(2024) 22:299
Mohammed et al. BMC Medicine
https://doi.org/10.1186/s12916-024-03506-9

BMC Medicine

RESEARCH ARTICLE

Open Access

Is trofinetide a future treatment for Rett
syndrome? A comprehensive systematic review
and meta-analysis of randomized controlled
trialsHazem E. Mohammed1†, Zeyad Bady1†, Mohamed E. Haseeb2, Heba Aboeldahab3,4,5,Wessam E. Sharaf‑Eldin6 and Maha S. Zaki7,8*   

Abstract
Background Rett syndrome (RTT) is a rare, life-threatening, genetic neurodevelopmental disorder. Treatment
in RTT encounters many challenges. Trofinetide, a modified amino-terminal tripeptide of insulin-like growth factor 1,
has demonstrated clinically promising results in RTT. In this study, trofinetide efficacy and safety in RTT are systemati‑
cally reviewed and meta-analyzed.
Methods A systematic search of five electronic databases was conducted until January 2024. Review Manager
5.4 software was used for the analysis. The analysis was based on a weighted mean difference and standard error
with a confidence interval (CI) of 95%, and a statistically significant P-value was considered if it was < 0.05. The study
was registered on PROSPERO with registration number CRD42024499849. Quality of evidence was assessed using
GRADE.
Results Three randomized controlled trials (RCTs) with 276 patients were included in the analysis. Trofinetide
improved both caregiver outcomes and clinical scales by improving the Rett Syndrome Behavior Questionnaire(RSBQ) (mean difference (MD): − 3.46 points, 95% CI: − 5.63 to − 1.27, P = 0.0002) and Clinical Global Impression
Scale–Improvement (CGI-I) (MD: − 0.35, 95% CI: − 0.51 to − 0.18, P < 0.0001), respectively. However, trofinetide nei‑
ther improved the Caregiver Top 3 Concerns Visual Analog Scale nor the Rett Motor Behavioral Assessment. Regarding
safety, trofinetide was significantly associated with vomiting compared to placebo (odds ratio (OR): 3.17, 95% CI: 1.57
to 6.43, P = 0.001). After solving heterogeneity, results showed a statistically significant incidence of diarrhea in the tro‑
finetide (200 mg) group compared to placebo (OR: 18.51, 95% CI: 9.30 to 36.84, P ≤ 0.00001).
Conclusions Trofinetide demonstrated statistically significant improvements in CGI-I and RSBQ in pediatrics
and adult patients with Rett. Side effects are limited to vomiting and diarrhea. Although diarrhea yielded an insignifi‑
cant result in our analysis, it emerged as a cause for treatment discontinuation in the participating trials, and a statisti‑cally significant risk for diarrhea emerged when excluding the study using a lower dose of the drug, hence causing
heterogeneity, in the meta-analysis. Given the diverse genetic landscape of RTT, future RCTs investigating correlations†

Hazem E. Mohammed and Zeyad Bady contributed equally to this work andwere designated as co-first authors.
*Correspondence:
Maha S. Zaki
dr_mahazaki@yahoo.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Mohammed et al. BMC Medicine(2024) 22:299

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between RTT genotype and phenotypic improvements by trofinetide will be beneficial. RCTs encompassing malepatients with larger and longer cohorts are recommended.
Keywords Rett syndrome, RTT​, Trofinetide, Neurodevelopmental disorders, Systematic review, Meta-analysis
Graphical Abstract

Background
Rett syndrome (RTT) is a rare neurodevelopmental
disease that predominantly affects girls and is mainly
caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, responsible for the synthesis of
the MeCP2 protein [1]. MeCP2 binds to methylated
genomic DNA, which is crucial for various physiological functions, including normal neurologicaldevelopment [2]. Consequently, patients with RTT suffer from several neurological disruptions, including
speech difficulty, motor dysfunction, hand stereotypies,
abnormal gait, and epilepsy [1]. Besides neurologicalmanifestations, RTT affects other systems, leading toabnormalities in growth, the gastrointestinal tract,
breathing, and pubertal development [3]. The prevalence of RTT is nearly 5 to 10 cases per 100,000 females
worldwide [4]. The cumulative incidence is 1.09 per
10,000 girls by the time they are 12 years old [1].
The primary management approaches to RTT involve
providing symptomatic and supportive treatment, comprising behavioral, physical, occupational, and speechtherapy, as well as medication to control seizures andother symptoms. Occupational therapy seeks to lessen
stereotypic movements, enhance appropriate hand use,

