Clinical trial NCT04181723
Conditions: Rett Syndrome
Sponsor: ACADIA Pharmaceuticals Inc.
Start date: 2019-11-06; Completion date: 2021-10-28
Enrollment: 187 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
To investigate the efficacy of treatment with oral trofinetide versus placebo in females with Rett syndrome

Detailed description:

Arms: arm 1: Trofinetide solution of 30-60 mL based on the subject's weight at Baseline, administered twice daily by mouth or gastrostomy tube (G-tube) arm 2: Trofinetide placebo solution of 30-60 mL based on the subject's weight at Baseline, administered twice daily by mouth or gastrostomy tube (G-tube)
Interventions: intervention 1: Trofinetide intervention 2: Placebo
Intervention descriptions: intervention 1: Trofinetide solution administered based on the subject's weight at Baseline, twice daily for 12 weeks intervention 2: Trofinetide placebo solution administered based on the subject's weight at Baseline, twice daily for 12 weeks

Protocol documents:
Prot_000.pdf: 
Official Title: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study of Trofinetide for 
the Treatment of Girls and Women with Rett Syndrome 
NCT Number: NCT04181723
Document Date: 07 August 2020


Study: ACP-2566-003 
Final Version: 1.0 
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Date: 07 August 2020 
    Confidential and Proprietary Information of ACADIA Pharmaceuticals Inc. 
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CLINICAL STUDY PROTOCOL 
A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study of 
Trofinetide for the Treatment of Girls and Women with Rett Syndrome  
Protocol Number: ACP-2566-003 
Amendment 2 
Original Protocol Date: 02 April 2019 
Protocol Amendment 1 Date: 27 April 2020 
Protocol Amendment 2 Date: 07 August 2020 
Protocol Template Version: 1.0 
Confidentiality Statement 
This protocol is the confidential information of ACADIA Pharmaceuticals Inc. and is intended solely for the 
guidance of the clinical investigation. This protocol may not be disclosed to parties not associated with the clinical 
investigation or used for any purpose without the prior written consent of ACADIA Pharmaceuticals Inc. 




Signature Page for ACP-2566-003 Protocol Amendment 2
Signature Page for ACP-2566-003 Protocol Amendment 2
Approve
13-Aug-2020 11:16:09 GMT+0000
Approve
20-Aug-2020 13:26:13 GMT+0000


Study: ACP-2566-003 
 
Final Version: 1.0 
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Date: 07 August 2020 
 
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DECLARATION OF INVESTIGATOR 
I confirm that I have read the above protocol. I understand it, and I will work according to the 
moral, ethical, and scientific principles governing clinical research as set out in the principles 
of Good Clinical Practice, as required by International Council for Harmonisation of 
Technical Requirements for Pharmaceuticals for Human Use (ICH) Guideline E6 and as 
described in the United States (US) Code of Federal Regulations (CFR) 21 CFR parts 50, 54, 
56, 312, and according to applicable local requirements. 
Confidentiality Statement 
The confidential information in this document is provided to you as a Principal Investigator 
or Consultant for review by you, your staff, and the applicable institutional review 
board/ethics committee. Your acceptance of this document constitutes agreement that you 
will not disclose the information contained herein to others without written authorization 
from the Sponsor. 
Principal Investigator 
 
 
Signature 
 
Date 
 
 
 
Name (printed) 
 
 
 
 




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Other Secondary Objectives 
• To investigate the benefit of treatment 
with oral trofinetide versus placebo on 
overall quality of life for girls and 
women with Rett syndrome 
• To investigate the efficacy of 
treatment with oral trofinetide versus 
placebo in girls and women with Rett 
syndrome on: 
o Hand function 
o Ambulation and other gross 
motor skills  
o Ability to communicate choices 
and preferences  
o Ability to communicate verbally 
• To investigate the efficacy of 
treatment with oral trofinetide versus 
placebo on a global assessment of the 
severity of illness in girls and women 
with Rett syndrome 
• To investigate the benefit of treatment 
with oral trofinetide versus placebo on 
the burden on caregivers of girls and 
women with Rett syndrome 
• To investigate the benefit of treatment 
with oral trofinetide versus placebo on 
the impact of the disability on the 
child’s and family’s everyday life 
Other Secondary Endpoints 
Change from Baseline to Week 12 in: 
• Overall Quality of Life Rating of the 
Impact of Childhood Neurologic Disability 
(ICND) Scale 
• Rett Syndrome Clinician Rating of Hand 
Function (RTT-HF)  
• Rett Syndrome Clinician Rating of 
Ambulation and Gross Motor Skills 
(RTT-AMB) 
• Rett Syndrome Clinician Rating of Ability 
to Communicate Choices (RTT-COMC) 
• Rett Syndrome Clinician Rating of Verbal 
Communication (RTT-VCOM) 
• Clinical Global Impression–Severity 
(CGI-S)  
• Rett Syndrome Caregiver Burden 
Inventory (RTT-CBI) Total Score 
(items 1-24) 
• Impact of Childhood Neurologic Disability 
(ICND) Scale Total Score 
Safety Objective 
• To investigate the safety and 
tolerability of treatment with oral 
trofinetide versus placebo in girls and 
women with Rett syndrome  
Safety Endpoints 
• Treatment-emergent adverse events 
(TEAEs) 
• Serious adverse events (SAEs) 
• Withdrawals due to adverse events 
• Potentially clinically important changes in 
other safety assessments 
Pharmacokinetic Objectives 
Pharmacokinetic Endpoints 
• Whole blood concentration of trofinetide 
and possible metabolites 


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• To characterize the pharmacokinetics 
(PK) of trofinetide in girls and women 
with Rett syndrome 
• To assess the 
pharmacokinetic/pharmacodynamic 
(PK/PD) relationship using safety and 
efficacy endpoints in girls and women 
with Rett syndrome 
• Trofinetide PK parameters using the 
population PK approach 
• PK/PD using appropriate PK/PD analysis 
methods 
Number of Study 
Sites 
Approximately 28 sites will participate in this study. 
Number of 
Subjects Planned 
184 subjects are expected to be randomized (with a minimum of 
12 subjects randomized for three age ranges [5-10 years old, 
11-15 years old, and 16-20 years old]) with a total of 92 subjects per 
treatment arm. 
Test Product, 
Dose, and 
Administration 
Subjects will receive an oral dose of trofinetide or placebo, for up to 
12 weeks. Dose will be based on the subject’s weight at Baseline, as 
outlined below in Table S–1. Doses may be administered by 
gastrostomy (G) tube (doses administered via gastrojejunal [GJ] tubes 
must be administered through the G-port). 
Table S–1 
Dosing Schedule Based on Weight at Baseline 
Weight 
Dose  
Total Daily Dose 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 
Abbreviations: BID=twice daily 
If the subject cannot tolerate administration of the full assigned dose 
(for example, if the subject experiences diarrhea) any time before the 
Week 6 visit, the Investigator may instruct the caregiver to reduce 
study drug to a dose as low as half the assigned dose. In addition, up 
to four doses (in total, consecutive or non-consecutive) may be 
withheld within the first 6 weeks. The Investigator must attempt to 
increase the dose as soon as it is possible based on the clinical 
situation. The aim is to return to the originally assigned dose. 
If the originally assigned dose cannot be reached, or the subject is 
again unable to tolerate that dose, the Investigator will continue 
treatment on the highest dose the subject can tolerate which should be 
no lower than half the assigned dose. The final dose must be given 


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BID, once in the morning and once in the evening, unless the subject 
is eligible and is continuing in the open-label extension study. 
Subjects who continue into the extension study will take their final 
dose for the current study the morning of Visit 5.  
The dose cannot be changed after the Week 6 visit. 
Coronavirus 
Disease 2019 
(COVID-19) 
Update 
As a result of the COVID-19 public health emergency, this protocol 
has incorporated revisions to study methodology in accordance with 
the FDA Guidance on Conduct of Clinical Trials of Medical Products 
during the COVID-19 Public Health Emergency. Specifically, 
alternative methods of performing safety and efficacy assessments 
include provisions for off-site assessments. The implementation of 
alternative processes were to be consistent with the protocol to the 
extent possible, and clinical investigators were to document the reason 
for any contingency measures implemented.  
Study Design 
This is a 12-week, multicenter, randomized, double-blind, 
placebo-controlled, parallel group study. The study will compare one 
active treatment group receiving weight-banded doses of trofinetide 
with a placebo group. Subjects will be stratified according to age 
stratum (5-10 years old, 11-15 years old, and 16-20 years old) and 
Baseline RSBQ severity (<35 total score and ≥35 total score). 
The Sponsor, subjects, caregivers, and Investigators will be blinded 
to treatment assignment.  
The study will have 3 periods: 
• Screening period: up to 3 weeks 
• Double-blind Treatment period: 12 weeks   
• Safety follow-up period: 30 days 
 
Screening Period (Up to 3 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. 
Investigators must not withdraw a subject’s prohibited medication for 
the purpose of enrolling them into the study. Medications will be 
discontinued only if it is deemed clinically appropriate to do so and in 
consultation with the treating physician.   
Subjects will be evaluated for the diagnosis of Rett syndrome. In 
addition, there must be verified documentation of a MECP2 mutation. 
Genotyping may be done as part of the study if documentation is not 
adequate.      


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Caregivers will begin to keep a semi-structured caregiver diary during 
the screening period. 
Double-blind Treatment Period (12 Weeks) 
The Baseline visit (Visit 2) may occur after screening procedures are 
completed and have not ruled the subject out of eligibility for the 
study. At Visit 2, and upon confirmation of eligibility, subjects will be 
randomized in a 1:1 ratio to trofinetide oral solution or matching 
placebo. All efficacy assessments (except the CGI-I) will be 
completed at the Baseline visit prior to administration of the first dose 
of study drug (Table S–2). 
Dose will be based on weight as outlined in Table S–1. The dose may 
be decreased during the study as discussed in the Test Product, Dose, 
and Administration section above. 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 of dosing. A 
triplicate ECG must be performed 2-3 hours after the first dose and a 
PK sample will be taken upon completion of the ECG.  
Study drug must be discontinued at any time during the study in the 
event that a post-randomization 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. 
Dosing is twice a day, once in the morning and once in the evening. 
Investigational product will be shipped directly to the subject. 
Confirmation of any delivery to the subject will be made by a visiting 
nurse. Study drug shipment, return, and accountability will be 
performed in accordance with the drug distribution plan. In addition, 
study drug will be dispensed at the site during the Baseline visit when 
the visit is conducted in the clinic. 
Study visits may be done off-site rather than in the clinic with the 
prior approval of the Sponsor or Medical Monitor. Screening, 
Baseline, and EOT visits should be done in the clinic whenever 
possible. 
Safety Follow-up Period (30 Days) 
Subjects who discontinue prematurely, or who complete but do not 
continue into the open-label extension study will receive a follow-up 
telephone call to assess safety 30 days after the last dose of study 
drug.  
The study schematic is provided in Figure S–1. 


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The schedule of assessments is provided in Table S–2. 
Study Duration 
The duration of participation for individual study subjects will be 
approximately 19 weeks, consisting of a screening period of up to 
3 weeks, a treatment period of 12 weeks, and a safety follow-up 
period of 30 days.  
The study completion date is defined as the date the final subject, 
across all sites, completes their final protocol-defined assessment. 
Main Criteria for 
Inclusion and 
Exclusion 
To be eligible for this study, subjects must meet all of the inclusion 
criteria and none of the exclusion criteria. 
Inclusion Criteria: 
1. Informed consent prior to the conduct of any study procedures 
is required as follows: 
a. For subjects who are minors: written informed 
consent will be obtained from the legally acceptable 
representative (LAR). The subject should provide 
written or oral assent if deemed able by the 
Investigator. The process of obtaining informed 
consent will be conducted in accordance with 
institutional review board (IRB) or ethics committee 
(EC) policy and applicable local law. 
b. For subjects who are not minors: written informed 
consent will be obtained from the LAR or the subject 
if deemed able by the Investigator. If the subject is 
deemed not able to provide consent, the subject 
should provide written or oral assent if deemed able 
by the Investigator. The process of obtaining informed 
consent will be conducted in accordance with IRB or  
EC policy and applicable local law. 
c. The subject’s caregiver must also provide written 
informed consent regarding their participation in the 
study prior to participating in any study procedures. 
2. Female subjects 5 to 20 years of age, inclusive, at Screening 
3. Body weight ≥12 kg at Screening  
4. Can swallow the study medication provided as a liquid 
solution or can take it by gastrostomy tube  
5. The subject’s caregiver is English-speaking and has sufficient 
language skills to complete the caregiver assessments 
Diagnosis 
6. Has classic/typical Rett syndrome (RTT) (Appendix A) 
7. Has a documented disease-causing mutation in the MECP2 
gene  


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8. Is post-regression at Screening, defined as: 
a. No loss or degradation of ambulation (including gait, 
coordination, independence of walking/standing) 
within 6 months of Screening 
b. No loss or degradation of hand function within 
6 months of Screening 
c. No loss or degradation of speech (including babbling, 
words or previously developed communicative 
vocalizations) within 6 months of Screening 
d. No loss or degradation of non-verbal communicative 
or social skills (including eye gaze, using body to 
indicate communicative intent, social attentiveness) 
within 6 months of Screening   
9. Has a severity rating of 10-36, inclusive, on the Rett 
Syndrome Clinical Severity Scale at Screening 
10. Has a CGI-S score of ≥4 at Screening and Baseline 
Concomitant Treatment  
11. If the subject is taking or was taking an anticonvulsant or any 
other psychoactive 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 
12. If the subject is taking or was taking any other medication 
daily for a chronic illness (not including antibiotics, pain 
relievers, and laxatives): 
a. the treatment regimen of the medication 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 
13. 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 
4 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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14. If the subject is receiving or was receiving non-pharmacologic 
treatments such as an educational, behavioral, physical, 
occupational, or speech therapy: 
a. the treatment regimen has been stable for at least 
4 weeks before Baseline and there is no current plan 
to change the treatment (Note: changes to a treatment 
regimen that are due to school schedules or are 
otherwise seasonally related are not exclusionary), 
OR 
b. if the treatment was discontinued, the discontinuation 
has occurred no fewer than 2 weeks before Baseline 
Seizures 
15. Has a stable pattern of seizures, or has had no seizures, within 
8 weeks of Screening 
Childbearing Potential 
16. Subjects of childbearing potential must abstain from sexual 
activity for the duration of the study and for at least 30 days 
thereafter. If a subject is sexually active or becomes sexually 
active during the study, she must use 2 clinically acceptable 
methods of contraception (e.g., oral, intrauterine device 
[IUD], diaphragm plus spermicide, injectable, transdermal or 
implantable contraception) for the duration of the study and 
for at least 30 days thereafter. Subject must not be pregnant or 
breastfeeding. 
Place of Residence 
17. Subject and caregiver(s) must reside at a location to which 
study drug can be delivered and have been at their present 
residence for at least 3 months prior to 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 


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Medical Conditions Other Than Rett Syndrome 
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 a clinically significant abnormal laboratory value at 
Screening. Laboratory testing may be repeated during the 
Screening period with agreement of the Medical Monitor. 
9. Has serum potassium below the normal range for the subject 
(according to the central laboratory) at Screening. Serum 
potassium may be repeated during the Screening period with 
the agreement of the Medical Monitor. 
10. Has a hemoglobin A1C (HbA1c) >7.0% at Screening 
11. Has a thyroid stimulating hormone (TSH) value outside the 
normal range for the subject (according to the central 
laboratory) at Screening 
12. Has clinically significant abnormality in vital signs at 
Screening or Baseline 
13. Has any of the following: 
a. QTcF interval of >450 ms at Screening or Baseline 
(before dosing) 
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 
14. Has any other clinically significant finding on ECG at 
Screening or Baseline (before dosing) 
15. Has a positive pregnancy test at Screening 
Other Criteria  
16. Has a significant sensitivity or allergic reaction to trofinetide 
or its excipients 


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17. Has participated in another interventional clinical study within 
30 days prior to Screening  
18. 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 5 timepoints for trofinetide 
concentration measurements at the Baseline visit (both before dosing 
and approximately 2-3 hours after dosing) and at Visit 3, Visit 4, and 
Visit 5, or upon early termination (ET).  
The second PK sample taken at the Baseline visit (Visit 2), 
approximately 2-3 hours after dosing, will take place as soon as 
possible after the postdose ECG is performed. 
PK samples at Visits 3, 4, and 5 should be collected at one of the 
following time intervals: 
• 2-3 hours after dosing 
• 4-6 hours after dosing 
• 7-11 hours after dosing 
Every effort should be made to collect PK samples at discrete time 
intervals during Visits 3, 4, and 5. However, if the interval is the same 
across these visits, then the collection time should vary within that 
interval. The following scenario is only for illustrative purposes, a 
number of other scenarios are possible.  
• For example, if the time interval of 4-6 hours after dosing is 
used for Visits 3, 4, and 5, then every effort should be made to 
collect PK samples at 4, 5, and 6 hours after dosing, 
respectively. 
Pharmacokinetic samples will also be collected, if possible, at any 
ET visit or the visit immediately following any SAE or following any 
AE leading to discontinuation. 
For all scheduled PK samples (and for unscheduled samples if 
possible), the dates and times of administration of the study drug, the 
dates, times and content of the meals, and the dates and times of the 
administration of concomitant medications over the 2 days prior to 
and on the morning of the PK sample draw, as well as the date and 
time of the sample draw, will be recorded. For samples collected from 
subjects who experience any SAE or experience an AE leading to 
discontinuation, the date and time of the last dose of study drug prior 
to the SAE or AE leading to discontinuation will also be recorded.  
Optional 
Biomarker 
Analysis 
Participation in the effort to identify biomarkers is an optional 
component of the study. Subjects for whom separate informed consent 
for the identification of biomarkers of response to trofinetide is 
provided will have blood samples taken at Baseline (before dosing) 


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and at Visit 5, or upon early termination. Blood samples will be used 
to investigate differences between responders and non-responders in 
both trofinetide-treated and placebo-treated subjects in RNA 
transcripts (transcriptomics), proteins (proteomics), and metabolites 
(metabolomics). 
Sample Size 
Calculations 
The sample size calculation was performed for the co-primary 
endpoints as a family of two hypothesis tests at an overall two-sided 
significance level of 0.05. A total sample size of 174 subjects in a 1:1 
ratio to trofinetide or placebo was estimated to provide at least 90% 
power for the hypothesis testing family assuming the following 
treatment differences (SD) estimated from Phase 2 study data: -4.4 (8) 
for the mean change from Baseline to Week 12 in the RSBQ total 
score and -0.5 (0.7) for the CGI-I mean score at Week 12.  
The sample size of 174 subjects will provide at least 95% power at a 
two-sided significance level of 0.05 for each individual hypothesis 
test within the family. Trofinetide will be claimed to be superior to 
placebo if both hypothesis tests within the family are shown to be 
statistically significant at 0.05. Therefore, the overall power to detect 
a treatment difference on both of the co-primary endpoints will be at 
least 90% (0.952). 
Adjusting for an anticipated discontinuation rate of up to 5%, 
approximately 184 subjects will be randomized in a 1:1 ratio to 
trofinetide or placebo. 
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 randomized subjects who 
received at least one dose of study medication. The Safety Analysis 
Set will be analyzed according to the actual treatment received.  
Full Analysis Set (FAS) 
The FAS will consist of all randomized subjects who received at least 
one dose of study medication and who have both a Baseline value and 
at least one post-Baseline value for the RSBQ total score or who have 
at least one post-Baseline value for the CGI-I score. The FAS will be 
analyzed according to the treatment they were assigned regardless of 
the actual treatment received.  
Per-protocol (PP) Analysis Set 
The PP Analysis Set will consist of the subjects in FAS who did not 
have a major protocol violation that would affect interpretation of the 


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efficacy data. The PP Analysis Set will be defined prior to study 
unblinding. 
Pharmacokinetic (PK) Analysis Set 
The PK Analysis Set will consist of subjects in the Safety Analysis Set 
with at least one measurable trofinetide whole blood concentration. 
General Statistical Approach 
Unless stated otherwise, all statistical tests will be 2-sided using a 
5% significance level, leading to 95% (2-sided) confidence intervals. 
Trofinetide will be claimed to be superior to placebo if both 
co-primary endpoints are shown to be statistically significant in favor 
of trofinetide. 
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. 
A hierarchical approach will be used to control for the multiple 
endpoints (co-primary and secondary). Details will be provided in the 
SAP. 
Primary Analysis 
The co-primary efficacy endpoints will be analyzed using a mixed 
model for repeated measures (MMRM). An unstructured covariance 
matrix will be used and the Kenward-Roger approximation will be 
used to adjust the denominator degrees of freedom. The treatment 
comparisons will be based on the difference in least squares means at 
Week 12. 
For the change from Baseline in the RSBQ total score, the MMRM 
model will include effects for treatment group, age group (5-10 years 
old, 11-15 years old, and 16-20 years old), Baseline RSBQ severity 
(<35 total score and ≥35 total score), visit, Baseline RSBQ total score, 
and interactions for treatment group by visit and Baseline RSBQ total 
score by visit. 
For the CGI-I score, the MMRM model will include effects for 
treatment group, age group (5-10 years old, 11-15 years old, and 
16-20 years old), visit, Baseline RSBQ severity (<35 total score and 
≥35 total score), Baseline CGI-S score, and interactions for treatment 
group by visit and Baseline CGI-S score by visit. 
Sensitivity analyses will be performed to assess the impact of missing 
data, including analyses based on a missing not at random 
assumption.  


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Complete details of the efficacy analyses will be specified in the 
statistical analysis plan (SAP). 
Secondary Analyses 
The key secondary endpoint, change from Baseline to Week 12 in the 
CSBS-DP-IT Social Composite Score, will be analyzed using a 
MMRM method with effects for treatment group, age group (5-10 
years old, 11-15 years old, and 16-20 years old), visit, Baseline RSBQ 
severity (<35 total score and ≥35 total score), Baseline CSBS-DP-IT 
Social score, and interactions for treatment group by visit and 
Baseline CSBS-DP-IT Social score by visit. An unstructured 
covariance matrix will be used and the Kenward-Roger approximation 
will be used to adjust the denominator degrees of freedom. The 
treatment comparisons will be based on the difference in least squares 
means at Week 12. 
For the other secondary endpoints that are assessed at multiple 
post-Baseline visits, the change from Baseline will be analyzed using 
a MMRM analysis similar to those described above for the co-primary 
endpoints. The MMRM model will include effects for treatment 
group, age group (5-10 years old, 11-15 years old, and 16-20 years 
old), visit, Baseline RSBQ severity (<35 total score and ≥ 35 total 
score), Baseline score, and interactions for treatment group by visit 
and Baseline score by visit. 
For the other secondary endpoints that are assessed at a single 
post-Baseline visit (i.e. Week 12 only), the change from Baseline will 
be analyzed using an analysis of covariance (ANCOVA) model with 
effects for treatment group, age group (5-10 years old, 11-15 years 
old, and 16-20 years old), Baseline RSBQ severity (<35 total score 
and ≥35 total score), and Baseline score. 
Safety Analyses 
Safety results will be summarized by treatment group using 
descriptive statistics. No formal statistical testing will be performed 
for any of the safety endpoints. Adverse events will be classified into 
standard terminology using the Medical Dictionary for Regulatory 
Activities (MedDRA). Treatment-emergent adverse events (TEAEs), 
TEAEs leading to discontinuation, TEAEs related to study drug, 
TEAEs by maximum severity, fatal TEAEs, serious adverse events 
(SAEs), and SAEs related to study drug 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 intervals 
and changes in QTc intervals in accordance with International Council 
on Harmonisation (ICH) guidelines. 


