weight as a significant factor, 
with effect on clearance and volume of distribution and consequently on the overall systemic 
exposure to trofinetide. 
Formal clinical studies assessing the effect of renal or hepatic impairment on the PK of 
trofinetide have not been performed. 
Extrinsic Factors 
Coadministered drugs that are inhibitors or inducers of CYP450 are not likely to significantly 
affect the plasma concentrations of trofinetide. 
At the target clinical concentrations, the potential of trofinetide to affect the PK of 
coadministered drug by inhibition or induction of CYP450 enzymes is low. Because of the 
relatively high clinical dose, the intestinal concentration of trofinetide is anticipated to be 
significantly high; trofinetide has the potential to inhibit CYP3A4 in the intestine, thus 
affecting coadministered drugs that have low bioavailability and are CYP3A4 substrates, 
such as midazolam. 
Coadministered drugs that are inhibitors or inducers of drug transporter are not likely to 
significantly affect the plasma concentrations of trofinetide. 
At the target clinical concentrations, the potential of trofinetide to affect the PK of 
coadministered drug by inhibition or induction of hepatic drug transporter is low. Because the 
intestinal concentration of trofinetide is anticipated to be significantly high, trofinetide has 
the potential to inhibit P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) 
transporter in the intestine. Similarly, because of the projected high renal concentration of 
trofinetide, it has the potential to inhibit multidrug and toxin extrusion protein 2-K 
(MATE2-K), which may decrease the renal excretion of endogenous and exogenous organic 
cations, such as metformin. 


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1.3.1.8 
Overview of Clinical Pharmacokinetic/Pharmacodynamic Relationships 
Exposure-response relationships were assessed in subjects with RTT using selected measures 
of efficacy endpoints. In subjects with RTT, a significant relationship was observed between 
systemic exposure and changes in the Rett Syndrome Behaviour Questionnaire (RSBQ), the 
RTT Domain Specific Visual Analog Scale (RTT-DSC), and the Clinical Global Impressions–
Improvement (CGI-I) scale, where improvements were observed with higher exposure. 
Pharmacokinetic/pharmacodynamic (PK/PD) analysis of the drug exposure and efficacy data 
from all trofinetide-treated subjects demonstrated a relationship between exposure (AUC) 
and magnitude of response for the RSBQ, RTT-DSC, and CGI-I. 
1.3.2 
Phase 2 Studies 
Two Phase 2, double-blind, randomized, placebo-controlled, dose-escalation clinical studies 
were conducted in girls and women with RTT: Study Neu-2566-RETT-001 and 
Study Neu-2566-RETT-002. 
In Study Neu-2566-RETT-001 (Study 001), 56 adolescent and adult RTT subjects (15 to 
44 years of age) received oral administration of placebo, trofinetide 35 mg/kg twice daily 
(BID), or trofinetide 70 mg/kg BID for up to 28 days. Results from the group-level analysis 
by individual cohort showed that trofinetide at 70 mg/kg BID demonstrated biological 
activity and preliminary evidence of efficacy. Trofinetide was associated with evidence of 
benefit over placebo based on prespecified criteria (p-value <0.2 in three core variables from 
three different efficacy domains): the Rett Syndrome Motor Behavior Assessment 
(RTT-MBA) change index (p=0.146), suggesting likely improvement in major signs and 
symptoms of Rett syndrome; the CGI-I score (p=0.164), suggesting improvement in illness 
and general clinical presentation compared to baseline; and the Caregiver Top 3 Concerns 
total visual analogue scale (VAS) score (p=0.076), suggesting improvement in the most 
concerning aspects of the Rett syndrome symptomology, as identified by caregivers. 
Study Neu-2566-RETT-002 (Study 002) was subsequently conducted to assess 42 days of 
double-blind treatment with trofinetide 50 mg/kg BID, 100 mg/kg BID, and 200 mg/kg BID 
versus placebo in 82 female children and adolescents with RTT (5 to 15 years of age). For the 
200 mg/kg BID dose group, three of the five core endpoints showed a statistically significant 
difference from placebo: the RSBQ total score (p=0.042), the clinician-rated RTT-DSC total 
score (p=0.025), and the CGI-I scale (p=0.029). The remaining two core outcome endpoints, 
the Caregiver Top 3 Concerns assessment and the RTT-MBA were directionally positive. The 
50 mg/kg BID and 100 mg/kg BID groups showed some improvement but did not reach 
statistical significance. Across the measures, improvement was seen in a number of symptom 
areas of the RTT phenotype that seriously impair the quality of life of women and girls with 
RTT: breathing problems, repetitive movements (including hand function), mood dysfunction 


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(including nighttime behaviors), ambulation, and seizures. On the RSBQ, all but one of the 
subscales were directionally in favor of the 200 mg/kg BID treatment group, with notable 
improvement in the General Mood subscale (p=0.007), the Breathing subscale (p=0.095), and 
the Repetitive Face Movement subscale (p=0.047). On the RTT-DSC, improvements were 
notable in two domains, Ambulation (p=0.040) and Seizures (p=0.057), and were 
directionally in favor of the 200 mg/kg BID treatment for Attentiveness and Social 
Interaction. 
Overall, these two Phase 2 studies demonstrated an acceptable safety and tolerability profile 
across the doses and treatment durations studied and a proof-of-concept efficacy profile to 
support initiation of Phase 3 clinical development. 
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 TBI have also been completed and are described in the Investigator’s brochure. 
1.3.3 
Phase 3 Studies 
Study ACP-2566-003 (Study 003) is a 12-week, multicenter, randomized, double-blind, 
placebo-controlled, parallel group Phase 3 study to evaluate efficacy and safety of a single 
trofinetide treatment group relative to placebo in girls and women 5 to 20 years of age with 
RTT. The starting dose of study drug is based on the individual’s weight as the population PK 
(PopPK) model included body weight as a significant covariate on both clearance (CL) and 
volume of distribution (Vd). Dosing is 6 g BID, 8 g BID, 10 g BID, or 12 g BID depending 
the weight ranges (12-20 kg, >20-35 kg, >35-50 kg, and > 50 kg, respectively). 
The co-primary efficacy endpoints in Study 003 are change from Baseline to Week 12 in 
RSBQ total score and CGI-I Score at Week 12. The key secondary endpoint is change from 
Baseline to Week 12 in Communication and Symbolic Behavior Scales Developmental 
Profile™ Infant-Toddler Checklist–Social Composite Score (CSBS-DP-IT Social). 
Approximately 184 subjects will be randomized in a 1:1 ratio to trofinetide or placebo. 
Subjects will be stratified according to age (5-10 years old, 11-15 years old, and 16-20 years 
old) and Baseline RSBQ severity (<35 total score and ≥35 total score). A minimum of 
12 subjects are required to be randomized for each age stratum. 
Additionally, ACP-2566-004 (Study 004) is a 40-week, multicenter, open-label extension 
(OLE) to Study 003 that gives RTT patients 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, 
tolerability, and efficacy of trofinetide in this population. Approximately 180 RTT patients 
are expected to be enrolled in Study 004. Upon completion of Study 004, subjects will have 


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the option to continue trofinetide open-label treatment in Study ACP-2566-005 (Study 005) 
for a further 32 months, or until such time as trofinetide may be approved and becomes 
commercially available, or development is discontinued in which case the study would be 
terminated. 
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, pharmacokinetics, efficacy, and safety. 
1.4 
Study Rationale 
Study 003 evaluates trofinetide treatment in girls and women with RTT who are 5 to 20 years 
of age and weigh ≥12 kg. However, In the US the diagnosis of RTT is commonly made as 
early as 18 months to 2 years of age. Study 009 would provide data on safety, tolerability, and 
PK that could inform trofinetide dosing recommendations in these patients as well as data on 
long-term safety and efficacy. 
The primary reason for maintaining an age cut-off of ≥5 years of age in Study 003 was in 
consideration of the variable clinical phenotype and developmental regression in the early 
presentation of RTT that could confound evaluation of the efficacy of interventional 
treatment. Enrollment of patients <5 years of age into clinical studies could not begin before 
analysis of animal juvenile toxicity data. Those data are now available and support 
administration across the intended pediatric age range. Please refer to the trofinetide 
Investigator’s Brochure for further details. 
1.4.1 
Rationale for Study Design 
A placebo-controlled study design is not feasible in this population because of the small 
numbers of patients with Rett syndrome in this restricted age range. It would be necessary to 
randomize a large number of patients in order to make any conclusions about differences 
between patients treated with active drug and those treated with placebo. In addition, 
interpretation of the efficacy data for an interventional treatment during the regression phase 
of RTT would present a challenge. The pattern and timing of the regression period varies 
among patients and would be difficult to characterize even in a large controlled study. The 
design of the Phase 3 Study 003 was deliberate in attempting to reduce the impact of disease 
variability in the evaluation of efficacy of trofinetide in RTT: an age cut-off of ≥5 years was 
selected for both the Phase 2 Study 002 and the Phase 3 Study 003 and both studies have an 
inclusion criterion requiring the patients to be post-regression at Screening. 
Because demonstrating efficacy is not the primary focus of this study, the starting dose is 
relatively low and increases toward the target dose are made each week in a stepwise fashion, 
taking into account how well the subject tolerates the medication (see Section 1.4.2). PK data 


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collected during treatment will provide information on the exposure to drug achieved by the 
doses the subjects reach, and can later be compared to the exposures associated with benefit 
in the other studies of trofinetide in RTT. Data from 10 patients is sufficient to provide the 
PK and tolerability data that may be compared to the data from studies of older patients with 
RTT. 
Period A is 12 weeks long to mirror the 12-week length of Study 003. Period B mirrors the 
open-label studies, Study 004 and Study 005.   
Like Study 005, Period B is designed to give subjects in the study the ability to receive 
open-label treatment with trofinetide until it becomes commercially available in the US, or 
until development of trofinetide for RTT is discontinued. 
It is estimated that the length of the study will provide enough time on treatment from the 
time the first subject enters the study until trofinetide is available, or until it becomes 
apparent that trofinetide will not be marketed in the US. 
1.4.2 
Rationale for Dose Selection 
As was done to determine the appropriate doses for the Phase 3 program based on the Phase 
2 study experience in girls and women with RTT, virtual patient simulations have been used 
to predict doses that would achieve the “target exposure” for the population in this study. 
As in the ongoing Phase 3 studies, PK simulations indicated a dose of 6 g BID would achieve 
the target exposure expected to achieve efficacy for patients who weigh 12 to <20 kg. PK 
simulations indicated a dose of 5 g BID would achieve the targeted exposure in patients who 
weigh ≥9 to <12 kg. This study will initiate with a lower trofinetide dose of 2 g BID to best 
ensure initial tolerability. The trofinetide dose will be increased to 5 g BID or 6 g BID for the 
respective weight ranges. The dose will be increased to 6 g BID only for subjects who weigh 
12 to <20 kg and who enroll under the present amended protocol.  
In each case, the dose will be increased only if the Investigator deems that the subject is 
showing acceptable tolerability of the treatment. If the assigned dose is not tolerated, the dose 
may be reduced to as low as 1 g BID. In addition, up to four doses (in total, consecutive or 
non-consecutive) may be withheld within the first 6 weeks. The Investigator will increase the 
dose as tolerated and the subject will continue treatment on the highest tolerated dose. 
1.5 
Benefit/Risk Assessment 
1.5.1 
Known Potential Risks 
Individuals with RTT frequently experience chronic constipation, and many have taken 
medications to treat constipation for a long time as well. Gastrointestinal side effects such as 


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diarrhea and vomiting are the most notable tolerability issue in clinical studies with oral 
trofinetide. 
In Study 002, in which subjects 5 to 15 years of age were enrolled, diarrhea was the most 
frequently reported TEAE and was more frequently reported as a TEAE in trofinetide-treated 
subjects than in the placebo group, and frequency appeared to be dose-related (Table 1–1). 
This included diarrhea, intermittent diarrhea, and loose stools. The majority of cases were 
mild or moderate in severity and resolved on their own or with treatment per standard clinical 
practice (such as treatment with loperamide). In addition, the incidence of vomiting was 
higher in the highest-dose trofinetide group than with placebo. Diarrhea was not as frequent 
in the trofinetide treatment groups in the other Phase 2 study of oral trofinetide, but it was 
generally more frequently reported as a TEAE in trofinetide-treated subjects than in 
placebo-treated subjects. Adverse events (AEs) of diarrhea have also occurred frequently in 
Study 003, the ongoing double-blind, placebo-controlled, study and Study 004, the ongoing 
open-label extension. 
A detailed summary of the safety data from clinical studies is available in the trofinetide 
Investigator’s Brochure. 
Table 1–1  
Gastrointestinal TEAEs Reported in ≥2 Trofinetide-Treated 
Subjects During or Following Double-Blind Treatment: Study 
Neu-2566-RETT-002 – ITT Population 
MedDRA System Organ Class 
Preferred Term 
Placebo 
BID 
(N=24) 
n (%) 
Trofinetide 
50 mg/kg 
BID 
(N=15) 
n (%) 
100 mg/kg 
BID 
(N=16) 
n (%) 
200 mg/kg 
BID 
(N=27) 
n (%) 
Gastrointestinal disorders 
4 (16.7) 
5 (33.3) 
4 (25.0) 
15 (55.6) 
Diarrhoea 
1 (4.2) 
4 (26.7) 
2 (12.5) 
15 (55.6) 
Vomiting 
3 (12.5) 
1 (6.7) 
2 (12.5) 
6 (22.2) 
Constipation 
- - 
- - 
- - 
2 (7.4) 
Gastroenteritis 
- - 
- - 
1 (6.3) 
1 (3.7) 
Source: Neu-2566-RETT-002 CSR, Table 12-3 
Abbreviations: BID=twice daily; ITT=Intention-to-treat; MedDRA= Medical Dictionary for Regulatory Activities 
 