Mohammed et al. BMC Medicine(2024) 22:299

and facilitate daily activity performance. Also, physicaland language therapies are required to improve mobility and social life [5]. Studies have assessed the effectiveness of specific drugs in controlling the symptoms of
RTT. Naltrexone, an opioid antagonist, was evaluated asa potential treatment for periodic breathing. However,it was associated with significant deterioration in motor
function and disorder progression [6]. The discovery of
MECP2 mutations and the possible significance of DNA
methylation led to the implementation of a folate-betaine
trial. Although parents reported improvements, noobjective evidence was found [7].
Trofinetide (chemical name: glycyl-l-2-methylprolyl-lglutamic acid), an analog of insulin-like growth factor-1(IGF1), was considered a potential treatment for RTT.
Phase II and III clinical trials, during which it showed
improvement in various RTT symptoms at maximum
dose [8]. Research conducted on mice with MECP2
mutations showed that the amino-terminal tripeptide ofinsulin-like growth factor 1 (IGF-1), also known as glypromate, can significantly enhance physiological behavior and improve survival rates [9]. However, it had low
bioavailability due to its rapid degradation [10, 11]. Trofinetide (brand name: Daybue), the synthetic analog ofglypromate, which showed more resistance to degradation [12], has been approved by the US Food and Drug
Administration (FDA) as a treatment for RTT in patients
2 years of age and older [13]. Various potential mechanisms of action have been suggested for trofinetide. The
stimulation of synaptic maturation and function is one
possible mode of action. It also restores dendritic morphology, neuronal signaling, and synaptic protein synthesis to normal, which are all essential for healthy neuronal
function. In addition, it shields neurons from damage
caused by oxidative stress via its antioxidant response[12, 14–17].
Trials conducted by Glaze et al. [18, 19] and Neul et al.
[20] evaluated trofinetide’s efficacy, tolerability, andsafety in RTT patients. Additionally, a study conductedby Neul et al. showed that trofinetide exhibited a significant change compared to placebo in the communication
ability measuring scales, the caregiver-rated Communication and Symbolic Behavior Scales Developmental
Profile™ Infant–Toddler Checklist (CSBS-DP-IT) Social
Composite score and the Rett syndrome clinician ratingof ability to communicate choices (RTT-COMC) [21].
Furthermore, an exposure–response (E-R) efficacy model
by Darwish et al. [22] demonstrated that high trofinetide
exposure improved the Rett Syndrome Behavior Questionnaire (RSBQ), CSBS-DP-IT, and RTT-COMC scores.
Trofinetide was much better than placebo in reducing
RSBQ total scores, with five to seven times greater reductions assuming target trofinetide with area under the

Page 3 of 15

concentration–time curve for the dosing interval 0 to
12 h (AUC​0–12) values of 800–1200 μg·h/mL [22]. LILAC
study by Percy et al. [23], a phase III open-label extension study of Neul et al. study [20], assessed the safety
and efficacy of trofinetide after 40 weeks of treatment in
154 females with RTT, aged 5–21 years. Trofinetide treatment in this trial was found to sustain improvement in
RTT symptoms as measured by several scales like RSBQ
[23, 24].
In the present study, we aimed to provide class-one evidence demonstrating the efficacy and safety of trofinetide
in RTT patients. We addressed several domains regarding RTT, including caregiver-completed assessments(Rett Syndrome Behavior Questionnaire (RSBQ), andCaregiver Top 3 Concerns), clinician-completed global
assessment (Clinical Global Impression–Improvement(CGI-I)), and clinician-completed syndrome-specificassessments (Motor Behavior Assessment (MBA)). Furthermore, we aimed to provide insights into the safety
profile of the drug. Besides, we investigated sources ofheterogeneity whenever possible using sensitivity analysis and provided assessment of quality of evidence using
GRADE.

Methods
This systematic review and meta-analysis followed thecriteria of the Preferred Reporting Items for Systematic
Review and Meta-analysis (PRISMA) statement [25]. The
protocol held a registration number of CRD42024499849
on PROSPERO.
Eligibility criteria

We included studies meeting these criteria: (1) randomized controlled trials (RCTs); (2) studies including
patients diagnosed with RTT; (3) the intervention group
was trofinetide and the comparator was placebo; (4) English-language studies only. However, any studies meeting the following criteria: (1) observational studies, casereports, and conference abstracts; (2) studies with duplicated or overlapping populations; (3) uncontrolled studies; and (4) studies not written in English were excluded.
Search strategy

Rigorous and comprehensive research was conductedfrom inception until January 2024 in Scopus, PubMed,
WOS, Clinical trials.gov, and Cochrane Central Register of Controlled Trials (CENTRAL) databases. The
search strategy comprised specific keywords and MeSH
terms, including the following: “Trofinetide,” “Glycyl-l2-methylprolyl-l-glutamic acid,” “RTT,” “Rett syndrome,”and “MECP2.” The search strategy used in the aforementioned databases is summarized in Additional file 1:
Table S1.