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Additional safety analysis details will be specified in the SAP. 
Pharmacokinetic Analyses 
Pharmacokinetic (PK) and efficacy (i.e., PD) measures will be 
collected from all subjects at the Baseline (Week 0) visit before 
dosing, at the Baseline (Week 0) visit after dosing, and after dosing at 
Weeks 2, 6, and 12/EOT.  
Whole blood concentration and possible metabolites data for 
trofinetide will be listed and summarized using descriptive 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. 
Trofinetide whole blood concentration data will remain blinded until 
the unblinding of the clinical database at the end of the study. 
The details of the PK and PK/PD analysis will be presented in a 
separate population PK report and PK/PD report in accordance with a 
separate data analysis plan (DAP).  
Date 
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Figure S–1 
Schematic of Study Design for ACP-2566-003 
 
 
 
R=Randomization 
a 
If the subject continues into the open-label extension (OLE) from the current study, she will not complete the follow-up visit and will roll over into the OLE


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Table S–2 
Schedule of Events and Assessments for Study ACP-2566-003 
Period 
Screening Baseline Double-blind Treatment Period 
Safety 
Follow-upb 
Visit Week 
 
0 
2a 
6 
12/EOT/ET 
EOT/ET+ 
30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
N/A 
±3 
±4 
+3 
+4 
Type of Visitk 
Clinic or Off-site 
Telephone or 
Telemedicine 
Informed consent 
X 
 
 
 
Xc 
 
Inclusion/exclusion criteria 
X 
X 
 
 
 
 
Medical history and demographics 
X 
 
 
 
 
 
Confirm documented                            
Rett diagnosis and MECP2 mutation 
X 
 
 
 
 
 
Rett syndrome history 
X 
 
 
 
 
 
Rett Syndrome Clinical Severity 
Scale  
X 
 
 
 
 
 
Physical examinationk 
X 
X 
X 
X 
X 
 
Vital signsd 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
X 
 
Weight 
X 
X 
Xk 
Xk 
Xk 
 
12-lead electrocardiogram (ECG)e 
X 
Xe 
X 
X 
X 
 
Clinical laboratory tests (hematology, 
chemistry) 
X 
X 
X 
X 
X 
 
Urinalysis 
X 
X 
X 
 
X 
 
TSH, Free T3, Free T4 
X 
X 
 
 
X 
 
HbA1c 
X 
 
 
 
 
 
Serum pregnancy testf 
X 
 
X 
X 
X 
 
Blood samples for pharmacokinetics 
 
Xg 
Xh 
Xh 
Xh 
 
Blood sample for optional analysis for 
biomarkersi 
 
X 
 
 
X 
 
Rett Syndrome Behaviour 
Questionnaire (RSBQ) 
X 
X 
X 
X 
X 
 
Clinical Global Impression–
Improvement (CGI-I) 
 
 
X 
X 
X 
 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
X 
 
Communication and Symbolic 
Behavior Scales-Developmental 
Profile™ Infant-Toddler (CSBS-DP-
IT) Checklist  
 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Hand Function (RTT-HF) 
 
X 
X 
X 
X 
 
Table continued on next page 


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Table S–2 
Schedule of Events and Assessments for Study ACP-2566-003 
(Continued) 
Period 
Screening Baseline 
Double-blind Treatment Period 
Safety  
Follow-upb 
Visit Week 
 
0 
2a 
6 
12/EOT/ET 
EOT/ET+ 
30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
N/A 
±3 
±4 
+3 
+4 
Type of Visitk 
Clinic or Off-site 
Telephone or 
Telemedicine 
Rett Syndrome Clinician Rating of 
Ability to Communicate Choices 
(RTT-COMC)k 
 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Ambulation and Gross Motor Skills 
(RTT-AMB) 
 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Verbal Communication 
(RTT-VCOM) 
 
X 
X 
X 
X 
 
Rett Syndrome Caregiver Burden 
Inventory (RTT-CBI) 
 
X 
 
 
X 
 
Impact of Childhood Neurologic 
Disability Scale (ICND) 
 
X 
 
 
X 
 
Dispensing and review of semi-
structured caregiver diary 
X 
X 
X 
X 
X 
 
Concomitant medications 
X 
X 
X 
X 
X 
X 
Assessment of adverse events 
X 
X 
X 
X 
X 
X 
Randomization 
 
X 
 
 
 
 
Study drug dispensedj 
 
X 
 
 
 
 
Authorization of study drug 
dispensationj 
 
X------------------------X 
 
 
Study drug returnj 
 
 
X------------------------------X 
 
Study drug accountabilityj 
 
 
X 
X 
X 
 
Abbreviations: EOT=end of treatment; ET=early termination; HbA1c=glycosylated hemoglobin; MECP2=methyl-CpG-
binding protein 2 gene; TSH=thyroid stimulating hormone 
a 
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 [±3 days] after the first day of dosing). 
b 
Subjects who roll over into the OLE study will not have the safety follow-up telephone call. 
c  
For subjects who decide to continue into the open-label extension (OLE) study, informed consent for the OLE must be 
obtained prior to performing the Week 12/EOT procedures.  
d  
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure will be measured after the subject has been sitting for ≥3 minutes. 
e 
ECGs will be completed in triplicate at Visit 1 (Screening), at Visit 2 (Baseline) both before dosing and 2-3 hours after 
dosing, and at Visit 5 (Week 12/EOT/ET). A single ECG will be completed at Visit 3 (Week 2) and Visit 4 (Week 6). 
f 
For subjects who have reached menarche and have not had surgical sterilization.  
g 
A predose PK blood sample must be collected before administration of study drug. A postdose PK blood sample will be 
collected at the end of ECG assessment 2-3 hours after study drug administration. 


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h 
PK samples at Visits 3, 4, and 5 will be collected at one of the following time intervals: 1) 2-3 hours after dosing OR 2) 
4-6 hours after dosing OR 3) 7-11 hours after dosing. Every effort should be made to collect the PK samples at discrete 
time intervals during Visits 3, 4, and 5. However, if the interval is the same across these visits, then the collection time 
should vary within that interval. 
i 
Participation in the effort to identify biomarkers is an optional component of the study requiring a separate informed 
consent, which may be obtained at any time during the study. If consent is obtained after Baseline, only the sample at 
Visit 5 (or upon early termination) will be taken. 
j  
Investigational product will be shipped directly to the subject. Confirmation of any delivery to the subject will be made 
by a visiting nurse. Study drug shipment, return, and accountability will be performed in accordance with the drug 
distribution plan. In addition, study drug will be dispensed at the site during the Baseline visit when the visit is 
conducted in the clinic. 
k 
Study visits may be done off-site rather than in the clinic with the prior approval of the Sponsor or Medical Monitor. 
Screening, Baseline, and EOT visits 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. The 
RTT-COMC should be completed if possible, but it is not required. 


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TABLE OF CONTENTS 
SPONSOR SIGNATURE PAGE ............................................................................................... 2 
DECLARATION OF INVESTIGATOR ................................................................................... 4 
PROTOCOL SYNOPSIS ........................................................................................................... 5 
TABLE OF CONTENTS ......................................................................................................... 23 
LIST OF TABLES ................................................................................................................... 28 
LIST OF FIGURES ................................................................................................................. 28 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................................. 29 
1 
INTRODUCTION ...................................................................................................... 31 
1.1 
Background Information ............................................................................................ 31 
1.2 
Investigational Product ............................................................................................... 32 
1.3 
Previous Clinical Experience ..................................................................................... 33 
1.4 
Study Rationale .......................................................................................................... 36 
2 
STUDY OBJECTIVES AND ENDPOINTS .............................................................. 37 
2.1 
Primary Objective....................................................................................................... 37 
2.1.1 
Co-Primary Endpoints ................................................................................................ 37 
2.2 
Key Secondary Objective ........................................................................................... 38 
2.2.1 
Key Secondary Endpoint ............................................................................................ 38 
2.3 
Other Secondary Objectives ....................................................................................... 38 
2.3.1 
Other Secondary Endpoints ........................................................................................ 38 
2.4 
Safety Objective ......................................................................................................... 39 
2.4.1 
Safety Endpoints......................................................................................................... 39 
2.5 
Pharmacokinetic Objectives ....................................................................................... 39 
2.5.1 
Pharmacokinetic Endpoints ........................................................................................ 39 
3 
STUDY DESCRIPTION ............................................................................................ 39 
3.1 
Overview of Study Design ......................................................................................... 39 
3.1.1 
Screening Period (Up to 3 Weeks) ............................................................................. 40 
3.1.2 
Double-Blind Treatment Period (12 Weeks) .............................................................. 41 
3.1.3 
Safety Follow-Up Period (30 Days) ........................................................................... 42 
4 
SUBJECT ELIGIBILITY AND WITHDRAWAL CRITERIA .................................. 42 
4.1 
Inclusion Criteria ........................................................................................................ 42 
4.2 
Exclusion Criteria ....................................................................................................... 44 


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4.3 
Subject Withdrawal of Consent .................................................................................. 45 
4.4 
Subject or Study Discontinuation ............................................................................... 46 
4.4.1 
Post-Baseline QTcF Interval Stopping Criteria .......................................................... 46 
4.4.2 
Handling of Subject Discontinuation During the Treatment Period .......................... 47 
4.5 
Subject Lost to Follow-Up ......................................................................................... 47 
4.6 
Prior and Concomitant Therapy ................................................................................. 47 
4.6.1 
Permitted and Prohibited Medications ....................................................................... 48 
5 
INVESTIGATIONAL PRODUCT ............................................................................. 49 
5.1 
Investigational Product Description ........................................................................... 49 
5.1.1 
Formulation, Appearance, and Packaging .................................................................. 49 
5.1.2 
Product Storage and Stability ..................................................................................... 49 
5.1.3 
Dosing and Administration ......................................................................................... 49 
5.1.3.1 Dosing ........................................................................................................................ 49 
5.1.3.2 Administration of Study Drug .................................................................................... 51 
5.1.4 
Method of Assigning Subjects to Treatment Groups.................................................. 51 
5.1.5 
Blinding ...................................................................................................................... 51 
5.1.6 
Study Drug Compliance ............................................................................................. 52 
5.1.7 
Overdose ..................................................................................................................... 52 
5.2 
Investigational Product Accountability Procedures ................................................... 52 
6 
STUDY PROCEDURES ............................................................................................ 52 
6.1 
Screening Assessments ............................................................................................... 53 
6.1.1 
Confirm Diagnosis of Rett Syndrome and MECP2 Mutation .................................... 53 
6.1.2 
Medical History, Including Rett Syndrome History, and Demographics ................... 53 
6.1.3 
The Rett Syndrome Clinical Severity Scale (RTT-CSS) ............................................ 53 
6.2 
Efficacy Assessments ................................................................................................. 54 
6.2.1 
Rett Syndrome Behaviour Questionnaire (RSBQ) ..................................................... 54 
6.2.2 
Clinical Global Impression–Improvement (CGI-I) and Clinical Global Impression–
Severity (CGI-S)......................................................................................................... 55 
6.2.3 
Communication and Symbolic Behavior Scales Developmental Profile 
Infant-Toddler (CSBS-DP-IT) Checklist .................................................................... 55 
6.2.4 
Impact of Childhood Neurologic Disability (ICND) Scale ........................................ 56 
6.2.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF) .................................. 57 
6.2.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills (RTT-AMB)
 .................................................................................................................................... 57 
6.2.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices (RTT-COMC)
 .................................................................................................................................... 57 
6.2.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM) .............. 57 


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6.2.9 
Rett Syndrome Caregiver Burden Inventory (RTT-CBI) ........................................... 58 
6.3 
Safety Assessments..................................................................................................... 58 
6.3.1 
Physical Examination ................................................................................................. 58 
6.3.2 
Vital Signs .................................................................................................................. 58 
6.3.3 
Height, Weight, and Body Mass Index ....................................................................... 59 
6.3.4 
Electrocardiograms ..................................................................................................... 59 
6.3.4.1 Post-Baseline QTcF Interval Stopping Criteria .......................................................... 60 
6.3.5 
Laboratory Evaluations .............................................................................................. 60 
6.4 
Caregiver Diary .......................................................................................................... 61 
6.5 
Pharmacokinetic Assessments .................................................................................... 61 
6.5.1 
Specimen Preparation, Handling, Storage, and Shipment.......................................... 63 
6.6 
Identification of Biomarkers of Response to Trofinetide in Rett Syndrome .............. 63 
7 
ADVERSE EVENTS ................................................................................................. 64 
7.1 
Specification of Safety Parameters............................................................................. 64 
7.1.1 
Definition of Adverse Event ....................................................................................... 64 
7.1.2 
Definition of Serious Adverse Event .......................................................................... 65 
7.2 
Classification of an Adverse Event ............................................................................ 66 
7.2.1 
Severity of Event ........................................................................................................ 66 
7.2.2 
Relationship to Study Drug ........................................................................................ 66 
7.2.2.1 Duration ...................................................................................................................... 67 
7.2.2.2 Frequency ................................................................................................................... 67 
7.2.2.3 Action Taken with Study Drug ................................................................................... 67 
7.2.2.4 Therapy ....................................................................................................................... 67 
7.2.2.5 Outcome ..................................................................................................................... 68 
7.2.2.6 Seriousness ................................................................................................................. 68 
7.2.3 
Definition of Unexpectedness .................................................................................... 68 
7.3 
Time Period and Frequency for Event Assessment and Follow-Up ........................... 68 
7.3.1 
Adverse Event Reporting ........................................................................................... 68 
7.3.2 
Serious Adverse Event Reporting............................................................................... 69 
7.3.3 
Reporting of Pregnancy .............................................................................................. 70 
7.3.3.1 Reporting Paternal Drug Exposure............................................................................. 70 
7.3.4 
Reporting of Overdose ............................................................................................... 70 
8 
CLINICAL MONITORING ....................................................................................... 70 
9 
STATISTICAL METHODS AND DATA ANALYSIS .............................................. 71 
9.1 
Statistical and Analytical Plans .................................................................................. 71 
9.2 
Statistical Hypotheses................................................................................................. 71 


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9.3 
Sample Size Determination ........................................................................................ 71 
9.4 
Subject Populations for Analysis ................................................................................ 71 
9.5 
Statistical Analyses ..................................................................................................... 72 
9.5.1 
General Approach ....................................................................................................... 72 
9.5.2 
Primary Analyses ........................................................................................................ 72 
9.5.3 
Secondary Analyses .................................................................................................... 73 
9.5.4 
Safety Analyses .......................................................................................................... 73 
9.5.5 
Pharmacokinetic Analyses .......................................................................................... 74 
9.5.6 
Subgroup Analyses ..................................................................................................... 74 
9.6 
Interim Analyses ......................................................................................................... 74 
9.7 
Data and Safety Monitoring Board ............................................................................ 74 
9.8 
Measures to Minimize Bias ........................................................................................ 74 
9.9 
Breaking the Study Blind/Subject Code ..................................................................... 75 
10 
STUDY MANAGEMENT AND DATA COLLECTION .......................................... 75 
10.1 
Data Collection and Management Responsibilities.................................................... 75 
10.2 
Source Documents ...................................................................................................... 76 
10.3 
Case Report Forms ..................................................................................................... 76 
10.4 
Confidentiality ............................................................................................................ 76 
10.5 
Study Records Retention ............................................................................................ 76 
10.6 
Protocol Exceptions and Deviations........................................................................... 77 
10.7 
Protocol Amendments ................................................................................................ 77 
11 
STUDY MONITORING, AUDITING, AND INSPECTING .................................... 77 
11.1 
Quality Control and Quality Assurance...................................................................... 77 
11.2 
Risk Management ....................................................................................................... 78 
12 
ETHICAL CONSIDERATIONS ................................................................................ 79 
12.1 
Ethical Standard ......................................................................................................... 79 
12.2 
Institutional Review Board/Ethics Committee ........................................................... 79 
12.3 
Informed Consent Process .......................................................................................... 79 
12.3.1 Consent and Other Informational Documents Provided to Subjects .......................... 80 
12.3.2 Consent Procedures and Documentation .................................................................... 80 
13 
PUBLICATION PLAN .............................................................................................. 81 
14 
CONFLICT OF INTEREST POLICY ....................................................................... 81 
14.1 
Finance, Insurance, and Indemnity............................................................................. 81 


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15 
LITERATURE REFERENCES .................................................................................. 82 
16 
APPENDICES ............................................................................................................ 87 
Appendix A Rett Syndrome Diagnostic Criteria ................................................................... 87 
 


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LIST OF TABLES 
Table S–1 
Dosing Schedule Based on Weight at Baseline ................................................. 7 
Table S–2 
Schedule of Events and Assessments for Study ACP-2566-003 ..................... 20 
Table 1–1 
Change from Treatment Baseline (Day 14) to End of Treatment  (Day 54) 
in Core Efficacy Outcomes (mITT) (Neu-2566-RETT-002) ........................... 34 
Table 5-1 
Dosing Schedule Based on Weight at Baseline ............................................... 50 
Table 6–1 
PK Sampling Times ......................................................................................... 63 
 
LIST OF FIGURES 
Figure S–1 
Schematic of Study Design for ACP-2566-003 ............................................... 19 
Figure 1–1 
Comparison of Change from Treatment Baseline on the RSBQ at 
Follow-Up Assessments Between 200 mg/kg BID and Placebo BID 
Treatment Groups ............................................................................................ 35 
 


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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Term 
Definition 
AE 
adverse event(s) 
BID 
bis in die; twice daily 
CGI-I 
Clinical Global Impression–Improvement 
CGI-S 
Clinical Global Impression–Severity 
CSBS-DP-IT 
Communication and Symbolic Behavior Scales-Developmental 
Profile™ Infant-Toddler  
DSMB 
Data and Safety Monitoring Board 
EC 
ethics committee 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
EOT 
end of treatment 
ET 
early termination 
GCP 
Good Clinical Practice 
GJ 
gastrojejunal 
GPE 
glycine-proline-glutamate 
HbA1c 
glycosylated hemoglobin 
ICF 
informed consent form 
ICH 
International Council for Harmonisation  
ICND 
Impact of Childhood Neurologic Disability Scale 
IGF-1 
insulin-like growth factor 1 
IRB 
institutional review board 
IRT 
interactive response technology 
LAR 
legally acceptable representative 
MeCP2 
methyl-CpG binding protein 2 
MeCP2 
gene encoding methyl-CpG binding protein 2 (in animals) 
MECP2 
gene encoding methyl-CpG binding protein 2 (in humans) 
MMRM 
mixed model for repeated measures 
PD 
pharmacodynamic 
PK 
pharmacokinetic(s) 
PR interval 
PR interval of ECG 
QRS interval 
QRS interval of ECG 
QT interval 
QT interval for heart rate of ECG 
QTc 
corrected QT interval of ECG for heart rate 
QTcB 
corrected QT interval using Bazett’s correction method 
QTcF 
corrected QT interval using Fridericia’s correction method 


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Term 
Definition 
RSBQ 
Rett Syndrome Behaviour Questionnaire 
RTT 
Rett syndrome 
RTT-AMB 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
RTT-CBI 
Rett Syndrome Caregiver Burden Inventory 
RTT-COMC 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
RTT-CSS 
Rett Syndrome Clinical Severity Scale 
RTT-DSC 
RTT Domain Specific Visual Analog Scale 
RTT-HF 
Rett Syndrome Clinician Rating of Hand Function 
RTT-VCOM 
Rett Syndrome Clinician Rating of Verbal Communication 
SAE 
serious adverse event(s) 
TEAE 
treatment emergent adverse event(s) 
US 
United States 


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


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other symptoms and disrupt activities of daily living including educational, recreational and 
treatment opportunities (Epstein et al. 2016; Perry et al. 1991; Thompson and Iwata 2001; 
Iemmi et al. 2015). Autonomic manifestations, which include abnormalities in cardiac and 
respiratory function, as well as in peripheral circulation, are considered to be predominantly 
of CNS origin. RTT is also characterized by impaired growth affecting the brain and other 
organ systems.  
Loss of purposeful hand use is a defining aspect of RTT during the onset of regression and at 
least 30% of individuals remain without any type of purposeful hand use (Downs et al. 2010). 
Individuals with some purposeful hand movements vary in their level of ability to reach for 
and grasp objects. The ability to self-feed is observed in 25-43% (Cass et al. 2003; Larsson et 
al. 2005; Downs et al. 2011). Poorer levels of hand function are associated with both age and 
severity of mobility impairment (Downs et al. 2011). 
In adulthood a late motor deterioration stage occurs, characterized by worsening dystonia, 
rigidity, and in some cases deterioration in the ability to walk and parkinsonian symptoms. 
Affected individuals have a yearly death rate between 1 and 2%, with 25% of all deaths 
characterized as sudden and unexpected (Kerr et al. 1997; Samaco and Neul 2011). 
Women with RTT can survive into their fifties and occasionally longer (Samaco and Neul 
2011). 
There are no medicines approved for the treatment of Rett syndrome. Treatment focuses on 
the management of each patient’s symptoms and, even in that regard, is often unsatisfactory 
and has only a limited effect on functional improvement. Accordingly, the burden on families 
and other caregivers is very great (Palacios-Ceña et al. 2018). 
1.2 
Investigational Product 
Trofinetide is a synthetic analog of glycine-proline-glutamate (also known as glypromate or 
GPE), a peptide that occurs naturally in the brain. GPE is the n-terminal tripeptide of the 
insulin-like growth factor 1 (IGF-1) protein. Trofinetide crosses the blood-brain barrier 
following oral administration. In the brain, it is believed to normalize decreased 
bioavailability of IGF-1 and GPE, as well as having an anti-inflammatory effect on 
pathologically activated glial cells. Both conditions contribute to deficits in synaptic 
development and functional maturation of synaptic plasticity that are fundamental to the 
wide-ranging effects of RTT. Tropea et al. (2009) observed that treatment with GPE reversed 
Rett-like symptoms in MeCP2 mutant mice. Therefore, the aim of treatment with trofinetide 
is to exert an effect on brain structure and function such as dendritic length and branching 
and long-term potentiation, which would be expected to lead to improvements across a wide 
range of symptoms of RTT. 


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1.3 
Previous Clinical Experience 
Trofinetide showed linear pharmacokinetics across the dose range tested in pediatric RTT 
patients. These pharmacokinetic results are in agreement with the data obtained previously in 
healthy subjects and in adult RTT patients. From a drug metabolism perspective, there was no 
accumulation, metabolic inhibition, or induction observed during treatment. For subjects 
treated with 50 mg/kg BID, median Cmax was 17.7 μg/mL and median AUC(0-12ss) was 
139.4 µg/mL·h. For subjects treated with 100 mg/kg BID, median Cmax was 52.6 μg/mL and 
median AUC(0-12ss) was 338.6 µg/mL·h. For subjects treated with 200 mg/kg BID, median 
Cmax was 82.2 μg/mL and median AUC(0-12ss) was 505.1 µg/mL·h. The geometric mean of the 
apparent terminal elimination half-life (T1/2) varied from 5.3 hr to 6.1 hr across the three 
dosing groups. 
Trofinetide has exhibited a favorable safety and tolerability profile in two studies in subjects 
with Rett syndrome. Study Neu-2566-RETT-001 (NCT01703533) was a Phase 2 study of 
trofinetide in 56 adolescent and adult females with RTT (Glaze et al. 2017). Subjects were 
randomized 2:1 to 35 mg/kg trofinetide BID or placebo for 14 or 28 days (Cohorts 0 and 1, 
respectively) or 70 mg/kg trofinetide BID or placebo for 28 days (Cohort 2). Both dose levels 
were well-tolerated in this study, and no time- or dose-dependent adverse events (AEs) were 
apparent. The most common AE across all treatment groups was diarrhea (39% in the 
combined 35 mg/kg BID group, 11% in the 70 mg/kg BID group and 15% in the placebo 
group). The most common AE in the 70 mg/kg BID group was somnolence (17%; versus 5% 
in the placebo group and 0% in the 35 mg/kg BID group). Four serious adverse events 
(SAEs) involving three subjects were reported during the study. In each of these instances, 
the adverse events were deemed unrelated to the study medication. One subject withdrew 
from the study due to these events while two subjects had already finished the study 
medication when their SAEs occurred. Each of these SAEs was deemed to be resolved by 
study conclusion. The results of this study also provided preliminary evidence of a treatment 
effect in the 70 mg/kg BID treatment group. 
A second Phase 2 study of oral trofinetide in pediatric and adolescent females with RTT, 
Study Neu-2566-RETT-002, assessed three dose levels of trofinetide (50 mg/kg BID, 
100 mg/kg BID, and 200 mg/kg BID versus placebo) (Glaze et al. 2019). The study 
population consisted of female subjects between 5 and 15 years of age with a diagnosis of 
RTT and a proven mutation of the MECP2 gene. The primary analysis of efficacy was change 
from Treatment Baseline (Visit 3, Day 14, end of placebo run-in) to End-of-Assessment 
(Visit 7, Day 54) between placebo and the 200 mg/kg BID treatment groups.  
At the time the study was initiated, there was no consensus upon the most appropriate and 
sensitive endpoint(s) for detecting beneficial drug effect in RTT, so the study examined 


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


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


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200 mg/kg BID group compared with 4% in the placebo group). The diarrhea associated with 
trofinetide may have been due to an improvement in gastrointestinal motility added to 
ongoing treatment with laxatives. Only three subjects experienced SAEs: one subject in the 
placebo BID group had an SAE of pneumonia, one subject in the 100 mg/kg BID group had 
an SAE of worsening of tonic cluster seizures, and one subject in the 200 mg/kg BID group 
had SAEs of strep infection and pneumonia. In each of these instances, the SAEs were 
deemed not related to study medication and resolved by the end of the study. 
No changes in QTc interval attributable to treatment were seen in adolescent and adult 
subjects with RTT in study Neu-2566-RETT-001, nor in pediatric subjects in Neu-2566-
RETT-002.   
Phase 2 studies of the oral formulation of trofinetide in adolescent and adult subjects with 
Fragile X syndrome and of the intravenous formulation in adult subjects with moderate to 
severe traumatic brain injury (TBI) have also been completed and are described in the 
Investigator’s brochure. 
Always refer to the latest version of the trofinetide Investigator’s Brochure for the overall 
benefit/risk assessment and the most accurate and current information regarding drug 
metabolism, pharmacokinetics, efficacy, and safety. 
1.4 
Study Rationale 
As described above, Rett syndrome is a devastating disorder for which there is as yet no 
treatment beyond symptomatic care resulting in a great unmet medical need. In the two 
studies of trofinetide in girls and women with RTT, trofinetide has been well tolerated. The 
highest dose assessed, 200 mg/kg BID, was observed to provide benefit compared to placebo 
even in a relatively small study, as described above. The rationale for the present study is to 
assess whether treatment with trofinetide does show a statistically significant benefit 
compared to placebo in a well-powered study.   
Accordingly, the present study is designed to assess whether the effects of administration of 
trofinetide seen in the two Phase 2 studies, are confirmed in a larger population of children, 
adolescents and young adults. 
This study will investigate effects in females only, and only in subjects who have typical RTT 
and a confirmed mutation of the MECP2 gene. The enrollees will be in a stable phase of their 
RTT; that is, they will not be undergoing active neurological regression. The exclusion of 
subjects with atypical RTT, subjects without a documented disease-causing MECP2 
mutation, and male subjects is based on considerations of study design that will allow for a 
more homogeneous study population and, it is hoped, reduce variability that will allow 


