Repurposing Mirtazapine in Rett Syndrome: a Multicentric Open Label Phase II Study
Phase: PHASE2
Conditions: RETT Syndrome With Proven MECP2 Mutation
Sponsor: University of Trieste

 
 
 
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Clinical Study Protocol 
Repurposing mirtazapine in Rett syndrome: 
a multicentric open label Phase II study 
 
Protocol Code 
MirtaRett 
Investigational Product Name/Number 
Mirtazapine 
Short title 
Repurposing mirtazapine in Rett 
syndrome: a multicentric open label 
Phase II study  
Sponsor Name and Legal Registered 
Address 
University of Trieste 
Dipartimento di Scienze della Vita (DSV) 
Via via Edoardo Weiss, 2 34128Trieste 
(ITALY) 
Telephone: +39 040 558 8724 (Office) –  
+39 040 558 2443 (Lab)  
Fax: +39 040 558 2445 
 
EU CT No. 
2024-515411-21-00 
Version Number 
2.0 
Date 
20/12/2024 
 
 
 
 
 

 
 
 
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Statement of confidentiality  
Information in this protocol and accompanying documents contains privileged or 
confidential information that is the property of University of Trieste. It is understood that 
the information will not be used, divulged, or published without prior written consent of 
University of Trieste, except to the extent such disclosure is required by applicable laws 
and regulations. 

 
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Protocol Signature Sheet and Acknowledgement 
 
The Università degli Studi di Trieste and the Sponsor representative have discussed 
this protocol version and agreed that it contains all necessary details for carrying out the 
trial. The investigations will be performed as by protocol except in the case of medical 
emergency.  
Problems related to this trial should be referred to the Scientific Coordinator, Prof 
Enrico Tongiorgi. 
The Scientific Coodinator agrees to conduct and supervise the clinical study. 
The sponsor hereby confirms that all investigators and institutions participating in this 
clinical trial agree to permit clinical trial-related monitoring, audits, and regulatory 
inspections. This includes providing direct access to source data and documents.  
 
Telephone: +39 040 558 8724 (Office) –  
+39 040 558 2443 (Lab)  
Fax: +39 040 558 2445 
 
 
 
Sponsor Signature: 
 
 
 
Signature:  _________________________  
           Date: 16/07/2024 
 
       Prof. Enrico Tongiorgi, Scientific Coordinator  
              
 
 
 

 
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Study Personnel 
 
SPONSOR 
 
Contact Name: Prof Enrico Tongiorgi   
Position: Scientific Coordinator MirtaRett 
Address: Università degli Studi di Trieste, Via L. Giorgieri 5, Building Q - 34127 Trieste - 
ITALY 
Tel: +39 040 558 8724 (Office) - +39 040 558 2443 (Lab) 
Fax: +39 040 52445 
Email: tongi@units.it  
 
PHARMACOVIGILANCE 
Contact Name: CVBF Safety Management Team 
Address: Consorzio per Valutazioni Biologiche e Farmacologiche (CVBF), Viale Cesare 
Battisti 17, Pavia, Italy 
Tel.: +39 373 8530614 
Fax: +39 080 9909321 
Email: pharmacovigilance@cvbf.net  
 
INVESTIGATIONAL SITES 
 
The full list of investigational sites will be kept in the Trial Master File and reported 
below: 
 
• 
Partner 1. Centro Epilessia – Unità Neurologia Pediatrica, ASST Ospedale 
Santi Carlo Paolo - Dipartimento Scienze della Salute, Università di Milano.  
• 
Partner 2. Unità di Neuropsichiatria Infantile, IRCCS, Istituto Giannina Gaslini, 
Genova.  
• 
Partner 3. Unità di Pediatria, Dipartimento della Donna e dei Bambini – 
Policlinico S. M. alle Scotte. Siena.  
• 
Partner 4. Dipartimento di Patologia Umana dell'Adulto e dell'Età Evolutiva 
"Gaetano Barresi", Policlinico Universitario "G. Martino, University of Messina. 
Messina. 
 