Mohammed et al. BMC Medicine(2024) 22:299

Study selection data extraction

After conducting the search strategy, two authors blindly
performed studies screening on Rayyan online software[26]. We began initially with title-abstract screening
and followed it with the screening of full text. A third
reviewer was consulted to resolve any conflict between
the two authors in the inclusion decision.
Two authors have extracted data independently on
an online Excel sheet for easier access and connection
between authors. The online sheet was divided into study
characteristics, population baseline characteristics, andoutcome measures data. Study characteristics included
study name and year, sample size, design, duration oftreatment, population, and key findings. Population
baseline characteristics included sample size, age, bodymass index (BMI) (kg/cm2), race, and ethnicity. Outcome
measures involved:
1) Clinician-completed syndrome-specific global measures: Clinical Global Impression Scale–Improvement (CGI-I) is an assessment by the clinician of how
much the patient’s illness severity has changed compared to baseline. A standard rubric specific to RTT
was used in all participating studies, as described by
Neul et al. [27]. A seven-point Likert scale is used,with a maximum score of seven, which indicates theworst stage of RTT.
2) Clinician-completed syndrome-specific measures:Rett Motor Behavioral Assessment (MBA): a scale
composed of 34 items, captured on a four-point Likert scale, grouped into 3 subscales: behavior/social,orofacial/respiratory, and motor/physical signs.
3) Caregiver-completed assessments like (A) Rett Syndrome Behavior Questionnaires (RSBQ) total score:a forty-five-item questionnaire, thirty-eight of which
are divided into eight domains/subscales that represent the fundamental characteristics of RTT: generalmood, breathing problems, hand behaviors, repetitive face movements, body rocking and expressionless face, night-time behaviors, fear/anxiety, andwalking/standing. (B) Caregiver Top 3 Concerns
Total Visual Analog Scale: a Rett-syndrome-specificmeasure comprising three signs or symptoms thatcaregivers individually determined as their top concerns they would like the intervention to improve.
The severity of each concern is scored using a 10-cm
visual analog scale (VAS) using the methodology
described in Glaze et al. [18] and Glaze et al. [19].
Quality assessment

We evaluated the risk of bias using Cochrane Collaboration’s risk of bias tool (RoB), which involved seven

Page 4 of 15

domains: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data,
selective reporting, and any other biases [28]. Studies
were denoted as high risk, low risk, or unclear risk ineach domain mentioned. A study was then judged basedon the total risks in all domains. Any discrepancies wereresolved by the opinion of a third reviewer.
Statistical analysis

Review Manager 5.4 software (RevMan) was used forthe statistical analysis [29]. We used RevMan to synthesize forest plots in both continuous and dichotomous
outcomes. A random-effect model was considered in all
outcomes due to the diversity of the genotypes of theinvestigated populations across the participating studies. The analysis was based on a weighted mean difference and standard error, with a confidence interval (CI)of 95%, and a statistically significant P-value was considered if it was < 0.05. Using the Higgins score (I2), theheterogeneity of the included studies was evaluated,
I-square values ≥ 50% were indicative of high heterogeneity [30]. Adverse events were reported as the numberof events per study arm and pooled as odds ratio (OR).
A chi-square P value less than 0.1 was considered significant heterogeneity. Furthermore, sensitivity analysis was
conducted to assess the heterogeneity and robustness of
the results whenever possible.
Quality of evidence

The level of certainty of the generated evidence wasassessed using the Grading of Recommendations, Assessment, Development and Evaluation criteria (GRADE)[31, 32] by the GRADEpro Guideline Development Tool
(GDT) online tool [33]. GRADE tool assesses the evidence and classifies it into four levels of certainty: very
low, low, moderate, and high, taking into consideration
the following domains of evaluation: risk of bias, inconsistency, indirect evidence, imprecision, publication bias,
and other domains like dose–response effect and plausible confounding.

Results
Literature search

After we searched the three databases, we extracted 189
records. We identified 40 records as duplicates, whichwere removed. A total of 149 records underwent titleabstract screening, yielding five eligible records. Tworecords were eliminated as they did not meet the inclusion criteria. Finally, three RCTs [18–20] (n = 276) were
included in both qualitative and quantitative analyses.
The PRISMA flow diagram is shown in Fig. 1.

Mohammed et al. BMC Medicine(2024) 22:299

Studies and population baseline characteristics

All three included studies are RCTs where patients wererandomized to receive either trofinetide or placebo. Twostudies, Glaze et al. [18, 19], were phase 2, while Neulet al. [20] study was phase 3. All remaining study characteristics, including sample size, duration of treatment,
and key findings, are summarized in Table 1. Moreover,the characteristics of the studies’ population are represented in Table 2.
Quality assessment

According to Cochrane’s risk of bias assessment tool, thestudies’ quality assessment was attributed to each andrepresented in both the graph and summary of the risk ofbias as shown in Fig. 2.