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observation of a difference between treatment with trofinetide and treatment with placebo in 
this relatively small sample of the population.   
The upper limit of the allowed age range has been increased to 20 years of age as compared 
to 15 years of age in the most recent Phase 2 study. The age range is not over 20 years in 
large part because in the United States school districts generally provide services until 20 or 
21 years of age and the availability of services after that age is not consistent across the 
states.  
The study will compare the efficacy and safety of a single trofinetide treatment group to 
treatment with placebo. Based on the results of the previous study treatment with trofinetide, 
200 mg/kg BID was targeted. However, it was observed that subjects with lower body 
weights tended to have lower than expected exposure than did subjects who weighed more. 
Identification of body weight as a covariate has often been observed with drugs showing 
similar distributional properties, especially when considering adolescents or other groups 
with different demographic characteristics (Jusko et al. 1982; Kersting et al. 2012; Piana et al. 
2014). A best practice approach to addressing this covariate is the development of a dosing 
model that accounts for the effect of weight on exposure. A model of simulated dose levels 
was developed based on the Neu-2566-RETT-002 data to characterize the dose levels 
required to reach a minimal target exposure level of orally administered trofinetide at 
different weights in a pediatric population. The dose simulation modeling showed that a 
four-level model of weight dosing bands with fixed doses of 6 g BID, 8 g BID, 10 g BID or 
12 g BID (Table 5-1) would result in an optimal percentage of subjects with exposures within 
the target range at weights 12 kg-100 kg. 
2 STUDY OBJECTIVES AND ENDPOINTS 
2.1 
Primary Objective 
The primary objective of this study is to investigate the efficacy of treatment with oral 
trofinetide versus placebo in girls and women with Rett syndrome. 
2.1.1 
Co-Primary Endpoints 
There are 2 primary endpoints. The co-primary endpoints are:  
• Rett Syndrome Behaviour Questionnaire (RSBQ) total score – change from Baseline 
to Week 12  
• Clinical Global Impression–Improvement (CGI-I) score at Week 12 


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2.2 
Key Secondary Objective 
The key secondary objective is to investigate the efficacy of treatment with oral trofinetide 
versus placebo on ability to communicate in girls and women with Rett syndrome. 
2.2.1 
Key Secondary Endpoint 
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). 
2.3 
Other Secondary Objectives 
Additional secondary objectives for this study are: 
• To investigate the benefit of treatment with oral trofinetide versus placebo on overall 
quality of life for girls and women with Rett syndrome 
• To investigate the efficacy of treatment with oral trofinetide versus placebo in girls 
and women with Rett syndrome on: 
o Hand function  
o Ambulation and other gross motor skills 
o Ability to communicate choices and preferences  
o Ability to communicate verbally 
• To investigate the efficacy of treatment with oral trofinetide versus placebo on a 
global assessment of the severity of illness in girls and women with Rett syndrome 
• To investigate the benefit of treatment with oral trofinetide versus placebo on the 
burden on caregivers of girls and women with Rett syndrome 
• To investigate the benefit of treatment with oral trofinetide versus placebo on the 
impact of the disability on the child’s and family’s everyday life 
2.3.1 
Other Secondary Endpoints 
The secondary endpoints for this study are change from Baseline to Week 12 in: 
• Overall Quality of Life Rating of the Impact of Childhood Neurologic Disability 
(ICND) Scale 
• Rett Syndrome Clinician Rating of Hand Function (RTT-HF) 
• Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills (RTT-AMB) 
• Rett Syndrome Clinician Rating of Ability to Communicate Choices (RTT-COMC) 


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• Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM)  
• Clinical Global Impression–Severity (CGI-S)  
• Rett Syndrome Caregiver Burden Inventory (RTT-CBI) Total Score (items 1-24) 
• Impact of Childhood Neurologic Disability (ICND) Scale Total Score 
2.4 
Safety Objective 
The safety objective of this study is to investigate the safety and tolerability of treatment with 
oral trofinetide versus placebo in girls and women with Rett syndrome.  
2.4.1 
Safety Endpoints 
The safety endpoints of this study are: 
• Treatment-emergent adverse events (TEAEs) 
• Serious adverse events (SAEs) 
• Withdrawals due to AEs 
• Potentially clinically important changes in other safety assessments 
2.5 
Pharmacokinetic Objectives 
The pharmacokinetic objectives of this study are: 
• To characterize the pharmacokinetics (PK) of trofinetide in girls and women with Rett 
syndrome 
• To assess the pharmacokinetic/pharmacodynamic (PK/PD) relationship using safety 
and efficacy endpoints in girls and women with Rett syndrome 
2.5.1 
Pharmacokinetic Endpoints 
The pharmacokinetic endpoints for this study are: 
• Whole blood concentration of trofinetide and possible metabolites 
• Trofinetide PK parameters using the population PK approach 
• PK/PD using appropriate PK/PD analysis methods  
3 STUDY DESCRIPTION 
3.1 
Overview of Study Design 
This study will be conducted as a multicenter, randomized, double-blind, placebo-controlled, 
parallel-group study in girls and women with Rett syndrome. The study will compare one 
active treatment group receiving weight-banded doses of trofinetide (Table 5-1) with a 


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placebo group. Subjects will be stratified according to age stratum (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. 
The Sponsor, subjects, caregivers, and Investigators will be blinded to treatment assignment. 
The duration of participation for individual study subjects will be approximately 19 weeks. 
Approximately 28 sites will participate in this study. 
The study will have 3 periods (Figure S–1): 
• Screening period: up to 3 weeks 
• Double-blind Treatment period: 12 weeks   
• Safety follow-up period: 30 days  
The schedule of events and assessments is provided in Table S–2. As shown in the schedule 
of events, study visits may be done off-site rather than in the clinic with the prior approval of 
the Sponsor or Medical Monitor. Screening, Baseline, and EOT visits should be done in the 
clinic whenever possible. Approval for off-site visits at Screening, Baseline, and EOT will be 
given only under special circumstances.  
The study completion date is defined as the date the final subject, across all sites, completes 
their final protocol-defined assessment. An individual subject is considered to have 
completed the study on the date of her final protocol-defined assessment. Please note that the 
‘final protocol-defined assessment’ includes the follow-up visit or contact, whichever is later. 
Procedures for when a subject is lost to follow-up are provided in Section 4.5. 
3.1.1 
Screening Period (Up to 3 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. 
Investigators must not withdraw a subject’s prohibited medication for the purpose of 
enrolling them into the study. Medications must be discontinued only if it is deemed 
clinically appropriate to do so and in consultation with the treating physician. 
Subjects will be evaluated for the diagnosis of Rett syndrome. In addition, there must be 
verified documentation of a MECP2 mutation. Genotyping may be done as part of the study 
if documentation is not adequate.      
Caregivers will begin to keep a semi-structured caregiver diary during the screening period. 


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3.1.2 
Double-Blind Treatment Period (12 Weeks) 
The Baseline visit (Visit 2) may occur after screening procedures are completed and have not 
ruled the subject out of eligibility for the study. At Visit 2, and upon confirmation of 
eligibility, subjects will be randomized in a 1:1 ratio to trofinetide oral solution or matching 
placebo. All efficacy assessments (except the CGI-I) will be completed at the Baseline visit 
prior to administration of the first dose of study drug (Table S–2). 
Dose will be based on the subject’s weight at Baseline as outlined in Table 5-1. The dose may 
be decreased during the study as discussed in Section 5.1.3. Doses may be administered by 
gastrostomy (G) tube (doses administered via gastrojejunal [GJ] tubes must be administered 
through the G-port).  
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 of dosing. A triplicate ECG must be 
performed 2-3 hours after first dose and a PK sample will be taken upon completion of the 
ECG. 
Study drug must be discontinued at any time during the study in the event that a 
post-randomization 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.  
Dosing is twice a day, once in the morning and once in the evening.  
Investigational product will be shipped directly to the subject. Confirmation of any delivery 
to the subject will be made by a visiting nurse. Study drug shipment, return, and 
accountability will be performed in accordance with the drug distribution plan. In addition, 
study drug will be dispensed at the site during the Baseline visit when the visit is conducted 
in the clinic. 
Study visits may be done off-site rather than in the clinic with the prior approval of the 
Sponsor or Medical Monitor. Screening, Baseline, and EOT visits should be done in the clinic 
whenever possible. 
For the 2 days before scheduled visits and the morning of the scheduled visit, the dates and 
times of study drug dosing, concomitant medication dosing, and meals will be recorded in the 
caregiver diary. Daily study drug dosing, including any dose modifications, missed or partial 
doses, will also be recorded in the caregiver diary. 


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3.1.3 
Safety Follow-Up Period (30 Days) 
A 30-day safety follow-up telephone or telemedicine contact is to be completed for subjects 
who complete the treatment period of the study and decide not to continue into the open-label 
study or are not eligible for the open-label study, as well as those who discontinue 
prematurely from the study. The telephone contact includes assessment of concomitant 
medications and treatments and assessment of AEs. 
4 SUBJECT ELIGIBILITY AND WITHDRAWAL CRITERIA 
To be eligible for this study, subjects must meet all of the inclusion criteria and none of the 
exclusion criteria. 
4.1 
Inclusion Criteria 
A subject must meet all of the following inclusion criteria to be eligible for participation in 
the study: 
1. Informed consent prior to the conduct of any study procedures is required as follows: 
a. For subjects who are minors: written informed consent will be obtained from 
the legally acceptable representative (LAR). The subject should provide 
written or oral assent if deemed able by the Investigator. The process of 
obtaining informed consent will be conducted in accordance with institutional 
review board (IRB) or ethics committee (EC) policy and applicable local law. 
b. For subjects who are not minors: written informed consent will be obtained 
from the LAR or the subject if deemed able by the Investigator. If the subject 
is deemed not able to provide consent, the subject should provide written or 
oral assent if deemed able by the Investigator. The process of obtaining 
informed consent will be conducted in accordance with IRB or EC policy and 
applicable local law. 
c. The subject’s caregiver must also provide written informed consent regarding 
their participation in the study prior to participating in any study procedures. 
2. Female subjects 5 to 20 years of age, inclusive, at Screening 
3. Body weight ≥12 kg at Screening  
4. Can swallow the study medication provided as a liquid solution or can take it by 
gastrostomy tube 
5. The subject’s caregiver is English-speaking and has sufficient language skills to 
complete the caregiver assessments 
Diagnosis 


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6. Has classic/typical Rett syndrome (RTT) (Appendix A) 
7. Has a documented disease-causing mutation in the MECP2 gene 
8. Is post-regression at Screening, defined as: 
a. No loss or degradation of ambulation (including gait, coordination, 
independence of walking/standing) within 6 months of Screening 
b. No loss or degradation of hand function within 6 months of Screening 
c. No loss or degradation of speech (including babbling, words or previously 
developed communicative vocalizations) within 6 months of Screening 
d. No loss or degradation of nonverbal communicative or social skills 
(including eye gaze, using body to indicate communicative intent, social 
attentiveness) within 6 months of Screening   
9. Has a severity rating of 10-36, inclusive, on the Rett Syndrome Clinical Severity 
Scale at Screening 
10. Has a CGI-S score of ≥4 at Screening and Baseline 
Concomitant Treatment 
11. If the subject is taking or was taking an anticonvulsant or any other psychoactive 
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 
12. If the subject is taking or was taking any other medication daily for chronic illness 
(not including antibiotics, pain relievers, and laxatives): 
a. the treatment regimen of the medication 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 
13. 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 4 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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14. If the subject is receiving or was receiving non-pharmacologic treatments such as an 
educational, behavioral, physical, occupational, or speech therapy: 
a. the treatment regimen has been stable for at least 4 weeks before Baseline 
and there is no current plan to change the treatment (Note: changes to a 
treatment regimen that are due to school schedules or are otherwise 
seasonally related are not exclusionary), OR 
b. if the treatment was discontinued, the discontinuation has occurred no fewer 
than 2 weeks before Baseline 
Seizures 
15. Has a stable pattern of seizures, or has had no seizures, within 8 weeks of Screening 
Childbearing Potential 
16. Subjects of childbearing potential must abstain from sexual activity for the duration of 
the study and for at least 30 days thereafter. If a subject is sexually active or becomes 
sexually active during the study, she must use 2 clinically acceptable methods of 
contraception (e.g., oral, intrauterine device [IUD], diaphragm plus spermicide, 
injectable, transdermal or implantable contraception) for the duration of the study and 
for at least 30 days thereafter. Subject must not be pregnant or breastfeeding. 
Place of Residence 
17. Subject and caregiver(s) must reside at a location to which study drug can be 
delivered and have been at their present residence for at least 3 months prior to 
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 Rett Syndrome 
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 


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7. Has a history of, or current, malignancy 
Laboratory Studies, Vital Signs, and Electrocardiogram 
8. Has a clinically significant abnormal laboratory value at Screening. Laboratory 
testing may be repeated during the Screening period with agreement of the Medical 
Monitor. 
9. Has serum potassium below the normal range for the subject (according to the central 
laboratory) at Screening. Serum potassium may be repeated during the Screening 
period with the agreement of the Medical Monitor. 
10. Has a hemoglobin A1C (HbA1c) >7% at Screening 
11. Has a thyroid stimulating hormone (TSH) value outside the normal range for the 
subject (according to the central laboratory) at Screening 
12. Has clinically significant abnormality in vital signs at Screening or Baseline 
13. Has any of the following: 
a. QTcF interval of >450 ms at Screening or Baseline (before dosing) 
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 
14. Has any other clinically significant finding on ECG at Screening or Baseline 
(before dosing) 
15. Has a positive pregnancy test at Screening 
Other Criteria  
16. Has a significant sensitivity or allergic reaction to trofinetide or its excipients 
17. Has participated in another interventional clinical study within 30 days prior to 
Screening  
18. Is judged by the Investigator or the Medical Monitor to be inappropriate for the study 
for any reason 
4.3 
Subject Withdrawal of Consent 
In accordance with the Declaration of Helsinki and other applicable regulations, a subject and 
legally acceptable representatives consenting on behalf of subjects have the right to withdraw 
from the study at any time, and for any reason, without prejudice to his or her future medical 
care. 


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Should a subject (or LAR) request or decide to withdraw consent, every reasonable effort will 
be made to complete and report observations as thoroughly as possible up to the date of 
withdrawal, including the evaluations specified at the ET or safety follow-up (whichever is 
applicable), as outlined in Table S–2. 
4.4 
Subject or Study Discontinuation 
Subjects may be discontinued from the study for a number of reasons, including, but not 
limited to, those listed below: 
• Adverse event 
• Death 
• Increase in post-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 
• Pregnancy 
• Protocol deviation 
• Study terminated by sponsor 
• Use of prohibited medication 
• Other 
The Sponsor reserves the right to discontinue the study at any time for any reason. Such 
reasons may be any of, but not limited to, the following: 
• Occurrence of AEs unknown to date in respect of their nature, severity, and duration 
or the unexpected incidence of known AEs 
• Medical, ethical or business reasons affecting the continued performance of the study 
Regulatory authorities also have the right to terminate the conduct of the study in their region 
for any reason. 
4.4.1 
Post-Baseline QTcF Interval Stopping Criteria 
Study drug must be discontinued in the event that a post-randomization QTcF duration of 
≥500 ms or an increase of ≥60 ms compared to the average QTcF interval at Baseline (before 


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dosing) is observed. For visits at which more than one ECG is completed, the average QTcF 
interval of all legible ECGs will be used to determine the QTcF interval for that visit. 
4.4.2 
Handling of Subject Discontinuation During the Treatment Period 
Unless the subject has withdrawn consent (or the LAR has withdrawn consent on behalf of 
the subject) to be contacted for this study, every reasonable effort should be made to 
complete Visit 5/early termination (ET) and the safety follow-up (as outlined in Table S–2) if 
a subject discontinues prematurely for any reason. All information will be reported on the 
applicable pages of the electronic case report form (eCRF). 
If a subject is discontinued from the study because of an AE, every reasonable attempt should 
be made to follow the subject until the AE resolves or until the Investigator deems the AE to 
be chronic or stable. For subjects who continue to be followed for safety, SAEs should 
continue to be reported as described in Section 7.3.2. All SAEs will continue to be followed 
until such events have resolved or the Investigator deems them to be chronic or stable. 
Pharmacokinetic samples will also be collected, if possible, at any ET visit or the visit 
immediately following any SAE or following any AE leading to discontinuation, even if it is 
an unscheduled visit. 
4.5 
Subject Lost to Follow-Up 
A subject will be considered lost to follow-up if they fail to attend a scheduled visit 
(excluding the safety follow-up telephone call) and the study site is unable to contact the 
subject or caregiver.  
Every reasonable effort should be made to contact the caregiver and will include a minimum 
of 3 documented telephone calls 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. 
In order to ensure that appropriate concomitant therapy is administered, it is essential that 
caregivers be instructed not to administer any medication to the subject without prior 
consultation with the Investigator (unless the subject is receiving treatment for a medical 
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The Investigator may prescribe appropriate medication to treat AEs. The Sponsor and 
Investigator or designee will confer to determine whether it is appropriate to continue such a 
subject in the trial if a prohibited medication is prescribed. 
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. 
4.6.1 
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 5. Medications that can prolong 
QT interval are not prohibited but must be used with caution. Any use of medications that 
could interfere with study conduct must be discussed with the Medical Monitor. 
Use of medications that could interfere with study conduct or any questions regarding 
concomitant medications must be reviewed and/or discussed with the Medical Monitor. 
Psychoactive 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 current treatment with a prohibited medication will be withdrawn from 
the study. 
Subjects who have previously taken a prohibited medication during the study will be 
withdrawn from the study unless: 
• the prohibited medication has been discontinued AND 
• withdrawal from the study presents an unacceptable medical risk to the subject 
The justification to allow the subject to continue in the trial will be made by the 
Sponsor/Medical Monitor, with medical input from the Investigator, and will be documented. 
If a subject is allowed to remain in the trial, this will be reported as a major protocol 
deviation and not a waiver. 


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5 INVESTIGATIONAL PRODUCT 
5.1 
Investigational Product Description 
The investigational product will be trofinetide oral solution or matching placebo for 
trofinetide oral solution. Dose will be based on weight as outlined in Table 5-1. Trofinetide 
will be provided in a ready-to-use aqueous solution for oral administration. Doses will be 
administered orally or by G-tube (doses administered by GJ tubes must be administered 
through the G-port). 
5.1.1 
Formulation, Appearance, and Packaging 
The Sponsor will supply trofinetide oral solution and matching placebo for 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. 
The placebo aqueous solution does not contain trofinetide active pharmaceutical ingredient 
(API), but contains purified water, acetic acid, caramel color, citric acid, lemon flavor, 
maltitol, methylparaben sodium, natural quinine flavor, propylparaben sodium, FD&C Red 
#40, strawberry flavor, sucralose, xanthan gum, and D&C Yellow #10.  
Trofinetide and matched placebo are manufactured under current Good Manufacturing 
Practices. 
During the treatment period, study drug will be distributed in a quantity sufficient to ensure 
the subject has an adequate supply of study drug between study visits. 
5.1.2 
Product Storage and Stability 
Investigational product will be shipped refrigerated at a temperature between 2°C and 8°C 
(36°F and 46°F) and will be stored at this temperature. Do not freeze. 
5.1.3 
Dosing and Administration 
5.1.3.1 
Dosing 
Dosing of study drug is based on weight. In the Phase 2 study, Study RETT-002, the dose for 
each subject was assigned based on weight using a single value in mg/kg for each subject in a 
particular dose arm. As a result, each subject received a unique dose of study drug to be 
administered twice a day. Instead of assigning a unique dose of study drug for the dose based 
on each individual’s weight, in the present study the subject is assigned one of four standard 


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doses, also based on the weight of the individual. The assigned dose is based on the range of 
weight in which the subject falls at Baseline (see Table 5-1).   
This approach is intended to balance: 1) simplification of assigning the dose and of 
packaging and dispensation of the study drug with 2) giving each subject a dose that is 
expected to be effective for subjects at all weights (see Section 1.4).   
The dose assigned to each subject is based on the subject’s weight at Baseline. The dose will 
not be increased or decreased if the subject’s weight at a postbaseline visit puts them in a new 
weight category. The dose may be decreased during the study for poor tolerability as 
discussed in Section 5.1.3.2. 
Study drug is administered twice a day, once in the morning and once in the afternoon or 
evening. There should be at least 8 hours between doses.  
Table 5-1 
Dosing Schedule Based on Weight at Baseline 
Weight 
Dose  
Total Daily Dose 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 
Abbreviations: BID=twice daily 
 
The first dose of study drug will be administered after all Baseline assessments are 
completed. If the Investigator judges that it is too late in the day to administer study drug, it 
may be administered on the following day. The day the first dose is taken will be considered 
Day 1 of dosing. An ECG must be performed 2-3 hours after the first dose and a PK sample 
will be taken upon completion of the ECG. Off-site dosing at Baseline may only be done if it 
is possible to record an ECG 2-3 hours after the first dose and draw blood for a PK sample 
upon completion of the ECG. The investigator must review the ECG readings with special 
attention to the QTcF interval before the subject can receive another dose of study drug. 
Off-site dosing at Baseline must first be approved by the Sponsor or Medical Monitor. 
Investigational product will be shipped directly to the subject. Confirmation of any delivery 
to the subject will be made by a visiting nurse. Study drug shipment, return, and 
accountability will be performed in accordance with the drug distribution plan. In addition, 
study drug will be dispensed at the site during the Baseline visit when the visit is conducted 
in the clinic. 


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5.1.3.2 
Administration of Study Drug 
Study drug is supplied in 500 mL (16 oz) bottles with a child-resistant closure. A press-in 
bottle adapter and syringe for accurately measuring the dose are supplied separately.  
The study drug must not be mixed with any food or liquid, including water.  
Doses may be taken orally or via gastrostomy tube.  
• When study drug is given orally, it may be administered directly from a syringe or 
transferred into another container to facilitate administration. 
• For gastrojejunal tubes, medication should be given via the gastric port. The tube 
must be flushed with water after study drug administration to clear the tube of study 
drug. No more than 250 mL of water should be used. Doses should be taken over a 
10-minute period. 
If the subject cannot tolerate administration of the full assigned dose (for example, if the 
subject experiences diarrhea) any time before the Week 6 visit, the Investigator may instruct 
the caregiver to reduce study drug to a dose as low as half the assigned dose. In addition, up 
to four doses (in total, consecutive or non-consecutive) may be withheld within the first 
6 weeks. The Investigator must attempt to increase the dose as soon as it is possible based on 
the clinical situation. The aim is to return to the originally assigned dose. 
If the originally assigned dose cannot be reached, or the subject is again unable to tolerate 
that dose, the Investigator will continue treatment on the highest dose the subject can tolerate. 
The final dose, i.e., the highest tolerated dose, must be given BID, once in the morning and 
once in the evening, unless the subject is eligible and is continuing in the open-label 
extension study. Subjects who continue into the extension study will take their final dose for 
the current study the morning of Visit 5. 
The dose cannot be changed after the Week 6 visit.  
5.1.4 
Method of Assigning Subjects to Treatment Groups 
At Visit 2, eligible subjects who meet inclusion and do not meet exclusion criteria will be 
randomized in a 1:1 ratio to receive either trofinetide or placebo. 
5.1.5 
Blinding 
Treatment assignments will be blinded to all study subjects, caregivers, Investigators, raters, 
site personnel, and Sponsor personnel. In the event of a potential suspected unexpected 
serious adverse reaction (SUSAR), in accordance with current health authority guidance, 
treatment assignments for the affected subject may be unblinded to a controlled group of the 
Sponsor’s Safety and/or Regulatory personnel for reporting purposes.  


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Details regarding medical emergency unblinding procedures are provided in Section 9.9. 
5.1.6 
Study Drug Compliance 
If a subject misses one dose of study drug, she must not take an extra dose the next day. 
5.1.7 
Overdose 
An overdose is a deliberate or inadvertent administration of a treatment at a dose higher than 
the maximum recommended dose per protocol. It must be reported, irrespective of outcome, 
even if toxic effects were not observed (Section 7.3.4). All events of overdose are to be 
captured as protocol deviations. 
5.2 
Investigational Product Accountability Procedures 
The Investigator or designee will keep current and accurate records of the study drug product 
dispensed, used, and returned for each subject to assure the health authority and the Sponsor 
that the study drug is being handled appropriately. Caregivers must be instructed to return all 
packaging to the Investigator or designee at regularly scheduled clinic or home nursing visits 
as appropriate.  
At appropriate intervals during the study, study drug reconciliation will be performed by the 
Sponsor (or designee) who may return appropriate unused study drug and used and unused 
packaging to the Sponsor’s designee for destruction. 
At the conclusion of the study, final study drug reconciliation will be conducted at the site. 
Final study drug accountability documentation will be maintained at both the site and by the 
Sponsor or designee. Any remaining unused study drug and all used and unused packaging 
will be sent back to the Sponsor’s designee for destruction or destroyed at the site, as allowed 
by country-specific regulations. Documentation of study drug destruction will be recorded 
and maintained by both the Sponsor and the Sponsor’s designee. 
The Investigator or designee is responsible for taking an inventory of each shipment of study 
drug received and comparing it with the accompanying material shipping form. The 
Investigator or designee will verify the accuracy of the information on the form, sign and date 
it, and provide a copy of it to the Sponsor or designee. Any study drug supplied is for use in 
this study only and must not be used for any other purpose. 
6 STUDY PROCEDURES 
Study-specific procedures are detailed below. All assessments will be completed according to 
the schedule described in Table S–2. Every effort should be made to complete the required 
procedures and evaluations at the designated visits and times. 