 
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1. Synopsis  
 
Study Title  
Repurposing Mirtazapine in Rett syndrome: a 
multicentre, open label, single- arm, phase II study 
(MirtaRett Study).  
Study code 
EU CT NUMBER 
MirtaRett 
2024-515411-21-00 
Study drug  
Mirtazapine (MTZ), a noradrenergic and specific 
serotonergic tetracyclic antidepressant (NaSSA) with an 
excellent safety profile.  
Background  
Rett Syndrome (RTT) is a rare disease caused by a 
specific genetic mutation in the MECP2 gene located on 
the female X chromosome and therefore particularly 
affects female subjects. It is characterised by an altered 
development of the nervous system and therefore it 
leads to an intellectual deficit.  
Some of the common symptoms associated with the Rett 
syndrome include: 
a) Learning problems and delayed development of 
motor and cognitive skills 
b) Loss of already acquired language skills 
c) Involuntary hand movements such as twisting, 
tapping or pushing, and hand-washing type 
movements 
d) Coordination and balancing problems 
e) Difficulties 
in 
verbal 
and 
non-verbal 
communication 
f) Repetitive behaviour, such as hand flapping or 
biting 
g) Breathing problems, such as sleep apnoea or 
hypoventilation 
h) Gastrointestinal problems, such as constipation or 
gastro-oesophageal reflux 
i) Alterations in mood and social behaviour, such as 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 6 of 29  
irritability, anxiety or estrangement 
j) Sleep problems, such as insomnia or restless 
sleep. 
Due to the fact that Rett syndrome is a progressive 
degenerative condition, symptoms may get worse over 
time and may be different from person to person. 
Particularly in childhood, the syndrome results in severe 
disability that makes impossible the active participation 
in physical activities and affects the ability of conception. 
Background data for dose selection 
Antidepressant drugs are typically administered using a 
scalar dosing approach, starting with low doses and 
gradually increasing them. This method allows for 
monitoring individual responses to the medication, as 
higher initial doses can lead to more severe side effects. 
Antidepressants generally take 2-4 weeks to show 
clinical effects, necessitating a careful titration schedule. 
For instance, the typical starting dose of mirtazapine in 
adults is around 15 mg/day, with a maximum of 50 
mg/day, while studies have also examined its use in 
specific 
populations, 
such 
as 
those 
with 
major 
depressive disorder or respiratory issues. In children and 
adolescents, lower doses are recommended due to 
potential side effects like gastrointestinal issues and self-
harm, which are particularly relevant for patients with 
Rett syndrome. 
Hypothesis and 
preliminary results  
The 
reduced 
in 
monoamine 
levels 
(serotonin, 
noradrenaline 
and 
dopamine) 
observed 
in 
RTT, 
suggested 
the 
possibility 
to 
use 
antidepressant 
treatments such as MTZ, to improve symptoms. RTT 
Mirtazapine (MTZ) is a tetracyclic noradrenergic and 
serotonergic specific antidepressant (NaSSA) with a 
favourable safety profile. In young male MeCP2-null 
mice, MTZ induced a marked improvement in cardiac 
and respiratory rhythm deficits, restored glutamatergic 
and GABAergic transmission and fully recovered 
microcephaly; in female MeCP2 heterozygous mice, it 
reduced sensory hypersensitivity and improved motor 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 7 of 29  
skills.  
In adult patients, MTZ promoted improved sleep, mood, 
hand stereotypies, social interactions and aggression. 
Therefore, considering these preliminary results in 
humans, we hypothesise that MTZ could be beneficial in 
RTT children.  
Study design  
We propose a multicentre, open label, single-arm, phase 
II study to evaluate safety and efficacy of MTZ in the 
treatment of mood, sleep quality, motor symptoms in 
particular hand control, in Rett syndrome children and 
adults. Other outcomes such as autonomic function, 
behavioural, caregiver burden and overall clinical 
severity will be examined along with neuroplasticity and 
metabolism biomarkers.  
Number of study 
participants 
 
Subjects included in the study are female patients with a 
diagnosis of RTT, confirmed by mutation of the MECP2 
gene which is responsible for the development of the 
disease, and who meet the inclusion/exclusion criteria of 
the study. 
The total number of participating patients is 54, aged 
between 5 and 40 years, divided into three groups of 18 
patients each (5-10 years, 11-17 years and 18-40 years).
Sample size justification  The minimum sample size (15 patients per age group) 
has been estimated considering the rarity of the disease 
under study and the preliminary results obtained in an 
open label test of mirtazapine in adult RTT patients.  
Primary endpoint: Motor-Behavior Assessment Scale 
(MBAS) change from Baseline.  
Summary Statement: A sample size of 15 achieves 
90.5% power to detect a difference of 18.0% between 
the null hypothesis (H0) of MBAS change from Baseline 
mean of -20.0% and the alternative hypothesis (Ha) of 
MBAS change from Baseline mean of -38.0% with an 
estimated standard deviation of 19.8% and with a 
significance level (alpha) of 0.05 using a two-sided one-