Fig. 1 PRISMA flowchart of study selection

Page 5 of 15

Efficacy of trofinetide

A. Clinician-completed syndrome-specific global measures (CGI-I)
Trofinetide exhibited a statistically significant reduction in CGI-I score compared to placebo (MD: − 0.35,
95% CI: − 0.51 to − 0.18, P < 0.0001), as shown in Fig. 3.
B. Clinician-completed(MBA)

syndrome-specificmeasures

The analysis showed no statistically significant difference between the drug and placebo in terms of MBAscore (MD: − 1.05, 95% CI: − 2.55 to 0.46, P = 0.17), as
shown in Fig. 4.

82

187

Randomized double-blind, phase 3, placebo-controlled
study

187 females between 5 and 20 years of age with RTT,
were assigned randomly to receive trofinetide (n = 93)
or placebo (n = 94)

Oral or via gastrostomy tube trofinetide

200 mg/ml

PlaceboTwelve weeks

CGI-I and RSBQ

Trofinetide resulted in a significant improvement
in both RSBQ and CGI-I compared to placebo, with P
values equal to 0.0175 and 0.0030 respectively. The mostcommon side effects were diarrhea and vomiting

Sample size

DesignPopulation

Intervention

Dose

Comparator

Study duration

Outcome measures

Key findings

56

Glaze et al. (2017) [18]Oral trofinetide

56 female patients with RTT. All of them were includedin the safety analysis, while the efficacy analysis included
55 of them

Trofinetide at a dose of 70 mg/kg showed efficacy com‑pared to a placebo in MBA (P = 0.146), CGI-I (P = 0.164),
and Caregiver Top 3 Concerns Total Visual Analog Scale(P = 0.076). The most common adverse effects were diar‑rhea, irritability, and somnolence

MBA, CGI-I, Caregiver Top 3 Concerns Total VAS score,and ABC

Twenty-eight days for 70 mg/kg and 35 mg/kg cohorts
and 14 days for another 35 mg/kg cohort

Placebo

RTT​Rett syndrome, RSBQ The Rett Syndrome Behavior Questionnaire, CGI-I Clinical Global Impression Scale–Improvement, RTT-DSC The Rett Syndrome-Clinician Domain Specific Concerns, MBA Rett Motor Behavioral
Assessment, ABC Aberrant Behavior Checklist, VAS visual analog score, MECP2 methyl CpG binding protein 2

Trofinetide at 200 mg/kg bid demonstrated significant
clinical improvements in RSBQ, CGI-I, and RTT-DSC com‑pared to placebo with P values equal to 0.042, 0.029,
and 0.029 respectively. The most commonly reported
adverse effects were diarrhea and vomiting

MBA, CGI-I, Caregiver Top 3 Concerns Total VAS score,
RSBQ, and RTT-DSC

Fifty-six days

Placebo

50, 100, or 200 mg/kg of trofinetide for 42 days. Another 35 mg/kg twice a day and 70 mg/kg twice a day
20 participants were randomized to the placebo
or 200 mg bid

Oral or via gastrostomy tube trofinetide

82 girls aged 5 to 15 years were diagnosed with RTT
and had molecular evidence of a pathogenic MECP2
variant

Exploratory phase 2, Multicenter, double-blind, placebo- Exploratory phase 2, Multicenter, double-blind, placebocontrolled, parallel-group trial
controlled, dose-escalation study

Glaze et al. (2019) [19]Study name and year Neul et al. (2023) [20]Table 1 Summary of the included study characteristics

Mohammed et al. BMC Medicine(2024) 22:299
Page 6 of 15

5 (5.4%)

1 (1.1%)1 (1.1%)2 (2.1%)

White

Asian

Black orAfricanAmericanRace, number (%)

> 10

≤ 10
42 (44.7%) 44 (47.3%)

52 (55.3%) 49 (52.7%)

N/A

Not Hispanic N/A

N/A not available, SD standard deviation

Age category, number (%)

N/A

5 (5.4%)

N/A

Ethnicity, Number (%) Hispanic

Other

90 (95.7%) 82 (88.2%)
1 (1.1%)

N/A

BMI, kg/cm2, mean (SD)
N/A

10.9 (4.57) 11.00 (4.69)

93

94

Age, years, mean (SD)10.06 (3.18)

15

9 (38%)

15 (63%)24 (100%)

0 (0%)

1 (4%)

0 (0%)

1 (4%)

22 (92%)5 (33%)

10 (67%)14 (93%)

1 (7%)

0 (0%)

0 (0%)

0 (0%)

15 (100%)

16.00 (2.85) 16.50 (3.61)

9.38 (3.26)

24

Trofinetide (50 mg/
kg)

Placebo

Placebo

Trofinetide(200 mg/kg)

Glaze et al. (2019) [19]Neul et al. (2023) [20]Sample size

Study names and groups

Table 2 Baseline characteristics of study population

6 (38%)