At the target clinical concentrations, the potential of trofinetide to affect the PK of 
coadministered drug by inhibition or induction of cytochrome P450 (CYP) enzymes, or by 
inhibition or induction of hepatic drug transporters is low. Because of the relatively high 
clinical dose, the intestinal concentration of trofinetide is anticipated to be significantly high; 
trofinetide has the potential to inhibit CYP 3A4 enzyme (CYP3A4) in the intestine. Similarly, 


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trofinetide has the potential to inhibit P-gp or BCRP transporter in the intestine. A 
physiologically based PK (PBPK) model showed that concomitant administration of 12 g oral 
trofinetide increases the exposure of orally administered midazolam, a CYP3A4 probe 
substrate, by 1.37- and 1.24-fold for AUC and Cmax, respectively. Therefore trofinetide is 
considered a weak inhibitor of CYP3A4. 
Similarly, because of the projected high renal concentration of trofinetide, it has the potential 
to inhibit MATE2-K, which may decrease the renal excretion of endogenous and exogenous 
organic cations, such as metformin. 
Coadministered drugs that are inhibitors or inducers of CYP enzymes, or that are inhibitors or 
inducers of a drug transporter are not likely to significantly affect the blood concentrations of 
trofinetide. 
Trofinetide is considered unlikely to adversely affect the cardiovascular system following 
oral dosing in humans. 
No clinically significant QTc prolongation was observed following administration of 
trofinetide to healthy subjects and there was no signal of increased risk of QT prolongation 
with trofinetide treatment in patients with RTT or FXS. 
Based on the results of the nonclinical and clinical studies, the risk of clinically significant 
QTc prolongation in humans is estimated to be low. However, the Investigator should be 
cautious when subjects in a trofinetide study take a medication that may prolong the 
QT interval. 
1.5.2 
Known Potential Benefits 
Consistent with the evidence from nonclinical studies of GPE and trofinetide, there is 
evidence from clinical trials that trofinetide may be effective in the treatment of 
Rett syndrome (see Section 1.3). Trofinetide is not authorized for use in any indication. 
A detailed summary of the potential risks and benefits is available in the trofinetide 
Investigator’s Brochure. 
2 STUDY OBJECTIVES AND ENDPOINTS 
2.1 
Primary Objectives 
The primary objectives of this study are: 
• To investigate the safety and tolerability of treatment with oral trofinetide in girls 
two to five years of age who have Rett syndrome 
• To characterize the pharmacokinetics of oral trofinetide in girls two to five years of 
age who have Rett syndrome 


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2.1.1 
Primary Endpoints 
The safety endpoints are as follows: 
• treatment-emergent adverse events (TEAEs) 
• serious adverse events (SAEs) 
• withdrawals due to adverse events (AEs) 
• potentially clinically important changes in other safety assessments 
The pharmacokinetic (PK) endpoints are as follows: 
• whole blood concentration of trofinetide 
• trofinetide PK parameters using the population PK approach 
2.2 
Exploratory Efficacy Objectives 
The exploratory efficacy objectives of this study are: 
• To investigate the efficacy of treatment with oral trofinetide in girls two to five years 
of age who have Rett syndrome 
• To investigate the benefit of treatment with oral trofinetide on overall quality of life 
for girls two to five years of age who have Rett syndrome 
2.2.1 
Exploratory Efficacy Endpoints 
The exploratory efficacy endpoints for this study are: 
• Clinical Global Impression–Improvement (CGI-I) score at Weeks 2, 4, 8, 12, 24, 52, 
78, and end of treatment (EOT) 
• Change from Baseline to Weeks 2, 4, 8, 12, 24, 52, 78, and EOT in Clinical Global 
Impression–Severity (CGI-S) score  
• Caregiver Global Impression–Improvement (CaGI-I) score at Weeks 12, 24, 52, 78, 
and EOT  
• Change from Baseline to Weeks 12, 24, 52, 78, and EOT in Overall Quality of Life 
Rating of the Impact of Childhood Neurologic Disability (ICND) Scale 
3 STUDY DESCRIPTION 
3.1 
Overview of Study Design 
This is a multicenter, open-label study of trofinetide for the treatment of girls 2 to 5 years of 
age with RTT. Participating girls 2 to 4 years of age must weigh ≥9 kg and <20 kg. Girls 
5 years of age who weigh ≥9 kg and <12 kg can also be enrolled. 


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Approximately 10 to 15 subjects are expected to be enrolled, at least 1 subject who weighs 
≥9 to <11 kg, and at least 4 subjects who are less than 4 years of age at Screening, including 
at least one subject who is 2 years of age. Approximately 10 sites will participate in this 
study. 
The duration of the study will be 26 months and will have three main periods (Figure S–1): 
• Screening period: up to 4 weeks 
• Treatment period 
o Period A: 12 weeks 
o Period B: up to approximately 21 months   
• Safety follow-up period: 30 days  
The schedule of events and assessments is provided in Table S–1 (Screening, Baseline, and 
Treatment Period A) and Table S–2 (Treatment Period B, and Safety Follow-Up). 
Study drug will be dispensed at the site during the Baseline visit when the visit is conducted 
in the clinic. For the remainder of the study, investigational product will be shipped directly 
to the subject. Study drug shipment, return, and accountability will be performed in 
accordance with the drug distribution plan. 
The study start date is defined as the date the first subject is enrolled, which is the baseline 
visit date for the first subject. 
The primary completion date is the last date that subject data was collected for the primary 
outcome measure. 
The study completion date is defined as the last date that subject data was collected for the 
primary outcome measure, secondary outcome measure(s), and adverse events, which 
includes the safety follow-up telephone call visit. 
Procedures for when a subject is lost to follow-up are provided in Section 4.5. 
The Sponsor may discontinue the study for any reason. 
3.1.1 
Screening Period (Up to 4 Weeks) 
During the Screening period, subjects will be assessed for study eligibility and prohibited 
medications will be discontinued if medically appropriate. Subject eligibility will be assessed 
by the site and the Sponsor through an eligibility review process. Only those subjects who 
meet all inclusion and no exclusion criteria will be eligible for the study. 
Subjects will be evaluated for the diagnosis of RTT. In addition, there must be verified 
documentation of a MECP2 mutation. 


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Investigators should not withdraw a subject’s prohibited medication for the purpose of 
enrolling them into the study. Medications should be discontinued only if it is deemed 
clinically appropriate to do so and in consultation with the treating physician. The 
investigator should consider decreasing or discontinuing laxatives before the baseline visit to 
mitigate possible gastrointestinal side effects from trofinetide administration. 
Caregivers will begin to keep a semi-structured caregiver diary during the Screening period. 
3.1.2 
Treatment Period A (12 Weeks) 
Treatment Period A is designed for evaluation of the dosing, tolerability, and 
pharmacokinetics of trofinetide in this population. Exploratory efficacy assessments will also 
be conducted. The duration of Treatment Period A is approximately 12 weeks (Table S–1), 
the same length as the double-blind, placebo controlled study in girls and women 5 to 
20 years of age. 
The Baseline and Week 12 visits must be done at the clinic. All other study visits may be 
done in the clinic or off-site. Study visits may be done off-site at the discretion of the 
Principal Investigator and only 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. 
The Baseline visit (Visit 2) may occur after screening procedures are completed and have not 
ruled the subject out of eligibility for the study. 
Dosing is twice a day, once in the morning and once in the evening. The study drug must not 
be mixed with any food or liquid, including water. Immediately after administration of the 
study drug, food or liquid may be consumed if required. 
The first dose of study drug will be administered after all Baseline assessments are 
completed, or, if the Investigator judges that it is too late in the day, on the following day. In 
that case, the second day is still considered part of the Baseline visit. 
The day the first dose is taken will be considered Day 1. A triplicate ECG must be performed 
2 hours (±15 minutes) after the first dose. Two PK samples will also be collected after the 
first dose: the first, 2 hours (±15 minutes) after dosing, as close as possible to the postdose 
ECG, and the second, at least 1 hour after the first. Study drug must be discontinued in the 
event that a post-Baseline QTcF (QT interval corrected using Fridericia’s correction method) 
duration of ≥500 ms or an increase of ≥60 ms compared to the average QTcF interval at 
Baseline (before dosing) is observed. 


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After Treatment Period A (and the 30-day follow-up period, if applicable) is completed by all 
participants, an interim clinical study report of those data will be written. A final clinical 
study report will be written after the entire study is completed. 
3.1.3 
Treatment Period B (Up to Approximately 21 Months) 
Treatment Period B is designed to assess the safety and exploratory efficacy of long-term 
treatment with trofinetide. Treatment Period B is similar in design and objectives to the 
open-label extension studies in girls and women 5 to 20 years of age. 
During Treatment Period B, subjects will have assessments completed at 24 weeks and 
52 weeks after the Baseline visit and then every 26 weeks (6 months) thereafter until the 
study concludes or is terminated. Assessments may be completed in the clinic or off-site. 
Study visits may be done off-site at the discretion of the Principal Investigator and only with 
the prior approval of the Sponsor or Medical Monitor. 
3.1.4 
Safety Follow-Up Period (30[+4] Days) 
A 30(+4)-day safety follow-up telephone or telemedicine contact is to be completed for 
subjects who complete the treatment period of the study or discontinue prematurely from the 
study (and do not begin to take marketed trofinetide within the 30 days after completing the 
treatment period of the study). The telephone or telemedicine contact includes assessment of 
concomitant medications and treatments and assessment of AEs. 
The study schematic is provided in Figure S–1. 
The schedule of assessments is provided in Table S–1 (Screening, Baseline, and Treatment 
Period A) and Table S–2 (Treatment Period B, and Safety Follow-Up). 
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. Written informed consent will be obtained from the legally acceptable 
representative (LAR). 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. The subject’s caregiver must also provide informed consent regarding their 
participation in the study prior to participating in any study procedures. 
2. Female subject  
a. 2 to 4 years of age and body weight ≥9 kg and <20 kg at Screening OR 
b. 5 years of age and body weight ≥9 kg and <12 kg at Screening 
3. Can swallow the study medication provided as a liquid solution or can take it by 
gastrostomy tube 
4. The subject’s caregiver is English-speaking and has sufficient language skills to 
complete the caregiver assessments 
Diagnosis 
5. Has classic/typical Rett syndrome (RTT) or possible RTT according to the Rett 
Syndrome Diagnostic Criteria (Appendix A) 
6. Has a documented disease-causing mutation in the MECP2 gene 
7. Has a CGI-S score of ≥4 at Screening and Baseline 
Concomitant Treatment 
8. If the subject is taking or was taking an anticonvulsant or any other CNS-active 
medication (including cannabinoids):  
a. the treatment regimen has been stable for at least 4 weeks before Baseline 
and there is no current plan to change the dose, OR 
b. if the medication was discontinued, the discontinuation has occurred no 
fewer than 2 weeks or 5 half-lives (whichever is greater) before Baseline 
9. If the subject is taking or was taking any other medication daily for chronic illness 
(not including antibiotics, pain relievers, or laxatives): 
a. the treatment regimen of the medication has been stable for at least 2 weeks 
before Baseline and there is no current plan to change the dose, OR 
b. if the medication was discontinued, the discontinuation has occurred no 
fewer than 2 weeks or 5 half-lives (whichever is greater) before Baseline 
10. If the subject is receiving or was receiving a non-pharmacologic somatic treatment 
(e.g., a ketogenic diet or vagal nerve stimulation): 
a. the treatment regimen has been stable for at least 2 weeks before Baseline 
and there is no current plan to change the treatment, OR 
b. if the treatment was discontinued, the discontinuation has occurred no fewer 
than 2 weeks before Baseline 