 
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sample t-test.  
Assuming approximately 15% of drop-out rate, 54 
patients are expected to be enrolled. 
Inclusion criteria 
Patients can only be included in the study if all the 
following conditions are satisfied: 
1. Female aged 5 to 39 years inclusive, at the time 
of signing the informed consent. 
2. Girls of childbearing age negative to pregnancy 
test. 
3. Body weight > 10 kg. 
4. Diagnosis of RTT based on consensus clinical 
criteria (Neul, 2010) and a confirmed mutation in 
MECP2 gene. 
5. Breathing dysfunction (at least one of the 
following): period apnoea, intermittent 
hyperventilation, breath holding spells, air 
swallowing, forced expulsion of air and /or saliva. 
6. Ten episodes or more/day of breathing 
dysfunction during wakefulness in the week prior 
to the screening visit (parents report). 
7. Stable medication regimen for 4 weeks prior to 
beginning the study (if receiving services - 
physical, occupational, or speech therapy - 
subjects must be on a stable regimen of these 
services for 3 months prior to beginning the 
study). 
8. Female patients of childbearing potential must 
use a highly effective contraceptive method such 
as combined hormonal contraception (containing 
estrogen and progestin) associated with ovulation 
suppression (oral, intravaginal, transdermal); 
progestin-only hormonal contraception associated 
with ovulation suppression (oral, injectable, 
implantable); intrauterine device; hormone-
releasing intrauterine system. Sexual abstinence 
is considered a highly effective contraceptive 
method if it aligns with the individual's usual 
lifestyle. 
9. Written consent signed by parent/legal 
guardian/representative prior to screening visit 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 9 of 29  
10. Patient is cooperative, willing to complete the 
study, and capable of doing so with assistance of 
a caregiver. 
11. Caregiver is able to understand the instructions 
and fully participate. 
Exclusion criteria 
The female patients cannot be included in the study if 
they meet one of the following exclusion criteria: 
1. they are participating in another investigational 
clinical trial; 
2. Hypersensitivity to MTZ; 
3. Clinically significant (as determined by the 
investigator) cardiovascular, respiratory, 
gastrointestinal, renal, hepatic, haematological 
pathologies or other pathologies, in addition to 
those directly related to RTT. In particular, 
patients with the following parameters will be 
excluded: leucocyte count is < 4000/mm2; 
neutrophil count is < 2000/mm3; hyponatremia (< 
125 mmol/L); renal dysfunction (creatinine > 2 X 
ULN), hepatic dysfunction (AST, ALT, bilirubin > 2 
X ULN); or if severe diabetes mellitus is present; 
4. QTcF interval at ECG > 450 msec; 
5. Scheduled surgery during the study; 
6. Severe diabetes mellitus (hyperglycaemia with 
values above 250/300 mg/dL);  
7. Pregnancy, breastfeeding. 
8. Evidence of clinically significant malnutrition with 
BMI (or BMI) (kg/m2) < 13. 
9. Patients who manifested prior suicidal ideation. 
Population assessment 
A total of three populations will be used for treatments 
and analyses. Study participants who have satisfied the 
enrolment criteria will be classified in the designated 
population and will only be included in analyses for 
which they have available data.  
Data analysis will be performed using the following 
analysis sets:  
 The Enrolled (ENR) population will consist of all 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 10 of 29  
patients who are enrolled into the study (i.e., 
signed ICF and met the eligibility criteria) and may 
or may not receive the study treatment. 
 The Intent to Treat (ITT) population will consist of 
all patients who received at least one dose of 
study medication and have at least Baseline (Day 
0) and End of Treatment (week 24) assessments 
with primary efficacy data. The ITT population will 
be used to present efficacy data.  
 The Per-Protocol (PP) set would include all ITT 
patients who met all inclusion/exclusion criteria 
liable to affect the primary assessment and did 
not present major protocol deviations. The 
detailed reasons for excluding patients from the 
PP set will be fully defined and documented 
before database lock. The PP population will be 
used to present efficacy data.  
 The Safety population will consist of all patients 
who received at least one dose of study 
medication. The Safety population will be used to 
present the demographic, baseline data and all 
safety data.  
The ITT will be the primary efficacy analysis set; the 
same analysis will be additionally performed on the PP 
set: any discrepancy between results obtained on both 
analysis sets will be discussed in the Clinical Study 
Report. All population sets for analysis will be discussed 
during data review meeting and authorization of subject’s 
inclusion/exclusion from each analysis set will be defined 
prior to the database lock.  
Patient disposition and withdrawals, including count and 
percentage of patients screened, screen failures, 
completed the study, discontinued the study, patient 
meets newly developed or not previously recognized and 
reason for discontinuation will be presented for the ENR 
population. Number of patients in each population, 
including reasons for exclusion, will be presented for 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 11 of 29  
ENR population.  
Subjects who withdraw prematurely can be part of any 
population (Safety, ITT or PP) depending on the 
individual follow-up characteristics and according to 
population derivation. Data collected on study patients 
up to the time of withdrawal must remain in the trial 
database.  
Drug administration  
From the day of the baseline visit (referred to as T0) to 
day 14 of the study, all enrolled subjects will start taking 
the drug with minimum dosage (Dose level 1) per age 
group: 
- 3.75 mg/per day (0.25 ml) oral solution for group 5-10 
years; 
- oral solution of 7.5 mg/day (0.50 ml) for group 11-17 
years old  
- oral solution of 15 mg/day (1 ml) for group 18-40 years. 
Afterwards from Day 15 until Week 24, the optimal 
dosage to be achieved (Dose level 2) will be: 
- oral solution of7.5 mg/ per day (0.50 ml) for group 5-10 
years; 
- 15 mg/ oral solution per day (1 ml) for group 11-17 
years;  
- oral solution of 30 mg/ per day (2 ml) for group 18-40 
years. 
Primary objective 
 
The study aims to evaluate the efficacy of MTZ to induce 
improving changes in social 
behaviour,orofacial/respiratory and motor/physical area 
and to assess the safety of MTZ. 
This study will provide information on what symptoms 
will MTZ be able to correct, depending on the age and 
severity of the subject. 
Secondary objectives 
1. To assess the effectiveness of MTZ in improving 
anxiety, depression and mood.  
2. To assess the effectiveness of MTZ in improving 
sleep quality.  
3. To assess the effectiveness of MTZ in improving 
hand function.  