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6.1 
Screening Assessments 
6.1.1 
Confirm Diagnosis of Rett Syndrome and MECP2 Mutation 
The site will confirm that the subject meets criteria for typical/classic Rett syndrome 
(Appendix A) and that there is documentation of disease-causing mutation of the MECP2 
gene. 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 Rett syndrome, 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 Rett syndrome, 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 Rett Syndrome History, and Demographics  
A complete medical history, including history of symptoms associated with Rett syndrome, 
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 
Rett syndrome 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 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 


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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 4- or 5-point Likert scale. Individual subscale scores and a 
total score are calculated. 
The RTT-CSS will be administered at Screening only. 
6.2 
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 
Rett Syndrome Behaviour Questionnaire (RSBQ) 
The Rett Syndrome Behaviour Questionnaire (RSBQ) is a 45 item caregiver-completed rating 
scale assessing a wide range of neurobehavioral symptoms known to be impaired in RTT 
(Mount et al. 2002). The RSBQ is a well-validated instrument that has been used in the 
Phase 2 study, Neu-2566-RETT-002, as well as in other observational and interventional 
studies in RTT (Glaze et al. 2019; Khwaja et al. 2014; O’Leary et al. 2018). The RSBQ has 
been correlated with functioning and quality of life and has been characterized and validated 
across a range of ages and genetic variations in RTT (Cianfaglione et al. 2015, 2016; 
Robertson et al. 2006; Barnes et al. 2015). The scale includes 45 items, including 8 subscales, 
whose ratings reflect the severity and frequency of symptoms. The caregiver rates items as 
“0” (Not True), “1” (somewhat or sometimes true) or “2” (very true). The eight subscales 
include: 
1. General Mood 
2. Breathing problems 
3. Hand Behavior 
4. Face movements 
5. Body rocking/expressionless face 
6. Night-time behaviors 


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7. Fear/anxiety 
8. Walking/standing 
 
The RSBQ will be administered at Screening, Baseline (Visit 2), and Visits 3, 4, and 5. 
As much as possible, caregiver raters will remain the same throughout the study. At the start 
of the study all caregiver raters will be required to complete a standardized training on how to 
complete the scale.  
6.2.2 
Clinical Global Impression–Improvement (CGI-I) and Clinical Global 
Impression–Severity (CGI-S) 
The CGI-I scale will be administered at Visits 3, 4, and 5. Completion of this scale requires 
the clinician to rate how much the subject’s illness has improved or worsened relative to a 
baseline state. A 7-point scale is used from 1=very much improved, 2=much improved, 
3=minimally improved, 4=no change, 5=minimally worse, 6=much worse, 7=very much 
worse.   
The CGI-S assessment will be made at Screening, Baseline, and Visits 3, 4, and 5. The CGI-S 
is a 7-point scale that requires the clinician to rate the severity of the subject’s illness at the 
time of assessment, relative to the clinician’s experience with subjects who have the same 
diagnosis. Considering total clinical experience, a subject is assessed on severity of illness at 
the time of rating: 1, normal, not at all ill; 2, borderline ill; 3, mildly ill; 4, moderately ill; 5, 
markedly ill; 6, severely ill; or 7, extremely ill.  
In this study, the illness being assessed is Rett syndrome as a whole. 
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). 
6.2.3 
Communication and Symbolic Behavior Scales Developmental Profile 
Infant-Toddler (CSBS-DP-IT) Checklist  
The Communication and Symbolic Behavior Scales-Developmental Profile™ (CSBS-DP) is 
standardized screening scale for assessing communication and pre-linguistic skills in young 
children 12-24 months (Wetherby et al. 2002) and can be used with older children with 
developmental delay (Anagnostou et al. 2015; Urbanowicz et al. 2016). The CSBS-DP 
includes a suite of three separate measures: The Infant-Toddler Checklist, a follow-up 
Caregiver Questionnaire and a Behavior Sample. In this study only the Infant-Toddler 
(CSBS-DP-IT) Checklist will be used. 


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Given the limited communication abilities of individuals with Rett Syndrome, the 
CSBS-DP-IT Checklist was assessed and a subset of items were found to be appropriate for 
assessing communication skills of individuals with Rett syndrome 8 to 19 years of age 
(Urbanowicz et al. 2016). The CSBS-DP-IT was assessed in a Phase 2 trial of mecasermin 
(recombinant human IGF-1) a compound related to trofinetide, in children with Rett 
syndrome 2 to 10 years of age (O’Leary et al. 2018). In that study, the first 16 items were 
completed, which allowed for calculation of the Social composite score. The CSBS-DP-IT 
demonstrated evidence of benefit in subjects in the active treatment group compared to those 
in the placebo treatment group (O’Leary et al. 2018). The CSBS-DP Social composite score 
has also shown evidence of sensitivity to change in behavioral intervention studies in other 
developmental disorders (e.g., Wetherby et al. 2014; Anagnostou et al. 2015). 
The CSBS-DP-IT Checklist is a 24-item rating scale completed by the caregiver. Each item is 
scored using a three-level rating of frequency: “not yet”, “sometimes” and “often”. Three 
composite scores assessing 7 skill areas can be calculated: 1) Social Composite (including 
Emotion and Eye Gaze, Communication Rate and Function, and Gestures); 2) Speech 
Composite (including Sounds and Words); 3) Symbolic Composite (including Understanding 
and Object Use). 
All 24 items on the Infant-Toddler Checklist are to be completed by the caregiver. The 
question after item 24 (“Do you have any concerns about your child’s development?”) is not 
to be completed. At each administration, the study staff will review the instructions and 
scoring rubric with the caregiver. The Social Composite raw score, comprised of items 1 to 
13, is used for the key secondary endpoint. 
The CSBS-DP-IT Checklist will be administered at Baseline, Visit 3, Visit 4, and Visit 5. 
6.2.4 
Impact of Childhood Neurologic Disability (ICND) Scale 
The Impact of Childhood Neurologic Disability (ICND) scale was developed to evaluate the 
impact that a child’s condition has on the child’s and the family’s everyday life at the present 
time and during the previous 3 months (Camfield et al. 2003). The parent or other caregiver 
evaluates the effect of four conditions or health problems on 11 aspects of the child’s or the 
family’s life as “A lot”, “Some”, “A little”, “Not at all”, or “Does not apply”. The four 
conditions or health problems are 1) inattentiveness, impulsivity, or mood, 2) ability to think 
and remember, 3) neurologic or physical limitations, and 4) epilepsy. 
The caregiver then rates overall quality of life of the subject by responding to the following: 
“Please rate your child’s overall ‘Quality of Life’ on the scale below. Choose the number 
which you feel is best and circle it”. The choices range from 1 (“Poor”) to 6 (“Excellent”). 
The assessment will be administered at Baseline and Visit 5. 


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6.2.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF)  
The Rett Syndrome Clinician Rating of Hand Function is a clinician completed clinical 
assessment of the subject’s ability to use her hands for functional purposes (such as reaching 
for and grasping objects, self-feeding or drawing). The assessment is made on an 8-point 
Likert scale (0-7) with 0 denoting normal functioning and 7 the most severe impairment. 
This rating is a further development of the RTT-DSC Hand Use Rating used in Study 
Neu-2566-RETT-002. 
The assessment will be administered at Baseline, Visit 3, Visit 4, and Visit 5. 
6.2.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
(RTT-AMB)  
The Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills is a clinician 
completed clinical assessment of the subject’s ability to sit, stand, and ambulate (e.g., 
walking, running, climbing stairs). The assessment is made on an 8-point Likert scale (0-7) 
with 0 denoting normal functioning and 7 the most severe impairment. This rating is a further 
development of the RTT-DSC Ambulation Rating used in Study Neu-2566-RETT-002. 
The assessment will be administered at Baseline, Visit 3, Visit 4, and Visit 5. 
6.2.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
(RTT-COMC)  
The Rett Syndrome Clinician Rating of the Ability to Communicate Choices is a clinician 
completed clinical assessment of the subject’s ability to communicate her choices or 
preferences, which can include the use of nonverbal means such as eye contact or gestures. 
The assessment is made on an 8-point Likert scale (0-7) with 0 denoting normal functioning 
and 7 the most severe impairment. This rating is a further development of the RTT-DSC 
Language/Communication Rating used in Study Neu-2566-RETT-002. 
The assessment will be administered at Baseline, Visit 3, Visit 4, and Visit 5 when the visit 
takes place in the clinic. When a study visit takes place off-site, the RTT-COMC should be 
completed if possible, but it is not required. 
6.2.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM)  
The Rett Syndrome Clinician Rating of Verbal Communication is a clinician completed 
clinical assessment of the subject’s ability to communicate verbally (e.g., words and phrases). 
The assessment is made on an 8-point Likert scale (0-7) with 0 denoting normal functioning 
and 7 the most severe impairment. This rating is a further development of the RTT-DSC 
Language/Communication Rating used in Study Neu-2566-RETT-002. 


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The assessment will be administered at Baseline, Visit 3, Visit 4, and Visit 5. 
6.2.9 
Rett Syndrome Caregiver Burden Inventory (RTT-CBI) 
The RTT-CBI is a syndrome-specific, caregiver-completed questionnaire that is based on the 
Caregiver Burden Inventory designed for Alzheimer’s disease (Lane et al. 2017; Novak and 
Guest 1989). The scale is intended to directly address caregiver burden and indirectly assess 
the significance of treatment effects on function in the context of activities of daily living. 
Caregivers rate how often a given statement describes their feeling or experience. Frequency 
ratings are on a 5-point Likert scale including: 0-never; 1-rarely; 2-sometimes; 3-frequently 
and 4-nearly always. As in the original Caregiver Burden Inventory, the RTT-CBI has 
24 negatively worded items (items 1 through 24) yielding a total score up to 96. The 
RTT-CBI also includes 2 positively worded items (items 25 and 26) that comprise the 
Optimism Index (Lane et al. 2017). In this study, as in the 2 previous studies of trofinetide in 
Rett syndrome, the total score is defined as the total Burden score (items 1-24). 
The RTT-CBI will be completed at Baseline and Visit 5. 
6.3 
Safety Assessments  
6.3.1 
Physical Examination 
A general physical examination will be conducted at Screening, Baseline, Visit 3, Visit 4, and 
Visit 5. 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 for ≥3 minutes. 


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Vital signs to be measured at Screening, Baseline, Visit 3, Visit 4, and Visit 5. 
6.3.3 
Height, Weight, and Body Mass Index 
Height will be measured at Screening and at Visit 5. 
Weight will be measured at Screening, Baseline, Visit 3, Visit 4, and Visit 5 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 12-lead ECGs will be complete, standardized recordings, whenever possible. ECGs will 
be completed in triplicate at Visit 1 (Screening), at Visit 2 (Baseline) both before dosing and 
2-3 hours after dosing, and at Visit 5 (Week 12/EOT/ET). A single ECG will be completed at 
Visit 3 (Week 2) and Visit 4 (Week 6). 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, QRS, 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 (before dosing) 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. 


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6.3.4.1 
Post-Baseline QTcF Interval Stopping Criteria 
In the event that a post-randomization 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–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:  
• 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 HbA1c 
 HbA1c will only be performed at Visit 1 (Screening) 
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), 
Visit 2 (Baseline), and Visit 5 
• Pregnancy test 
o A serum pregnancy test will be performed at the designated visits (Table S–2) for 
subjects of childbearing potential 


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• 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. 
For the 2 days before scheduled visits as well as the morning of the scheduled visit, the dates 
and times of study drug dosing, concomitant medication dosing, and meals will be recorded 
in the caregiver diary. Daily study drug dosing, including any dose modifications, missed or 
partial doses, will also be recorded in the caregiver diary. 
The caregiver diary will be completed and collected on an ongoing basis throughout the study 
from Screening to Visit 5. The clinician will verbally ask about AEs (see Section 7.3.1), 
review the events recorded in the diary with the caregiver and will make a clinical evaluation, 
including an evaluation of whether an adverse event will be reported. 
6.5 
Pharmacokinetic Assessments 
Pharmacokinetic blood samples will be collected for measurement of whole blood 
concentrations of trofinetide and possible metabolites identified. 
PK blood samples will be collected at 5 timepoints for trofinetide concentration 
measurements at the Baseline visit (both before dosing and approximately 2-3 hours after 


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dosing) and at Visit 3, Visit 4, and Visit 5 in accordance with the sampling schedule outlined 
below (Table 6–1). 
The second PK sample to be taken at the Baseline visit (Visit 2), approximately 2-3 hours 
after dosing, will take place as soon as possible after the postdose ECG is performed.  
PK samples at Visits 3, 4, and 5 should be collected at one of the following time intervals: 
• 2-3 hours after dosing 
• 4-6 hours after dosing 
• 7-11 hours after dosing 
Every effort should be made to collect PK samples at discrete time intervals during Visits 3, 
4, and 5. However, if the interval is the same across these visits, then the collection time 
should vary within that interval. The following scenario is only for illustrative purposes, a 
number of other scenarios are possible. 
• For example, if the time interval of 4-6 hours after dosing is used for Visits 3, 4, and 
5, then every effort should be made to collect PK samples at 4, 5, and 6 hours after 
dosing, respectively. 
Pharmacokinetic samples will also be collected, if possible, at any ET visit or the visit 
immediately following any SAE or following any AE leading to discontinuation, even if it is 
an unscheduled visit. 
For all scheduled PK samples (and for unscheduled samples if possible), the dates and times 
of administration of the study drug, the dates, times and content of the meals, and the dates 
and times of the administration of concomitant medication over the 2 days prior to and on the 
morning of the PK sample draw, as well as the date and time of the sample draw, will be 
recorded. For samples collected from subjects who experience any SAE or experience an AE 
leading to discontinuation, the date and time of the last dose of study drug prior to the SAE or 
AE leading to discontinuation will also be recorded. 


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Table 6–1 
PK Sampling Times 
Visit 
Timing of Sample  
(relative to start of dosing on the visit day) 
Week of Treatment 
2 (Baseline) 
Any time before dosing 
and 
2 – 3 hours after dosing (after ECG) 
Week 0 (Baseline) 
3, 4, and 5 
2 – 3 hours after dosing  
or 
4 – 6 hours after dosing 
or 
7 – 11 hours after dosing 
Weeks 2, 6, and 12/EOT 
Abbreviations: ECG=electrocardiogram; EOT=end of treatment; PK=pharmacokinetic 
 
6.5.1 
Specimen Preparation, Handling, Storage, and Shipment 
PK blood samples may be collected from a cannula port or via venipuncture. Pre-prepared 
PK sampling tubes will be provided to each site within the lab visit kits for collection and 
storage of PK samples. Blood samples will be processed for determination of trofinetide 
whole blood concentrations (and of concentrations of 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. 
A laboratory manual will be provided for sample processing, storage, and shipping 
procedures. 
When possible, an additional PK sample will be collected from subjects who experience any 
SAE or experience an AE leading to discontinuation as soon as possible after the occurrence 
of that event. 
6.6 
Identification of Biomarkers of Response to Trofinetide in Rett Syndrome 
Subjects for whom separate informed consent for the identification of biomarkers of response 
to trofinetide is provided (where local regulations permit) will have blood drawn and stored 
for future investigations. Blood samples will be taken at Baseline (before dosing) and at 
Visit 5, or upon early termination. Participation in the effort to identify biomarkers is an 
optional component of the study requiring a separate informed consent, which may be 
obtained at any time during the study. If consent is obtained after Baseline, only the sample at 
Visit 5, or upon early termination will be taken.   
Blood samples will be used to investigate differences between responders and non-responders 
in both trofinetide-treated and placebo-treated subjects in RNA transcripts (transcriptomics), 
proteins (proteomics), and metabolites (metabolomics). Unbiased analyses and targeted 
analyses will test candidate molecular pathways based on the available knowledge of RTT 


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and trofinetide at the time of the investigation (Erhart et al. 2016; Shovlin and Tropea 2018; 
West et al 2014; Buchovecky et al. 2013). The analysis of biomarkers does not include any 
DNA, genomic, or genetic testing or analysis.   
Stored samples and relevant clinical data will be made non-identifiable after the clinical 
study report has been issued. Any personal identifiers will be removed, and each study 
subject identifier will be replaced with a new number to limit the possibility of linking 
genetic data to a subject’s identity. 
7 ADVERSE EVENTS 
7.1 
Specification of Safety Parameters 
7.1.1 
Definition of Adverse Event 
An AE is defined as “any untoward medical occurrence in a patient or clinical study 
participant, temporally associated with the use of study drug, whether or not considered 
related to study drug”. 
An AE can therefore be any unfavorable and unintended sign (e.g., an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a drug, without any 
judgment about causality or seriousness. An AE can arise from any use of the drug 
(e.g., off-label use, use in combination with another drug) and from any route of 
administration, formulation, or dose, including an overdose. 
A suspected adverse reaction is any AE for which there is a reasonable possibility that the 
drug caused the AE. 
AEs do not include the following: 
• Stable or intermittent chronic conditions (such as myopia requiring eyeglasses) 
that are present prior to Baseline and do not worsen during the study 
• Medical or surgical procedures (e.g., surgery, endoscopy, tooth extraction, 
transfusion). The condition that leads to the procedure is an AE if not present at 
Baseline. 
• Overdose of concomitant medication without any signs or symptoms unless the 
subject is hospitalized for observation 
• Hospitalization for elective surgery planned prior to study (situation where an 
untoward medical occurrence has not occurred) 
• Pregnancy will not be considered an AE, but if it occurs, it will be reported on a 
pregnancy form 


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For subjects who enroll into the open-label extension study, AEs will be recorded from the 
time informed consent is obtained in the present study until the first dose of study drug in the 
open-label study. 
For subjects who discontinue from the study or do not enroll into the open-label extension, 
AEs will be recorded from the time informed consent is obtained until 30 days after the last 
dose of study drug. 
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. 
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 


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• 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 
• 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). 


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Consider the following when assessing causality: 
• Temporal associations between the agent and the event 
• Response to cessation (de-challenge) or re-challenge 
• Compatibility with known class effect 
• Known effects of concomitant medications 
• Pre-existing risk factors 
• A plausible mechanism 
• Concurrent illnesses 
7.2.2.1 
Duration 
The start and stop dates for AEs will be recorded using the following criteria: 
• Start: Date of the first episode of the AE or date of significant sustained worsening in 
severity 
• Stop: Date when AE either ceased permanently or changed in severity 
For AEs of diarrhea, the start date and stop date of a decrease in severity will also be 
recorded. 
7.2.2.2 
Frequency 
The frequency of the AE should be indicated according to the following definitions: 
• Single: Experienced once, without recurrence 
• Recurrent: More than one discrete episode with the same severity 
7.2.2.3 
Action Taken with Study Drug 
• Dose not changed: No change in study drug 
• Drug interrupted: Study drug temporarily stopped 
• Drug withdrawn: Study drug discontinued permanently 
• Dose decreased: Dose of study drug reduced 
7.2.2.4 
Therapy 
• None: No new treatment instituted 
• Medication: New treatment initiated as a direct result of AE 
• Other: Other action required 


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7.2.2.5 
Outcome 
• Recovered/resolved: Recovered or resolved 
• Recovered/resolved with sequelae: Recovered or resolved with sequelae 
• Not recovered/not resolved: Not recovered or not resolved 
• Fatal: Death due to an AE 
• Unknown: Unknown 
7.2.2.6 
Seriousness 
• Not serious 
• Serious 
7.2.3 
Definition of Unexpectedness 
An AE, the nature or severity of which is not consistent with the information provided in the 
Reference Safety Information section of the current trofinetide Investigator’s brochure. 
7.3 
Time Period and Frequency for Event Assessment and Follow-Up 
Adverse events will be recorded from the time informed consent is obtained through the 
safety follow-up period. All AEs must be either resolved or stable at the end of the safety 
follow-up period. If ongoing at the end of the safety follow-up period, the subject should be 
referred for appropriate treatment. 
In the event that a subject discontinues and has an ongoing AE at the time of discontinuation 
(Section 4.4.2) or is withdrawn from the study because of an AE, the subject should be 
followed and treated by the Investigator until the AE has resolved, stabilized, or a new 
chronic baseline has been established. 
7.3.1 
Adverse Event Reporting 
The Investigator must record all observed AEs and all reported AEs. At each visit, the 
Investigator should ask the subject a nonspecific question (e.g., “Have you noticed anything 
different since your last visit?”) to assess whether any AEs have been experienced since the 
last report or visit. 
Note that any use of medication (and specifically any newly prescribed medication) during 
the course of a study may indicate the occurrence of an AE that may need to be recorded on 
both the AE and the concomitant medication page. 


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All AEs, serious and not serious, will be recorded on the AE eCRF page using appropriate 
medical terminology. Severity and relationship to study drug will be assessed by the 
Investigator. 
When possible, clinical AEs should be described by diagnosis and not by symptoms 
(e.g., “cold” or “seasonal allergies” instead of “runny nose”). 
All AEs, whether or not related to the study drug, must be fully and completely documented 
on the AE eCRF and in the subject’s notes. 
7.3.2 
Serious Adverse Event Reporting 
The reporting of SAEs by the Sponsor or designee to the regulatory authorities is a regulatory 
requirement. Each regulatory authority has established a timetable for reporting SAEs based 
upon established criteria. 
Serious AEs must be reported within 24 hours of discovery to the Sponsor or its designee; 
use the appropriate form for initial and/or follow-up reporting.  
At a minimum, events identified by the Sponsor to require expedited reporting as serious, 
unexpected, and related to study drug must be brought to the attention of the responsible 
Institutional Review Board/Ethics Committee (IRB/EC), as per applicable regulations. These 
will be provided by the Sponsor after their assessment. For European Union member states, 
the Sponsor or its designee will provide reports of suspected unexpected serious adverse 
reactions (SUSARs) directly to the ECs, as required by local legislation. In all other 
countries, it is the Investigator’s responsibility to provide these expedited reports to the 
responsible IRB/EC. It is also the Investigator’s responsibility to notify the responsible 
IRB/EC regarding any new and significant safety information. 
When an SAE occurs, Investigators will review all documentation related to the event and 
will complete the paper SAE form (for initial and/or follow-up information) and fax or email 
(within 24 hours of discovery) to the contact information provided on the SAE form. 
Subjects will be followed through the safety follow-up period for 30 days after last dose of 
study drug for any SAEs and/or other reportable information until such events have resolved 
or the Investigator, in conjunction with the Sponsor, deems them to be chronic or stable. 
In the event of any SAE (other than death), the study subject will be instructed to contact the 
Investigator (or designee) using the telephone number provided in the 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. 


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Serious AEs occurring after the study follow-up period (i.e., 30 days after last dose of study 
drug) should be reported if in the judgment of the Investigator there is “a reasonable 
possibility” that the event may have been caused by the product. 
SAEs should also be reported to the IRB/EC according to local regulations. 
7.3.3 
Reporting of Pregnancy 
Any subject who becomes pregnant during the study (with or without AEs) must be 
withdrawn from the study and the pregnancy must be reported on the Pregnancy form within 
24 hours of discovery to the Sponsor or its designee. Any subject who becomes pregnant 
during the study will be followed through the pregnancy outcome. 
Any AEs that are the consequence of pregnancy and which meet the criteria for serious 
should also be reported via the SAE form. 
7.3.3.1 
Reporting Paternal Drug Exposure 
Paternal drug exposure is defined as a father’s exposure to a medicinal product before or 
during his partner’s pregnancy. Any paternal drug exposure cases must be reported to the 
Sponsor within 24 hours of discovery via the Pregnancy form. Any AEs that are the 
consequence of paternal drug exposure and which meet the criteria for serious must also be 
reported to the Sponsor within 24 hours of discovery via the SAE form. Since no males are 
enrolling in this study, paternal drug exposure would occur only if a male who was not a 
study subject ingested study drug. 
7.3.4 
Reporting of Overdose 
An overdose is a deliberate or inadvertent administration of a treatment at a dose higher than 
the maximum recommended dose per protocol. It must be reported to the Sponsor or 
designee on the Overdose form within 24 hours of discovery. In addition, all events of 
overdose are to be captured as protocol deviations. 
8 CLINICAL MONITORING  
Routine monitoring of study sites is described in Section 11. 
Clinical site monitoring is conducted to ensure that the rights and well-being of human 
subjects are protected, that the reported study data are accurate, complete, and verifiable, 
and that the conduct of the study is in compliance with the currently approved protocol and 
amendment(s) as applicable, with GCP, and with applicable regulatory requirements. Details 
of the study site monitoring process are described in a separate clinical monitoring plan 
document. 