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Seizures 
11. Has a stable pattern of seizures, or has had no seizures, within 8 weeks prior to 
Screening 
Place of Residence 
12. 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 4 weeks prior to 
Screening 
4.2 
Exclusion Criteria 
A subject must meet none of the following exclusion criteria to be eligible for the study: 
Concomitant Treatment 
1. Has been treated with growth hormone within 12 weeks of Baseline 
2. Has been treated with IGF-1 within 12 weeks of Baseline 
3. Has been treated with insulin within 12 weeks of Baseline 
Medical Conditions Other Than RTT 
4. Has current 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. Has a history of, or current, cerebrovascular disease or brain trauma 
6. Has significant, uncorrected visual or uncorrected hearing impairment 
7. Has a history of, or current, malignancy 
Laboratory Studies, Vital Signs, and Electrocardiogram 
8. Has one or more clinical laboratory test value(s) outside the range specified below at 
Screening:  
a. hemoglobin value below the normal range 
b. aspartate aminotransferase (AST) or alanine aminotransferase (ALT) value 
more than 1.5 × the upper limit of the normal range (ULN) for that subject’s 
age and gender 
c. total bilirubin value ≥1.5 × ULN 
d. serum creatinine ≥ULN  
e. serum potassium below the normal range; serum potassium may be repeated 
during the Screening period with the agreement of the Medical Monitor 
f. thyroid stimulating hormone (TSH) value outside the normal range 


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9. Has a clinically significant abnormal laboratory value at Screening. Laboratory 
testing may be repeated during the Screening period with agreement of the Medical 
Monitor.  
10. Has clinically significant abnormality in vital signs at Screening or Baseline 
11. Has any of the following: 
a. QTcF interval of >450 ms at Screening or Baseline 
b. History of a risk factor for torsades de pointes (e.g., heart failure or family 
history of long QT syndrome) 
c. History of clinically significant QT prolongation that is deemed to put the 
subject at increased risk of clinically significant QT prolongation 
d. Other clinically significant finding on ECG at Screening or Baseline 
Other Criteria 
12. Has a significant sensitivity or allergic reaction to trofinetide or its excipients 
13. Has participated in another interventional clinical study within 30 days prior to 
Screening  
14. 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, legally 
acceptable representatives (LARs) consenting on behalf of subjects have the right to 
withdraw the subject from the study at any time, and for any reason, without prejudice to her 
future medical care. 
If the LAR decides to withdraw consent on behalf of the subject from all components in the 
study, this must be documented and no additional assessments will be performed. The 
Sponsor may retain and continue to use any data collected before such a withdrawal of 
consent. The LAR may request destruction of any samples taken and not tested, prior to their 
withdrawal of consent, and the investigator must document this in the site study records. 
If the LAR wants to discontinue treatment on behalf of the subject and agrees to the 
evaluations specified at the EOT/ET visit and/or at safety follow up (whichever is 
applicable), as outlined in Table S–2, the agreed assessments should be conducted. The 
subject’s reason for wanting to discontinue treatment and the agreement to continue with the 
applicable assessments for study termination must be documented. 


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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-Baseline QTcF interval (defined below and in Section 6.3.4) 
• Lack of efficacy 
• Lost to follow-up (Section 4.5) 
• Non-compliance with study drug 
• Physician decision 
• Protocol deviation 
• Study terminated by sponsor 
• Use of prohibited medication 
• Withdrawal of consent by LAR 
• 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-Baseline 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. 


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4.4.2 
Handling of Subject Discontinuation During the Treatment Period 
Unless 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 10 or the early termination (ET) 
visit and the safety follow-up (as outlined in Table S–2) if a subject discontinues during the 
treatment period of the study 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 and appropriately treat 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 7.4.2. All SAEs will 
continue to be followed and appropriately treated until such events have resolved or the 
Investigator deems them to be chronic or stable. 
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 
(including the safety follow-up visit) and the study site is unable to contact the caregiver. 
Every reasonable effort should be made to contact the caregiver and will include a minimum 
of 3 documented telephone calls (each performed at different times of the day) and, if 
necessary, a certified letter to the caregiver’s last known mailing address or local equivalent 
methods. All contact attempts are to be documented in the source documents. 
4.6 
Prior and Concomitant Therapy 
All medications used up to 12 weeks prior to Baseline through completion of the safety 
follow-up visit or ET are to be recorded. 
4.6.1 
Prior Medication 
Prior medication is defined as any medication taken before the date of the first dose of study 
drug. 
4.6.2 
Concomitant Medication 
Concomitant medication is defined as any medication taken on or after the date of the 
first dose of study drug. 
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 


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consultation with the Investigator (unless the subject is receiving treatment for a medical 
emergency). 
The Investigator may prescribe appropriate medication to treat AEs. 
Every effort should be made to maintain stable regimens of concomitant medications and 
allowed non-medicine based therapies throughout the course of the study, with the 
understanding that there will be some changes to a treatment regimen that are due to school 
schedules or are otherwise seasonally related. Special cases are medications taken for the 
treatment of constipation or that have diarrhea as an acute side effect. These may be adjusted 
as needed if diarrhea occurs. 
4.6.2.1 
Permitted and Prohibited Medications 
Prohibited medications are IGF-1, growth hormone, and insulin. Prohibitions for concomitant 
medications will be followed between Visit 2 and Visit 10. 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. 
CNS-active concomitant medications should remain at a stable dose throughout the study if 
possible. Any non-pharmacologic somatic treatment regimen (e.g., a ketogenic diet or vagal 
nerve stimulation) that has CNS effects should remain stable throughout the study if possible. 
Treatment of constipation may be changed as needed. 
Subjects who require initiation of treatment with a prohibited medication will be withdrawn 
from the study. 
Subjects who are discovered to 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 
Trofinetide oral solution will be provided in a ready-to-use aqueous solution for oral 
administration. Doses will be administered orally or by gastrostomy (G) tube (G-tube) 
(doses administered by gastrojejunal [GJ] tubes must be administered through the G-port). 


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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 must be stored at this temperature. Do not freeze. 
5.1.3 
Dosing and Administration 
5.1.3.1 
Dosing 
Note: Subjects who began treatment with trofinetide under the original protocol dated 
08 December 2020 will continue to have trofinetide titration according to the dosing schedule 
in the original protocol.  
Subjects Who Began Treatment Under Original Protocol Dated 08 December 2020 
Subjects will begin treatment with trofinetide 2 g (in 10 mL) BID (Table 5–1). The dose will 
be increased to 3 g (in 15 mL) BID at the Week 2 visit, 4 g (in 20 mL) BID at the Week 4 
visit, and 5 g (in 25 mL) BID at the Week 8 visit. In each case, the dose will be increased 
only if the Investigator judges that the subject is showing acceptable tolerability of the 
treatment. An ECG must be performed 2 hours (±15 minutes) after the first dose, and a 
PK sample is to be taken as close as possible to the postdose ECG. A second PK sample will 
be taken at least 1 hour after the first PK sample. 


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 Table 5–1 
Dosing Schedule for Trofinetide for Subjects Who Began 
Treatment Under Original protocol Dated 08 December 2020 
Dose Commences (Visit) 
Dose  
Total Daily Dose 
Day 1 a 
10 mL (2 g) BID 
20 mL (4 g) 
Week 2 (Visit 3) 
15 mL (3 g) BID 
30 mL (6 g) 
Week 4 (Visit 4) 
20 mL (4 g) BID 
40 mL (8 g) 
Week 8 (Visit 5) 
25 mL (5 g) BID 
50 mL (10 g) 
a 
The first dose of study drug will be administered after all Baseline assessments are completed, or, if the 
Investigator judges that it is too late in the day, on the following day. The day the first dose is taken will be 
considered Day 1. 
 
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. 
At any point in the study, if the subject is not able to tolerate administration of the assigned 
dose, the Investigator may instruct the caregiver to reduce the dose of study drug to a dose as 
low as 1 g BID. In addition, up to four doses (in total, consecutive or non-consecutive) may 
be withheld within the first 6 weeks. 
After the subject is able to tolerate treatment, the Investigator will increase the dose as 
tolerated and will continue treatment on the highest dose the subject can tolerate (up to 5 g 
BID). This final assigned dose, i.e., the highest tolerated dose, must be given BID and the 
morning and evening doses must be identical. 
Study drug will be dispensed at the site during the Baseline visit when the visit is conducted 
in the clinic. For the remainder of the study, investigational product will be shipped directly 
to the subject. Study drug shipment, return, and accountability will be performed in 
accordance with the drug distribution plan. 
Subjects Enrolled Under Protocol Amendment 1 Dated 30 June 2021  
Subjects will begin treatment with trofinetide 2 g (in 10 mL) BID (Table 5–2). The dose will 
be increased to 4 g (in 20 mL) BID at the Week 2 visit. At the Week 4 visit, the dose will be 
increased to 5 g (in 25 mL) BID for subjects who weigh ≥9 to <12 kg, or 6 g (in 30 mL) BID 
for subjects who weigh 12 to <20 kg. In each case, the dose will be increased only if the 
Investigator judges that the subject is showing acceptable tolerability of the treatment. An 
ECG must be performed 2 hours (±15 minutes) after the first dose, and a PK sample is to be 
taken as close as possible to the postdose ECG. A second PK sample will be taken at least 
1 hour after the first PK sample. 


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 Table 5–2 
Dosing Schedule for Trofinetide, Based on Weight at Baseline, for 
Subjects Who Enrolled Under Protocol Amendment 1 Dated 
30 June 2021  
Dose Commences 
(Visit) 
Weight at Baseline 
Dose  
Total Daily Dose 
Day 1 a 
All subjects 
10 mL (2 g) BID 
20 mL (4 g) 
Week 2 (Visit 3) 
All subjects 
20 mL (4 g) BID 
40 mL (8 g) 
Week 4 (Visit 4) 
≥9 to <12 kg 
25 mL (5 g) BID 
50 mL (10 g) 
12 to <20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
a 
The first dose of study drug will be administered after all Baseline assessments are completed, or, if the 
Investigator judges that it is too late in the day, on the following day. The day the first dose is taken will be 
considered Day 1. 
 
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. 
At any point in the study, if the subject is not able to tolerate administration of the assigned 
dose, the Investigator may instruct the caregiver to reduce the dose of study drug to a dose as 
low as 1 g BID. In addition, up to four doses (in total, consecutive or non-consecutive) may 
be withheld within the first 6 weeks. 
After the subject is able to tolerate treatment, the Investigator will increase the dose as 
tolerated and will continue treatment on the highest dose the subject can tolerate (up to 5 g 
BID for subjects who weigh ≥9 to <12 kg, or up to 6 g BID for subjects who weigh 12 to 
<20 kg). This final assigned dose (i.e., the highest tolerated dose) must be given BID and the 
morning and evening doses must be identical. 
Study drug will be dispensed at the site during the Baseline visit when the visit is conducted 
in the clinic. For the remainder of the study, investigational product will be shipped directly 
to the subject. Study drug shipment, return, and accountability will be performed in 
accordance with the drug distribution plan. 
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. 
Dosing is twice a day, once in the morning and once in the evening. The study drug must not 
be mixed with any food or liquid, including water. Immediately after administration of the 
study drug, food or liquid may be consumed if required. 


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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. 
5.1.4 
Method of Assigning Subjects to Treatment Groups 
This is an open-label study. All subjects will receive treatment with trofinetide. Details of 
trofinetide dosing and administration is provided in Section 5.1.3. 
5.1.5 
Blinding 
Not applicable; 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 7.4.3). 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, as allowed by country-specific 


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regulations. Documentation of study drug destruction will be recorded and maintained by 
both the Sponsor and the Sponsor’s designee. 
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–1 and Table S–2. Every effort should be made to complete 
the required procedures and evaluations at the designated visits and times. 
6.1 
Screening Assessments 
6.1.1 
Confirm Diagnosis of RTT and MECP2 Mutation 
The site will confirm that the subject meets criteria for typical/classic RTT or possible RTT 
(Appendix A) and that there is documentation of disease-causing mutation of the MECP2 
gene. The diagnosis of “possible” RTT is given to those individuals under 3 years old who 
have not lost any skills but otherwise have clinical features suggestive of RTT (Appendix A). 
The genotyping must have been performed at a laboratory certified by the College of 
American Pathologists (CAP), or under the Clinical Laboratory Improvement 
Act/Amendment (CLIA), or by an equivalent organization. If documentation of the mutation 
is not adequate, genomic testing for a mutation in the MECP2 gene may be conducted as part 
of Screening. 
The documented mutation must be associated with RTT, such as according to the MECP2 
mutation database RettBASE (http://mecp2.chw.edu.au/cgi-
bin/mecp2/search/search.cgi?form=combined) (Krishnaraj et al. 2017). If there is any 
question regarding the association of the mutation with RTT, the Medical Monitor must be 
consulted. The disease-causing mutation will be documented in the source documentation 
and eCRF. 
6.1.2 
Medical History, Including RTT History, and Demographics  
A complete medical history, including history of symptoms associated with RTT and history 
of regression, will be performed at Screening to document all current medical conditions, and 
previous major medical events and conditions. For subjects who were already receiving care 
for RTT at the study site, summary documents from the medical record (such as clinician’s 
summaries) should be available as source documentation of major medical conditions or 