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 12 of 29  
4. To assess the improvement or worsening of the 
subject's general clinical condition over time.  
5. To assess the effectiveness of the drug in reducing 
the severity and number of symptoms associated 
with Rett syndrome.  
6. To assess the effectiveness in improving lung 
function. This will be assessed by measuring thoracic 
and abdominal respiratory movements, airflow, 
arterial oxyhaemoglobin saturation (HbSaO2), heart 
frequency and posture. 
7. To assess the improvement in parental/carer stress. 
The rating scale used will be the Parenting Stress 
Index (PSI-SF). 
8. To assess the improvement in the levels of 
neurotrophic factors involved in the regulation of 
mood and cognition.  
Primary endpoint  
The rating scale used is the Motor-Behavior Assessment 
Scale (MBAS). 
The drug will be considered effective if the treatment 
decreases 
the 
Motor-Behavior 
Assessment 
Scale 
(MBAS) score (maximum score=68) by at least 8.5 
points (12.5%) compared to baseline.  
Adverse events (AEs) and serious adverse events 
(SAEs), both  related and non-related.   
Secondary endpoints  
1. The drug will be considered effective if the treatment 
reduces the overall Anxiety, Depression, and Mood 
Scale (ADAMS) score (maximum score=84) by at 
least 10.6 points (12.5%) and reduces the overall 
Rett Syndrome Behaviour Questionnaire (RSBQ) 
score by at least 11.25 points (12.5%). 
 
2. The drug will be considered effective if the treatment 
improves the measurements obtained from the 
medical devices for remote sensing (Youcare Smart 
T-shirt 
and 
the 
Actigraphy), 
and 
the 
Sleep 
disturbances scale for children (SDSC) scores by at 
least 20% compared to the initial value. 
 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 13 of 29  
3. The drug will be considered effective if the 
Purposeful Hand Function scale (PHF) score 
increases at least 2 points above the baseline score. 
 
4. The drug will be considered effective if the Clinical 
Global Impression of Change scale (CGI-C) has a 
score decrease at least 1 point. 
 
5. The drug will be considered effective if the Rett 
Syndrome Severity Scale (RCSS) scores decrease 
at least 3%, i.e. by 2 points. 
 
6. The 
assessment 
will 
be 
performed 
through 
measurements of thoracic and abdominal respiratory 
movements, 
airflow, 
arterial 
oxyhaemoglobin 
saturation (HbSaO2), heart rate and posture. 
 
7. The rating scale used will be the Parenting Stress 
Index (PSI-SF). An improvement in parental stress 
will be considered as such, if the Parenting Stress 
Index (PSI-SF) shows a reduction of at least 20%. 
 
8.  The effectiveness of the drug will be assessed by 
considering a 20% increase from baseline in serum 
levels of the biomarkers BDNF, GDNF and PDGF. 
Study duration 
Total duration of the study: 10 months 
Duration of the screening phase: 3 months 
Duration of treatment phase: 6 months  
Duration of follow-up: 1 month 
Visits scheduled 
Number of visits or contacts scheduled:  
1. Screening (from Day -90 to Day 0)  
2. T0 Baseline (Day 0),  
3. I phone follow-up (2 weeks ± 1 day),  
4. T1 (4 weeks ± 1 week from T0),  
5. II phone follow-up (8 weeks ± 1 week from T0), 
6. T2 (12 weeks ± 1 week from T0), 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 14 of 29  
7. III phone follow-up (16 weeks ± 1 week from T0), 
8. T3 (24 weeks ± 1 week from T0) 
9. T4 follow-up visit (1 months ± 1 week from the last 
drug administration) 
Study timeline in details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1. Screening (Day -90, Day 0): the screening phase 
will be carried out in the three months leading up 
to the baseline visit. Subjects will be considered 
for the study if fulfilling inclusion criteria. During 
the 
Screening 
visit, 
the 
following 
procedures/assessments will be performed and 
data collected: 
a) Obtaining signed informed consent (prior to any 
study procedure) 
b) Collection of demographic data  
c) Medical history  
d) Clinical assessment 
e) Neurological assessment 
f) Pregnancy test (only for post-puberty patients) 
g) Body measurement for ordering the Youcare 
Smart T-shirt wearable device that will be 
delivered at the hospital at T0. 
h) Delivery of the wrist actigraph (Motionlogger) to 
the patient.  
 
2. T0 Baseline (Day 0):  
During 
the 
day 
0 
visit, 
the 
following 
procedures/assessments will be performed, and data 
collected: 
a) Confirmation of study inclusion/exclusion criteria 
b) Vital 
signs 
(body 
temperature, 
heart 
rate, 
respiratory rate and blood pressure) 
c) Standard 12-lead electrocardiogram 
d) Clinical assessment 
e) Neurological assessment 
f) Laboratory tests (haematology, biochemistry, 
TSH, metabolism biomarkers, serum prolactin, 
ACTH, and cortisol) 
g) Pregnancy tests (only for post-puberty patients) 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 15 of 29  
Study timeline in details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
h) Biomarkers of neuronal plasticity (BDNF, GDNF 
PDGF) 
i) Anthropometric assessment 
j) Polysomnographic assessment (only for patients 
enrolled in the Genova and Milano centres) 
k) Delivery of wrist actigraph (Motionlogger) to the 
patient. 
l) Delivery of the Youcare Smart T-shirt wearable 
device 
and 
instructions 
on 
its 
use. 
Cardiorespiratory and nighttime sleep quality 
evaluation is performed at home for 24 hours, 
during the first week after the T0 visit 
m) Completion/evaluation of clinical questionnaires 
(RCSS, MBAS, ADAMS, RSBQ, PHF, SDSC, 
CGI-C and PSI-SF 
n) Dispensing of study drug according to minimum 
dosage per age group 
o) Data collection/evaluation of adverse events 
(AEs) - reported by the patient/caregiver or 
observed by the investigator 
p) Data collection/evaluation of serious adverse 
events (SAEs) - reported by the patient/caregiver 
or observed by the investigator 
q) Collection of concomitant medication data 
r) Handing over of the patient's diary in which the 
caregiver should record the dispensing data of the 
experimental drug and any undesirable effects 
and concomitant drugs. 
 
