Pharmacological inhibition of the CB1 cannabinoid receptor restores abnormal brain mitochondrial CB1 receptor expression and rescues bioenergetic and cognitive defects in a female mouse model of Rett syndrome.

BACKGROUND: Defective mitochondria and aberrant brain mitochondrial bioenergetics are consistent features in syndromic intellectual disability disorders, such as Rett syndrome (RTT), a rare neurologic disorder that severely affects mainly females carrying mutations in the X-linked MECP2 gene. A pool of CB1 cannabinoid receptors (CB1R), the primary receptor subtype of the endocannabinoid system in the brain, is located on brain mitochondrial membranes (mtCB1R), where it can locally regulate energy production, synaptic transmission and memory abilities through the inhibition of the intra-mitochondrial protein kinase A (mtPKA). In the present study, we asked whether an overactive mtCB1R-mtPKA signaling might underlie the brain mitochondrial alterations in RTT and whether its modulation by systemic administration of the CB1R inverse agonist rimonabant might improve bioenergetics and cognitive defects in mice modeling RTT. METHODS: Rimonabant (0.3 mg/kg/day, intraperitoneal injections) was administered daily to symptomatic female mice carrying a truncating mutation of the Mecp2 gene and its effects on brain mitochondria functionality, systemic oxidative status, and memory function were assessed. RESULTS: mtCB1R is overexpressed in the RTT mouse brain. Subchronic treatment with rimonabant normalizes mtCB1R expression in RTT mouse brains, boosts mtPKA signaling, and restores the defective brain mitochondrial bioenergetics, abnormal peripheral redox homeostasis, and impaired cognitive abilities in RTT mice. LIMITATIONS: The lack of selectivity of the rimonabant treatment towards mtCB1R does not allow us to exclude that the beneficial effects exerted by the treatment in the RTT mouse model may be ascribed more broadly to the modulation of CB1R activity and distribution among intracellular compartments, rather than to a selective effect on mtCB1R-mediated signaling. The low sample size of few experiments is a further limitation that has been addressed replicating the main findings under different experimental conditions. CONCLUSIONS: The present data identify mtCB1R overexpression as a novel molecular alteration in the RTT mouse brain that may underlie defective brain mitochondrial bioenergetics and cognitive dysfunction.

Effect of positive allosteric modulation and orthosteric agonism of dopamine D2-like receptors on respiration in mouse models of Rett syndrome.

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the methyl-CPG-binding protein 2 (Mecp2) gene. Frequent apneas and irregular breathing are prevalent in RTT, and also occur in rodent models of the disorder, including Mecp2Bird and Mecp2R168X mice. Sarizotan, a serotonin 5-HT1a and dopamine D2-like receptor agonist, reduces the incidence of apneas and irregular breathing in mouse models of RTT (Abdala et al., 2014). Targeting the 5HT1a receptor alone also improves respiration in RTT mice (Levitt et al., 2013). However, the contribution of D2-like receptors in correcting these respiratory disturbances remains untested. PAOPA, a dopamine D2-like receptor positive allosteric modulator, and quinpirole, a dopamine D2-like receptor orthosteric agonist, were used in conjunction with whole-body plethysmography to evaluate whether activation of D2-like receptors is sufficient to improve breathing disturbances in female heterozygous Mecp2Bird/+ and Mecp2R168X/+ mice. PAOPA did not significantly change apnea incidence or irregularity score in RTT mice. PAOPA also had no effect on the ventilatory response to hypercapnia (7 % CO2). In contrast, quinpirole reduced apnea incidence and irregularity scores and improved the hypercapnic ventilatory response in Mecp2R168X/+ and Mecp2Bird/+ mice, while also reducing respiratory rate. These results suggest that D2-like receptors could contribute to the positive effects of sarizotan in the correction of respiratory abnormalities in Rett syndrome. However, positive allosteric modulation of D2-like receptors alone was not sufficient to evoke these effects.

Is trofinetide a future treatment for Rett syndrome? A comprehensive systematic review and meta-analysis of randomized controlled trials.

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 systematically 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 neither 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 trofinetide (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 insignificant result in our analysis, it emerged as a cause for treatment discontinuation in the participating trials, and a statistically 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 between RTT genotype and phenotypic improvements by trofinetide will be beneficial. RCTs encompassing male patients with larger and longer cohorts are recommended.