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events (e.g., surgeries). For subjects who were not a part of the clinical site’s practice, the 
study team should make every effort to obtain summary medical records from their other 
providers in preparation of the screening visit. 
6.1.3 
The Rett Syndrome Clinical Severity Scale (RTT-CSS) 
The RTT-CSS has been evaluated in over 1200 RTT children, adolescents and adults enrolled 
in the NIH-sponsored Natural History of Rare Diseases Project. This scale has been used as a 
measure of severity as reported in studies of genotype/phenotype correlations and epilepsy in 
RTT (Glaze et al. 2010) and was evaluated as an outcome measure in the Neu-2566-RETT-
001 study of trofinetide in adolescent and adults with RTT. The scale was derived from that 
reported in Amir et al. (2000) and Monrós et al. (2001). 
The RTT-CSS is a clinician-completed rating scale that measures the severity of core 
symptoms of RTT. The CSS consists of 13 items, 3 of which measure historical or static 
characteristics (age of onset of regression, age of onset of stereotypes, head growth) and 
10 of which measure current function (somatic growth, independent sitting, ambulation, hand 
use, scoliosis, language, nonverbal communication, respiratory dysfunction, autonomic 
symptoms, and epilepsy/seizures) at the time of assessment, i.e., during the study visit.  
All items are scored during a clinical interview and examination by the Investigator or 
qualified designee using either a 5- or 6-point Likert scale. Individual subscale scores and a 
total score are calculated. 
The RTT-CSS will be administered at Screening only. 
6.2 
Exploratory 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. 
6.2.1 
Clinical Global Impression–Improvement (CGI-I) and Clinical Global 
Impression–Severity (CGI-S) 
The CGI-I will be administered at Visits 3, 4, 5, 6, 7, 8, 9, and 10. 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 


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improved, 3=minimally improved, 4=no change, 5=minimally worse, 6=much worse, 
7=very much worse. 
The CGI-S assessment will be made at Screening, Baseline, and Visits 3, 4, 5, 6, 7, 8, 9, and 
10. 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 RTT as a whole. 
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). 
6.2.2 
Caregiver Global Impression–Improvement (CaGI-I)  
The question of the CaGI-I is identical to the question utilized in the Rett Natural History 
Study regarding Overall Function and Well-Being and asks “How would you describe your 
child’s overall function?” The CaGI-I will be administered at Visits 6, 7, 8, 9, and 10. 
Completion of this scale requires the caregiver to rate how much the subject’s illness has 
improved or worsened relative to a baseline state. A 5-point scale is used with the following 
response options: much improved, improved, unchanged, worse, and much worse. The data 
will be presented both by text response and also by numeric value in which 1= much 
improved, 2= improved, 3=unchanged, 4=worse, 5=much worse. 
In this study, the illness being assessed is RTT as a whole. 
6.2.3 
Overall Quality of Life Rating of the 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). 
In this study, the caregiver will complete only the Overall Quality of Life rating of the ICND. 
The caregiver 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, Visits 6, 7, 8, 9, and 10. 


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6.3 
Safety Assessments  
6.3.1 
Physical Examination 
A general physical examination will be conducted at Screening, Baseline, and Visits 3, 4, 5, 
6, 7, 8, 9, and 10. 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 
6.3.2 
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 or supine for ≥3 minutes. 
Vital signs to be measured at Screening, Baseline, Visits 3, 4, 5, 6, 7, 8, 9, and 10. 
6.3.3 
Height, Weight, and Body Mass Index 
Height will be measured at Screening and at Visits 6 and 10. 
Weight will be measured at Screening, Baseline, and Visits 3, 4, 5, 6, 7, 8, 9, and 10 when the 
visit takes place in the clinic. 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. 
6.3.4 
Electrocardiograms 
All ECGs will be complete, standardized recordings appropriate for the age of the subject, 
whenever possible. ECGs will be completed in triplicate at Visit 1 (Screening), at Visit 2 
(Baseline) predose and at 2 hours (±15 minutes) after dosing, and at Visit 6 (Week 12). 
A single ECG will be completed at Visit 3 (Week 2), and Visit 4 (Week 4), Visit 5 (Week 8), 


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Visit 7 (Week 24), Visit 8 (Week 52), Visit 9 (Week 78), and Visit 10 (Week 104). 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 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 interval of 
ECG, QRS interval of ECG, QT, QTcF, and QTcB intervals) will be included in the subject’s 
study records. 
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. At Screening and Baseline, the average QTcF interval of all 
legible ECGs will be used to determine eligibility. 
If the average QTcF value from the set of ECGs done at Screening is prolonged due to an 
identifiable cause, and it is medically appropriate to address that cause, a repeat set of 
triplicate ECGs may be performed during the Screening period and before the Baseline visit 
with the agreement of the Medical Monitor. In this case, the repeat ECGs will be used in 
determination of subject eligibility. 
6.3.4.1 
Post-Baseline QTcF Interval Stopping Criteria 
In the event that a post-Baseline QTcF duration of ≥500 ms or an increase of ≥60 ms 
compared to the average QTcF interval at Baseline (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.  
6.3.5 
Laboratory Evaluations 
Laboratory evaluations will be completed according to the schedule presented in Table S–1 
and Table S–2, and procedures detailed in the laboratory manual. Additional safety testing 
may be performed at the discretion of the Investigator or designee. Laboratory testing may be 
repeated during the Screening period with agreement of the Medical Monitor if the 
Investigator suspects that a laboratory abnormality is a temporary or reversible finding. 
Clinical laboratory sample collection is not required to be completed under fasting 
conditions. The laboratory evaluations will include the following: 


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• Clinical chemistry serum tests 
o Sodium (Na), potassium (K), chloride (Cl), phosphorus (P), calcium (Ca), 
magnesium (Mg), carbon dioxide (CO2), blood urea nitrogen (BUN), creatinine 
(CR), uric acid 
 Mg will only be performed at Visit 1 (Screening) 
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 (Screening) 
• 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. 
6.4 
Caregiver Diary 
A semi-structured caregiver diary will be completed during screening and treatment in which 
caregivers will record seizures and seizure-like spells if they are present.  
At the Screening visit, the clinician will review the subject’s seizure profile (if applicable) 
and will help the family to characterize the subject’s typical seizure types. These are recorded 
in the caregiver diary. 


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Study drug dosing and concomitant medication dosing will be recorded in the caregiver diary. 
Instances of missed or partial doses that occur on any day of the study will also be recorded 
in the caregiver diary. 
The caregiver diary will be completed and collected on an ongoing basis throughout the study 
beginning at Screening. The clinician will verbally ask about AEs (see Section 7.4.1), review 
the events recorded in the diary with the caregiver and will make a clinical evaluation, 
including an evaluation of whether an adverse event will be reported. 
6.5 
Caregiver Interview 
An optional qualitative interview with caregivers of study subjects will be conducted 
remotely by telephone. Only caregivers of subjects for whom separate informed consent for 
the caregiver interview is completed will be interviewed. Interviews will be conducted by 
trained moderators from a 3rd party and are anticipated to be approximately up to 60 minutes 
in duration. The interviews are planned to elicit a better understanding of the observable signs 
as well as the impact of study drug and RTT both on the study subject and on the caregiver. 
The interviews will document changes observed by the caregivers over the course of 
participation in the study. The interviews will also provide an opportunity to describe the 
meaningfulness of any improvements that may have occurred from the perspective of the 
caregiver. Interviews will be audio-recorded and transcribed for analysis. Any personally 
identifiable health information mentioned during the interview will be redacted by the 
interviewer prior to transcription of the interview. Furthermore, if any potential adverse 
events are revealed during the interview, the interviewer will inform the investigator of the 
potential adverse event. Audio recordings of the interviews will be destroyed following 
transcription.  
Following the analysis of the qualitative data, a summary report that describes the objectives, 
methods, participants, and results of the qualitative interviews will be prepared and reported 
separately from the clinical study report. 
The caregiver interview will be administered during Treatment Period B, at or before the 
final study visit (Week 104 [EOT/ET]). 
6.6 
Pharmacokinetic Assessments 
PK blood samples will be collected at each visit during Treatment Period A. 
At the Baseline visit, a PK sample is drawn predose. After the first dose of study drug is 
administered, two PK samples will be collected: the first, 2 hours (±15 minutes) after dosing, 
as close as possible to the postdose ECG, and the second, at least 1 hour after the first. 


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In addition to the two PK samples taken at Baseline, two PK samples will be taken at each 
subsequent visit (or upon early termination [ET]) in Treatment Period A, for a total of ten 
postdose PK samples over the duration of the study. At Visit 3, Visit 4, Visit 5, and Visit 6, 
the two PK samples will be collected at one of the following time intervals: 
• 1-3 hours after dosing 
• 4-7 hours after dosing 
• 8-11 hours after dosing 
Every subject should provide at least two PK samples from each of the specified time 
intervals over the course of Visits 3 through 6 (1-3 hours after dosing, 4-7 hours after dosing, 
and 8-11 hours after dosing). The two PK samples taken within a time interval should be 
collected at least one hour apart. 
Pharmacokinetic samples will also be collected, if possible, at any ET visit during Treatment 
Period A, or the visit immediately following any SAE, or following any AE leading to 
discontinuation. 
Pharmacokinetic samples will not be collected at the Week 104 visit, or at an ET visit during 
Treatment Period B. 
For all scheduled PK samples (and for unscheduled samples if possible), the dates and times 
of administration of the study drug 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. 
6.6.1 
Blood Sampling 
Table 6–1 tabulates the maximum allowable blood collection volumes by weight for affected 
children and Table 6–2 summarizes the blood volumes to be drawn in the present study in 
both a 24-hour and a 30-day period. The total amount of blood to be obtained from each 
subject should not exceed the allowable limits for affected children. 
Each PK sample requires collection of 2 mL of venous blood. A total of three 2-mL venous 
blood samples will be collected for PK sampling from each subject at the Baseline visit for 
the measurement of trofinetide concentrations in blood (Table 6–2). A total of two 2-mL 
venous blood samples will be collected from each subject on each subsequent day of 
PK sampling. 


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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 (see Section 7.4.2). 
Additional information for specimen preparation, handling, storage and shipment is provided 
in Section 6.6.2. 
Table 6–1 
Blood Collection Volume Limits in Affected Children 
Weight 
9 kg 
10 kg 
11 kg 
12 kg 
Maximum allowable blood volume in a 24-hour 
period 
18 mL 
20 mL 
22 mL 
24 mL 
Maximum allowable blood volume in a 30-day 
period 
36 mL 
40 mL 
44 mL 
48 mL 
Sources: North Shore LIJ Human Subject Protection Program Guidance Document, Maximum Blood Draw 
Limits, Version 11/24/14 
Note: Subjects must weigh ≥9 kg and <20 kg at Screening. 
 
Table 6–2 
Blood Collection Volumes for MECP2 Mutation Testing, Safety 
Labs and Pharmacokinetic Samples 
 
Screening 
Visit 
Baseline 
Visita 
Visit 3 
(Week 2) 
Visit 4 
(Week 4) 
Visit 5 
(Week 8) 
Visit 6 
(Week 12) 
Testing for MECP2 Mutation (if 
necessary) 
3.0 mL 
- - 
- - 
- - 
- - 
- - 
Safety Labs 
Chemistry and TSH/free T3/free 
T4 
3.7 mL 
 
 
 
 
 
Chemistry 
- - 
2.2 mL 
2.2 mL 
2.2 mL 
2.2 mL 
2.2 mL 
Hemoglobin/reticulocyte 
1.2 mL 
1.2 mL 
1.2 mL 
1.2 mL 
1.2 mL 
1.2 mL 
Pharmacokinetic Samples 
Predose 
- - 
2.0 mL 
- - 
- - 
- - 
- - 
Postdose 
- - 
4.0 mL 
4.0 mL 
4.0 mL 
4.0 mL 
4.0 mL 
Total volume in any 24-hour 
period  
7.9 mL 
9.4 mL 
7.4 mL 
7.4 mL 
7.4 mL 
7.4 mL 


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Table 6–2 
Blood Collection Volumes for MECP2 Mutation Testing, Safety 
Labs and Pharmacokinetic Samples (Continued) 
Total volume in any 30-day period 
Screening and Baseline 
---------17.3 mL-------
---
Screening, Baseline, and Week 2 
---------------24.7 mL-------------- 
Baseline, Week 2, and Week 4 
---------------24.2 mL--------------
-
Week 4 and Week 8 
---------14.8 mL--------- 
Week 8 and Week 12 
--------14.8 mL--------- 
Abbreviations: TSH=thyroid stimulating hormone; T3=triiodothyronine; T4=thyroxine 
a 
Baseline visit may take place over 2 days (see Section 3.1.2). 
6.6.2 
Specimen Preparation, Handling, Storage, and Shipment 
An indwelling intravenous cannula may be used for collection of serial PK blood samples 
(saline is to be used to flush the indwelling cannula); otherwise, PK blood samples will be 
obtained via standard venipuncture procedures. 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 possible metabolites identified). 
At each time point, blood will be collected, processed as appropriate, and samples will be 
shipped to the central laboratory for storage and to the bioanalytical laboratory for analysis. 
The date and actual time of PK blood collection must be collected and recorded in the 
subject’s eCRF. Each label will state the study number, subject number, analyte (whole 
blood), study day, and scheduled sample time. 
A laboratory manual will be provided for sample processing, storage, and shipping 
procedures. 
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., 


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off-label use, use in combination with another drug) and from any route of administration, 
formulation, or dose, including an overdose. 
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; if an overdose occurs, it will be reported on an 
overdose form. 
• Hospitalization for elective surgery planned prior to study (situation where an 
untoward medical occurrence has not occurred) 
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 
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. 