3. I phone follow-up (Week 2)  
The investigator will contact the family to obtain 
information about the data in the patient diary in order to 
assess the occurrence of any drug-related undesirable 
effects, and to check the tolerability of the experimental 
drug dose administered. During the call, the following 
data will be collected: 
a) 
Adverse 
events 
(AEs) 
- 
reported 
by 
the 
patient/caregiver;  
b) Serious adverse events (SAEs) - reported by the 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 16 of 29  
Study timeline in details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
patient/caregiver; 
c) Concomitant medications taken by the patient; 
d) Compliance with experimental drug; 
e) Guidance on increasing the dosage of experimental 
drug to be administered to patients. 
4. T1 (4 weeks ± 1 week from T0) 
During 
the 
visit 
T1, 
the 
following 
procedures/assessments will be performed and data 
collected: 
a) 
Vital 
signs 
(body 
temperature, 
heart 
rate, 
respiratory rate and blood pressure) 
b) Standard 12-lead electrocardiogram 
c) Clinical assessment 
d) Neurological assessment 
e) Laboratory tests (haematology, biochemistry, TSH, 
metabolism biomarkers, serum prolactin, ACTH, and 
cortisol) 
f) Delivery of wrist actigraph (Motionlogger) to the 
patient.  
g) Assessment of cardiorespiratory parameters and 
nighttime sleep quality performed at home for 24h 
within the first week after  the T1 visit (YouCare T-
shirt) 
h) Completion/assessment of clinical questionnaires 
(RCSS, MBAS, ADAMS, RSBQ, PHF, SDSC, CGI-C 
and PSI-SF) 
i) Dispensing of study drug (if necessary) and 
accounting of drug used by patients 
(j) Data collection/evaluation of adverse events (AEs) - 
reported by the patient/caregiver or observed by the 
investigator 
(k) Data collection/assessment of serious adverse 
events (SAEs) - reported by the patient/caregiver or 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 17 of 29  
Study timeline in details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
observed by the investigator 
l) Collection of concomitant medication data 
m) Evaluation of patient diary 
n) Pregnancy test (only for post-puberty patients). 
 
5. II phone follow-up (8 weeks ± 1 week after T0) 
The investigator will contact the family to obtain 
information about the data recorded in the patient diary 
in order to assess the occurrence of any drug-related 
undesirable effects, and to check the tolerability of the 
dose of experimental drug administered. During the call, 
the following data will be collected: 
a) 
Adverse 
events 
(AEs) 
- 
reported 
by 
the 
patient/caregiver  
b) Serious adverse events (SAEs) - reported by the 
patient/caregiver  
c) Concomitant medications taken by the patient 
d) Compliance with experimental drug 
 
6. T2 (12 weeks ± 1 week from T0)  
During 
the 
VISIT 
T3, 
the 
following 
procedures/assessments will be performed and data 
collected: 
a) 
Vital 
signs 
(body 
temperature, 
heart 
rate, 
respiratory rate and blood pressure) 
b) Standard 12-lead electrocardiogram 
c) Clinical assessment 
d) Neurological assessment 
e) Laboratory tests (haematology, biochemistry, TSH, 
metabolism biomarkers, serum prolactin, ACTH, and 
cortisol) 
f) Biomarkers of neuronal plasticity (BDNF, GDNF 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 18 of 29  
Study timeline in details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PDGF) 
g) Delivery of wrist actigraph (Motionlogger) to the 
patient.  
h) Assessment of cardiorespiratory parameters and 
nighttime sleep quality performed for 24h at home 
within first week after the T2 visit (YouCare T-shirt) 
i) Completion/assessment of clinical questionnaires 
(RCSS, MBAS, ADAMS, RSBQ, PHF, SDSC, CGI-C 
and PSI-SF) 
j) Dispensing of study drug (if necessary) and 
accounting of drug used by patients 
k) Data collection/evaluation of adverse events (AEs) - 
reported by the patient/caregiver or observed by the 
investigator 
l) Data collection/assessment of serious adverse 
events (SAEs) - reported by the patient/caregiver or 
observed by the investigator 
m) Collection of concomitant medication data 
n) Evaluation of patient diary 
o) Pregnancy test (only for post-puberty patients). 
 
7. III phone follow-up (16 weeks ± 1 week after 
T0)  
The investigator will contact the family to obtain 
information about the data in the patient diary in order to 
assess the onset of any drug-related adverse reactions, 
and to check the tolerability of the administered dose of 
the experimental drug. During the call, the following data 
will be collected: 
a) 
Adverse 
events 
(AEs) 
- 
reported 
by 
the 
patient/caregiver  
b) Serious adverse events (SAEs) - reported by the 
patient/caregiver  
c) Concomitant medications taken by the patient 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 19 of 29  
Study timeline in details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
d) Compliance with the experimental drug 
 