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9 STATISTICAL METHODS AND DATA ANALYSIS 
9.1 
Statistical and Analytical Plans 
Statistical methods will be documented in detail in a statistical analysis plan (SAP) to be 
approved by the Sponsor prior to database lock. 
9.2 
Statistical Hypotheses 
The co-primary endpoints are change from Baseline to Week 12 in RSBQ total score and 
CGI-I score at Week 12. 
Let ΔRSBQ and ΔCGI-I be the difference between the trofinetide and placebo groups in the mean 
change from Baseline to Week 12 in RSBQ total score and in the mean CGI-I score at 
Week 12 respectively. 
RSBQ: The null hypothesis is: ΔRSBQ = 0 and the alternative hypothesis is: ΔRSBQ ≠ 0. 
CGI-I: The null hypothesis is: ΔCGI-I = 0 and the alternative hypothesis is: ΔCGI-I ≠ 0. 
9.3 
Sample Size Determination 
The sample size calculation was performed for the co-primary endpoints as a family of two 
hypothesis tests at an overall two-sided significance level of 0.05. A total sample size of 
174 subjects in a 1:1 ratio to trofinetide or placebo was estimated to provide at least 90% 
power for the hypothesis testing family assuming the following treatment differences (SD) 
estimated from Phase 2 study data: -4.4 (8) for the mean change from Baseline to Week 12 in 
the RSBQ total score and -0.5 (0.7) for the CGI-I mean score at Week 12. 
The sample size of 174 subjects will provide at least 95% power at a two-sided significance 
level of 0.05 for each individual hypothesis test within the family. Trofinetide will be claimed 
to be superior to placebo if both hypothesis tests within the family are shown to be 
statistically significant at 0.05. Therefore, the overall power to detect a treatment difference 
on both of the co-primary endpoints will be at least 90% (0.952). 
Adjusting for an anticipated discontinuation rate of up to 5%, approximately 184 subjects 
will be randomized in a 1:1 ratio to trofinetide or placebo. 
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 randomized subjects who received at least one 
dose of study medication. The Safety Analysis Set will be analyzed according to the actual 
treatment received. The Safety Analysis Set will be used for the safety analysis. 


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The Full Analysis Set (FAS) will consist of all randomized subjects who received at least one 
dose of study medication and who have both a Baseline value and at least one post-Baseline 
value for the RSBQ total score or who have at least one post-Baseline value for the CGI-I 
score. The FAS will be analyzed according to the treatment they were assigned regardless of 
the actual treatment received. The Full Analysis Set will be used for the efficacy analysis. 
The Per-protocol (PP) Analysis Set will consist of the subjects in the Full Analysis Set who 
did not have a major protocol violation that would affect interpretation of the efficacy data. 
The Per-protocol Analysis Set will be defined prior to study unblinding.  
The Pharmacokinetic Analysis Set will consist of subjects in the Safety Analysis Set with at 
least one measurable trofinetide whole blood concentration. 
9.5 
Statistical Analyses 
9.5.1 
General Approach 
Unless stated otherwise, all statistical tests will be 2-sided using a 5% significance level, 
leading to 95% (2-sided) confidence intervals. Trofinetide will be claimed to be superior to 
placebo if both of the hypothesis tests with respect to the co-primary endpoints are shown to 
be statistically significant in favor of trofinetide. 
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. 
A hierarchical approach will be used to control for the multiple endpoints (co-primary and 
secondary). Details will be provided in the SAP. 
9.5.2 
Primary Analyses 
The co-primary efficacy endpoints will be analyzed using a mixed model for repeated 
measures (MMRM). An unstructured covariance matrix will be used and the Kenward-Roger 
approximation will be used to adjust the denominator degrees of freedom. The treatment 
comparisons will be based on the difference in least squares means at Week 12. 
For the change from Baseline in the RSBQ total score, the MMRM model will include effects 
for treatment group, age group (5-10 years old, 11-15 years old, and 16-20 years old), 
Baseline RSBQ severity (<35 total score and ≥35 total score), visit, Baseline RSBQ total 
score, and interactions for treatment group by visit and Baseline RSBQ total score by visit. 
For the CGI-I score, the MMRM model will include effects for treatment group, age group 
(5-10 years old, 11-15 years old, and 16-20 years old), visit, Baseline RSBQ severity 


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(<35 total score and ≥35 total score), Baseline CGI-S score, and interactions for treatment 
group by visit and Baseline CGI-S score by visit. 
Sensitivity analyses will be performed to assess the impact of missing data, including 
analyses based on a missing not at random assumption.  
Complete details of the efficacy analyses will be specified in the statistical analysis plan 
(SAP). 
9.5.3 
Secondary Analyses 
The key secondary endpoint, change from baseline to Week 12 in the CSBS-DP-IT Social 
Composite Score, will be analyzed using a MMRM method with effects for treatment group, 
age group (5-10 years old, 11-15 years old, and 16-20 years old), visit, Baseline RSBQ 
severity (<35 total score and ≥35 total score), Baseline CSBS-DP-IT Social score, and 
interactions for treatment group by visit and Baseline CSBS-DP-IT Social score by visit. An 
unstructured covariance matrix will be used and the Kenward-Roger approximation will be 
used to adjust the denominator degrees of freedom. The treatment comparisons will be based 
on the difference in least squares means at Week 12. 
For the other secondary endpoints that are assessed at multiple post-Baseline visits, the 
change from Baseline will be analyzed using a MMRM analysis similar to those described 
above for the co-primary endpoints. The MMRM model will include effects for treatment 
group, age group (5-10 years old, 11-15 years old, and 16-20 years old), visit, Baseline 
RSBQ severity (<35 total score and ≥ 35 total score), Baseline score, and interactions for 
treatment group by visit and Baseline score by visit. 
For the other secondary endpoints that are assessed at a single post-Baseline visit 
(i.e. Week 12 only), the change from Baseline will be analyzed using an analysis of 
covariance (ANCOVA) model with effects for treatment group, age group (5-10 years old, 
11-15 years old, and 16-20 years old), Baseline RSBQ severity (<35 total score and ≥35 total 
score), and Baseline score. 
9.5.4 
Safety Analyses 
Safety results will be summarized by treatment group using descriptive statistics. No formal 
statistical testing will be performed for any of the safety endpoints. Adverse events will be 
classified into standard terminology using the Medical Dictionary for Regulatory Activities 
(MedDRA). Treatment-emergent adverse events (TEAEs), TEAEs leading to discontinuation, 
TEAEs related to study drug, TEAEs by maximum severity, fatal TEAEs, SAEs, and SAEs 
related to study drug will all be summarized. 


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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 intervals and 
changes in QTc intervals in accordance with International Council on Harmonisation (ICH) 
guidelines. 
Additional safety analysis details will be specified in the SAP. 
9.5.5 
Pharmacokinetic Analyses 
Pharmacokinetic (PK) and efficacy (i.e., PD) measures will be collected from all subjects at 
the Baseline (Week 0) visit before dosing, at the Baseline (Week 0) visit after dosing, and 
after dosing at Weeks 2, 6, and 12/EOT.  
Whole blood concentration and possible metabolites data for trofinetide will be listed and 
summarized using descriptive 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. Trofinetide whole blood 
concentration data will remain blinded until the unblinding of the clinical database at the end 
of the study. 
The details of the PK and PK/PD analysis will be presented in a separate population 
PK report and PK/PD report in accordance with a separate data analysis plan (DAP). 
9.5.6 
Subgroup Analyses 
Selected analyses may be performed in subgroups. Details will be provided in the SAP. 
9.6 
Interim Analyses 
No interim analysis is planned in this study. 
9.7 
Data and Safety Monitoring Board 
An independent Data and Safety Monitoring Board (DSMB) will review safety information 
on a regular basis throughout the study. The DSMB will be independent of the Sponsor and 
will be empowered to recommend stopping the study due to safety concerns. The DSMB may 
review blinded, unblinded, or partially unblinded data, but the Sponsor and the Investigators 
will remain blinded to the data provided to the DSMB until the official unblinding of the 
database at the completion of the study. The membership, activities, responsibilities, and 
frequency of meetings will be described separately in the DSMB charter. 
9.8 
Measures to Minimize Bias 
Eligible subjects will be randomized into one of two treatment groups (trofinetide or placebo) 
in a 1:1 ratio using an interactive response technology (IRT) system. The randomization will 


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be stratified by age group (5-10 years old, 11-15 years old, and 16-20 years old) and Baseline 
RSBQ severity (<35 total score and ≥35 total score). The assignments will be based on a 
pre-generated permuted-block randomization schedule. Blinding will be assured by 
restricting access of Investigators and Sponsor personnel and/or designee to the treatment 
codes and providing identical packaging for the trofinetide and placebo treatments. 
9.9 
Breaking the Study Blind/Subject Code 
For the final analysis, the treatment codes for all subjects will be released to the Sponsor after 
all subjects have completed the study and the clinical database is locked.  
For DSMB safety reviews, the treatment codes will be released to an independent 
statistician/programmer to produce unblinded statistical outputs. The Sponsor and the 
Investigators will remain blinded. 
Unblinding of individual treatment assignment during the study is discouraged. The 
Investigator may break the blind in the event of a medical emergency if it is considered 
necessary for the care of the subject. The Investigator should attempt whenever possible to 
contact the Medical Monitor before unblinding a subject’s treatment to discuss the event. 
Lack of Medical Monitor contact does not preclude the Investigator from unblinding the 
subject. In an emergency situation, the subject’s treatment assignment may be obtained by the 
Investigator from the IRT system. Details of the process to be followed are provided in a 
separate IRT manual. In the event that the IRT system is used to perform a code break, the 
Sponsor or designee will be notified immediately via an automated notification from the 
IRT system that an unblinding has occurred. The notification only alerts the Sponsor or 
designee that the unblinding occurred and does not include any information about the 
unblinded subject’s treatment assignment. 
10 STUDY MANAGEMENT AND DATA COLLECTION 
10.1 Data Collection and Management Responsibilities 
All documents required for the conduct of the study as specified in the ICH GCP guidelines 
will be maintained by the Investigator in an orderly manner and made available for 
monitoring and/or auditing by the Sponsor and regulatory authorities. 
The Investigator and institution must permit authorized representatives of the Sponsor or 
designees (including monitors and auditors), regulatory authorities (including inspectors), 
and the IRB/EC direct access to source documents (such as original medical records). Direct 
access includes permission to examine, analyze, verify, and reproduce any records and 
reports that are needed for the evaluation of the study. The Investigator must ensure the 


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


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required by applicable law or regulation. Only the Sponsor can notify an Investigator or 
vendor when any records may be discarded. Investigators should contact the Sponsor before 
destroying any files. 
10.6 Protocol Exceptions and Deviations 
No prospective entry criteria protocol deviations are allowed; all subjects must meet all 
eligibility criteria in order to participate in the study. 
Protocol waivers for eligibility will not be granted by the Sponsor under any circumstances. 
If, during the course of a subject’s post-enrollment participation in the trial it is discovered 
that the subject did not meet all eligibility criteria, he or she will be discontinued, unless the 
discontinuation presents an unacceptable medical risk. The justification to allow the subject 
to continue in the trial will be made by the Sponsor, with medical input from the Investigator, 
and will be documented. If allowed to remain in the trial, this will be reported as a major 
protocol deviation and not a waiver. All follow-up safety assessments must be completed and 
documented as outlined in the protocol (Section 3.1.3). The Investigator must report any 
protocol deviation to the Sponsor and, if required, to the IRB/EC in accordance with local 
regulations, within reasonable time. 
10.7 Protocol Amendments 
Changes to the protocol may be made only by the Sponsor (with or without consultation with 
the Investigator). All protocol modifications must be submitted to the site IRB/EC in 
accordance with local requirements and, if required, to regulatory authorities, as either an 
amendment or a notification. Approval for amendments must be awaited before any changes 
can be implemented, except for changes necessary to eliminate an immediate hazard to trial 
subjects, or when the changes involve only logistical or administrative aspects of the trial. 
No approval is required for notifications. 
11 STUDY MONITORING, AUDITING, AND INSPECTING 
11.1 Quality Control and Quality Assurance 
The Sponsor or designees and regulatory authority inspectors are responsible for contacting 
and visiting the Investigator for the purpose of inspecting the facilities and, upon request, 
inspecting the various records of the trial (e.g., eCRFs and other pertinent data) provided that 
subject confidentiality is respected. 
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 


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research. The monitor should have access to subject medical records and other study-related 
records needed to verify the entries on the eCRFs. 
The Investigator agrees to cooperate with the monitor to ensure that any problems detected in 
the course of these monitoring visits are resolved. 
In accordance with ICH guidance on GCP and the Sponsor’s audit plans, a certain percentage 
of sites participating in this study will be audited. These audits may include a review of site 
facilities (e.g., pharmacy, drug storage areas, and laboratories) and review of study-related 
records may occur in order to evaluate the trial conduct and compliance with the protocol, 
ICH guidance on GCP, and applicable regulatory requirements. 
The Sponsor’s or designee’s representatives, regulatory authority inspectors and IRB/EC 
representatives who obtain direct access to source documents should also respect subject 
confidentiality, taking all reasonable precautions in accordance with applicable regulatory 
requirements to maintain the confidentiality of subjects’ identities. 
11.2 Risk Management 
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 
activities with a focus on critical processes and critical study data:  
• 
Risk Identification: risks to critical trial processes, governing systems, investigational 
product, trial design, data collection, and recording are identified. 
• Risk Evaluation: identified risks are evaluated by considering the following factors: 
(a) likelihood of occurrence, (b) impact on human subject protection and data 
integrity, and (c) detectability of errors. 
• Risk Control: risks that can be reduced (e.g., mitigating) or can be accepted are 
differentiated. Risk mitigation activities are incorporated in protocol design and 
implementation, study plans, training, processes, and other documents governing the 
oversight and execution of study activities. Where possible, predefined quality 
tolerance limits are to be defined to identify systematic issues that can impact subject 
safety or data integrity and deviations from the predefined quality tolerance limits will 
trigger an evaluation and possibly an action. Contingency plans are developed for 
issues with a high risk factor that cannot be avoided. 


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


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authorized under applicable law to consent on behalf of a prospective subject to the subject’s 
participation in the procedures involved in the research’ (US CFR 21 part 50). 
For subjects who are minors, written informed consent will be obtained from the LAR. For 
subjects who are not minors, written informed consent will be obtained from the LAR or the 
subject if deemed able by the Investigator. When consent is being provided by an LAR, 
subject assent for participation should be documented, when possible. Assent is the 
affirmative agreement to participate in the research of a minor or of an adult who does not 
have the capacity to consent. If written assent is not possible, verbal assent is allowed and 
must be documented. If subject assent is not possible, then the site must document rationale 
for not being able to provide written or documented subject assent.  
If a subject’s 18th birthday takes place during the study and the subject is deemed able to 
consent by the Investigator, the subject should sign the informed consent. Reconsenting 
should take place if required by and in accordance with IRB or EC policy and applicable 
local law. 
The subject’s caregiver must also provide informed consent regarding their participation in 
the study prior to participating in any study procedures. 
The informed consent must, at a minimum, include the elements of consent described in the 
ICH guidance on GCP and the US CFR 21 part 50.25. A copy of the ICF planned for use will 
be reviewed by the Sponsor or designee for acceptability and must be submitted by the 
Investigator or designee together with the protocol, to the appropriate IRB/EC for review and 
approval prior to the start of the study at that investigational site. Consent forms must be in a 
language fully comprehensible to the LAR of the prospective subject. The Investigator must 
provide the Sponsor or designee with a copy of the IRB/EC letter approving the protocol and 
the ICF before the study drug supplies will be shipped and the study can be initiated. 
The consent form must be revised if new information becomes available during the study that 
may be relevant to the subject. Any revision must be submitted to the appropriate IRB/EC for 
review and approval in advance of use. 
12.3.1 
Consent and Other Informational Documents Provided to Subjects 
The subject/LAR must be given a copy of the signed informed consent and the original 
maintained in the designated location at the site. 
12.3.2 
Consent Procedures and Documentation 
It is the Investigator or designee’s responsibility to obtain written informed consent from the 
subject/LAR after adequate explanation of the aims, methods, anticipated benefits, and 
potential hazards of the study. The LAR must be given ample time to decide about study 


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


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Lee JY, Leonard H, Piek JP, Downs J. Early development and regression in Rett syndrome. 
Clin Genet. 2013;84(6):572–576. 
Monrós E, Armstrong J, Aibar E, Poo P, Canos I, Pineda M. Rett syndrome in Spain: 
mutation analysis and clinical correlations. Brain & Development. 2001;23:S251-S253. 
Motil KJ, Caeg E, Barrish JO, et al. Gastrointestinal and nutritional problems occur 
frequently throughout life in girls and women with Rett syndrome. J Pediatr Gastroenterol 
Nutr. 2012;55(3):292-298. 
Mount RH, Charman T, Hastings RP, Reilly S, Cass H. Features of autism in Rett syndrome 
and severe mental retardation. J Autism Dev Disord. 2003;33(4):435-442. 
Mount RH, Charman T, Hastings RP, Reilly S, Cass H. The Rett Syndrome Behaviour 
Questionnaire (RSBQ): Refining the behavioural phenotype of Rett syndrome. J Child 
Psychol Psychiatry. 2002;43(8):1099–1110. 
Mount RH, Hastings RP, Reilly S, Cass H, Charman T. Behavioural and emotional features in 
Rett syndrome. Disabil Rehabil. 2001;23(3-4):129-138. 
Neul JL, Benke TA, Marsh ED, et al. The array of clinical phenotypes of males with 
mutations in Methyl-CpG binding protein 2. Am J Med Genet B Neuropsychiatr Genet. 
2019;180(1):55-67.  


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Neul JL, Glaze D, Percy A, et al. Improving Treatment Trial Outcomes for Rett Syndrome: 
the development of Rett-specific anchors for the Clinical Global Impression Scale. J Child 
Neurol. 2015;30(13):1743-1748. 
Neul JL, Lane JB, Lee HS, et al. Developmental delay in Rett syndrome: data from the 
natural history study. J Neurodevelop Dis. 2014;(30):2-9. 
Neul JL, Kaufmann WE, Glaze DG, et al. Rett syndrome: revised diagnostic criteria and 
nomenclature. Ann Neurol. 2010;68(6):944-950. 
Neul JL, Fang P, Barrish J, et al. Specific mutations in methyl-CpG-binding protein 2 confer 
different severity in Rett syndrome. Neurology. 2008;70(16):1313-1321. 
Novak M, Guest C. Application of a multidimensional Caregiver Burden Inventory. 
Gerontologist. 1989;29(6):798-803. 
O’Leary HM, Kaufmann WE, Barnes KV, et al. Placebo controlled crossover efficacy 
assessment of mecasermin for the treatment of Rett syndrome. Ann Clin Transl Neurol 
2018;5(3):323-332. 
Oosterholt SP, Horrigan J, Jones N, Glass L, Della Pasqua O. Population pharmacokinetics of 
NNZ-2566 in healthy subjects. Eur J Pharm Sci. 2017;15;109S:S98-107. 
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genes other than MECP2. Transl Sci Rare Dis. 2018;3(1):49-53. 
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Neurol. 2013;55(4):364-371. 


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Samaco RC, Neul JL. Complexities of Rett syndrome and MeCP2. J Neurosci. 
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Tropea D, Giacomettim E, Wilson NR, et al. Partial reversal of Rett Syndrome-like 
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communication and symbolic behavior scales developmental profile with very young 
children. J Speech Lang Hear Res. 2002;45(6):1202-1218. 
Woodyatt GC, Ozanne AE. A longitudinal study of cognitive skills and communication 
behaviours in children with Rett syndrome. J Intellect Disabil Res. 1993;37(Pt 4):419-435. 
Yasui DH, Xu H, Dunaway KW, Lasalle JM, Jin LW, Maezawa I. MeCP2 modulates gene 
expression pathways in astrocytes. Mol Autism. 2013;4(1):3. 
Young DJ, Bebbington A, Anderson A, et al. The diagnosis of autism in a female: could it be 
Rett syndrome? Eur J Pediatr. 2008;167(6):661-669. 


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16 APPENDICES 
Appendix A Rett Syndrome Diagnostic Criteria 
RTT Diagnostic Criteria 2010 
1. 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: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study of 
Trofinetide for the Treatment of Girls and Women with Rett Syndrome 
NCT Number: NCT04181723
Document Date: 20 October 2021


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STATISTICAL ANALYSIS PLAN 
Protocol No.: 
ACP-2566-003 
Protocol Title: 
A Randomized, Double-Blind, Placebo-Controlled, 
Parallel-Group Study of Trofinetide for the Treatment 
of Girls and Women with Rett Syndrome 
Drug: 
trofinetide oral solution 
Sponsor: 
Acadia Pharmaceuticals Inc. 
Version No. and Date 
Version 3.0,  20 October 2021 


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SIGNATURE/APPROVAL PAGE 
AUTHORS 
 
________________________________________                                    ___________         
                                                                                                        Date 
Associate Director, Biostatistics 
Acadia Pharmaceuticals Inc. 
 
 
APPROVERS 
 
 
_____________________________________________                               ____________    
                                                                                   Date 
VP, Clinical Development 
Acadia Pharmaceuticals Inc.                                                                                        
 
 
_____________________________________________                               ____________    
                                                                                                     Date 
Senior Director, Biostatistics 
Acadia Pharmaceuticals Inc. 
 
 
 
 
 




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TABLE OF CONTENTS 
LIST OF TABLES ........................................................................................................... 7 
LIST OF FIGURES ......................................................................................................... 7 
ABBREVIATIONS ......................................................................................................... 8 
1. 
INTRODUCTION ............................................................................................. 10 
2. 
OBJECTIVES .................................................................................................... 11 
2.1 
Primary Objective .............................................................................................. 11 
2.2 
Key Secondary Objective .................................................................................. 11 
2.3 
Other Secondary Objectives .............................................................................. 11 
2.4 
Safety Objective ................................................................................................ 11 
2.5 
Pharmacokinetic Objectives .............................................................................. 12 
3. 
STUDY DESIGN .............................................................................................. 13 
3.1 
General Study Design ........................................................................................ 13 
3.2 
Schedule of Assessments ................................................................................... 14 
3.3 
Randomization ................................................................................................... 14 
3.4 
Blinding ............................................................................................................. 14 
3.5 
Determination of Sample Size ........................................................................... 14 
3.6 
Coronavirus Disease 2019 ................................................................................. 15 
4. 
ANALYSIS SETS ............................................................................................. 16 
5. 
DATA HANDLING CONVENTIONS ............................................................ 17 
5.1 
General Data Reporting Conventions ................................................................ 17 
5.2 
Derived Efficacy Variables ............................................................................... 17 
5.2.1 
Rett Syndrome Behaviour Questionnaire (RSBQ) ..................................... 18 
5.2.2 
Clinical Global Impression–Improvement (CGI-I) .................................... 20 
5.2.3 
Communication and Symbolic Behavior Scales Developmental Profile™ 
Infant-Toddler  Checklist – Social Composite Score (CSBS-DP-IT Social)
..................................................................................................................... 20 
5.2.4 
Overall Quality of Life Rating of the Impact of Childhood Neurologic 
Disability (ICND) Scale .............................................................................. 20 
5.2.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF) .................. 20 
5.2.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
(RTT-AMB) ................................................................................................ 21 
5.2.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
(RTT-COMC) ............................................................................................. 21 


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5.2.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM)
..................................................................................................................... 21 
5.2.9 
Clinical Global Impression–Severity (CGI-S) ............................................ 21 
5.2.10 Rett Syndrome Caregiver Burden Inventory (RTT-CBI) ........................... 21 
5.2.11 Impact of Childhood Neurologic Disability (ICND) Scale Total Score ..... 22 
5.3 
Study Day .......................................................................................................... 22 
5.4 
Baseline Definition ............................................................................................ 22 
5.5 
Analysis Visit Windows .................................................................................... 23 
5.5.1 
Unscheduled Assessments .......................................................................... 23 
5.5.2 
Multiple Measurements within Visit Windows .......................................... 23 
5.6 
Missing or Incomplete Date for Last Dose of Study Drug ................................ 23 
5.7 
Missing or Incomplete Dates for Prior or Concomitant Medications ............... 24 
5.8 
Missing or Incomplete Date for Adverse Events .............................................. 24 
5.9 
Missing Severity Assessment for Adverse Events ............................................ 24 
5.10 
Missing Relationship to Study Drug for Adverse Events ................................. 24 
5.11 
Character Values of Clinical Laboratory Variables .......................................... 24 
6. 
SUBJECT DISPOSITION ................................................................................. 25 
7. 
PROTOCOL DEVIATIONS ............................................................................. 26 
8. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS ........................ 27 
9. 
MEDICAL HISTORY ....................................................................................... 28 
10. 
EXTENT OF EXPOSURE AND TREATMENT COMPLIANCE .................. 29 
10.1 
Exposure to Study drug ..................................................................................... 29 
10.2 
Measurement of Treatment Compliance ........................................................... 29 
11. 
PRIOR, CONCOMITANT, AND POST-TREATMENT MEDICATION ...... 31 
12. 
EFFICACY ANALYSES .................................................................................. 32 
12.1 
Efficacy Variables ............................................................................................. 32 
12.2 
Adjustment for Covariates ................................................................................. 32 
12.3 
Handling of Missing Data ................................................................................. 33 
12.4 
Multiple Comparisons / Multiplicity ................................................................. 33 
12.5 
Examination of Subgroups ................................................................................ 33 
12.5.1 MECP2 Mutation Groupings ...................................................................... 34 
13. 
METHODS OF EFFICACY ANALYSES ....................................................... 35 
13.1 
Primary Efficacy Endpoints .............................................................................. 35 
13.1.1 Primary Analysis ......................................................................................... 35 