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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. 
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 


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• 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 
7.2.3 
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 worsened in severity 
For AEs of diarrhea, the start date and stop date of a decrease in severity will also be 
recorded. 
7.2.4 
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 
7.2.5 
Action Taken with Study Drug 
• Dose not changed: No change in study drug 
• Dose decreased: Study drug dose decreased 
• Drug interrupted: Study drug temporarily stopped 


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• Drug withdrawn: Study drug discontinued permanently 
• Not applicable 
• Unknown 
7.2.6 
Therapy 
• None: No new treatment instituted 
• Medication: New treatment initiated as a direct result of AE 
• Other: Other action required 
7.2.7 
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 
7.2.8 
Seriousness 
• Not serious 
• Serious (see Section 7.1.2) 
7.2.9 
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 will be recorded from the time informed consent is obtained through the 
study safety follow-up period. If an AE is ongoing at the end of the study safety follow-up 
period, every reasonable attempt should be made to follow and appropriately treat the subject 
until the AE resolves or until the Investigator deems the AE to be chronic or stable. 
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, a blood sample should be 
collected and the subject should be followed and appropriately treated until the AE resolves 
or until the Investigator deems the AE to be chronic or stable. 


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7.4 
Reporting Procedures 
7.4.1 
Adverse Event Reporting 
The Investigator must record all observed AEs and all reported AEs. At each visit, the 
Investigator should ask the subject and/or caregiver 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. 
7.4.2 
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. 
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.  
In the event an SAE or an AE leading to withdrawal is reported, a blood sample for 
determination of whole blood concentrations of trofinetide will be collected. 
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. 


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When an SAE occurs, Investigators will review all documentation related to the event and 
will complete the paper SAE form with all required information (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 (i.e., 30[+4] days after last 
dose of study drug) for any SAEs and/or other reportable information until such events have 
resolved or the Investigator deems them to be chronic or stable. 
In the event of any SAE (other than death), the study subject’s caregiver will be instructed to 
contact the Investigator (or designee) using the telephone number provided in the 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[+4] 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. 
7.4.3 
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 11.2. 
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. Deviations from the approved SAP will be 
described and justified in the final clinical study report. 


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9.2 
Statistical Hypotheses 
This is an open-label and single arm study. All statistical analyses will be descriptive. No 
statistical hypothesis will be tested. 
9.3 
Sample Size Determination 
Approximately ten (10) to fifteen (15) subjects will be enrolled in this study, at least 1 subject 
who weighs ≥9 to <11 kg, and at least 4 subjects who are less than 4 years of age at 
Screening, including at least one subject who is 2 years of age. The sample size is not based 
on statistical considerations and is believed to be adequate to characterize the PK of 
trofinetide in this population. 
9.4 
Subject Populations for Analysis 
The following populations will be defined and used in the analysis: 
The Safety Analysis Set will consist of all enrolled subjects who received at least one dose of 
study medication. This analysis set will be used for all safety as well as any descriptive 
efficacy analyses. 
The PK Analysis Set will consist of all subjects who receive at least 1 dose of study drug and 
have sufficient blood concentration data to calculate at least one PK parameter. 
9.5 
Statistical Analyses 
9.5.1 
General Approach 
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 number and percentage of subjects in each 
category will be reported. 
9.5.2 
Safety Analyses 
Safety results will be summarized using descriptive statistics. Adverse events will be coded 
into standard terminology using the Medical Dictionary for Regulatory Activities 
(MedDRA). Treatment-emergent adverse events (TEAEs), TEAEs leading to discontinuation, 
TEAEs considered related to study drug, TEAEs by maximum severity, and serious adverse 
events (SAEs) reported after study medication start, will all be summarized. 
Descriptive statistics for ECG, vital signs and weight, and clinical laboratory parameters, 
including changes from Baseline, will be tabulated by timepoint. Additionally, categorical 
analyses will be conducted on the incidence of subjects with prolonged QTc (QT interval of 
ECG corrected for heart rate) intervals and changes in QTc intervals in accordance with 
International Council for Harmonisation (ICH) guidelines. 


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Additional safety analysis details will be specified in the SAP. 
9.5.3 
Pharmacokinetic Analyses 
Pharmacokinetic (PK) and exploratory efficacy 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, 4, 8, and 12. 
Whole blood concentration data for trofinetide will be listed and summarized using 
descriptive statistics. Population PK analyses will be performed to characterize the PK in 
children 2 to 5 years of age. The details of the PK analysis will be presented in a separate 
population PK report in accordance with a separate data analysis plan (DAP). 
9.5.4 
Efficacy Analyses 
Descriptive summary statistics for the CGI-I and CaGI-I (observed value only), and for the 
CGI-S and ICND QoL score (observed values and change from Baseline) will be summarized 
by visit using the Safety Analysis Set. 
9.6 
Interim Analyses 
Interim analyses will be conducted after completion of Treatment Period A (including safety 
follow-up period if applicable), and during Treatment Period B if required for regulatory 
purposes. 
9.7 
Measures to Minimize Bias 
Not applicable; this is an open-label study. 
9.8 
Breaking the Study Blind/Subject Code 
Not applicable; this is an open-label study. 
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) as 
allowed by local regulations. Direct access includes permission to examine, analyze, verify, 
and reproduce any records and reports that are needed for the evaluation of the study. The 


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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 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 
(unless eCRF is considered the source) at the site. 
10.4 Confidentiality 
The Investigator must ensure that each subject’s anonymity is maintained as described below. 
On the eCRFs, medical records, 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.  
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, audio recordings, 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 2 years after the drug investigational program is discontinued, 
unless a longer period is required by applicable law or regulation. Only the Sponsor can 


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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. 
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, this will be reported as a major protocol 
deviation and not a waiver. In this situation, the subject 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. All follow-up safety assessments must be completed and 
documented as outlined in the protocol (Section 3.1.4). 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 QUALITY MANAGEMENT 
11.1 Risk Management 
The Sponsor 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 Sponsor’s risk management approach includes the following 
documented activities: 
• Critical Process and Data Identification: during protocol development, risks of 
processes and data that are critical to ensure human subject protection and the 
reliability of trial results are identified and assessed. 


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• 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 avoided, reduced (i.e., mitigated), or 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 defined to identify systematic issues that can impact subject safety 
or data integrity and deviations from the predefined quality tolerance limits will 
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 
during trial execution and risk outcome reporting in the clinical study report (CSR). 
11.2 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. 
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, sites participating in 
this study may 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 


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confidentiality, taking all reasonable precautions in accordance with applicable regulatory 
requirements to maintain the confidentiality of subjects’ identities. 
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 European Union General Data Protection Regulation 
[EU GDPR]). 
The study will be performed in accordance with current US Health Insurance Portability and 
Accountability Act (HIPAA) regulations, US FDA GCP Regulations (US 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.  
Properly executed, informed consent (and assent, if applicable) must be obtained from each 
LAR/subject prior to any screening procedures. The LAR is defined as “An individual or 
judicial or other body authorized under applicable law to consent on behalf of a prospective 


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subject to the subject’s participation in the procedures involved in the research” (US CFR 21 
part 50). 
Written informed consent will be obtained from the LAR. 
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 prospective subject’s LAR. 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 LAR’s willingness for the subject to continue participation. Any 
revision must be submitted to the appropriate IRB/EC for review and approval in advance of 
use. 
12.3.1 
Consent and Other Informational Documents Provided to Subjects 
The 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 Other Informational Documents Provided to Subjects 
It is the Investigator or designee’s responsibility to obtain written informed consent from the 
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 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 screening visit procedures being performed indicating their agreement to 
participate in the study in the caregiver role. 


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Participation in the caregiver interview is optional. Informed consent must be obtained by the 
Investigator (or designee), as appropriate, prior to the interviewer contacting the caregiver 
and conducting the optional caregiver interview. 
Where applicable and permitted, informed consent forms may be administered remotely in 
accordance with ICH E6 and applicable regulations. 
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 
Amir RE, Van den Veyver IB, Schultz R, et al. Influence of mutation type and X chromosome 
inactivation on Rett syndrome phenotypes. Ann Neurol. 2000;47:670-679. 
Busner J, Targum SD. The clinical global impressions scale: applying a research tool in 
clinical practice. Psychiatry. 2007;Jul;4(7):28-37. 
Camfield C, Breau L, Camfield P. Assessing the impact of pediatric epilepsy and concomitant 
behavioral, cognitive, and physical/neurologic disability: Impact of Childhood Neurologic 
Disability Scale. Dev Med Child Neurol. 2003;45:152-159. 
Centers for Disease Control and Prevention. https://www.cdc.gov/nchs/fastats/births.htm 
Accessed October, 2020. 
Glaze DG, Neul JL, Kaufmann WE, et al. Double-blind, randomized, placebo-controlled 
study of trofinetide in pediatric Rett syndrome. Neurology. 2019;92(16):e1912-e1925. 
Glaze DG, Neul JL, Percy A, et al. A double-blind, randomized, placebo-controlled clinical 
study of trofinetide in the treatment of Rett syndrome. Pediatr Neurol. 2017;76:37-46. 
Glaze DG, Percy AK, Skinner S, et al. Epilepsy and the natural history of Rett syndrome. 
Neurology. 2010;74:909-912. 
Krishnaraj R, Ho G, Christodoulou J. RettBASE: Rett syndrome database update. 
Hum Mutat. 2017;38(8):922-931. 
Monrós E, Armstrong J, Aibar E, Poo P, Canos I, Pineda M. Rett syndrome in Spain: 
mutation analysis and clinical correlations. Brain Dev. 2001;23:S251-S253. 
Neul JL, Chang Q. Rett syndrome and MECP2-related disorders. In: Neurodevelopmental 
Disorders. Academic Press; 2020;269-284. 
Neul JL, Glaze DG, Percy AK, 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, Kaufmann WE, Glaze DG, et al. Rett syndrome: revised diagnostic criteria and 
nomenclature. Ann Neurol. 2010;68(6):944-950. 
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. Int 
J Environ Res Public Health. 2018;16(1):41. 
Samaco RC, Neul JL. Complexities of Rett syndrome and MeCP2. J Neurosci. 
2011;31(22):7951-7959. 


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16 APPENDICES 
Appendix A Rett Syndrome Diagnostic Criteria 
RTT Diagnostic Criteria 2010 
Consider diagnosis when postnatal deceleration of head growth observed. 
Required for typical or classic RTT: 
1. A period of regression followed by recovery or stabilizationa 
2. All main criteria and all exclusion criteria 
3. Supportive criteria are not required, although often present in typical RTT 
Required for atypical or variant RTT: 
1. A period of regression followed by recovery or stabilizationa 
2. At least 2 out of the 4 main criteria 
3. 5 out of 11 supportive criteria 
Main Criteria: 
1. Partial or complete loss of acquired purposeful hand skills. 
2. Partial or complete loss of acquired spoken languageb 
3. Gait abnormalities: Impaired (dyspraxic) or absence of ability. 
4. Stereotypic hand movements such as hand wringing/squeezing, clapping/tapping, mouthing and 
washing/rubbing automatisms 
Exclusion Criteria for typical RTT: 
1. Brain injury secondary to trauma (peri- or postnatally), neurometabolic disease, or severe infection 
that causes neurological problemsc 
2. Grossly abnormal psychomotor development in first 6 months of lifed 
Supportive Criteria for atypical RTT:e 
1. Breathing disturbances when awake 
2. Bruxism when awake 
3. Impaired sleep pattern 
4. Abnormal muscle tone 
5. Peripheral vasomotor disturbances 
6. Scoliosis/kyphosis 
7. Growth retardation 
8. Small cold hands and feet 
9. Inappropriate laughing/screaming spells 
10. Diminished response to pain 
11. Intense eye communication - “eye pointing” 
Table footnotes provided on next page 
Source: Neul et al. 2010 


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a  
Because MECP2 mutations are now identified in some individuals prior to any clear evidence of regression, 
the diagnosis of “possible” RTT should be given to those individuals under 3 years old who have not lost 
any skills but otherwise have clinical features suggestive of RTT. These individuals should be reassessed 
every 6–12 months for evidence of regression. If regression manifests, the diagnosis should then be 
changed to definite RTT. However, if the child does not show any evidence of regression by 5 years, the 
diagnosis of RTT should be questioned. 
b 
Loss of acquired language is based on best acquired spoken language skill, not strictly on the acquisition of 
distinct words or higher language skills. Thus, an individual who had learned to babble but then loses this 
ability is considered to have a loss of acquired language. 
c 
There should be clear evidence (neurological or ophthalmological examination and MRI/CT) that the 
presumed insult directly resulted in neurological dysfunction. 
d 
Grossly abnormal to the point that normal milestones (acquiring head control, swallowing, developing 
social smile) are not met. Mild generalized hypotonia or other previously reported subtle developmental 
alterations during the first six months of life is common in RTT and do not constitute an exclusionary 
criterion. 
e 
If an individual has or ever had a clinical feature listed it is counted as a supportive criterion. Many of these 
features have an age dependency, manifesting and becoming more predominant at certain ages. Therefore, 
the diagnosis of atypical RTT may be easier for older individuals than for younger. In the case of a younger 
individual (under 5 years old) who has a period of regression and ≥2 main criteria but does not fulfil the 
requirement of 5/11 supportive criteria, the diagnosis of “probably atypical RTT” may be given. Individuals 
who fall into this category should be reassessed as they age, and the diagnosis revised accordingly. 
 