10 (63%)14 (88%)1 (6%)

0 (0%)

1 (6%)

0 (0%)

15 (94%)17.70 (5.06)

10.81 (3.10)

16

Trofinetide(100 mg/kg)

10 (37%)17 (63%)21 (78%)6 (22%)

0 (0%)

0 (0%)

2 (7%)

25 (93%)16.31 (3.57)

9.23 (3.88)

27

Trofinetide(200 mg/kg)
18

Trofinetide (35 mg/
kg)

N/A

N/A

17 (85%)

3 (15%)

0 (0%)

1 (5%)

0 (0%)

19 (95%)

N/A

N/A

18 (100%)

0 (0%)

0 (0%)

3 (16.7%)

0 (0%)

15 (83.3%)

21.54 (5.27) 23.45 (7.34)

27.41 (8.33) 23.74 (6.60)

20

Placebo

Glaze et al. (2017) [18]

N/A

N/A

15 (88%)2 (12%)

0 (0%)

1 (6%)

1 (6%)

15 (88%)20.5 (6.77)

24.52 (5.85)

17

Trofinetide (70 mg/kg)

Mohammed et al. BMC Medicine(2024) 22:299
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Mohammed et al. BMC Medicine(2024) 22:299

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Fig. 2 The risk of bias summary and risk of bias graph

C. Caregiver-completed syndrome-specific measures(RSBQ and Caregiver Top 3 Concerns VAS)
Compared to placebo, the drug showed a statistically significant reduction regarding RSBQ total score(MD: − 3.46, 95% CI: − 5.63 to − 1.27, P = 0.0002), as
shown in Fig. 5A. However, no statistically significant
reduction was found between trofinetide and placebowith respect to the caregiver’s top 3 concerns VAS outcome (MD: − 17.93, 95% CI: − 48.89 to 13.03, P = 0.26),
as shown in Fig. 5B.
Safety of trofinetide

Trofinetide was significantly associated with a higher
incidence of vomiting compared to placebo (OR: 3.17,
95% CI: 1.57 to 6.43, P = 0.001), as shown in Fig. 6A. The

analysis revealed no significant difference between trofinetide and placebo with regard to diarrhea (OR: 7.53,
95% CI: 0.99 to 57.41, P = 0.05, Fig. 6B), pyrexia (OR: 1.07,
95% CI: 0.26 to 4.46, P = 0.92, Fig. 6C), upper respiratory
tract infection (OR: 1.78, 95% CI: 0.48 to 6.64, P = 0.39,
Fig. 6D), seizures (OR: 1.62, 95% CI: 0.58 to 4.53, P = 0.36,
Fig. 6E), and irritability (OR: 1.77, 95% CI: 0.12 to 27.27,
P = 0.68, Fig. 6F).
Sensitivity analysis

We conducted a sensitivity analysis for diarrhea outcome
in order to investigate sources of heterogeneity and totest the stability and robustness of the result. The heterogeneity in diarrhea was resolved by removing Glaze et al.
[18]. Remarkably, the pooled results showed a statisticallysignificant incidence of diarrhea in the trofinetide group

Mohammed et al. BMC Medicine(2024) 22:299

Page 9 of 15

Fig. 3 Comparison of trofinetide vs placebo in terms of CGI-I

Fig. 4 Comparison of trofinetide vs placebo in terms of MBA

Fig. 5 Comparison of trofinetide vs placebo in terms of A RSBQ and B Caregiver Top 3 Concerns VAS

compared to placebo (OR: 18.51, 95% CI: 9.30 to 36.84,
P ≤ 0.00001, Fig. 7).
Quality of evidence

The quality of evidence regarding trofinetide efficacy
and safety in the most important and relevant outcomes versus placebo was assessed using GRADE.
Regarding efficacy, CGI-I and RSBQ were not downgraded at any level and yielded high-quality evidence.
MBA was downgraded in the imprecision domain. Caregiver Top 3 Concerns VAS was downgraded in two

domains: imprecision and inconsistency. Concerning
safety, both vomiting and diarrhea were downgraded.
The former was downgraded in the imprecision domain
only, while the latter was downgraded in two domains:imprecision and inconsistency. A summary of the findings and a GRADE evaluation of the outcomes are represented in Table 3.