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13.1.2 Sensitivity Analyses .................................................................................... 37 
13.1.3 Supportive Analysis .................................................................................... 41 
13.2 
Key Secondary Efficacy Endpoints ................................................................... 41 
13.3 
Other Secondary Efficacy Endpoints ................................................................ 41 
13.4 
Exploratory Efficacy Analyses .......................................................................... 42 
13.4.1 CGI-I Responder Analysis .......................................................................... 42 
13.4.2 Hypothetical Strategy Estimand ................................................................. 42 
14. 
SAFETY ANALYSES ...................................................................................... 43 
14.1 
Adverse Events .................................................................................................. 43 
14.2 
Clinical Laboratory Variables ........................................................................... 44 
14.3 
Vital Signs ......................................................................................................... 48 
14.4 
Electrocardiogram (ECG) .................................................................................. 49 
14.5 
Physical Examination ........................................................................................ 51 
14.6 
Other Safety Endpoints ...................................................................................... 51 
15. 
CLINICAL PHARMACOKINETIC AND PHARMACODYNAMIC 
ANALYSES ...................................................................................................... 52 
16. 
INTERIM ANALYSIS ...................................................................................... 53 
17. 
DATA MONITORING/REVIEW COMMITTEE ............................................ 54 
18. 
COMPUTER METHODS ................................................................................. 55 
19. 
CHANGES TO ANALYSES SPECIFIED IN PROTOCOL ............................ 56 
20. 
REFERENCES .................................................................................................. 57 
21. 
APPENDICES ................................................................................................... 58 
21.1 
Schedule of Assessments ................................................................................... 58 
21.2 
Summary of Version Changes ........................................................................... 61 


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LIST OF TABLES 
Table 1 
Dosing Schedule Based on Weight at Baseline .................................................... 13 
Table 2 
Analysis Visit Windows ....................................................................................... 23 
Table 3 
Dosing Schedule and Total Expected Volume Based on Weight at Baseline ...... 29 
Table 4 
Conversion of Height of Remaining Liquid (cm) into Volume (mL) .................. 30 
Table 5 
Imputation Predictors ............................................................................................ 39 
Table 6 
Criteria for Potentially Clinically Important Laboratory Values – Hematology and 
Chemistry .............................................................................................................. 46 
Table 7 
Criteria for Potentially Clinically Important Laboratory Values - Urinalysis ...... 47 
Table 8 
Criteria for Potentially Clinically Important Vital Signs ...................................... 49 
Table 9  
Criteria for Potentially Clinically Important ECG Values .................................... 51 
 
LIST OF FIGURES 
Figure 1 
Schematic of Study Design ................................................................................... 14 
 


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ABBREVIATIONS 
AE 
adverse event 
ANCOVA 
analysis of covariance 
ATC 
Anatomical/Therapeutic/Chemical 
BMI 
body mass index 
CIs 
confidence intervals 
CGI-I 
Clinical Global Impression – Improvement  
CGI-S 
Clinical Global Impression – Severity  
COVID-19 
coronavirus disease 2019 
DSMB 
Data and Safety Monitoring Board  
eCRF 
electronic case report form 
ECG 
Electrocardiogram 
EDC 
electronic data capture 
FAS 
Full Analysis Set 
FDA 
Food and Drug Administration 
GJ 
Gastrojejunal 
GSD 
Guidance for Site Documentation and Data Management Querying of 
Data Impacted by COVID-19 
ICND 
Impact of Childhood Neurologic Disability Scale  
IRT 
interactive response technology 
MedDRA 
Medical Dictionary for Regulatory Activities 
MMRM 
mixed model for repeated measures 
OC 
observed cases 
PCI 
potentially clinically important 
PD 
pharmacodynamic(s) 
PHE 
public health emergency 
PK 
pharmacokinetic(s) 
PP 
Per Protocol 
QTcB 
QT Interval Corrected for Heart Rate using Bazett’s Formula 
QTcF 
QT Interval Corrected for Heart Rate using Fridericia’s Formula 


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RSBQ 
Rett Syndrome Behaviour Questionnaire 
RTT 
Rett Syndrome 
RTT-AMB 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
RTT-CBI 
Rett Syndrome Caregiver Burden Inventory 
RTT-COMC 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
RTT-CSS 
Rett Syndrome Clinical Severity Scale 
RTT-HF 
Rett Syndrome Clinician Rating of Hand Function 
RTT-VCOM 
Rett Syndrome Clinician Rating of Verbal Communication 
SAE 
serious adverse event 
SAP 
SD 
SE 
statistical analysis plan 
standard deviation 
standard error 
SOC 
system organ class 
TEAE   
treatment-emergent adverse event 
 
 
 


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


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2. 
OBJECTIVES 
2.1 
Primary Objective 
The primary objective of this study is to investigate the efficacy of treatment with oral 
trofinetide versus placebo in girls and women with Rett syndrome. 
2.2 
Key Secondary Objective 
The key secondary objective is to investigate the efficacy of treatment with oral trofinetide 
versus placebo on ability to communicate in girls and women with Rett syndrome. 
2.3 
Other Secondary Objectives 
Additional secondary objectives for this study are: 
• To investigate the benefit of treatment with oral trofinetide versus placebo on overall 
quality of life for girls and women with Rett syndrome 
• To investigate the efficacy of treatment with oral trofinetide versus placebo in girls 
and women with Rett syndrome on: 
o Hand function 
o Ambulation and other gross motor skills 
o Ability to communicate choices and preferences 
o Ability to communicate verbally 
• To investigate the efficacy of treatment with oral trofinetide versus placebo on a 
global assessment of the severity of illness in girls and women with Rett syndrome 
• To investigate the benefit of treatment with oral trofinetide versus placebo on the 
burden on caregivers of girls and women with Rett syndrome 
• To investigate the benefit of treatment with oral trofinetide versus placebo on the 
impact of the disability on the child’s and family’s everyday life 
2.4 
Safety Objective 
The safety objective of this study is to investigate the safety and tolerability of treatment with 
oral trofinetide versus placebo in girls and women with Rett syndrome. 


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2.5 
Pharmacokinetic Objectives 
The pharmacokinetic objectives of this study are: 
• To characterize the pharmacokinetics (PK) of trofinetide in girls and women with 
Rett syndrome 
• To assess the pharmacokinetic/pharmacodynamic (PK/PD) relationship using safety 
and efficacy endpoints in girls and women with Rett syndrome 
 
 


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3. 
STUDY DESIGN  
3.1 
General Study Design 
This study is a Phase 3, multicenter, randomized, double-blind, placebo-controlled, parallel-
group study in girls and women with Rett syndrome. 
The study drug is oral trofinetide or the matching placebo. Dose will be based on the 
subject’s weight at Baseline, as outlined in Table 1. Doses may be administered by 
gastrostomy tube (doses administered via gastrojejunal [GJ] tubes must be administered 
through the G-port). 
Table 1 
Dosing Schedule Based on Weight at Baseline 
Weight 
Dose  
Total Daily Dose 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 
Abbreviations: BID=twice daily 
Subjects will be stratified according to age stratum (5-10 years old, 11-15 years old, and 
16-20 years old) and Baseline Rett Syndrome Behaviour Questionnaire (RSBQ) severity 
(<35 total score and ≥35 total score). A minimum of 12 subjects are required to be 
randomized for each age stratum. The Sponsor, subjects, caregivers, and Investigators will be 
blinded to treatment assignment. 
The duration of participation for individual study subjects will be approximately 19 weeks, 
consisting of a screening period of up to 3 weeks, a 12-week double-blind treatment period, 
and a safety follow-up period of 30 days (for subjects who do not continue into the 
open-label extension study). The study completion date is defined as the date the final 
subject, across all sites, completes their final protocol-defined assessment. 
Approximately 28 sites will participate in this study. Approximately 184 subjects will be 
randomized (with a minimum of 12 subjects randomized for three age ranges [5-10 years old, 
11-15 years old, and 16-20 years old]) with a total of 92 subjects per treatment arm. 
The study design is summarized in Figure 1.  
 
 


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Figure 1 
Schematic of Study Design 
 
R=Randomization 
a If the subject continues into the open label extension (OLE) from the current study, she will not complete the 
follow-up visit and will roll over into the OLE. 
3.2 
Schedule of Assessments 
The schedule of assessments is in the Appendix (Section 21.1). 
3.3 
Randomization 
Eligible subjects will be randomized into one of two treatment groups (trofinetide or placebo) 
in a 1:1 ratio using an interactive response technology (IRT) system. The randomization will 
be stratified by age group (5-10 years old, 11-15 years old, and 16-20 years old) and Baseline 
RSBQ severity (<35 total score and ≥35 total score). The assignments will be based on a 
pre-generated permuted-block randomization schedule.  
3.4 
Blinding  
This is a double-blind study. Blinding will be assured by restricting access of Investigators 
and Sponsor personnel and/or designee to the treatment codes, and providing identical 
packaging for the trofinetide and placebo treatments.  
Unblinding of individual treatment assignment during the study is discouraged. The 
Investigator may break the blind in the event of a medical emergency if it is considered 
necessary for the care of the subject. The Investigator should attempt whenever possible to 
contact the Medical Monitor before unblinding a subject’s treatment to discuss the event. 
For the final analysis, the treatment codes for all subjects will be released to Acadia after all 
subjects have completed the study and the clinical database is locked. 
3.5 
Determination of Sample Size 
The sample size calculation was performed for the co-primary efficacy endpoints as a family 
of two hypothesis tests at an overall two-sided significance level of 0.05. A total sample size 
of 174 subjects in a 1:1 ratio to trofinetide or placebo was estimated to provide at least 90% 


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power for the hypothesis testing family assuming the following treatment differences (SD) 
estimated from Phase 2 study data: -4.4 (8) for the mean change from Baseline to Week 12 in 
the RSBQ total score and -0.5 (0.7) for the CGI-I mean score at Week 12. 
The sample size of 174 subjects will provide at least 95% power at a two-sided significance 
level of 0.05 for each individual hypothesis test within the family. Trofinetide will be claimed 
to be superior to placebo if both hypothesis tests within the family are shown to be 
statistically significant at 0.05.  The overall power to detect a treatment difference on both of 
the co-primary efficacy endpoints would be at least 90% (0.952).  
Adjusting for an anticipated discontinuation rate of up to 5%, approximately 184 subjects 
will be randomized in a 1:1 ratio to trofinetide or placebo to ensure 174 subjects who 
complete the study. 
3.6 
Coronavirus Disease 2019  
In March 2020, the emerging coronavirus disease 2019 (COVID-19) pandemic resulted in 
implementation of urgent safety measures designed to ensure subject safety. Mechanisms to 
record information on the potential impact of the COVID-19 pandemic on data itself, as well 
as data collection and integrity, were implemented (as detailed in the appendix of 
ACP-2566-003’s Data Management Plan titled “Guidance for Site Documentation and 
Data Management Querying of Data Impacted by COVID-19” [GSD] included in eTMF). 
Relationship to the public health emergency caused by the COVID-19 pandemic will be 
assessed for early terminations, protocol deviations, selected medications, and selected 
adverse events as detailed in the GSD. 
Subjects who were discontinued from Study ACP-2566-003 before completion because of 
the COVID-19 public health emergency and subjects who completed Study ACP-2566-003, 
but were prevented from entering Study ACP-2566-004 because of the COVID-19 public 
health emergency, may be re-evaluated for eligibility to enroll into the open-label extension 
study (Study ACP-2566-004). 
To assess the impact of COVID-19 PHE on the study population and data collection, 
summaries of demographics, baseline characteristics and Rett syndrome history by 
randomization date before or after the onset of COVID-19 (18 March 2020) and COVID-19 
PHE related intercurrent events will be summarized. 
 
 


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4. 
ANALYSIS SETS 
Randomized Analysis Set 
The Randomized Analysis Set consists of all subjects who were randomized. Subjects will be 
classified according to the randomized treatment assignment. 
Safety Analysis Set 
The Safety Analysis Set consists of all randomized subjects who received at least one dose of 
study drug. The Safety Analysis Set will be analyzed according to the actual treatment 
received. 
Full Analysis Set 
The Full Analysis Set (FAS) consists of all subjects who were randomized, received at least 
one dose of study drug, and have both a Baseline value and at least one post-Baseline value 
for the RSBQ total score or have at least one CGI-I score after taking study medication. The 
FAS will be analyzed according to the treatment they were randomized regardless of the 
actual treatment received. 
Per-protocol Analysis Set 
The Per-protocol Analysis Set consists of the subjects in the Full Analysis Set who do not 
have a major protocol violation that would affect interpretation of the efficacy data. The 
Per-protocol Analysis Set will be defined prior to study unblinding. The Per-protocol 
Analysis Set will be analyzed according to randomized treatment assignment. 
Pharmacokinetics (PK) Analysis Set 
The PK Analysis Set consists of the subjects in Safety Analysis Set with at least one 
measurable trofinetide whole blood concentration. Subjects will be classified according to the 
actual treatment received. 
 
 


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5. 
DATA HANDLING CONVENTIONS 
All data collected in the study will be listed. 
5.1 
General Data Reporting Conventions 
Continuous variables will be summarized using the following descriptive statistics: number 
of subjects, mean, median, standard deviation (SD), standard error (SE), minimum, and 
maximum. Unless specified otherwise, means, medians, and confidence intervals (CIs) will 
be presented to one more decimal place than the raw data, and the standard deviations and 
standard errors 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 percent of 
subjects in each category; the number of subjects and the percentage of subjects with missing 
data will be summarized for demographic and baseline characteristics (if applicable). 
Categories with zero counts will not have zero percentages displayed. For demographic 
summaries, percentages will be calculated by using the total number of subjects in the given 
treatment group as the denominator. Percentages will be presented with one decimal place. 
Duration in months will be calculated as ([the number of days / 365.25] *12). 
Unless specified otherwise, all statistical tests will be 2-sided hypothesis tests performed at 
the significance level of 5% for main effects and all CIs will be 2-sided 95% CIs. P-values 
will generally be presented to 4 decimal places; values less than 0.0001 will be presented as 
<0.0001. 
Clinical laboratory assessment values that are collected with “<” or “>” signs will generally 
be analyzed as the numerical value without the sign in tables and figures. In listings, these 
data will be reported as collected with the sign. 
5.2 
Derived Efficacy Variables  
In general, assessment total scores and subscores will be derived within the analysis datasets. 
In the event that total scores and/or subscores are also collected on the electronic case report 
from (eCRF), the derived values will be used for all analyses. Both the raw and derived 
scores will be presented in listings. 


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5.2.1 
Rett Syndrome Behaviour Questionnaire (RSBQ)  
The RSBQ is a 45-item caregiver-completed rating scale in which the caregiver rates items as 
“0” (Not True), “1” (somewhat or sometimes true) or “2” (very true). The RSBQ includes 
8 subscales:1) general mood, 2) breathing problems, 3) hand behavior, 4) face movements, 
5) body rocking/expressionless face, 6) night-time behaviors, 7) fear/anxiety, and 8) 
walking/standing.  The total score ranging from 0 to 90 will be calculated as the sum of the 
scores for all 45 items. Score for item 31 (Uses eye gaze to convey feelings, needs and 
wishes.) will be reversed (2-observed item score) in the calculation of total score and 
subscores.  Higher total scores indicate greater frequency of symptoms. If there are 9 or less 
missing item scores, the total score will be calculated by the arithmetic mean of non-missing 
items multiplied by 45, rounded to the nearest integer. The RSBQ total score will be 
considered missing if there are missing scores for 10 or more items. 
For the calculation of RSBQ subscores, the items under each RSBQ subscore will be 
summed. If an item is missing it will not be imputed and the subscore will be considered 
missing. 
RSBQ Subscores 
1) General mood consists of: 
a. Abrupt changes in mood (Item 14) 
b. Spells of screaming for no apparent reason during the day (Item 2) 
c. There are times when she appears miserable for no apparent reason (Item 16) 
d. Spells of inconsolable crying for no apparent reason during the day (Item 30) 
e. There are times when she is irritable for no apparent reason (Item 29) 
f. Screams hysterically for long periods of time and cannot be consoled (Item 
22) 
g. There are certain days/periods where she performs worse than usual (Item 15) 
h. Vocalises for no apparent reason (Item 36) 
2) Breathing problems consists of: 
a. There are times when breath is held (Item 5) 
b. Swallows air (Item 19) 
c. Abdomen fills with air and sometimes feels hard (Item 25) 
d. There are times when breathing is deep and fast (hyperventilation) (Item 1) 
e. Air or saliva expelled from mouth with force (Item 6) 
3) Hand behaviors consists of: 
a. Does not use hands for purposeful grasping (Item 18) 
b. Restricted repertoire of hand movement (Item 24) 
c. Hand movements uniform and monotonous (Item 20) 


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


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5.2.2 
Clinical Global Impression–Improvement (CGI-I) 
The CGI-I scale is used by the clinician to rate how much the subject’s illness has improved 
or worsened relative to a baseline state. A 7-point scale is used from 1=very much improved, 
2=much improved, 3=minimally improved, 4=no change, 5=minimally worse, 6=much 
worse, 7=very much worse. 
Higher CGI-I scores denote more severe illness and less improvement in the illness 
respectively. 
5.2.3 
Communication and Symbolic Behavior Scales Developmental Profile™ 
Infant-Toddler  Checklist – Social Composite Score (CSBS-DP-IT Social) 
The Checklist consists of 24 questions that range from 0 to 4 points within each of 7 Clusters. 
Credit of 0 points is given for items checked “Not Yet”, 1 point for items checked 
“Sometimes”, or 2 points for items checked “Often”. For items that describe a series of 
numbers or ranges, credit of 0 points is given for items checked “None” and 1 to 4 points for 
items containing numbered choices. 
Three composite scores assessing 7 skill areas can be calculated. The Social Composite score 
is comprised of 13 items in the skill areas “Emotion and Eye Gaze” (items 1 to 4), 
“Communication” (items 5 to 8), and “Gestures” (items 9 to 13). The Speech Composite 
score, including the skill areas “Sounds” and “Words” is based on items 14 to 18. The 
Symbolic Composite score, including the skill areas “Understanding” and “Object Use”, is 
based on items 19 to 24. 
The Social Composite raw score (comprised of items 1 to 13) ranging from 0 to 26 will be 
calculated as the sum of the scores for all 13 items. Higher Social Composite raw scores 
indicate better social communication development. If there are 2 or less missing item scores, 
the Social Composite raw score will be calculated by the arithmetic mean of the non-missing 
item scores multiplied by 13, rounded to the nearest integer. The Social Composite raw score 
will be considered as missing if there are missing scores for 3 or more items. 
5.2.4 
Overall Quality of Life Rating of the Impact of Childhood Neurologic 
Disability (ICND) Scale 
The overall quality of life score rating of the ICND will be analyzed as a separate endpoint. 
The numeric scale of the child’s overall quality of life ranges from 1 (“Poor”) to 
6 (“Excellent”); lower overall quality of life scores indicate lower quality of life. 
5.2.5 
Rett Syndrome Clinician Rating of Hand Function (RTT-HF) 
The RTT-HF is a clinician completed clinical assessment of the subject’s ability to use her 
hands for functional purposes. The assessment is made on an 8-point Likert scale (0-7) with 
0 denoting normal functioning and 7 the most severe impairment. 


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5.2.6 
Rett Syndrome Clinician Rating of Ambulation and Gross Motor Skills 
(RTT-AMB) 
The RTT-AMB is a clinician completed clinical assessment of the subject’s ability to sit, 
stand, and ambulate. The assessment is made on an 8-point Likert scale (0-7) with 0 denoting 
normal functioning and 7 the most severe impairment. 
5.2.7 
Rett Syndrome Clinician Rating of Ability to Communicate Choices 
(RTT-COMC) 
The RTT-COMC is a clinician completed clinical assessment of the subject’s ability to 
communicate her choices or preferences, which can include the use of nonverbal means such 
as eye contact or gestures. The assessment is made on an 8-point Likert scale (0-7) with 
0 denoting normal functioning and 7 the most severe impairment. 
5.2.8 
Rett Syndrome Clinician Rating of Verbal Communication (RTT-VCOM) 
The RTT-VCOM is a clinician completed clinical assessment of the subject’s ability to 
communicate verbally. The assessment is made on an 8-point Likert scale (0-7) with 
0 denoting normal functioning and 7 the most severe impairment. 
5.2.9 
Clinical Global Impression–Severity (CGI-S) 
The CGI-S is a 7-point scale that requires the clinician to rate the severity of the subject’s 
illness at the time of assessment, relative to the clinician’s experience with subjects who have 
the same diagnosis. Considering total clinical experience, a subject is assessed on severity of 
illness at the time of rating: 1=normal, not at all ill; 2=borderline ill; 3=mildly ill; 
4=moderately ill; 5=markedly ill; 6=severely ill; or 7=extremely ill. 
Higher CGI-S scores denote more severe illness and less improvement in the illness 
respectively. 
5.2.10 
Rett Syndrome Caregiver Burden Inventory (RTT-CBI) 
The RTT-CBI consists of 24 negatively worded items (Items 1 through 24). Frequency 
ratings are on a 5-point Likert scale including: 0-never; 1-rarely; 2-sometimes; 3-frequently 
and 4-nearly always. The RTT-CBI also includes 2 positively worded items (items 25 and 
26) that comprise the Optimism Index; this index will not be used for analysis. The total 
score ranging from 0 to 96 will be calculated as the sum of the scores for Items 1-24. If there 
are 4 or fewer missing item scores, the RTT-CBI total score will be calculated by  the 
arithmetic mean of the non-missing item scores multiplied by 24, rounded to the nearest 
integer. The RTT-CBI total score will be considered as missing if there are missing scores for 
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5.2.11 
Impact of Childhood Neurologic Disability (ICND) Scale Total Score 
The ICND scale evaluates the effect of four health problems on 11 aspects of the child’s or 
the family’s life which are scored 0 (“Not at all”), 1 (“A little”), 2 (“Some”), 3 (“A lot”), or 
“Does not apply”. The four health problems are 1) inattentiveness, impulsivity, or mood, 
2) ability to think and remember, 3) neurologic or physical limitations, and 4) epilepsy. The 
ICND total score ranging from 0 to 132 will be calculated as follows: 
1. For each health problem, the score will be calculated as the sum of the 11 item scores. 
The health problem score will be considered missing 
a.  if more than 5 item scores are either missing or “Does not apply” 
b. or if more than 3 item scores are missing for reasons other than the response 
of “Does not apply”. 
2. The ICND total score will be calculated as the sum of the average of each problem 
score multiplied by 11. 
𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆= ෍𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝_𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠_𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑒𝑒𝑘𝑘∗11
4
𝑘𝑘=1
 
where k indexes the four health problems. 
The ICND total score will be considered missing if at least 1 health problem score is 
missing. Imputed ICND total scores will be rounded to the nearest integer. 
Higher ICND total scores indicate worse health problems. The ICND total score does not 
include the Overall Quality of Life Rating. 
5.3 
Study Day 
If the date of assessment occurs on or after the first dose date, then study day will be 
calculated as (date of assessment – date of first dose) + 1. If the date of assessment occurs 
prior to the first dose date, then study day will be calculated as (date of assessment – date of 
first dose). There is no study day 0. 
5.4 
Baseline Definition 
Baseline data are defined as data collected which are prior to the administration of the first 
dose. If there is more than one value on or prior to Study Day 1, the value closest to and prior 
to the receipt of the first dose, whether scheduled or unscheduled, will be used as the 
Baseline value. 


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5.5 
Analysis Visit Windows  
Baseline of the study is defined as the last non-missing result, including results from repeated 
and unscheduled measurements, before first dosing. 
Efficacy, safety, and PK assessments will be summarized by analysis visit as presented in 
Table 2 below. 
Table 2 
Analysis Visit Windows 
Analysis Visit  
Target Study Day 
Study Day Interval 
Baseline (Day 1) 
1 
< 1 
Day 1 Post First Dose* 
1 
1 
Week 2 
15 
2 – 28 
Week 6 
43 
29 – 63 
Week 12 
85 
64 – 115 
*For ECG and PK assessments only; other assessments post-first dose on Day 1 will go to the 
Week 2 analysis visit window. 
5.5.1 
Unscheduled Assessments 
Both Scheduled and Unscheduled assessments, including the assessments at early termination 
visits, will be included for planned timepoint analyses based on the above analysis visit 
windowing rules. All assessments will be presented in data listings. 
5.5.2 
Multiple Measurements within Visit Windows 
In the event that more than one assessment falls within a given window, the assessment 
closest to the target study day will be selected for the by-visit analysis. If two assessments are 
equidistant from the target study day, then the chronologically last assessment will be used. 
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. 
5.6 
Missing or Incomplete Date for Last Dose of Study Drug  
For subjects with completely missing last dose date, the last dose date will be imputed by the 
last expected dosing date, defined as the earliest of the following dates: last drug kit dispense 
date + scheduled dosing interval per protocol, EOT/ET date and the return date of the last 
dispensed drug kit. 
For subjects with partial missing last dose date, the imputation will be compared against the 
last expected dosing date as defined above. Detailed algorithms will be documented in a 
separate programming specifications document. The missing or incomplete dates will be 
displayed in the data listings as reported on the eCRF rather than the imputed dates. 