SAP_001.pdf: 
Official Title: An Open-Label Study of Trofinetide for the Treatment of Girls Two to 
Five Years of Age who Have Rett Syndrome 
NCT Numbers: NCT04988867 
Document Date: 26 Jun 2023 
 


 
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STATISTICAL ANALYSIS PLAN 
Protocol No.: 
ACP-2566-009 
Protocol Title: 
An Open-Label Study of Trofinetide for the Treatment of 
Girls Two to Five Years of Age who Have Rett 
Syndrome 
Drug: 
Trofinetide oral solution 
Sponsor:  
Acadia Pharmaceuticals Inc. 
Version No. and Date 
Version 2.0, 26 June 2023 
 
 
 


Protocol ACP-2566-009 
 
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Statistical Analysis Plan 
 
26 June 2023 
 
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SIGNATURE/APPROVAL PAGE 
AUTHORS 
 
See appended electronic signature page 
____________________________________________                                    ___________       
                                                                                      Date 
Senior Director, Biostatistics 
Acadia Pharmaceuticals Inc. 
 
 
APPROVERS 
 
 
See appended electronic signature page 
_____________________________________________                               ____________       
                                                                                  Date 
VP, Clinical Development 
Acadia Pharmaceuticals Inc.                                                                                        
 
See appended electronic signature page 
_____________________________________________                               ____________       
                                                                                                    Date 
Senior Director, Biostatistics 
Acadia Pharmaceuticals Inc. 
 
 
 
 
 


Signature Page for ACP-2566-009 Statistical Analysis Plan - Amended
Signature Page for ACP-2566-009 Statistical Analysis Plan - Amended
Approve
Approve
Approve


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TABLE OF CONTENTS 
SIGNATURE/APPROVAL PAGE ............................................................................................ 2 
TABLE OF CONTENTS ............................................................................................................ 4 
LIST OF TABLES ...................................................................................................................... 7 
LIST OF FIGURES .................................................................................................................... 8 
ABBREVIATIONS .................................................................................................................... 9 
1. 
INTRODUCTION ........................................................................................................ 11 
2. 
OBJECTIVES ............................................................................................................... 12 
2.1 
Primary Objectives ....................................................................................................... 12 
2.2 
Exploratory Efficacy Objectives .................................................................................. 12 
3. 
STUDY DESIGN ......................................................................................................... 13 
3.1 
General Study Design ................................................................................................... 13 
3.2 
Randomization .............................................................................................................. 14 
3.3 
Blinding ........................................................................................................................ 14 
3.4 
Determination of Sample Size ...................................................................................... 14 
4. 
ANALYSIS SETS ........................................................................................................ 15 
5. 
DATA HANDLING CONVENTIONS ........................................................................ 16 
5.1 
General Data Reporting Conventions ........................................................................... 16 
5.2 
Derived Variables ......................................................................................................... 16 
5.2.1 
Rett Syndrome Clinical Severity Scale (RTT-CSS) .............................................. 16 
5.2.2 
Clinical Global Impression–Improvement (CGI-I) ................................................ 17 
5.2.3 
Clinical Global Impression–Severity (CGI-S) ....................................................... 17 
5.2.4 
Caregiver Global Impression–Improvement (CaGI-I) ........................................... 17 
5.2.5 
Overall Quality of Life Rating of the Impact of Childhood Neurologic 
Disability (ICND) Scale ......................................................................................... 17 
5.3 
Data Presentation Conventions ..................................................................................... 17 
5.4 
Study Day ..................................................................................................................... 18 
5.5 
Baseline Definition ....................................................................................................... 18 
5.6 
Analysis Visit Windows ............................................................................................... 18 
5.6.1 
Unscheduled Assessments ..................................................................................... 18 
5.6.2 
Multiple Measurements within Visit Windows ..................................................... 18 
5.7 
Missing or Incomplete Date for Last Dose of Study Drug ........................................... 19 
5.8 
Missing or Incomplete Date for Prior or Concomitant Medications ............................ 19 


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5.9 
Missing or Incomplete Date for Adverse Events .......................................................... 19 
5.10 
Missing Severity Assessment for Adverse Events ....................................................... 20 
5.11 
Missing Relationship to Study Drug for Adverse Events ............................................. 20 
5.12 
Character Values of Clinical Laboratory Variables ..................................................... 20 
6. 
STUDY ENROLLMENT ............................................................................................. 21 
7. 
SUBJECT DISPOSITION ............................................................................................ 22 
8. 
PROTOCOL DEVIATIONS ........................................................................................ 23 
9. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS ................................... 24 
10. 
MEDICAL HISTORY .................................................................................................. 25 
10.1 
MECP2 MUTATION GROUPINGS ........................................................................... 25 
11. 
EXTENT OF EXPOSURE AND TREATMENT COMPLIANCE ............................. 27 
11.1 
Exposure to Study drug ................................................................................................ 27 
11.2 
Measurement of Treatment Compliance ...................................................................... 27 
12. 
PRIOR, CONCOMITANT, AND POST-TREATMENT MEDICATION .................. 29 
13. 
EFFICACY ANALYSES ............................................................................................. 30 
13.1 
Exploratory Efficacy Endpoints ................................................................................... 30 
13.2 
Adjustment for Covariates ............................................................................................ 30 
13.3 
Handling of Missing Data ............................................................................................. 30 
13.4 
Multiple Comparisons / Multiplicity ............................................................................ 30 
14. 
METHODS OF EFFICACY ANALYSES ................................................................... 31 
14.1 
Analysis of Continuous Efficacy Endpoints ................................................................. 31 
15. 
SAFETY ANALYSES ................................................................................................. 32 
15.1 
Primary Safety Endpoints ............................................................................................. 32 
15.2 
Adverse Events ............................................................................................................. 32 
15.3 
Clinical Laboratory Variables ...................................................................................... 33 
15.4 
Vital Signs .................................................................................................................... 37 
15.5 
Electrocardiogram (ECG) ............................................................................................. 38 
15.6 
Physical Examination ................................................................................................... 39 
15.7 
Other Safety Endpoints ................................................................................................. 39 
16. 
CLINICAL PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSES.. 40 
17. 
INTERIM ANALYSIS ................................................................................................. 41 


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17.1 
Examination of Subject Disposition for Treatment Period A ....................................... 41 
17.2 
Examination of Subgroups for Adverse Events ........................................................... 41 
18. 
DATA MONITORING/REVIEW COMMITTEE ....................................................... 42 
19. 
COMPUTER METHODS ............................................................................................ 43 
20. 
CHANGES TO ANALYSES SPECIFIED IN PROTOCOL ....................................... 44 
21. 
APPENDICES .............................................................................................................. 45 
21.1 
Schedule of Events and Assessments for Study ACP-2566-009 (Screening, 
Baseline, and Treatment Period A) ............................................................................... 45 
21.1 
Schedule of Events and Assessments for Study ACP-2566-009 (Screening, 
Baseline, and Treatment Period A) (Continued) .......................................................... 46 
21.2 
Schedule of Events and Assessments for Study ACP-2566-009 (Treatment Period 
B, and Safety Follow-Up) ............................................................................................. 47 
21.3 
Summary of Version Changes ...................................................................................... 48 
 
 
 


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LIST OF TABLES 
Table 1 
Dosing Schedule for Subjects Who Enrolled Under Protocol Amendment 1 
Dated 30 June 2021 ................................................................................................ 14 
Table 2 
Analysis Visit Windows......................................................................................... 18 
Table 3 
Conversion of Height of Remaining Liquid (cm) into Volume (mL) .................... 28 
Table 4 
Criteria for Potentially Clinically Important Laboratory Values – Hematology 
and Chemistry ........................................................................................................ 36 
Table 5 
Criteria for Potentially Clinically Important Laboratory Values - Urinalysis ....... 37 
Table 6 
Criteria for Potentially Clinically Important Vital Signs ....................................... 38 
Table 7  
Criteria for Potentially Clinically Important ECG Values ..................................... 39 


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 LIST OF FIGURES 
Figure 1 
Schematic of Study Design .................................................................................... 13 


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ABBREVIATIONS 
AE 
adverse event 
ATC 
Anatomical/Therapeutic/Chemical 
BID 
bis in die; twice daily 
BLQ 
below the limit of quantification 
BMI 
body mass index 
CaGI-I 
Caregiver Global Impression – Improvement 
CGI-I 
Clinical Global Impression – Improvement  
CGI-S 
Clinical Global Impression – Severity  
COVID-19 
coronavirus disease 2019 
CSR 
clinical study report 
DSMB 
Data and Safety Monitoring Board  
eCRF 
electronic case report form 
ECG 
electrocardiogram 
EDC 
electronic data capture 
EOT 
end of treatment 
ET 
early termination 
ICND 
Impact of Childhood Neurologic Disability Scale  
MECP2 
gene encoding methyl-CpG binding protein 2 (in humans) 
MedDRA 
Medical Dictionary for Regulatory Activities 
PCI 
potentially clinically important 
PD 
pharmacodynamic(s) 
PHE 
public health emergency 
PK 
pharmacokinetic(s) 
PT 
preferred term 
QT interval 
QT interval for heart rate of ECG 
QTc 
QT interval of ECG corrected for heart rate 
QTcB 
QT interval corrected for heart rate using Bazett’s formula 
QTcF 
QT interval corrected for heart rate using Fridericia’s formula 
RTT 
Rett Syndrome 


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RTT-CSS 
Rett Syndrome Clinical Severity Scale 
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 for Study ACP-2566-009. This plan should be read in 
conjunction with the study protocol and the electronic case report forms (eCRFs). 
Specifications for tables, figures, and listings are contained in separate documents. Statistical 
analyses for population pharmacokinetic (PK) will be presented in a separate report and therefore 
will not be included in this SAP. 
The SAP version 1.0 is based on protocol Amendment 1, dated 30 June 2021. The SAP version 
2.0 is based on protocol Amendment 2, dated 19 December 2022. Main changes include:  
• added summary for number of caregivers consented and participated for optional exit 
interview 
• added summary for number of subjects who took commercially marketed trofinetide 
within 30 days after study completion 
• updated definition for treatment-emergent adverse events with Day 1 onset. 
 
Note that for the caregiver optional exit interviews, following the analysis of the qualitative data, a 
summary report that describes the objectives, methods, participants, and results of the qualitative 
interviews will be prepared and reported separately from the clinical study report. 
 