Discussion
The present meta-analysis provided class-one evidence
about trofinetide efficacy and safety in RTT patients. It
included three studies with 276 patients, aged 5–44 years,

Mohammed et al. BMC Medicine(2024) 22:299

Page 10 of 15

Fig. 6 Comparison of trofinetide vs placebo in terms of A vomiting, B diarrhea, C pyrexia, D upper respiratory tract infections, E seizures, and F
irritability

Mohammed et al. BMC Medicine(2024) 22:299

Page 11 of 15

Fig. 7 Comparison of trofinetide vs placebo in terms of diarrhea after removing Glaze et al. [18]in the pooled analysis. Regarding the clinician-completed
measures, trofinetide significantly improved RTT severity by reducing the CGI-I score by 0.35 points. Besides,
the caregiver-completed assessments were improvedby reducing the RSBQ total score by almost three anda half points. Both of the aforementioned outcomes arecomplementary and reflect functionally critical dimensions of RTT. As the most widely employed instrumentin RTT investigations, the RSBQ is validated over a broad
spectrum of ages (2–47 years) and has associations withfunctioning [34–36]. Furthermore, the CGI-I, which isalso a widely used clinical scale in RTT trials, adds clinical context to a care-giver outcome like RSBQ [18, 19,
37–41]. It is noteworthy that the changes in CGI-I and
RSBQ total scores in this meta-analysis are almost similar
to the changes exhibited across all the participating trials. Given that all the participating trials had a Cohen’s deffect size for their CGI-I and RSBQ outcomes ranging
from 0.4 to 0.6, this implies medium clinically meaningful improvements [42]. The most notable side effect ofthese improvements was vomiting (P = 0.001). Interestingly, diarrhea was not significant (P = 0.05).
It is worth mentioning that diarrhea was the mostfrequent and leading cause of treatment discontinuations, if present, among participants [19, 20]. However,it did not influence overall tolerability and resolved
shortly after trofinetide cessation. Even a subset of caregivers who experienced diarrhea with their patients
reported their willingness to continue treatment [43].
Although the diarrhea intensity exceeded the caregivers’ expectations, it was viewed as being difficult to manage. However, the caregivers reported some strategies
to deal with diarrhea like increasing dietary fiber, being
prepared for cleanup, and adjusting the drug dose [43].
Although lowering the dose improved diarrhea in somecandidates, a post-hoc analysis of the Neul et al. study(LAVENDER) revealed that the starting dose didn’t significantly affect the incidence of diarrhea. The rates were
69.2%, 88.0%, 83.3%, and 83.3% for initial doses of 150
to < 250, 250 to < 300, 300 to < 350, and 350 to < 500 mg/
kg administered twice daily, respectively [44]. However,
it is important to note that the aforementioned doses are
relatively large and were a source of heterogeneity in our

meta-analysis. Although diarrhea was statistically insignificant in our analysis (P = 0.05), sensitivity analysis byremoving Glaze et al. [18] yielded a statistically significant risk for developing diarrhea (OR: 18.51, 95% CI: 9.3
to 36.84, P = 0.001). Glaze et al. [18] used a lower doseof 70 mg/kg compared to 200 mg/kg in Glaze et al. [19]and Neul et al. [20]. The dosing effect might also explain
the low prevalence of diarrhea in other trials using trofinetide for fragile X syndrome that used doses of 35 and70 mg/kg daily [45]. In addition to dosing, RTT is associated with constipation [46, 47]. Constipation is managedin RTT using different medications [48, 49] as reported
in Neul et al. study [20] where about 60% of trofinetide group was taking drugs to treat constipation, whichmight be considered, in theory, a confounding element.
It is unclear whether incidence of diarrhea was similar
between RTT patients on or off drugs to treat constipation. However, it appears that RTT increases the susceptibility to trofinetide-induced diarrhea; in a phase 1 study,
41 healthy adults were administered trofinetide approximately 160 mg/kg once daily, and no treatment-emergent
adverse events (TEAEs) related to diarrhea were reported[50]. Therefore, it is recommended to stop or reduce all
drugs to treat constipation upon starting trofinetide andconsider an individualized approach for each patient [44].
An individualized approach is crucial in RTT treatment because a pivotal contributor to the phenotype
is the location of the mutation on the X chromosome.
The mutation location contributes to various degrees of
X chromosome inactivation and skewing [51, 52]. The
phenotype severity of RTT can also be correlated withthe genotype. Patients who carried either missense variants or late truncating variants seem to have a milder
phenotype. For instance, a study reported that patientswith the R133C variant exhibited a less severe phenotype than those with the R168X variant. Moreover, thelatter was associated with a more severe phenotype than
R294X variant-bearing patients [53]. As a result, Glazeet al. [18] randomized participants according to loci of
the MECP2 variant, proximal to R294X versus R294X
and distally, creating a similar distribution of variants
across the experimented cohorts. Nevertheless, noneof the involved clinical trials provided any information

Not serious

Not serious

Not serious

Not serious

Not serious

Not serious

Not serious

Not serious

Not serious

Not serious

Seriousa

Seriousa

Not serious

Not serious

Not serious

Not serious

Not serious

Not serious

Seriousb

Seriousb

Seriousb

Not serious

Seriousb

Not serious

None

None

None

None

None

None

⨁⨁⨁◯
Moderate

⨁⨁◯◯
Low

⨁⨁◯◯
Low

⨁⨁⨁⨁
High

⨁⨁⨁◯
Moderate

⨁⨁⨁⨁
High

44

120

44

137

-

-

-

-

12/138 (8.7%)