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5.7 
Missing or Incomplete Dates for Prior or Concomitant Medications 
Missing or incomplete medication start or stop dates will be imputed for the purpose of 
determining whether the medication is taken concomitantly or not (see Section 11 for 
definition). When the chronological order of medication use relative to the study drug 
treatment period is unclear due to missing or incomplete date(s), the medication will be 
considered as concomitant. The imputation algorithms will be detailed in the analysis dataset 
specification document. The missing or incomplete dates as captured on the eCRF will be 
displayed in the data listings. 
5.8 
Missing or Incomplete Date for Adverse Events 
Missing or incomplete adverse event (AE) start dates will be imputed for the purpose of 
determining whether the AEs are treatment-emergent or not (see Section 14.1 for definition). 
When the chronological order of an AE onset relative to the study drug treatment period is 
unclear due to missing or incomplete date(s), the AE will be considered as 
treatment-emergent. The imputation algorithms will be detailed in the analysis dataset 
specification document. The missing or incomplete dates captured on the eCRF will be 
displayed in the data listings. 
5.9 
Missing Severity Assessment for Adverse Events 
If the severity is missing for an AE starting on or after the date of the first dose of study drug, 
then a severity of “Severe” will be assigned. The imputed values for severity assessment will 
be used for incidence summaries, while the actual values will be used in data listings. 
5.10 
Missing Relationship to Study Drug for Adverse Events 
If the relationship to study drug is missing for an AE starting on or after the date of the first 
dose of study drug, a causality of “Related” will be assigned. The imputed values for 
relationship to study drug will be used for incidence summaries, while the actual values will 
be presented in data listings. 
5.11 
Character Values of Clinical Laboratory Variables  
If the reported value of a clinical laboratory variable cannot be used in a statistical analysis 
due to, for example, a character string reported for a numeric variable, an appropriately 
determined coded value may be used in the statistical analysis. The coding algorithms will be 
detailed in the analysis dataset specification document. The actual values as reported in the 
database will be presented in data listings. 
 
 


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6. 
SUBJECT DISPOSITION 
For subjects who participate in the screening phase but are not randomized (screen failures), 
their demographics information (including age, sex, and primary race), screen failure reasons 
(the specific inclusion/exclusion criterion (or criteria) not met or other reasons including the 
reason due to the COVID-19 public health emergency (PHE) and protocol version will be 
listed. If a subject is re-screened, then the re-screening subject ID and the final enrollment 
status (whether eventually enrolled) will also be displayed in this listing. In addition, the 
frequency that the screen failure reasons are reported will be summarized. Note that one 
subject may be deemed ineligible for multiple inclusion/exclusion criteria and may be 
allowed to rescreen with the permission of the Medical Monitor, provided the screen failure 
was due to a temporary condition that subsequently resolved.  
The number of sites that screened at least 1 subject, number of sites that randomized at least 
1 subject, number of subjects screened, and number of unique subjects screened will be 
tabulated. In addition, the number of subjects enrolled at each site will also be tabulated by 
Analysis Set and by treatment group and overall. 
The number and percentage of subjects who completed the study, discontinued early (all 
discontinued and by discontinuation reasons including reason due to the COVID-19 PHE), 
and the reason for discontinuation will be summarized by treatment group using the 
Randomized and Full Analysis Sets. All subjects excluded from the Safety, Full or 
Per-protocol Analysis Sets, and the reason(s) for exclusion will be listed. The number and 
percentage of subjects who are excluded from the Per-protocol Analysis Set will be presented 
in a summary table by reason, and by treatment group and overall. 
 
 


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


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8. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS 
Demographics and baseline characteristics will be summarized by treatment group and 
overall for Randomized Analysis Set and Full Analysis Set using descriptive statistics. 
Variables include, but are not limited to, age, age group 1 (5-10 years old, 11-15 years old, 
and 16-20 years old), age group 2 (5-12 years old, 13-17 years old, and ≥18 years old), race, 
ethnicity, height, weight, BMI, Baseline RSBQ total score, Baseline RSBQ severity (< 35 
total score and ≥ 35 total score), Baseline CGI-S score, and Rett Syndrome Clinical Severity 
Scale (RTT-CSS) score at Screening. 
Race will also be categorized by White vs. Non-White. The reported age reflects a subject’s 
age at the informed consent date. 
To assess the impact of COVID-19 PHE on study population, summaries of demographics and 
baseline characteristics will be presented for subjects randomized before and after the onset of 
COVID-19 PHE (18 March 2020). 
 
 


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9. 
MEDICAL HISTORY 
Medical history reported terms will be coded with Medical Dictionary for Regulatory 
Activities (MedDRA), version 23.0 or newer. The subject incidence will be summarized for 
each system organ class (SOC) and preferred term by treatment group and overall for the 
Safety Analysis Set. A subject will be counted only once per SOC or per preferred term for 
the summary. 
A listing of the SOC, preferred term, body system, verbatim for the medical history 
condition/event, start and stop dates (when available), and an indicator for whether or not the 
condition is ongoing will be provided. 
Rett Syndrome History 
The age at Rett syndrome diagnosis, the age at  first symptoms noticed, the number and 
percentage of subjects by MECP2 gene mutation, and the number and percentage of subjects 
with more than one MECP2 mutation (Yes, No) collected on the Rett History eCRF page will 
be summarized. 
To assess the impact of COVID-19 PHE on study population, summaries of Rett syndrome 
history will be presented for subjects randomized before and after the onset of COVID-19 PHE 
(18 March 2020). 
Rett syndrome history will be listed. A flag variable will indicate if a subject was randomized 
after the onset of COVID-19 PHE. 
 
 


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10. 
EXTENT OF EXPOSURE AND TREATMENT COMPLIANCE 
Extent of exposure and treatment compliance will be summarized as both continuous 
variables and categorical variables by treatment group for Safety Analysis set. 
10.1 
Exposure to Study drug 
Duration of exposure to study drug will be calculated for each subject as (last dose date – 
first dose date + 1). The number and percentage of subjects within each of the following 
exposure levels in terms of duration of exposure will also be tabulated: <2 week (1 to 13 
days), 2 to <4 weeks (14 to 27 days), 4 to <6 weeks (28 to 41 days), 6 to <8 weeks (42 to 55 
days), 8 to <10 weeks (56 to 69 days), 10 to <12 weeks (70 to 83 days), and >=12 weeks (84 
days or longer). 
10.2 
Measurement of Treatment Compliance 
The study drug is provided in liquid form supplied in a 500 mL bottle. Study drug 
compliance will be calculated based on the drug accountability and dose modification data as 
collected on the eCRF. The study drug compliance will be calculated as (the total volume of 
drug actually taken (in mL) divided by the total volume of drug expected to be taken)*100. 
The total volume of drug expected to be taken will be based on the duration of exposure and 
dosing schedule as in Table 3. However, if there is any dose modification prescribed by the 
investigator due to intolerance, the total drug expected to be taken will be adjusted 
accordingly to account for the modified prescribed dose schedule as recorded in the EDC. 
The total volume of drug actually taken will be calculated as (total drug dispensed – total 
drug returned (height in cm of drug remaining converted into volume in mL based on the 
following conversion Table 4). 
Treatment compliance will be summarized as a categorical variable. The number and 
percentage of subjects within each of the following compliance levels will be tabulated: 
<80%, 80 to 120% and >120%. 
Details of treatment compliance calculation are provided in a separate programming 
specifications document. 
Table 3 
Dosing Schedule and Total Expected Volume Based on Weight at Baseline 
Weight 
Dose 
Total Daily Dose 
Total Expected Volume 
12-20 kg 
30 mL (6 g) BID 
60 mL (12 g) 
60 mL x Number of Days Expected to be Dosed  
>20-35 kg 
40 mL (8 g) BID 
80 mL (16 g) 
80 mL x Number of Days Expected to be Dosed  
>35-50 kg 
50 mL (10 g) BID 
100 mL (20 g) 
100 mL x Number of Days Expected to be Dosed  
>50 kg 
60 mL (12 g) BID 
120 mL (24 g) 
120 mL x Number of Days Expected to be Dosed  
BID = twice daily 




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11. 
PRIOR, CONCOMITANT, AND POST-TREATMENT MEDICATION 
Prior medication is defined as any medication with stop dates prior to the date of the first 
dose of 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 2020 or newer version. 
The number and percentage of subjects taking prior, concomitant and post-treatment 
medications will be tabulated separately by each drug class (ATC Level 3) and medication 
preferred term, treatment group and overall for Safety Analysis Set. Multiple medication 
usage by a subject in the same category will be counted only once. Listings of the prior, 
concomitant and post-treatment medications will also be provided. 
COVID-19 Infection Related Medications 
Concomitant medication analyses described above will also be tabulated and listed by 
relationship to COVID-19 Infection (Not related to COVID-19 Infection vs. Related to 
COVID-19 Infection). 
 
 


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12. 
EFFICACY ANALYSES 
Unless otherwise specified, all efficacy analyses will be performed using the planned 
treatment assignments based on the randomization schedule for the Full Analysis Set. 
12.1 
Efficacy Variables 
Co-Primary Efficacy Endpoints 
The co-primary efficacy endpoints are: 
• RSBQ total score – change from Baseline to Week 12 
• CGI-I score at Week 12 
Key Secondary Efficacy Endpoints 
The key secondary endpoint is change from Baseline to Week 12 in CSBS-DP-IT Social 
Composite Score. 
Other Secondary Efficacy Endpoints 
The other secondary endpoints for this study are change from Baseline to Week 12 in: 
• Overall Quality of Life Rating of the ICND  
• RTT-HF score 
• RTT-AMB score 
• RTT-COMC score 
• RTT-VCOM score 
• CGI-S score 
• RTT-CBI total score (items 1-24) 
• ICND total score 
12.2 
Adjustment for Covariates 
The Baseline RSBQ total score will be included as a covariate for the analysis of change 
from baseline in the RSBQ total score using the mixed model for repeated measures 
(MMRM). The Baseline CGI-S score will be included as a covariate for the analysis of CGI-I 
score using the MMRM method. The Baseline CSBS-DP-IT Social Composite Score will be 
included as a covariate for the analysis of change from Baseline in the CSBS-DP-IT Social 
Composite Score using the MMRM method. Unless otherwise specified, the randomization 
stratification variables of age group (5-10 years old, 11-15 years old, and 16-20 years old) 


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and Baseline RSBQ severity (<35 total score and ≥35 total score) will also be included as 
fixed effects in the MMRM method. 
12.3 
Handling of Missing Data 
The primary analyses of 
• RSBQ total score – change from Baseline to Week 12 (co-primary efficacy endpoint) 
• CGI-I score at Week 12 (co-primary efficacy endpoint) 
• CSBS-DP-IT Social Composite Score – change from Baseline to Week 12 (key 
secondary endpoint) 
will be performed assuming missing at random (MAR) using the direct likelihood-based 
MMRMs and missing scores (after any imputation of individual missing items as described 
in Section 5.2) will not be imputed. The MMRM method is unbiased under the MAR 
assumptions in the estimation of treatment effect that could have been observed if all subjects 
had continued on treatment for the full study duration (EMA, 2009). 
12.4 
Multiple Comparisons / Multiplicity 
There are a total of three hypotheses, two primary hypotheses and a key secondary 
hypothesis, grouped in two families. The two primary hypotheses form one family and the 
key secondary, the other family. The family of the two primary hypotheses constitute the 
gatekeeper. Each hypothesis in the gatekeeper will be tested at the two-sided α=5% without 
multiplicity adjustment because both hypotheses in the family must be rejected to move 
forward to the next family. 
The family of the key secondary hypothesis will be tested if and only if trofinetide is superior 
to the placebo with respect to both co-primary efficacy endpoints. The key secondary 
hypothesis will be tested at the two-sided α=5%. This hierarchical testing of the family of 
primary hypotheses and that of the key secondary hypothesis will provide a strong control of 
the family-wise error rate at the nominal α=5%. 
12.5 
Examination of Subgroups 
Treatment effect will be examined with respect to the co-primary and key secondary efficacy 
endpoints within the subgroups of age group 1 (5-10 years old, 11-15 years old, and 16-20 
years old) , age group 2 (5-12 years old, 13-17 years old, and ≥18 years old), and Baseline 
RSBQ severity (<35 total score and ≥35 total score).  Subgroup analysis by MECP2 mutation 
groupings will also be performed; if a subgroup size is too small, categories may be pooled 
prior to analysis, and footnoted appropriately. 


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


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13. 
METHODS OF EFFICACY ANALYSES 
13.1 
Primary Efficacy Endpoints 
13.1.1 
Primary Analysis 
Estimand 
Target Population: Female subjects 5 to 20 years of age inclusive with Rett syndrome, as 
defined by the inclusion/exclusion criteria of the study. 
Variables: The co-primary endpoints in this study are the change from baseline to Week 12 
in the RSBQ total score and the CGI-I score at Week 12. 
Intercurrent events: Possible intercurrent events include: 
• Treatment Discontinuation not due to COVID-19 PHE 
• Treatment Discontinuation due to COVID-19 PHE 
• COVID-19 PHE events leading to intermediate missing data 
• Non-COVID-19 PHE events leading to intermediate missing data 
• Remote Assessments (regardless of due to COVID-19 PHE or not) 
Summary measure: Treatment difference in the mean change from Baseline to Week 12 in 
the RSBQ total score and treatment difference in the mean CGI-I score at Week 12. 
In general, the treatment policy strategy will be used to handle all intercurrent events in the 
primary analysis. That is, observations on the co-primary efficacy endpoints will be used 
regardless of the occurrence of intercurrent events. 
Alternative approaches to handling intercurrent events will be addressed in sensitivity 
analyses.  
Hypotheses 
Let Δ1 ( 𝝁𝝁𝑇𝑇𝑇𝑇𝑇𝑇1 −𝝁𝝁𝑃𝑃𝑃𝑃𝑃𝑃1) be the difference in the mean change from Baseline to Week 12 in 
the RSBQ total score and Δ2 ( 𝝁𝝁𝑇𝑇𝑇𝑇𝑇𝑇2 −𝝁𝝁𝑃𝑃𝑃𝑃𝑃𝑃2) be the difference in the mean CGI-I score at 
Week 12 between the trofinetide (µTRO) and placebo (µPBO) groups. 
The null hypothesis is: ∆𝑖𝑖= 0; 𝑖𝑖= 1, 2. 
The alternative hypothesis is: ∆𝑖𝑖≠0; 𝑖𝑖= 1, 2. 
Primary Estimator 
The hypothesis testing will be performed for Full Analysis Set using the direct likelihood 
MMRM method assuming missing data are missing at random (MAR). The MMRM for the 


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RSBQ analysis will include effects for treatment group, age group (5-10 years old, 11-15 
years old, and 16-20 years old), Baseline RSBQ severity (<35 total score and ≥35 total 
score), visit, treatment-by-visit interaction as fixed effects, Baseline RSBQ total score, and 
the Baseline RSBQ total score-by-visit interaction as covariates and subjects as random 
effect. The MMRM for the CGI-I analysis will include effects for treatment group, age group 
(5-10 years old, 11-15 years old, and 16-20 years old), Baseline RSBQ severity (<35 total 
score and ≥35 total score), visit, treatment-by-visit interaction as fixed effects, subjects as 
random effect, and Baseline CGI-S score, and the Baseline CGI-S score-by-visit interaction 
as covariates. An unstructured covariance matrix will be used and the Kenward-Roger 
approximation will be used to adjust the denominator degrees of freedom. 
The following is sample SAS code for the MMRM: 
For RSBQ: 
proc mixed data=xxx; 
    class trt subjid visit age-group RSBQ-severity; 
    model cfb-RSBQ-total-score = trt|visit age-group RSBQ-severity base-RSBQ 
    base-RSBQ*visit/ ddfm=kr2; 
    repeated visit / subject = subjid type=UN; 
    LSmeans trt*visit / pdiff cl OM; 
run; 
For CGI-I: 
proc mixed data=xxx; 
    class trt subjid visit age-group RSBQ-severity; 
    model CGI-I = trt|visit age-group RSBQ-severity base-CGI-S 
    base-CGI-S*visit/ ddfm=kr2; 
    repeated visit / subject = subjid type=UN; 
    LSmeans trt*visit / pdiff cl OM; 
run; 
In the event that the model fails to converge using the unstructured covariance matrix, the 
following covariance structures will be modeled in the order given (i.e., from least 
parsimonious to most parsimonious): heterogeneous Toeplitz, heterogeneous compound 
symmetry, heterogeneous autoregressive(1), Toeplitz, compound symmetry, 
autoregressive(1), variance components. The first covariance structure that allows for 
convergence will be selected for the final model. 
Summary statistics for the RSBQ total score (observed and change from Baseline) and the 
CGI-I score (observed), LS means, the between-group difference in LS mean with the 


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corresponding 95% confidence interval, p-value, and the effect size (Cohen’s d) will be 
presented at each post-Baseline visit for Full Analysis Set.  
The treatment effect size (Cohen’s d) is calculated using the following formula: 
Effect Size = LS mean difference / SD 
SD is the model-based estimate, i.e., the estimated standard deviation from the modeled 
covariance matrix. The sign (+ or -) of the effect size will be chosen so that a positive value 
favors trofinetide. 
LS mean ± SE from the MMRM models over time for the change from Baseline values by 
treatment group will be displayed in line plots for Full Analysis set.  
The analyses for co-primary endpoints will also be performed for the subgroups of age group 
1 (5-10 years old, 11-15 years old, and 16-20 years old), age group 2 (5-12 years old, 13-17 
years old, and ≥18 years old), and Baseline RSBQ severity (<35 total score and ≥35 total 
score). In addition, subgroup analysis by MECP2 mutation groupings will also be performed.  
13.1.2 
Sensitivity Analyses 
The following sensitivity analyses of the co-primary efficacy endpoints are planned to 
account for intercurrent events. 
13.1.2.1 Pattern-Mixture Models Assuming Missing Not At Random (PMM-MNAR) 
The sensitivity analysis is implemented for Full Analysis Set using multiple imputations that 
are based on the distribution of placebo group responses over time to account for the 
intercurrent event of treatment discontinuations and missing assessments. The underlying 
assumption is that subjects with missing data due to early withdrawal evolve in the same way 
as placebo subjects that remain in the study. 
The following steps are involved and the imputed values will be constrained to be within the 
limits of 0 - 90 for RSBQ total score and 1 - 7 for CGI-I score: 
 Non-monotone (intermediate) missing data at Baseline (not applicable for CGI-I 
score), Week 2, Week 6, and Week 12 will be multiply imputed using the Markov 
chain Monte Carlo (MCMC) method to create 50 monotone datasets. The imputation 
models for post baseline RSBQ total score and CGI-I score will include effects as 
follows:  
Post baseline RSBQ total score: age group (5-10 years old, 11-15 years old, and 16-20 
years old), Baseline RSBQ severity (<35 total score and ≥35 total score), Baseline 
RSBQ total score, and treatment group 


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CGI-I score: age group (5-10 years old, 11-15 years old, and 16-20 years old), 
Baseline RSBQ severity (<35 total score and ≥35 total score), Baseline CGI-S score, 
and treatment group. 
• The monotone missing data will be imputed using a parametric, sequential linear 
regression method in which the missing data are imputed only on data from the 
placebo arm. A single imputation will be performed sequentially at each visit for each 
of the 50 imputed datasets. The predictors and their order in the PROC MI VAR 
statement for each visit are summarized in Table 5. The following SAS codes are to 
impute missing observations using the control-based pattern imputation method. 
For RSBQ: 
proc mi data=xxxx seed=2566003 nimpute=1 out=outname; 
  by _Imputation_; 
  class Trt Baseline_RSBQ_severity Age_group; 
  monotone reg (/details); 
  mnar model( W2_RSBQ W6_RSBQ W12_RSBQ / modelobs= (Trt='Placebo')); 
  var Age_group Baseline_RSBQ_severity Baseline_RSBQ W2_RSBQ W6_RSBQ 
W12_RSBQ; 
run; 
 
For CGI-I: 
  by _Imputation_; 
  proc mi data=xxxx seed=2566003 nimpute=1 out=outname; 
  class Trt Baseline_RSBQ_severity Age_group; 
  monotone reg (/details); 
  mnar model( W2_CGI-I W6_CGI-I W12_CGI-I / modelobs= (Trt='Placebo')); 
  var Age_group Baseline_RSBQ_severity Baseline_CGI-S W2_CGI-I W6_CGI-I 
W12_CGI-I; 
run;


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 Table 5 
Imputation Predictors 
Visit 
RSBQ Total Score - Predictors 
CGI-I Score - Predictors 
Week 2 
Age group, Baseline RSBQ severity, 
Baseline RSBQ total score 
Age group, Baseline RSBQ severity, Baseline 
CGI-S score 
Week 6 
Age group, Baseline RSBQ severity, 
Baseline RSBQ total score, Week 2 RSBQ 
total score 
Age group, Baseline RSBQ severity, Baseline 
CGI-S score, Week 2 CGI-I score 
Week 12 
Age group, Baseline RSBQ severity, 
Baseline RSBQ total score, Week 2 RSBQ 
total score, Week 6 RSBQ total score 
Age group, Baseline RSBQ severity, Baseline 
CGI-S score, Week 2 CGI-I score, Week 6 
CGI-I score 
Note: Age group (5-10 years old, 11-15 years old, and 16-20 years old); Baseline RSBQ severity (<35 total 
score and ≥35 total score).  
• The change from Baseline to each post baseline visit values will then be calculated 
and analyzed for each of the 50 fully imputed datasets using the analysis of 
co-variance (ANCOVA) model. The ANCOVA model for RSBQ total score will have 
treatment group, Baseline RSBQ severity, and age group as fixed factors and Baseline 
RSBQ total score as a covariate; the ANCOVA model for CGI-I score will have 
treatment group, age group, and Baseline RSBQ severity as fixed factors and Baseline 
CGI-S score as a covariate. 
• The results will be summarized by post baseline visit using the SAS MIANALYZE 
procedure to yield a combined estimate for treatment effect with its associated 95% 
confidence interval (CI) and p-value. 
13.1.2.2 Sensitivity Analysis for Missing Data Due to COVID-19 PHE (PMM-COVID-
19-PHE-MAR Variant) 
The sensitivity analysis differs from Section 13.1.2.1 by using a different assumption for 
missing data due to COVID-19 PHE related withdrawal (after intermediate data has been 
imputed). This variant will operate under the assumption that missing data after withdrawal 
due to COVID-19 PHE related withdrawal is MAR, while missing data after withdrawal not 
due to COVID-19 PHE is MNAR and assumed to evolve in the same way as placebo subjects 
that remain in the study. 
This sensitivity analysis involves extra steps from the previously described sensitivity 
analysis. After the monotone missing data sets are imputed the data sets will be duplicated 
and made into two partitions 1) the original 50 monotone missing data sets 2) the 50 
monotone data sets consisting of only subjects that did not have COVID-19 PHE related 
withdrawal. 


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Using the original 50 monotone missing data sets (partition 1); the monotone missing data 
due to COVID-19 PHE withdrawal will be imputed using a parametric, sequential linear 
regression method based on data from the respective treatment arm. Then the subjects who 
had COVID-19 PHE related withdrawal will be merged into the partition of the 50 monotone 
data sets consisting of only subjects that did not have COVID-19 PHE related withdrawal 
(partition 2). The monotone missing data in this combined partition will be imputed using a 
parametric, sequential linear regression method based on data from the placebo arm. 
Then analysis will be performed as previously specified in Section 13.1.2.1. 
13.1.2.3 Sensitivity Analysis Accounting for Impact of Remote Assessments on the Co-
Primary Endpoints 
To assess the impact of the intercurrent events of remote assessments on the primary 
analysis, an indicator variable will be assigned to flag subjects that had assessments done 
remotely for the co-primary efficacy endpoints at any visit including baseline (Yes/No); the 
flag will be defined for each of the co-primary efficacy variables, separately. The remote visit 
flag indicator variable and the interaction between treatment group and remote visit flag 
indicator variable will be included as factors in the MMRM model. 
13.1.2.4 Additional Summaries Evaluating the Impact of COVID-19 on the Primary 
Endpoint 
To assess the impact of COVID-19 PHE on data collection, the following will be summarized: 
• The number and percentage of subjects impacted by at least one of the following 
COVID-19 PHE-related ICEs; and the number and percentage of subjects impacted by 
each of the following COVID-19 PHE-related ICEs: 
o Treatment Discontinuation due to COVID-19 PHE 
o Intermediate missing data due to COVID-19 PHE 
o Remote assessments due to COVID-19 PHE) 
• The number and percentage of subjects with remote assessments not due to COVID-19 
PHE 
• The number and percentage of subjects with missed assessments for the co-primary 
endpoints by visit 
o Due/not due to COVID-19 PHE 
• The number and percentage of subjects with remote assessments for the co-primary 
endpoints by visit 
o Due/not due to COVID-19 PHE 


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13.1.2.5 Derived Baseline RSBQ Randomization Strata 
Baseline RSBQ severity score (<35 total score and ≥35 total score) was used as randomization 
strata without reversing the score for item 31 “Uses eye gaze to convey feelings, needs and 
wishes.”. A sensitivity analysis will be performed using the same model described for the co 
primary endpoints and the key secondary endpoint, except the model will replace the Baseline 
RSBQ severity strata from IRT with a derived Baseline RSBQ severity strata score where item 
31 is reversed. 
13.1.3 
Supportive Analysis 
Supportive analysis of the co-primary endpoints will be analyzed, similarly to the primary 
analysis, using the Per-Protocol set. 
13.2 
Key Secondary Efficacy Endpoints  
The key secondary endpoint, change from baseline to Week 12 in the CSBS-DP-IT Social 
Composite Score for the Full Analysis Set, will be analyzed using the direct likelihood 
MMRM method assuming missing data are MAR. The MMRM model will include effects 
for treatment group, age group (5-10 years old, 11-15 years old, and 16-20 years old), 
Baseline RSBQ severity (<35 total score and ≥35 total score), visit, treatment-by-visit 
interaction as fixed effects, Baseline CSBS-DP-IT Social Composite Score, and the Baseline 
CSBS-DP-IT Social Composite Score -by-visit interaction as covariates and subjects as 
random effect. Supportive analyses of the key secondary endpoint will use the Per-Protocol 
set. 
13.3 
Other Secondary Efficacy Endpoints 
Analyses for the other secondary efficacy endpoints in this section will be performed for Full 
Analysis Set using the observed values. 
 