 


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2. 
OBJECTIVES 
2.1 
Primary Objectives 
The primary objectives of this study are: 
• To investigate the safety and tolerability of treatment with oral trofinetide in girls two to 
five years of age who have Rett syndrome 
• To characterize the pharmacokinetics of oral trofinetide in girls two to five years of age 
who have Rett syndrome 
2.2 
Exploratory Efficacy Objectives 
The exploratory efficacy objectives of this study are: 
• To investigate the efficacy of treatment with oral trofinetide in girls two to five years of 
age who have Rett syndrome 
• To investigate the benefit of treatment with oral trofinetide on overall quality of life for 
girls two to five years of age who have Rett syndrome 


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3. 
STUDY DESIGN  
3.1 
General Study Design 
This is a multicenter, open-label study of trofinetide for the treatment of girls 2 to 5 years of age 
with Rett syndrome. Participating girls 2 to 4 years of age must weigh ≥9 kg and <20 kg. Girls 
5 years of age who weigh ≥9 kg and <12kg can also be enrolled.  
Approximately 10 to 15 subjects are expected to be enrolled, at least 1 subject who weighs 
≥9 to <11 kg, and at least 4 subjects who are less than 4 years of age at Screening, including at 
least one subject who is 2 years of age. Approximately 10 sites will participate in this study. 
The duration of the study will be 26 months and will have three main periods (Figure 1): 
• Screening period: up to 4 weeks 
• Treatment period 
o Period A: 12 weeks 
o Period B: up to approximately 21 months 
• Safety follow-up period: 30 days 
The study design is summarized in Figure 1. 
Figure 1 
Schematic of Study Design  
 
The schedule of events and assessments is provided in appendices (21.1 and 21.2). 
All subjects are dosed under protocol amendment 1 dated 30 June 2021 and protocol amendment 
2 dated 19 December 2022, and will have trofinetide titration according to the dosing schedule in 
Table 1. At any point in the study, if the subject is not able to tolerate administration of the 
assigned dose, the Investigator may instruct the caregiver to reduce the dose of study drug to a 
dose as low as 1 g BID. In addition, up to 4 doses (in total, consecutive or non-consecutive) may 
be withheld within the first 6 weeks. After the subject is able to tolerate treatment, the 
Investigator will increase the dose as tolerated and will continue treatment on the highest dose 
the subject can tolerate (up to 5 g BID for subjects who weigh ≥9 to <12 kg, or up to 6 g BID for 


Protocol ACP-2566-009 
 
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subjects who weigh 12 to <20 kg). This final assigned dose (i.e. the highest tolerated dose) must 
be given BID, and the morning and evening doses must be identical.  
Table 1 
Dosing Schedule for Subjects Who Enrolled Under Protocol Amendment 1 
Dated 30 June 2021 
Dose Commences 
(Visit) 
Weight at Baseline 
Dose 
Total Daily Dose 
Day 1 a 
All subjects 
10 mL (2 g) BID 
20 mL (4 g) 
Week 2 (Visit 3) 
All subjects 
20 mL (4 g) BID 
40 mL (8 g) 
Week 4 (Visit 4) 
≥9 to <12 kg 
25 mL (5 g) BID 
 50 mL (10 g) 
12 to <20 kg 
30 mL (6 g) BID 
 60 mL (12 g) 
a 
The first dose of study drug will be administered after all Baseline assessments are completed, or, if the 
Investigator judges that it is too late in the day, on the following day. The day the first dose is taken will be 
considered Day 1. 
 
3.2 
Randomization 
Not Applicable. This is not a randomized study. 
3.3 
Blinding  
Not Applicable. This is an open-label study. 
3.4 
Determination of Sample Size 
Approximately 10 to 15 subjects will be enrolled in this study, at least 1 subject who weighs 
≥9 to <11 kg, and at least 4 subjects who are less than 4 years of age at Screening, including at 
least 1 subject who is 2 years of age. The sample size is not based on statistical considerations 
and is believed to be adequate to characterize the PK of trofinetide in this population.  


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4. 
ANALYSIS SETS 
Safety Analysis Set 
The Safety Analysis Set consists of all enrolled subjects who received at least one dose of study 
medication. This analysis set will be used for all safety as well as any descriptive efficacy 
analyses. 
Pharmacokinetics (PK) Analysis Set 
The PK Analysis Set consists of all subjects in the Safety Analysis Set with at least 
1 quantifiable PK concentration. 


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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. 
Unless specified otherwise, means and medians 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 be displayed as “- -”. Percentages will be presented with one decimal place. 
Duration in months will be calculated as ([number of days / 365.25] *12). 
Laboratory values that are collected with “<” or “>” signs will generally be analysed as the 
numerical value without the sign in tables and figures. In listings, these data will be reported as 
collected with the sign. 
For tables, all subjects will be pooled together as a “Total” group for summary.  
5.2 
Derived Variables  
In general, assessment of total scores will be derived within the analysis datasets. In the event 
that total scores are also collected on the electronic case report form (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 Clinical Severity Scale (RTT-CSS) 
The RTT-CSS is a clinician-completed rating scale that measures the severity of core symptoms 
of RTT. The CSS consists of 13 items, 3 of which measure historical or static characteristics: age 
of onset of regression, age of onset of stereotypes, and head growth; 10 of which measure current 
function at the time of assessment: somatic growth, independent sitting, ambulation, hand use, 
scoliosis, language, nonverbal communication, respiratory dysfunction, autonomic symptoms, 
and epilepsy/seizures. All items are scored during a clinical interview and examination by the 


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Investigator or qualified designee using either a 5- or 6-point Likert scale. The total score 
ranging from 0 to 58 will be calculated as the sum of scores for all 13 items. Higher total scores 
indicate greater severity of symptoms. If there are any missing item scores, the total score will be 
considered missing.  
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 less improvement in the illness. 
5.2.3 
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. 
5.2.4 
Caregiver Global Impression–Improvement (CaGI-I) 
The CaGI-I scale is a variation of the CGI scale used to ask caregivers for a global assessment of 
the subject’s illness. The scale requires the caregiver to rate how much the subject’s illness has 
improved or worsened relative to a baseline state. A 5-point scale is used from 1=much 
improved, 2=improved, 3=unchanged, 4=worse, 5=much worse. 
Higher CaGI-I scores denote less improvement in the illness. 
5.2.5 
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 evaluated. The numeric score 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. 
5.3 
Data Presentation Conventions 
• 1 year = 365.25 days. Year is calculated as (days/365.25) and will be rounded to 
1 digit after the decimal point (tenths) for presentation purposes. 


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• 1 month = 30.4375 days. Month is calculated as (days/30.4375) and will be 
rounded to 1 digit after the decimal point (tenths) for presentation purposes. 
• Body mass index (BMI) is calculated as [weight (kg)/height (m)2] 
5.4 
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. 
5.5 
Baseline Definition 
Baseline will be defined as the last non-missing assessment, including those from repeated and 
unscheduled measurements, before the first trofinetide dose is administered.  
5.6 
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  
Study Visit 
Target Study Day 
Study Day Interval 
Baseline (Day 1) 
2  
1 
≤ 1 
Week 2 
3  
15 
2 – 21 
Week 4 
4  
29 
22 – 42  
Week 8 
5  
57 
43 – 70  
Week 12 
6  
85 
71 – 126  
Week 24 
7 
169 
127 – 266  
Week 52 
8 
365 
267 – 455  
Week 78 
9 
547 
456 – 637  
Week 104 
10 
729 
638 – 759  
5.6.1 
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. 
5.6.2 
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 are 


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made for incomplete assessments, in which case, more complete assessments will be given 
priority.  
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 
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. 
5.7 
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 three dates:  
• the non-missing drug return date of the last dispensed drug kits,  
• the last drug dispensed date plus the number of days that the dosing would continue if 
given to the subject per protocol,  
• EOT/ET date.  
For subjects with partial missing last dose date, the imputation will be compared against the last 
expected dosing date as defined above. Detailed imputation 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.  
5.8 
Missing or Incomplete Date 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 12 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.   
5.9 
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 15.2 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. 


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5.10 
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. 
5.11 
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.  
5.12 
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. 
STUDY ENROLLMENT 
The number of subjects screened, subjects screen failed, and subjects dosed will be summarized. 
Data listings of subject enrollment and subject eligibility (i.e. inclusion/exclusion criteria) will be 
provided. 


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7. 
SUBJECT DISPOSITION 
The number and percentage of subjects who completed the study or discontinued early and the 
reason for study discontinuation will be summarized using the Safety Analysis Set. 
The number of caregivers who consented and participated in the optional exit interview, and the 
number of subjects who took commercially marketed trofinetide within 30 days after study 
completion will also be summarized using the Safety Analysis Set. 
  


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8. 
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. 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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9. 
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 at screening, sex, race, race 
subgroup (white vs non-white), ethnicity, height, weight, BMI, RTT-CSS total score at 
Screening, Baseline Overall Quality of Life score, and Baseline CGI-S score.  


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10. 
MEDICAL HISTORY 
Medical history reported terms will be coded with Medical Dictionary for Regulatory Activities 
(MedDRA) version 24.0. The subject incidence will be summarized for each system organ class 
(SOC) and preferred term (PT) for the Safety Analysis Set. A subject will be counted only once 
per SOC or per PT for the summary. 
A listing of the SOC, PT, 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 symptoms first 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. 
10.1 
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 
 


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Mutations  
Code in EDC 
Category 
Severity 
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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11. 
EXTENT OF EXPOSURE AND TREATMENT COMPLIANCE 
Extent of exposure and treatment compliance will be summarized as both continuous variables 
and categorical variables for the Safety Analysis Set.  
11.1 
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 <4 weeks (14 to 27 days), 4 to <8 weeks (28 to 55 days), 8 to <12 weeks (56 to 83 days), 
12 to <24 weeks (84 to 167 days), 24 to <52 weeks (168 to 363 days), 52 to <78 weeks (364 to 
545 days), 78 to <104 weeks (546 to 727 days), and >=104 weeks (728 days or longer). 
11.2 
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 (see Table 1). 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 3).  
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. 
 
 


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Table 3 
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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12. 
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 
study drug. 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 2021. 
The number and percentage of subjects taking concomitant and post-treatment medications will 
be tabulated separately by each drug class (ATC Level 3) and medication PT for the Safety 
Analysis Set. Multiple medication usage by a subject in the same category will be counted only 
once. Listing of prior, concomitant and post-treatment medications will also be provided. 
COVID-19 Infection Related Medications 
Concomitant medications related to COVID-19 Infection will also be listed. 


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13. 
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. 
13.1 
Exploratory Efficacy Endpoints 
The exploratory efficacy endpoints are: 
• CGI-I score at Weeks 2, 4, 8, 12, 24, 52, 78, and 104 
• Change from Baseline to Weeks 2, 4, 8, 12, 24, 52, 78, and 104 in CGI-S score 
• CaGI-I score at Weeks 12, 24, 52, 78, and 104 
• Change from Baseline to Weeks 12, 24, 52, 78, and 104 in Overall Quality of Life Rating 
of the ICND Scale 
13.2 
Adjustment for Covariates 
Not applicable. 
13.3 
Handling of Missing Data 
Missing data imputation rules are described in Section 5.2. 
13.4 
Multiple Comparisons / Multiplicity 
No hypothesis testing is planned. 


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14. 
METHODS OF EFFICACY ANALYSES 
14.1 
Analysis of Continuous Efficacy Endpoints 
Descriptive statistics for all efficacy endpoints listed in Section 13.1 will be tabulated by analysis 
visit (see Section 5.6). The summaries of observed values and change from baseline results will 
be presented. 


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15. 
SAFETY ANALYSES 
All safety analyses will be performed using the Safety Analysis Set. Safety summaries will be 
presented using descriptive statistics. For each continuous measure in clinical laboratory 
variables, vital signs, and electrocardiogram, change from baseline results will be displayed by 
analysis visit. 
15.1 
Primary Safety Endpoints 
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 
15.2 
Adverse Events  
Adverse events will be coded using MedDRA dictionary version 24.0. 
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. 
The event counts, the number, and percentage of subjects reporting TEAEs will be tabulated by 
system organ class (SOC) and PT; and by SOC, PT, and maximum severity. A subject is counted 
at most once per SOC and PT even if the subject has more than one TEAE. A subject is counted 
at most once per SOC and PT at the maximum severity for the summary by maximum severity. 
The display in these tables will be sorted alphabetically by SOC and then by descending subject 
frequency for the PTs within each SOC. 
The event counts, the number and percentage of subjects with any TEAEs will also be tabulated 
by PT without SOCs. This table will be sorted by descending subject frequency. 
Treatment-emergent SAEs, TEAEs leading to study drug withdrawn, and TEAEs related to study 
drug will be summarized by SOC and PT. The tables will be sorted alphabetically by SOC and 
then by descending subject frequency for the PTs within each SOC. In addition, the incidence of 
fatal treatment-emergent AEs (i.e., events that cause death) will be summarized separately by 
SOC and PT.  
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 PT. This 
listing will also include all relevant eCRF data associated with the event: date of onset, date 


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resolved, date of last dose, severity, frequency, outcome, relationship to study drug, and action 
taken with study drug. Separate listings will be presented for treatment-emergent SAEs, TEAEs 
leading to study drug withdrawn, fatal TEAEs (if any), and TEAEs related to COVID-19 
infection. 
15.3 
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 according to the schedule presented in appendices 
(21.1 and 21.2).  
• Clinical chemistry serum tests include the following: 
o Sodium (Na), potassium (K), chloride (Cl), phosphorus (P), calcium (Ca), 
magnesium (Mg), carbon dioxide (CO2), blood urea nitrogen (BUN), creatinine 
(CR), uric acid 
 Mg will only be performed at Visit 1 (Screening) 


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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 (Screening) 
• 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 
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 Table 4 and Table 5. The number and percentage of subjects with 
post-Baseline PCI values for each of the categories in Table 4 and Table 5 will be summarized 


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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 4 
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 (SGPT) 
U/L 
No lower 
limit  
≥3 X ULN 
U/L 
No lower 
limit  
≥3 X ULN 
AST (SGOT) 
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 5 
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. 
15.4 
Vital Signs 
Vital signs and weight will be collected throughout the study; height will be measured at 
Screening, Week 12, and Week 104/EOT/ET. 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 6. 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 
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 non-missing Baseline and at least 1 post-Baseline vital sign for the given 
parameter. A listing of subjects with any PCI vital sign values will be provided.  