33/138 (23.9%) OR 3.17
(1.57 to 6.43)

22/138 (15.9%) 92/138 (66.7%) OR 7.53
(0.99 to 57.41)

35

118

35

129

With
trofinetide

87 per 1000

159 per 1000

-

-

-

-

145 more per
1000(from 43 to 293
more)

429 more per
1000(from 1 fewer
to 756 more)

MD 17.93 points
lower(48.89 lower
to 13.03 higher)

MD 3.46 pointlower(5.63 lower
to 1.28 lower)

MD 1.05 pointlower(2.55 lower
to 0.46 higher)

MD 0.35 points
lower(0.51 lower
to 0.18 lower)

Relative effect Anticipated absolute effects(95% CI)
Risk with
Risk difference
placebowith trofinetide

Wide variance of point estimates across studies

Wide 95% confidence intervals, which include clinically important differencesb(2024) 22:299

a

RCT​ randomized controlled trial, CI confidence interval, MD mean difference, OR odds ratio, CGI-I Clinical Global Impression Scale–Improvement, MBA Rett Motor Behavioral Assessment, RSBQ Rett Syndrome Behavior
Questionnaires, None Not applicable because of the small number of included studies276(3 RCTs)

Vomiting

276(3 RCTs)

Diarrhea

79(2 RCTs)

Caregiver top 3 concerns visual analog scale

238(2 RCTs)

RSBQ

79(2 RCTs)MBA

266(3 RCTs)

CGI-I

With placebo

Study event rates (%)

Overallcertainty ofevidence

Outcomes/
number ofpatients(number ofstudies)

Risk of bias Inconsistency Indirectness Imprecision Publication
bias

Summary of findings

Certainty assessment

Table 3 Summary of findings and quality of evidence

Mohammed et al. BMC Medicine
Page 12 of 15

Mohammed et al. BMC Medicine(2024) 22:299

regarding the efficacy of trofinetide according to thedifferent types of variants. As a consequence, it is quite
challenging to determine any correlation between trofinetide and particular variants.
In the majority of participants, trofinetide positiveeffects were diminished after cessation of the drug. Thismight entail longer-term treatment to investigate suchsequelae and substantiate increased clinical benefits
with longer durations. Furthermore, the Glaze et al. [19]study showed more manifest improvements than that of
Glaze et al. [18]. These improvements could be attributed to some clinically crucial factors. First and foremost,younger age was clearer in Glaze et al. [19] with a mean
age of nine compared to 24 in Glaze et al. [18] (refer tobaseline characteristics table). Aging has been linked
to diminished neuroplasticity [54–56], and the lack of afunctional MECP2 protein—the most common cause of
RTT—prematurely closes a window of neuronal plasticity in a brain region involved in social memory. Since
trofinetide is believed to enhance synaptic functions andrestore synaptic structure [12, 14, 15], it is likely to leadto more significant improvements if administered at a
younger age. Second, drug doses were higher in the Glazeet al. (2019) study [19], corresponding to increased drug
exposure. Finally, the longer duration—42 days comparedto 28 days—might have contributed to the observed
increased improvements. Besides, heterogeneity wasevident in our pooled analysis of diarrhea and irritability(I2 = 80%, P = 0.007 and I2 = 58%, P = 0.09), respectively.
We assume that the aforementioned differences regarding drug doses (i.e., 70 mg in Glaze et al. [18] compared
to 200 mg in the others), different treatment durations,altered patients’ tolerability to the drug, administration of drugs to treat constipation, and a wide spectrumof genotype variations might account for the observed
heterogeneity.
Males diagnosed with RTT were not enrolled in anyof the clinical trials conducted until this time. MECP2
variants have been believed for a long time to be lethal
in males; however, such cases were identified with a wide
range spectrum of phenotypes, and nine variants thathave not been seen in females beforehand are reported
in males. Eventually, this led to the introduction a newentity called “Male RTT Encephalopathy” [57]. Moreover, males exhibit MECP2 duplication syndrome more
frequently than RTT, which both have many clinical features in common [58]. The aforementioned aspects highlight the profound difference in RTT diagnosis between
males and females accounting for the plausible exclusionof males from these clinical trials.
This study stands out at some points, including conducting a sensitivity analysis, which revealed that lower