RTT-HF, RTT-AMB, RTT-COMC, RTT-VCOM, and CGI-S  
The change from Baseline will be analyzed using the MMRM method similar to the primary 
analysis for the co-primary endpoints. The MMRM model will include effects for treatment 
group, age group (5-10 years old, 11-15 years old, and 16-20 years old), Baseline RSBQ 
severity (<35 total score and ≥ 35 total score), visit, the treatment-by-visit interaction, 
Baseline score, and the Baseline score-by-visit interaction. 
Summary statistics (observed and change from Baseline), LS means, the between-treatment 
difference in LS mean with the corresponding 95% confidence interval, p-value, and the 
effect size (Cohen’s d) will be presented at each post-Baseline visit. 


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Quality of Life Rating of the ICND, ICND Total Score, and RTT-CBI 
The change from Baseline to Week 12 will be summarized by treatment group and analyzed 
using the ANCOVA model with treatment group, age group (5-10 years old, 11-15 years old, 
and 16-20 years old), and Baseline RSBQ severity (<35 total score and ≥ 35 total score) as 
fixed factors and the corresponding Baseline value as a covariate. 
13.4 
Exploratory Efficacy Analyses 
13.4.1 
CGI-I Responder Analysis 
Responder analysis based on CGI-I  will be performed as exploratory analysis.  Responder is 
defined as CGI-I scores of 1 or 2 (much or very much improved). Responder analysis will be 
performed in two ways:1) Missing CGI-I will be imputed as non-responder and 2) using 
observed cases only (subjects with missing CGI-I will be excluded).  The proportion of 
responders will be presented at each visit.  Treatment comparison will be performed using 
logistic regression adjusted for baseline CGI-S, age group, and baseline RSBQ severity.  
Analysis for RSBQ subscales will also be performed similarly as that for the change from 
baseline in RSBQ total score. 
Histograms of CGI-I by visit will be presented. 
13.4.2 
Hypothetical Strategy Estimand 
An exploratory analysis will be performed for the co-primary endpoints that will differ from 
the primary analysis which uses the treatment policy estimand. For this exploratory analysis, 
the intercurrent event is permanently discontinuing the study treatment more than 2 and half 
days before final RSBQ/CGI-I assessment. The hypothetical strategy will be used to estimate 
the hypothetical effect if all subjects in the full analysis set adhered to the prescribed treatment 
until their final co-primary efficacy assessments. In this analysis, the final RSBQ/CGI-I 
assessment data observed after more than 5 doses from the last dose (i.e., missed the last 5 
scheduled doses which is defined as the morning and evening doses of the prior two days and 
the morning dose of the visit of the efficacy assessment) will be considered missing and 
excluded from the analysis. The same MMRM model as the primary analysis will be used after 
excluding the data observed beyond 5 doses from last dose.     
 
 


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14. 
SAFETY ANALYSES 
All safety analyses will be performed using the actual treatment for the Safety Analysis Set.  
14.1 
Adverse Events  
Adverse events will be coded using MedDRA dictionary, Version 23.0 or newer. 
An AE (classified by preferred term) will be considered a treatment-emergent AE (TEAE) if 
started after first dose administration and no later than last dose date + 30 days. AEs reported 
on Day 1 based on Baseline (pre-dose) findings (e.g., clinically significantly abnormal vital 
signs, laboratory test results, or electrocardiogram parameters) will not be considered as 
TEAEs. 
The event counts, the number, and percentage of subjects reporting TEAEs in each treatment 
group will be tabulated by system organ class (SOC) and preferred term; by SOC, preferred 
term, and maximum severity; and by SOC, preferred term, and relationship to study drug. If 
more than one TEAE occurs with the same preferred term for the same subject, then the 
subject will be counted only once for that preferred term using the most severe and most 
related occurrence for the summarization by severity and by relationship to study drug. The 
display in these tables will be sorted alphabetically by SOC and then by descending subject 
frequency for the preferred terms (counts from trofinetide group) within each SOC. 
The event counts, the number and percentage of subjects with any TEAEs will also be 
tabulated by preferred term without SOCs. This table will be sorted by descending subject 
frequency within trofinetide group. 
The incidence of most frequently reported (preferred terms reported by ≥ 5% of subjects in 
any treatment group) TEAEs, SAEs reported after treatment start, TEAEs leading to drug 
withdrawn, and TEAEs related to study drug will be summarized by SOC, preferred term, 
and treatment group. The tables will be sorted alphabetically by SOC and then by descending 
frequency within each SOC in trofinetide group. In addition, the incidence of fatal 
treatment-emergent AEs (i.e., events that cause death) will be summarized separately by SOC 
and preferred term. 
These summary tables except for the most frequently reported TEAEs tables will also be 
presented by relationship to COVID-19 Infection (Not related to COVID-19 Infection vs. 
Related to COVID-19 Infection). 
An AE listing by subject will display all events, including those which occur during 
screening, and will include the verbatim term in addition to the MedDRA SOC and preferred 
term. This listing will also include all relevant eCRF data associated with the event: date of 
onset, date resolved, date of last dose, severity, frequency, outcome, relationship to study 


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drug, and action taken with study drug. Separate listings will be presented for subjects with 
treatment-emergent SAEs, related TEAEs, TEAEs leading to drug withdrawn, fatal TEAEs 
(if any), and TEAEs Related to COVID-19 Infection. 
14.2 
Clinical Laboratory Variables 
Due to COVID-19 PHE related disruptions, it is possible that some test results may be 
collected from a local laboratory. Local laboratory results and the associated normal ranges 
will be converted to SI units; local laboratory results, in SI units, will then be normalized to 
central lab ranges to be  included in summary data analysis together with the central 
laboratory results. The normalization will be performed using the following scale 
transformation equation: 
𝑠𝑠= 𝐿𝐿𝑠𝑠+ (𝑥𝑥−𝐿𝐿𝑥𝑥) 𝑈𝑈𝑠𝑠−𝐿𝐿𝑠𝑠
𝑈𝑈𝑥𝑥−𝐿𝐿𝑥𝑥
 
where s is the normalized individual laboratory value to be used for summary; x is the 
original value from the local lab; Lx and Ux are the lower and upper limits from the local lab; 
Ls and Us are the lower and upper limits from the central lab. 
For labs with only a single upper (or lower) limit, the following scale transformation equation 
will be used: 
𝑠𝑠= 𝑥𝑥𝑈𝑈𝑠𝑠
𝑈𝑈𝑥𝑥
 
where s is the normalized individual laboratory value to be used for summary; x is the 
original value from the local lab; Us is the upper (or lower) limit from the central lab; Ux is 
the upper (or lower) limit from the local laboratory. Local laboratory results and normalized 
results will be included in data listings. Only central lab and normalized local lab results will 
be used for summary of change from baseline, shift, and potentially clinically important 
(PCI) analyses. 
Clinical laboratory assessments are performed at Screening Visit 1, Baseline (Week -3 to 0), 
Week 2, Week 6 (no urinalysis), and Week 12/EOT. 
• 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) 
o Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-
glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total bilirubin 
(TBIL), lactate dehydrogenase (LDH) 


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o HbA1c  
 HbA1c will only be performed at Visit 1 (Screening) 
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), Visit 2 
(Baseline), and Visit 5 
• Pregnancy test 
o A serum pregnancy test will be performed at all designated visits for subjects 
of childbearing potential 
• Hematology tests include the following: 
o Complete blood count (CBC) including: 
- White blood cell (WBC) count 
- Complete differential (relative and absolute) 
- Hematocrit (Hct), hemoglobin, red blood cells (RBC), platelets 
- Reticulocyte count 
• Urinalysis tests include the following: 
o Blood, RBCs, WBCs, protein, glucose, ketones, specific gravity, pH 
Clinical laboratory values (in Système International [SI] units) and the change from Baseline 
values will be summarized by treatment group 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, visit and treatment group. 
Laboratory values will also be summarized in shift tables by treatment group, 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 and treatment group. 
For the shift to the overall post-Baseline minimum or maximum, all post-Baseline values will 


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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 and treatment group. 
Clinical laboratory values are potentially clinically important (PCI) if they meet either the 
low or high PCI criteria listed in Tables 6 and 7. The number and percentage of subjects with 
post-Baseline PCI values for each of the categories in Tables 6 and 7 will be summarized by 
treatment group 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 treatment group, and the denominator is the number of subjects with at least 1 post-
Baseline laboratory value for the given parameter and treatment group.  
Table 6 
Criteria for Potentially Clinically Important Laboratory Values – 
Hematology and Chemistry 
Analyte 
Conventional 
Unit 
Low PCI 
Criteria 
High PCI 
Criteria 
 
SI Unit 
Low PCI 
Criteria 
High PCI 
Criteria 
Hematology (whole 
blood) 
 
 
 
 
 
 
Hemoglobin 
(female) 
g/dL 
<10 
>17 
g/L 
<100 
>170 
Hematocrit (female) 
% 
<30 
>50  
L/L 
<0.3 
>0.5  
Leukocyte (White 
Blood Cell Count) 
x 103/uL 
≤2.8 
≥15 
x 109/L 
≤2.8 
≥15 
Neutrophils 
x 103/uL 
≤1.5 
No upper 
limit 
x 109/L 
≤1.5 
No upper 
limit 
Platelet Count 
x 103/uL 
≤75 
≥700 
x 109/L 
≤75 
≥700 
Chemistry (serum 
or plasma) 
 
 
 
 
 
 
ALT 
U/L 
No lower 
limit  
≥3 X ULN 
U/L 
No lower 
limit  
≥3 X ULN 
AST 
U/L 
No lower 
limit  
≥3 X ULN 
U/L 
No lower 
limit  
≥3 X ULN 
Total Bilirubin 
mg/dL 
No lower 
limit 
≥1.5 ULN 
umol/L 
No lower 
limit 
≥1.5 ULN 
BUN 
mg/dL 
No lower 
limit 
≥30.0 
mmol/L 
No lower 
limit 
≥10.71 


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Table 6 
Criteria for Potentially Clinically Important Laboratory Values – 
Hematology and Chemistry (Continued) 
 
Analyte 
Conventional 
Unit 
Low PCI 
Criteria 
High PCI 
Criteria 
 
SI Unit 
Low PCI 
Criteria 
High PCI 
Criteria 
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 
Table 7 
Criteria for Potentially Clinically Important Laboratory Values - Urinalysis 
Urinalysis (qualitative dipstick) 
Low PCI Criteria 
High PCI Criteria 
Blood (occult blood) 
Not Applicable 
≥ Moderate 
Protein 
Not Applicable 
≥ 100 mg/dL 
Glucose 
Not Applicable 
≥ 500 mg/dL 
 
Clinical laboratory data will be displayed in data listings with date and study day of 
collection. All units will be displayed according to SI conventions for units. Out of range 
values will be flagged in the data listings (i.e., ‘L’ or ‘H’). A separate listing will be provided 
for a subset of the chemistry, hematology, and urinalysis analytes with values classified as 
PCI.  
At the initiation of the study, the main reference ranges (the reference ranges provided by the 
central laboratory validated for use for the general population) were used for the purpose of 
flagging abnormal laboratory values for the investigator to assess clinical significance. 
During the study, at the request of a leading investigator an alternative set of reference 
ranges, more specific to patients with Rett syndrome rather than to the general population, 


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was used for the purpose of flagging abnormal lab values for the investigator to assess 
clinical significance.  
All analysis will be done using the main reference ranges, not the alternative ranges. During 
the course of the study the main reference ranges were updated by the central laboratory. The 
latest version of the reference ranges provided by the central laboratory will be used for all 
analysis; PCI status and categorical labs (Low, Normal, High) and shifts will be derived from 
the latest version of the main reference range. Both of the reference ranges (main and 
alternative) and the corresponding categorical assessment will be listed. 
The pregnancy results (positive or negative) will be presented in a listing. 
14.3 
Vital Signs 
Vital signs will be collected throughout the study; height will be measured at Screening and 
Week 12/EOT; weight will be measured at Screening, Baseline, and Week 12/EOT. 
Observed vital signs including weight and BMI and the changes from Baseline at each post-
Baseline visit will be summarized by treatment group using descriptive statistics. 
Vital sign values will be considered PCI if they meet the criteria listed in Table 8. The 
number and percentage of subjects with post-Baseline vital signs that are PCI will be 
summarized by treatment group 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, visit and treatment group, and the denominator is the number of subjects 
with non-missing values for the given parameter, visit and treatment group. For the overall 
post-Baseline summary, the numerator for the percentage is the number of subjects with at 
least 1 post-Baseline PCI vital sign for the given parameter and treatment group, and the 
denominator is the number of subjects with at least 1 post-Baseline vital sign for the given 
parameter and treatment group. A listing of overall and of subjects with any PCI vital sign 
values will be provided. 
 
 


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Table 8 
Criteria for Potentially Clinically Important Vital Signs 
Vital Sign 
Parameter 
 
Unit 
Criteria 
Observed Value 
And/Or 
Change Relative 
to Baseline 
Change from Supine 
to Standing 
Systolic blood 
pressure 
(supine or sitting) 
mmHg 
≥180 
And 
Increase of ≥20 
- 
≤90 
And 
Decrease of ≥20 
- 
Diastolic blood 
pressure (supine or 
sitting) 
mmHg 
≥105 
And 
Increase of ≥15 
- 
≤50 
And 
Decrease of ≥15 
- 
Pulse (supine or 
sitting) 
bpm 
≥120 
And 
Increase of ≥15 
- 
≤50 
And 
Decrease of ≥15 
- 
Weight  
kg 
Not Applicable 
Increase of ≥7% 
- 
Decrease of ≥7% 
- 
14.4 
Electrocardiogram (ECG) 
12-lead ECGs are collected throughout the study at every visit.  
When ECG is collected multiple times (e.g. at Screening visit, Baseline visit both before 
dosing and 2-3 hours after dosing, and Week 12/EOT/ET visit), the average of all available 
values on the same day will be considered as one assessment for the summaries, except for 
Day 1 where averages will be by pre-dose and post-dose averages. The averages will be 
rounded to integer values. 
Observed (average) of ECG variables (e.g., heart rate, PR interval, QRS interval, QT interval, 
QTcB and QTcF interval) and the changes from Baseline at each assessment time point will 
be summarized by treatment group.  
Observed (average) of QTcB and QTcF will also be categorized into the following categories 
(msec) and the number and percentage of subjects in each category will be summarized by 
treatment group at each visit and for the overall post-Baseline maximum: 
• Observed: ≤450, 451 - ≤480, 481 - ≤500, and >500; >450; >480. 
• 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 


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non-missing cardiologist’s interpretation at Baseline and the given visit for the given 
treatment group. For the summaries of shift from Baseline to the overall post-Baseline worst 
interpretation, the denominator is the number of subjects with non-missing Baseline and at 
least 1 post-Baseline cardiologist’s interpretation for the given treatment group. 
Electrocardiogram variable average values will be considered PCI if they meet the criteria 
listed in Table 9. The number and percentage of subjects with post-baseline PCI values will 
be summarized by treatment group at each post-Baseline visit and for overall post-Baseline. 
For the by-visit summary, the numerator for the percentage is the number of subjects with a 
post-Baseline PCI ECG for the given parameter, visit and treatment group, and the 
denominator is the number of subjects with non-missing values for the given parameter, visit 
and treatment group. For the overall post-Baseline summary, the numerator for the 
percentage is the number of subjects with at least 1 post-Baseline PCI ECG for the given 
parameter and treatment group, and the denominator is the number of subjects with at least 
1 post-Baseline ECG value for the given parameter and treatment group. A listing of overall 
and of all subjects with any PCI ECG values will be provided. 
 
 


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Table 9  
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  
14.5 
Physical Examination 
Physical examinations are performed throughout the study at every visit in the clinic. 
Physical examination results (normal, abnormal, and not done) will be summarized in a 
frequency table by treatment group, body system and visit. A listing of overall physical 
examination data will be listed. 
14.6 
Other Safety Endpoints 
There are no other safety endpoints in this study. 
 
 


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


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16. 
INTERIM ANALYSIS 
No interim analysis is planned in this study. 
 
 


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17. 
DATA MONITORING/REVIEW COMMITTEE 
An independent Data and Safety Monitoring Board (DSMB) will review safety information 
on a regular basis throughout the study. The DSMB will be independent of the Sponsor and 
will be empowered to recommend stopping the study due to safety concerns. The DSMB may 
review blinded, unblinded, or partially unblinded data, but the Sponsor and the Investigators 
will remain blinded to the data provided to the DSMB until the official unblinding of the 
database at the completion of the study. The membership, activities, responsibilities, and 
frequency of meetings will be described separately in the DSMB charter. 
 
 


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


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


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20. 
REFERENCES 
EMA (2009). Guideline on Missing Data in Confirmatory Clinical Trials, European 
Medicines Agency, London, UK. 
ICH E9(R1): Addendum on Estimands and Sensitivity Analysis in Clinical Trials to the 
Guideline on Statistical Principles for Clinical Trials, Adopted on November 2019 
FDA Guidance on Conduct of Clinical Trials of Medical Products during COVID-19 
Pandemic Guidance for Industry, Investigators, and Institutional Review Boards (March 
2020) 
Points to consider on implications of Coronavirus disease 4 (COVID-19) on methodological 
aspects of ongoing clinical 5 trials, March 25 2020. 
 
 


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21. 
APPENDICES 
21.1 
Schedule of Assessments 
Period 
Screening Baseline Double-blind Treatment Period 
Safety 
Follow-upb 
Visit Week 
 
0 
2a 
6 
12/EOT/ET 
EOT/ET+ 
30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
N/A 
±3 
±4 
+3 
+4 
Type of Visitk 
Clinic or Off-site 
Telephone or 
Telemedicine 
Informed consent 
X 
 
 
 
Xc 
 
Inclusion/exclusion criteria 
X 
X 
 
 
 
 
Medical history and demographics 
X 
 
 
 
 
 
Confirm documented                            
Rett diagnosis and MECP2 mutation 
X 
 
 
 
 
 
Rett syndrome history 
X 
 
 
 
 
 
Rett Syndrome Clinical Severity 
Scale  
X 
 
 
 
 
 
Physical examinationk 
X 
X 
X 
X 
X 
 
Vital signsd 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
X 
 
Weight 
X 
X 
Xk 
Xk 
Xk 
 
12-lead electrocardiogram (ECG)e 
X 
Xe 
X 
X 
X 
 
Clinical laboratory tests (hematology, 
chemistry) 
X 
X 
X 
X 
X 
 
Urinalysis 
X 
X 
X 
 
X 
 
TSH, Free T3, Free T4 
X 
X 
 
 
X 
 
HbA1c 
X 
 
 
 
 
 
Serum pregnancy testf 
X 
 
X 
X 
X 
 
Blood samples for pharmacokinetics 
 
Xg 
Xh 
Xh 
Xh 
 
Blood sample for optional analysis 
for biomarkersi 
 
X 
 
 
X 
 
Rett Syndrome Behaviour 
Questionnaire (RSBQ) 
X 
X 
X 
X 
X 
 
Clinical Global Impression–
Improvement (CGI-I) 
 
 
X 
X 
X 
 
 


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21.1 
Schedule of Assessments (Continued) 
Period 
Screening Baseline Double-blind Treatment Period 
Safety 
Follow-upb 
Visit Week 
 
0 
2a 
6 
12/EOT/ET 
EOT/ET+ 
30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
N/A 
±3 
±4 
+3 
+4 
Type of Visitk 
Clinic or Off-site 
Telephone or 
Telemedicine 
Clinical Global Impression–Severity 
(CGI-S) 
X 
X 
X 
X 
X 
 
Communication and Symbolic 
Behavior Scales-Developmental 
Profile™ Infant-Toddler (CSBS-DP-
IT) Checklist  
 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Hand Function (RTT-HF) 
 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Ability to Communicate Choices 
(RTT-COMC)k 
 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Ambulation and Gross Motor Skills 
(RTT-AMB) 
 
X 
X 
X 
X 
 
Rett Syndrome Clinician Rating of 
Verbal Communication 
(RTT-VCOM) 
 
X 
X 
X 
X 
 
Rett Syndrome Caregiver Burden 
Inventory (RTT-CBI) 
 
X 
 
 
X 
 
Impact of Childhood Neurologic 
Disability Scale (ICND) 
 
X 
 
 
X 
 
Dispensing and review of semi-
structured caregiver diary 
X 
X 
X 
X 
X 
 
Concomitant medications 
X 
X 
X 
X 
X 
X 
Assessment of adverse events 
X 
X 
X 
X 
X 
X 
Randomization 
 
X 
 
 
 
 
Study drug dispensedj 
 
X 
 
 
 
 
Authorization of study drug 
dispensationj 
 
X------------------------X 
 
 
 


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21.1 
Schedule of Assessments (Continued) 
Period 
Screening Baseline Double-blind Treatment Period 
Safety 
Follow-upb 
Visit Week 
 
0 
2a 
6 
12/EOT/ET 
EOT/ET+ 
30 days 
Visit Number 
1 
2 
3 
4 
5 
 
Visit window (days) 
N/A 
N/A 
±3 
±4 
+3 
+4 
Type of Visitk 
Clinic or Off-site 
Telephone or 
Telemedicine 
Study drug returnj 
 
 
X------------------------------X 
 
Study drug accountabilityj 
 
 
X 
X 
X 
 
Abbreviations: EOT=end of treatment; ET=early termination; HbA1c=glycosylated hemoglobin; MECP2=methyl-CpG-
binding protein 2 gene; TSH=thyroid stimulating hormone 
 
a 
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 [±3 days] after the first day of dosing).  
b 
Subjects who roll over into the OLE study will not have the safety follow-up telephone call.  
c 
For subjects who decide to continue into the open-label extension (OLE) study, informed consent for the OLE 
must be obtained prior to performing the Week 12/EOT procedures.  
d 
Vital signs will include body temperature, resting respiration rate, sitting systolic and diastolic blood pressure, and 
pulse rate. The sitting blood pressure will be measured after the subject has been sitting for ≥3 minutes.  
e 
ECGs will be completed in triplicate at Visit 1 (Screening), at Visit 2 (Baseline) both before dosing and 2-3 hours 
after dosing, and at Visit 5 (Week 12/EOT/ET). A single ECG will be completed at Visit 3 (Week 2) and Visit 4 
(Week 6).  
f 
For subjects who have reached menarche and have not had surgical sterilization.  
g 
A predose PK blood sample must be collected before administration of study drug. A postdose PK blood sample 
will be collected at the end of ECG assessment 2-3 hours after study drug administration. 
h 
PK samples at Visits 3, 4, and 5 will be collected at one of the following time intervals: 1) 2-3 hours after dosing 
OR 2) 4-6 hours after dosing OR 3) 7-11 hours after dosing. Every effort should be made to collect the PK 
samples at discrete time intervals during Visits 3, 4, and 5. However, if the interval is the same across these visits, 
then the collection time should vary within that interval.  
i 
Participation in the effort to identify biomarkers is an optional component of the study requiring a separate 
informed consent, which may be obtained at any time during the study. If consent is obtained after Baseline, only 
the sample at Visit 5 (or upon early termination) will be taken.  
j 
Investigational product will be shipped directly to the subject. Confirmation of any delivery to the subject will be 
made by a visiting nurse. Study drug shipment, return, and accountability will be performed in accordance with the 
drug distribution plan. In addition, study drug will be dispensed at the site during the Baseline visit when the visit 
is conducted in the clinic.  
k 
Study visits may be done off-site rather than in the clinic with the prior approval of the Sponsor or Medical 
Monitor. Screening, Baseline, and EOT visits 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. The RTT-COMC should be completed if possible, but it is not required. 


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21.2 
Summary of Version Changes 
Version No: 
Document History 
Description of Update 
Author(s) 
Version Date 
1.0 
Original version 
 
04 January 2021 
2.0 
Added MECP2 Groupings; 
added definitions of RSBQ 
subscores; clarified analysis visit 
windowing; added additional 
sensitivity analyses; added 
additional descriptive analysis to 
assess COVID-19 PHE impact 
to data collection 
 
20 May 2021 
 
 
 
3.0 
Updated MECP2 Groupings; 
clarified reference ranges used 
over the course of the study, 
clarified text throughout. 
 
20 October 2021