8. T3 (24 weeks ± 1 week from T0): End of 
Treatment.  
During 
the 
visit 
T3 
the 
following 
clinical 
procedures/assessments will be performed and the 
following data will be collected: 
a) 
Vital 
signs 
(body 
temperature, 
heart 
rate, 
respiratory rate and blood pressure) 
b) Standard 12-lead electrocardiogram 
c) Clinical assessment 
d) Neurological assessment 
e) Laboratory tests (haematology, biochemistry, TSH, 
metabolism biomarkers, serum prolactin, ACTH, and 
cortisol) 
f) Pregnancy tests (only for post-puberty patients) 
g) Biomarkers of neuronal plasticity (BDNF, GDNF 
PDGF) 
h) Delivery of wrist actigraph (Motionlogger) to the 
patient.  
i) Polysomnographic evaluation (only for patients 
enrolled in the Genova and Milano centres) 
j) Assessment of cardiorespiratory parameters and 
nighttime sleep quality performed for 24h at home 
within first week after the 3 days T3 visit (YouCare T-
shirt) 
k) Clinical questionnaires completion/assessment 
(RCSS, MBAS, ADAMS, RSBQ, PHF, SDSC, CGI-C 
and PSI-SF) 
l) Collection of study drug bottles (used, unused or 
partially used)  
m) Data collection/evaluation of adverse events (AEs) 
- reported by the patient/caregiver or observed by the 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 20 of 29  
Study timeline in details 
investigator 
n) Data collection/assessment of serious adverse 
events (SAEs) - reported by the patient/caregiver or 
observed by the investigator 
o) Data collection about concomitant medication 
p) Evaluation of patient diary 
q) Return of study treatment kits and You Care T-shirt 
9. T4 Follow-up visit (1 month ± 1 week since last 
drug administration) 
During the follow-up visit, the following 
procedures/assessments will be performed and data 
collected: 
a) 
Vital 
signs 
(body 
temperature, 
heart 
rate, 
respiratory rate and blood pressure) 
b) Standard 12-lead electrocardiogram 
c) Clinical assessment 
d) Neurological assessment 
k) Completion/evaluation of CGI-C questionnaire   
m) Data collection/evaluation of adverse events (AEs) 
- reported by the patient/caregiver or observed by the 
investigator 
n) Data collection/evaluation of serious adverse 
events (SAEs) - reported by the patient/caregiver or 
observed by the investigator 
o) Data collection concomitant medication 
 
Trial interventions 
 
 
 
 
Study medication (3.75 mg, 7.5 mg, 15 mg, 30 mg of 
MTZ oral solution) will be given once daily at bedtime. 
During the first 14 days of the treatment period, the oral 
solution of the active drug at Dose Level 1 will be used to 
achieve the planned target daily dose, according to age 
(3.75 mg for 5-10 yrs, 7.5 mg for 11-17 yrs and 15 mg > 
18 yrs, from day 1 to 14). From Day 15 to the end of 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 21 of 29  
 
 
 
 
 
Trial interventions 
week 24, Dose Level 2 will be achieved: 7.5 mg for 5-10 
yrs, 15 mg for 11-17 yrs and 30 mg for > 18 yrs).  
The patients will receive at T0 a first supply of study 
medication, and then additional supplies at each visit to 
cover the 24 weeks (i.e. 6-months) treatment period. 
Dose titration at Dose Level 2 should be completed by 
Day 15. If there are no tolerability issues, patients will 
continue on their maximum tolerated dose (MTD) though 
the final dose. In case of need, patients will receive a 
supply of study medication at the subsequent visit. The 
date, time, and ml of solution administered by the 
caregiver will be recorder in a patient’s diary.  
If intolerance develops at any time during the study, the 
Investigator may reduce patient's dose to a lower level, 
with a single dose interruption and administration every 
other day. Patient unable to tolerate treatment even after 
a dose interruption should be discontinued from the 
study. Discontinuation of the treatment must be also 
considered if: white blood cell count < 4000/mm3 or an 
absolute neutrophil count < 2000/mm3; hyponatremia (< 
125 mmol/L); renal impairment (creatinine > 2 x ULN); 
hepatic impairment (AST, ALT, bilirubin > 2 x ULN); 
cardiovascular, respiratory, gastrointestinal, hematologic 
or other significant medical disorders (in addiction to 
other typical RTT); severe diabetes mellitus; QTcF 
interval on the ECG > 450 msec; severe hypotension.  
These medications are not permitted before and/or 
during the trial: concurrent antidepressants or anxiolytic 
treatment with selective serotonin reuptake inhibitors 
(SSRIs); Monoamine Oxidase Inhibitors (MAOIs); anti-
coagulant therapies (warfarin).  
In accordance with ICH requirements, the Investigator 
must all times be able to account for all study drugs 
furnished to the institution. The Pharmacist/Investigator 
must sign the Drug Receipt Form, confirming that the 
IMP will be handled and stored properly. At the end of 
the study, it must be possible to reconcile delivery 
records with those of usage and returned stocks. 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 22 of 29  
Account must be given of any discrepancy. Under no 
circumstances is the Investigator allowed to use the 
study medications other than those directed by the 
protocol.  
Study sites  
  