Page 13 of 15

doses were associated with a lower incidence of diarrhea. Furthermore, we performed a GRADE assessment
to evaluate the quality of the evidence, providing a more
rigorous and structured appraisal of the evidence. Our
study provided a detailed discussion on the incidenceof diarrhea in RTT patients in the context of trofinetide
treatment, aiming for a clearer understanding. The present study provided quantitative and qualitative evidence
on the efficacy and safety of trofinetide on a wide spectrum of RTT patients, ranging from 5 to 44 years old.
However, the optimism of this study must be titrated
by the lack of assessing the safety and efficacy of trofinetide in the long term. Moreover, the analyzed cohort
is small, which is understandable given the rarity of theinvestigated disease. Besides, males suffering from RTT
were not enrolled in any of the participating studies dueto phenotype variability in males. Moreover, the youngest
age investigated in the participating studies was 5 years.
We believe that earlier administration of the drug may
highlight new aspects about the efficacy and safety of the
drug. Another limitation is the absence of biomarkers
determining responders to trofinetide treatment. Therefore, we recommend that future studies should focus
on conducting more RCTs assessing both younger age
groups, (i.e., below 5 years of age), and longer-duration
treatments to evaluate their effect on treatment efficacy.
In addition, they have to consider genotype–phenotype
relationships by dividing participants into subgroups
based on their mutational type and evaluate the drug efficacy between different mutations. RCTs on males, exclusively, assessing a wide spectrum of mutations are also
recommended.

Conclusions
In conclusion, this meta-analysis of trofinetide demonstrated statistically significant, moderate clinical improvement in complementary scales of RTT
like CGI-I and RSBQ. The safety profile of trofinetide
revealed a significant increase in vomiting comparedto placebo. Although diarrhea yielded an insignificant
result in our analysis, it emerged as a cause for treatment discontinuation in the participating trials, and astatistically significant risk for diarrhea emerged whenexcluding the study using a lower dose of the drug,
hence causing heterogeneity, in the meta-analysis. We
underscored the importance of considering genotype–phenotype correlations in evaluating trofinetide’s efficacy, especially given the diverse genetic landscape of
RTT. Furthermore, younger ages, longer durations, andmale-limited RCTs are required.

Mohammed et al. BMC Medicine(2024) 22:299

Abbreviations
ABC	Aberrant Behavior Checklist
CGI-I	Clinical Global Impression Scale–Improvement
CI	Confidence intervalMBA	Rett Motor Behavioral Assessment
MD	Mean difference
MECP2	Methyl CpG binding protein 2
OR	Odds ratio
PRISMA	Preferred Reporting Items for Systematic Reviews and Meta-Analyses
RCTs	Randomized controlled trials
RevMan	Review Manager software
RoB	Cochrane risk-of-bias tool for randomized trials
RSBQ	The Rett Syndrome Behavior Questionnaires
RTT​	Rett syndrome
VAS	Visual analog score

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12916-​024-​03506-9.
Additional file 1: Table S1. Search strategy utilized in all databases.
Acknowledgements
None.
Human ethics and consent to participate statement
Our manuscript was not applied to human beings and thus requires no ethicalapproval.
Authors’ contributions
The authors meet the criteria for authorship as recommended by theInternational Committee of Medical Journal Editors (ICMJE). H.E.M. and Z.B.
contributed equally to this work and were designated as co-first authors.
H.E.M. and Z.B. contributed to the study’s conception and design. H.E.M., Z.B.,
M.E.H., and H.A. performed studies screening and data extraction. H.E.M., Z.B.,
M.E.H., W.E.S.E., and M.S.Z. wrote the initial draft of the manuscript. H.E.M., Z.B.,
H.A., W.E.S.E., and M.S.Z. revised and prepared the manuscript for submission.
All authors provided feedback on earlier drafts of the manuscript. The final
manuscript was read and approved by all authors.
Authors’ Twitter handles
Twitter handles: @Hazem_E_Mohamed (Hazem E. Mohammed), @Bady_Zeyad(Zeyad Bady), and @Mohamedhaseeb25 (Mohamed E. Haseeb).
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB).
Availability of data and materials
All data generated or analyzed during this study are included in this publishedarticle [and its supplementary information files].

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Faculty of Medicine, Assiut University, Assiut, Egypt. 2 Faculty of Medicine,
Minia University, Minia, Egypt. 3 Medical Research Group of Egypt (MRGE),
Negida Academy, Cairo, Egypt. 4 Clinical Research Department, El-Gomhoria

Page 14 of 15

General Hospital, MOHP, Alexandria, Egypt. 5 Biomedical Informatics and Medi‑
cal Statistics Department, Medical Research Institute, Alexandria University,
Alexandria, Egypt. 6 Medical Molecular Genetics Department, Human Genetics
and Genome Research Institute, National Research Centre, Cairo, Egypt. 7 Clini‑
cal Genetics Department, Human Genetics and Genome Research Institute,
National Research Centre, Cairo, Egypt. 8 Medical Genetics Department, Armed
Forces College of Medicine (AFCM), Cairo, Egypt.
Received: 10 February 2024 Accepted: 27 June 2024

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