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Table 6 
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 
≥140 
And 
Increase of ≥20 
- 
≤70 
And 
Decrease of ≥20 
- 
Diastolic blood 
pressure (supine or 
sitting) 
mmHg 
≥90 
And 
Increase of ≥15 
- 
≤50 
And 
Decrease of ≥15 
- 
Pulse (supine or 
sitting) 
bpm 
≥131 
And 
Increase of ≥15 
- 
≤50 
And 
Decrease of ≥15 
- 
Weight  
kg 
Not Applicable 
Increase of ≥7% 
- 
Decrease of ≥7% 
- 
15.5 
Electrocardiogram (ECG) 
ECGs are collected throughout the study at every visit. Observed values of ECG variables (e.g., 
heart rate, PR interval, QRS interval, QT interval, and QTc interval) and the changes from 
Baseline at each assessment time point will be summarized.  
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 at each visit and for the overall 
post-Baseline maximum: 
• Observed: ≤450, 451 - ≤480, 481 - ≤500, and >500; >450; >480. 
• 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 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. 


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Electrocardiogram variable values will be considered PCI if they meet the criteria listed in 
Table 7. The number and percentage of subjects with post-baseline PCI values will be presented 
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 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 ECG for the given 
parameter, and the denominator is the number of subjects with at least 1 post-Baseline ECG 
value for the given parameter. A listing of all subjects with any PCI ECG values will be 
provided.  
Table 7  
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  
15.6 
Physical Examination 
Physical examinations are performed throughout the study at every visit in the clinic. When a 
study visit takes place off-site, the physical examination is not required. Physical examination 
results (normal, abnormal, and not done) will be summarized in a frequency table by body 
system and analysis visit. A listing of abnormal physical examination data will be provided. 
15.7 
Other Safety Endpoints 
There are no other safety endpoints in this study. 


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16. 
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 
analysis 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.   


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17. 
INTERIM ANALYSIS 
All available data up to and including an assigned data cut date will be cleaned and included for 
an interim analysis to serve the purpose of submission of data as part of a New Drug Application, 
(NDA), including the writing of an interim synoptic clinical study report (CSR). An interim PK 
analysis including all available PK data by the date of interim analysis cut will be presented in a 
separate report.   
All pre-specified analyses (Sections 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16) will be performed. 
17.1 
Examination of Subject Disposition for Treatment Period A 
In addition to the analysis specified in Section 7, the number and percentage of subjects who are 
ongoing in Treatment Period A, who completed Treatment Period A (i.e. completed Week 12 
visit), who discontinued the study in Period A and the reason for study discontinuation will be 
summarized using the Safety Analysis Set.  
17.2 
Examination of Subgroups for Adverse Events 
In addition to the analysis specified in Section 15.2, the following summaries will be performed 
for adverse events with onset during Treatment Period A: 
• TEAEs with onset during Treatment Period A: by SOC and PT; by SOC, PT and 
maximum severity; by PT only 
• Treatment-emergent SAEs with onset during Treatment Period A: by SOC and PT 
• TEAEs with onset during Treatment Period A that lead to study drug withdrawn during 
Treatment Period A: by SOC and PT 
• TEAEs with onset during Treatment Period A that are related to study drug: by SOC and 
PT 
• Fatal TEAEs with onset during Treatment Period A: by SOC and PT 
  


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18. 
DATA MONITORING/REVIEW COMMITTEE 
Data from this study will be reviewed by the Data and Safety Monitoring Board (DSMB) that 
reviews all ongoing clinical studies of trofinetide. Details on the data review are provided in a 
separate DSMB charter.  


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19. 
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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20. 
CHANGES TO ANALYSES SPECIFIED IN PROTOCOL 
No changes are made to the analyses specified in the protocol.  
 


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21. 
APPENDICES 
21.1 
Schedule of Events and Assessments for Study ACP-2566-009 (Screening, 
Baseline, and Treatment Period A) 
Period 
Screening 
Baselinea 
Treatment Period A 
Visit 
Week 2b 
Week 4 
Week 8 
Week 12 
Visit Number 
1 
2 
3 
4 
5 
6 
Visit window (days) 
N/A 
N/A 
±3 
±3 
±3 
+3 
Type of Visitc 
Clinic or 
Off-site 
Clinic 
Clinic or Off-site 
Clinic 
Informed consent 
X 
Inclusion/exclusion criteria 
X 
X 
Medical history and demographics 
X 
Confirm documented diagnosis of RTT 
or possible RTT 
X 
Confirm documented MECP2 
mutationd 
X 
Rett syndrome history 
X 
Rett Syndrome Clinical Severity Scale 
X 
Physical examinationc 
X 
X 
X 
X 
X 
X 
Vital signse 
X 
X 
X 
X 
X 
X 
Height 
X 
X 
Weightc 
X 
X 
X 
X 
X 
X 
Electrocardiogram (ECG)f 
Xf 
Xf 
X 
X 
X 
Xf 
Clinical laboratory tests 
X 
X 
X 
X 
X 
X 
Urinalysis 
X 
X 
X 
X 
TSH, free T3, free T4 
X 
Blood samples for pharmacokineticsg 
X 
X 
X 
X 
X 
Clinical Global Impression– 
Improvement (CGI-I) 
X 
X 
X 
X 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
X 
X 
Caregiver Global Impression– 
Improvement (CaGI-I) 
X 
Overall Quality of Life Rating of the 
Impact of Childhood Neurologic 
Disability (ICND) Scale 
X 
X 
Dispensing and review of semi- 
structured caregiver diary 
X 
X 
X 
X 
X 
X 
Prior medications 
X 
Concomitant medications 
X 
X 
X 
X 
X 
 


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21.1 
Schedule of Events and Assessments for Study ACP-2566-009 (Screening, 
Baseline, and Treatment Period A) (Continued) 
Period 
Screening 
Baselinea 
Treatment Period A 
Visit 
Week 2b 
Week 4 
Week 8 
Week 12 
Visit Number 
1 
2 
3 
4 
5 
6 
Visit window (days) 
N/A 
N/A 
±3 
±3 
±3 
+3 
Type of Visitc 
Clinic or 
Off-site 
Clinic 
Clinic or Off-site 
Clinic 
Assessment of adverse events 
X 
X 
X 
X 
X 
X 
Authorization of study drug 
dispensationh 
X ----------------------------------------------------- X 
Study drug returnh 
X --------------------------------------- X 
Study drug accountabilityh 
X 
X 
X 
X 
Abbreviations: AE=adverse event; EOT=end of treatment; MECP2=methyl-CpG–binding protein 2 gene; N/A=not 
applicable; PK=pharmacokinetic; RTT=Rett syndrome; SAE=serious adverse event; T3=triiodothyronine; 
T4=thyroxine; TSH=thyroid stimulating hormone 
a 
The first dose of study drug will be administered after all Baseline assessments are completed, or, if the Investigator 
judges that it is too late in the day, on the following day. In that case, the second day is still considered part of the 
Baseline visit. The day the first dose is taken will be considered Day 1. 
b 
Timing of post-Baseline visits will be calculated from the first day of dosing (Day 1) (i.e., the Week 2 visit will occur 2 
weeks [14±3 days] after the first day of dosing). 
c 
The Baseline and Week 12 visits must be done at the clinic. All other study visits may be done in the clinic or off-site. 
Study visits may be done off-site at the discretion of the Principal Investigator and only 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. 
d 
If documentation of the mutation is not adequate, genomic testing for a mutation in the MECP2 gene may be conducted 
as part of Screening. 
e 
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure should be measured after the subject has been sitting or supine for ≥3 minutes. 
f 
ECGs will be completed in triplicate at Visit 1 (Screening), at Visit 2 (Baseline), both predose and at 2 hours 
(±15 minutes) after dosing, and at Visit 6 (Week 12). A single ECG will be completed at all other designated visits. 
g 
A predose PK blood sample must be collected before administration of study drug at Baseline (Visit 2). After the first 
dose of study drug is administered, two PK samples will be collected: the first, 2 hours (±15 minutes) after dosing, as 
close as possible to the postdose ECG, and the second, at least 1 hour after the first. 
In addition to the two PK samples taken at Baseline, two PK samples will be taken at each subsequent visit (or upon early 
termination [ET]) in Treatment Period A, for a total of ten postdose PK samples over the duration of the study. At Visit 3, 
Visit 4, Visit 5, and Visit 6, the two PK samples will be collected at one of the following time intervals: 1-3 hours after 
dosing, 4-7 hours after dosing, 8-11 hours after dosing. Every subject should provide at least two PK samples from each 
of the specified time intervals over the course of Visits 3 through 6 (1-3 hours after dosing, 4-7  hours after dosing, and 8-
11 hours after dosing). The two PK samples taken within a time interval should be collected at least one hour apart. 
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. 
h 
Study drug will be dispensed at the site during the Baseline visit when the visit is conducted in the clinic. For the 
remainder of the study, investigational product will be shipped directly to the subject. Study drug shipment, return, and 
accountability will be performed in accordance with the drug distribution plan. 


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21.2 
Schedule of Events and Assessments for Study ACP-2566-009 (Treatment Period 
B, and Safety Follow-Up) 
Period 
Treatment Period B 
Safety 
Follow-Upg 
Visit 
Week 24 
Week 52 
(12 months) 
Week 78 
(18 months) 
Week 104 
EOT/ET 
(24 months) 
EOT/ET 
+30 days 
Visit Number 
7 
8 
9 
10 
Visit window (days) 
±7 
±7 
±7 
±7 
+4 
Type of Visita 
Clinic or Off-site 
Telephone or 
Telemedicine 
Physical examinationa 
X 
X 
X 
X 
Vital signsb 
X 
X 
X 
X 
Height 
X 
Weighta 
X 
X 
X 
X 
Electrocardiogram (ECG)a 
X 
X 
X 
X 
Clinical laboratory tests 
X 
X 
X 
X 
Urinalysis 
X 
X 
X 
X 
Blood samples for pharmacokineticsc 
Xc 
Clinical Global Impression– 
Improvement (CGI-I) 
X 
X 
X 
X 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
Caregiver Global Impression– 
Improvement (CaGI-I) 
X 
X 
X 
X 
Overall Quality of Life Rating of the 
Impact of Childhood Neurologic 
Disability (ICND) Scale 
X 
X 
X 
X 
Dispensing and review of semi- 
structured caregiver diary 
X 
X 
X 
Xd 
Concomitant medications 
X 
X 
X 
X 
X 
Assessment of adverse events 
X 
X 
X 
X 
X 
Optional caregiver 
interviewe 
X 
Authorization of study drug 
dispensationf 
X ---------------------------------------------------- X 
Study drug returnf 
X ---------------------------------------------------- X 
Study drug accountabilityf 
X 
X 
X 
X 
Abbreviations: AE=adverse event; EOT=end of treatment; ET=early termination; SAE=serious adverse event 
a 
Study visits may be done in the clinic or off-site. Study visits may be done off-site at the discretion of the Principal 
Investigator and only with the prior approval of the Sponsor or Medical Monitor. The EOT visit should be done in the 
clinic whenever possible. 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. 
b 
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure should be measured after the subject has been sitting or supine for ≥3 minutes. 
c 
Pharmacokinetic samples will not be collected at the Week 104 visit or at an ET visit during Treatment Period B. 
Pharmacokinetic samples will be collected, if possible, at the visit immediately following any SAE or following any 


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AE leading to discontinuation. 
d 
At the Week 104 EOT/ET visit, the caregiver diary will be returned and reviewed, but no diary will be dispensed. 
e 
Caregiver interviews will be conducted remotely via telephone during Treatment Period B, at or before the final study visit 
(Week 104 [EOT/ET]). Participation in the caregiver interview is an optional component of the study requiring a separate 
informed consent. 
f 
Study drug will be dispensed at the site during the Baseline visit when the visit is conducted in the clinic. For the 
remainder of the study, investigational product will be shipped directly to the subject. Study drug shipment, return, and 
accountability will be performed in accordance with the drug distribution plan. 
g 
A 30(+4)-day safety follow-up telephone or telemedicine contact is to be completed for subjects who complete the 
treatment period of the study or discontinue prematurely from the study (and do not begin to take marketed trofinetide 
within the 30 days after completing the treatment period of the study). 
21.3 
Summary of Version Changes  
Version No: 
Document History 
Description of Update 
Author(s) 
Version Date 
1.0 
Original version 
 
15 February 2022 
2.0 
Amended version 
 
26 June 2023