Patients will be recruited by four RTT reference Italian 
hospitals (Partners 1-4) which collectively care >420 
RTT patients/year.  
 Coordinating centre - Dipartimento Scienze della 
Vita, Università di Trieste. Trieste.  
 Partner 1. Centro Epilessia – Unità Neurologia 
Pediatrica, ASST Ospedale Santi Carlo Paolo - 
Dipartimento Scienze della Salute, Università di 
Milano.  
 Partner 2. Unità di Neuropsichiatria Infantile, 
IRCCS, Istituto Giannina Gaslini, Genova.  
 Partner 3. Unità di Pediatria, Dipartimento della 
Donna e dei Bambini – Policlinico S. M. alle Scotte. 
Siena.  
 Partner 4. Dipartimento di Patologia Umana 
dell'Adulto e dell'Età Evolutiva "Gaetano Barresi", 
Policlinico Universitario "G. Martino, University of 
Messina. Messina.  
Statistical Analysis Plan  
A Statistical Analysis Plan (SAP) will be finalized prior to 
database lock. The SAP will describe in detail study 
endpoints and statistical analyses, including the analysis 
of the primary as well as additional endpoints. In case 
changes to the original primary endpoint or of the original 
primary analyses occurs during the study, these changes 
will be the subject of a substantial protocol amendment.  
All statistical analyses not prespecified and run after 
database lock will be considered additional/exploratory 
post-hoc analyses.  
Statistical Analysis  
The default significant level will be 0.05 (5%); confidence 
intervals will be 95% and all tests will be two-sided, 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 23 of 29  
unless otherwise specified in the description of the 
analyses.  
Summaries 
of 
continuous 
variables 
will 
include 
descriptive 
statistics 
(number 
of 
non-missing 
observations sample size [n], mean, standard deviation 
[SD], median, minimum and maximum) and for 
categorical variables (the number of non-missing 
observations frequency [n] and percentage), unless 
otherwise stated in the relevant section. Percentages will 
be based on the number of patients within the relevant 
analysis population, or the number of patients with data 
available where relevant.  
The primary efficacy analysis will be performed for the 
ITT population. 
The study hypothesis will be tested as following: change 
from baseline in Motor-Behavior Assessment Scale 
(MBAS) from prior and post mirtazapine therapy will be 
tested using paired t-test. The 2-sided p-value obtained 
from the paired t-test will be presented. Assumption of 
Normality will be investigated using Wilk-Shapiro test. If 
the p-value is greater than .05, it means we cannot reject 
the null hypothesis that a variable is normally distributed. 
If the data is found to be non-Normal, the paired t-test 
will be replaced by Wilcoxon signed rank test. The 
primary endpoint will be summarized descriptively using 
n, mean, median, SD, Q1, Q3, minimum, and maximum. 
Only those patients for whom we have MBAS 
assessment results available at both Baseline (Day 0) 
and End of Study (week 24) will be considered. If 
baseline (Day 0) or end of treatment (week 24) MBAS 
value is missing the patient is not included in the ITT 
population.  
A repeated measure mixed model (MMRM) will be 
performed on the primary efficacy endpoint to test the 
factor/covariate effects for the ITT population. The 
MIXED model will include change from baseline in mean 
MBAS as dependent variable. A MIXED procedure, with 
an unstructured covariance matrix, will be used for the 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 24 of 29  
analysis and the corresponding two-sided p-values for 
each of the covariates will be presented. If the fit of the 
unstructured covariance structure fails to converge, the 
following covariance structures will be tried in order until 
convergence is reached: Toeplitz with heterogeneity, 
compound symmetry, autoregressive with heterogeneity, 
Toeplitz, and autoregressive. Smaller value of AIC and 
BIC will be consider for selection of best fit for 
covariance structure.  
The study will follow the Consolidated Standards of 
Reporting Trials (CONSORT) reporting guideline. The 
statistical analyses will be performed using SPSS 
version 29.0 (IBM SPSS Statistics for Windows, Version 
29.0. Armonk, NY: IBM Corp).  
Trial Ethical 
considerations 
The study will start after the favourable opinion given by 
the Ethics Committee and approval from the regulatory 
authorities.  
The study will be conducted in accordance with Good 
Clinical Practice (GCP) guidelines and in accordance 
with the ethical principles of the Declaration of Helsinki.  
 
  

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 25 of 29  
2. Schedule of Activities (SoA) 
 
 
 
Screening 
(up to 90 
days 
before Day 
-1) 
T0
(Baseline 
day 0) 
I  Phone 
follow-up 
contact 
(2 weeks 
after T0) 
T1 
(4 weeks 
± 1 week 
from T0) 
 
II Phone 
follow-up 
contact 
(8 weeks 
after T0) 
T2
(12 
weeks ± 
1 week 
from T0) 
 
III Phone 
follow-up 
contact 
(16 weeks 
after T0) 
T3 
(24 
weeks ± 
1 week 
from T0) 
T4 
Follow-
up 
(1 month 
after the 
last dose) 
Informed consent 
X 
 
 
 
 
 
 
 
 
Inclusion / 
Exclusion criteria 
X 
X 
 
 
 
 
 
 
 
Demography 
X 
 
 
 
 
 
 
 
 
Medical history 
(includes 
substance usage 
and Family history 
of premature CV 
disease) and 
current medical 
conditions 
X 
 
 
 
 
 
 
 
 
Vital signs (body 
temperature, pulse 
rate, respiratory 
rate, and blood 
pressure) 
 
X 
 
X 
 
X 
 
X 
X 
ECG (12-lead) 
 
X 
 
X 
 
X 
 
X 
X 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 26 of 29  
 
 
Screening 
(up to 90 
days 
before Day 
-1) 
T0
(Baseline 
day 0) 
I  Phone 
follow-up 
contact 
(2 weeks 
after T0) 
T1 
(4 weeks 
± 1 week 
from T0) 
 
II Phone 
follow-up 
contact 
(8 weeks 
after T0) 
T2
(12 
weeks ± 
1 week 
from T0) 
 
III Phone 
follow-up 
contact 
(16 weeks 
after T0) 
T3 
(24 
weeks ± 
1 week 
from T0) 
T4 
Follow-
up 
(1 month 
after the 
last dose) 
Physical 
examination 
including 
anthropometric 
assessment 
X 
X 
 
X 
 
X 
 
X 
X 
Neurological 
examination 
X 
X 
 
X 
 
X 
 
X 
X 
Blood sample 
collection 
(Hematology, 
Biochemistry, and 
thyroid) 
 
X 
 
X 
 
X 
 
X 
 
Blood sample 
collection for 
Metabolism 
biomarkers 
 
X 
 
X 
 
X 
 
X 
 
Serum prolactin 
 
X 
 
X 
 
X 
 
X 
 
Serum ACTH and 
cortisol 
 
X 
 
X 
 
X 
 
X 
 
Serum pregnancy 
test (for post-
pubertal patients) 
X 
X 
 
X 
 
X 
 
X 
 
Blood sample for 
neuronal plasticity 
(BDNF, GDNF 
PDGF) 
 
X 
 
 
 
X 
 
X 
 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 27 of 29  
 
 
Screening 
(up to 90 
days 
before Day 
-1) 
T0
(Baseline 
day 0) 
I  Phone 
follow-up 
contact 
(2 weeks 
after T0) 
T1 
(4 weeks 
± 1 week 
from T0) 
 
II Phone 
follow-up 
contact 
(8 weeks 
after T0) 
T2
(12 
weeks ± 
1 week 
from T0) 
 
III Phone 
follow-up 
contact 
(16 weeks 
after T0) 
T3 
(24 
weeks ± 
1 week 
from T0) 
T4 
Follow-
up 
(1 month 
after the 
last dose) 
Polysomnographic 
exam (only in the 
centres of Genova 
and Milano) 
 
X 
 
 
 
 
 
X 
 
Actigraphy using a 
wrist sensor 
 
X 
 
X 
 
X 
 
X 
 
Biovital and 
cardiorespiratory 
monitoring at 
home (YouCare T-
shirt, 24 h) 
 
X 
 
X 
 
X 
 
X 
 
Assessment of the 
severity (RCSS) 
 
X 
 
X 
 
X 
 
X 
 
Motor-Behavior 
Assessment Scale 
(MBAS) 
 
X 
 
X 
 
X 
 
X 
 
Mood assessment 
(ADAMS) 
 
X 
 
X 
 
X 
 
X 
 
Rett Syndrome 
Behavior 
Questionnaire 
(RSBQ) 
 
X 
 
X 
 
X 
 
X 
 
Purposeful Hand 
Function Scale 
(PHF) 
 
X 
 
X 
 
X 
 
X 
 
Sleep quality scale 
(SDSC) 
 
X 
 
X 
 
X 
 
X 
 

 
[Ver. 2.0 Date 20/12//2024] CONFIDENTIAL                             Page 28 of 29  
 
 
Screening 
(up to 90 
days 
before Day 
-1) 
T0
(Baseline 
day 0) 
I  Phone 
follow-up 
contact 
(2 weeks 
after T0) 
T1 
(4 weeks 
± 1 week 
from T0) 
 
II Phone 
follow-up 
contact 
(8 weeks 
after T0) 
T2
(12 
weeks ± 
1 week 
from T0) 
 
III Phone 
follow-up 
contact 
(16 weeks 
after T0) 
T3 
(24 
weeks ± 
1 week 
from T0) 
T4 
Follow-
up 
(1 month 
after the 
last dose) 
Clinical Global 
Impression of 
Change  
(CGI-C) 
 
X 
 
X 
 
X 
 
X 
X 
Parenting Stress 
Index (PSI-SF) 
 
X 
 
 
 
X 
 
X 
 
Study treatment  
 
X 
X 
X 
 
X 
 
 
 
Study treatment 
compliance 
(patient’s diary 
review) 
 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
Return of study 
treatment kits and 
YouCare T-shirt 
 
 
 
 
 
 
 
X 
 
AE review 
 
X 
X
X 
X 
X 
X 
X 
X 
SAE review 
 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
medications 
 
X 
X 
X 
X 
X 
X 
X 
X 
 

 
 
 
 
[Ver. 2.0 Date 20/12/2024] 
CONFIDENTIAL 
Page 29 of 29 
Time Schedule 
MONTH 1-6 Study set-up and Regulatory actions (AIFA, EC)  
MONTH 7-9 Screening and Recruitment  
MONTH 9-15 Treatment phase 
MONTH 15-18 Follow-up phase and Data analysis 
 
GANNT Chart of activities 
Phase 
1
2 3 4 5
6
7
8
9 10 11
12
13
14
15
16 17
18
Study set-up 
and 
Regulatory 
x x x
x x
x
 
 
 
 
 
 
 
 
 
 
 
 
Screening 
and 
Recruitment 
 
 
 
 
 
 
X
x
x
 
 
 
 
 
 
 
 
 
Treatment 
phase 
 
 
 
 
 
 
 
 
x
x 
x 
x 
X 
x 
x 
 
 
 
Follow-up 
phase and 
Data 
analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x