Recommendations for the management of gastrointestinal comorbidities with or without trofinetide use in Rett syndrome.

INTRODUCTION: Although gastrointestinal (GI) comorbidities are experienced by over 90% of individuals with Rett syndrome (RTT), a neurodevelopmental disorder associated with mutations in the MECP2 gene, many neurologists and pediatricians do not rank the management of these comorbidities among the most important treatment goals for RTT. Trofinetide, the first approved pharmacologic treatment for RTT, confers improvements in RTT symptoms but is associated with adverse GI events, primarily diarrhea and vomiting. Treatment strategies for GI comorbidities and drug-associated symptoms in RTT represent an unmet clinical need. AREAS COVERED: This perspective covers GI comorbidities experienced by those with RTT, either with or without trofinetide treatment. PubMed literature searches were undertaken on treatment recommendations for the following conditions: constipation, diarrhea, vomiting, aspiration, dysphagia, gastroesophageal reflux, nausea, gastroparesis, gastritis, and abdominal bloating. EXPERT OPINION: The authors recommend a proactive approach to management of symptomatic GI comorbidities and drug-associated symptoms in RTT to enhance drug tolerance and improve the quality of life of affected individuals. Management strategies for common GI comorbidities associated with RTT are reviewed based on authors' clinical experience and augmented by recommendations from the literature.

Trofinetide for the treatment of Rett syndrome: Results from the open-label extension LILAC study.

BACKGROUND: Trofinetide was approved for the treatment of Rett syndrome based on the results of the phase 3, randomized, placebo-controlled, 12-week LAVENDER study. Rett syndrome is a chronic disorder requiring long-term treatment. We report the efficacy and safety results of LILAC, a 40-week, open-label extension study of LAVENDER. METHODS: Females with Rett syndrome aged 5-21 years received open-label treatment with trofinetide for 40 weeks. The primary endpoint was long-term safety of trofinetide; secondary endpoints included the change from baseline at week 40 in the Rett Syndrome Behaviour Questionnaire score and the Clinical Global Impression-Improvement score at week 40. FINDINGS: Overall, 154 participants were enrolled and treated with trofinetide in LILAC. The most common adverse events in LILAC were diarrhea (74.7%), vomiting (28.6%), and COVID-19 (11.0%). Diarrhea was the most common adverse event leading to treatment withdrawal (21.4%). The Rett Syndrome Behaviour Questionnaire mean score (standard error) improvement from the LAVENDER baseline to week 40 in LILAC was -7.3 (1.62) and -7.0 (1.61) for participants treated with trofinetide and placebo in LAVENDER, respectively. Mean Clinical Global Impression-Improvement scores (standard error) at week 40 rated from the LILAC baseline were 3.1 (0.11) and 3.2 (0.14) for participants treated with trofinetide and placebo in LAVENDER, respectively. CONCLUSIONS: Treatment with trofinetide for ≤40 weeks continued to improve symptoms of Rett syndrome. Trofinetide had a similar safety profile in LILAC as in LAVENDER. FUNDING: The study was supported by Acadia Pharmaceuticals Inc. (San Diego, CA, USA). This trial was registered at ClinicalTrials.gov (NCT04279314).

Potentiation of the muscarinic acetylcholine receptor 1 modulates neurophysiological features in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in the X chromosome-linked gene Methyl-CpG Binding Protein 2 (MECP2). Restoring MeCP2 expression after disease onset in a mouse model of RTT reverses phenotypes, providing hope for development of treatments for RTT. Translatable biomarkers of improvement and treatment responses have the potential to accelerate both preclinical and clinical evaluation of targeted therapies in RTT. Studies in people with and mouse models of RTT have identified neurophysiological features, such as auditory event-related potentials, that correlate with disease severity, suggesting that they could be useful as biomarkers of disease improvement or early treatment response. We recently demonstrated that treatment of RTT mice with a positive allosteric modulator (PAM) of muscarinic acetylcholine subtype 1 receptor (M1) improved phenotypes, suggesting that modulation of M1 activity is a potential therapy in RTT. To evaluate whether neurophysiological features could be useful biomarkers to assess the effects of M1 PAM treatment, we acutely administered the M1 PAM VU0486846 (VU846) at doses of 1, 3, 10 and 30 â€‹mg/kg in wildtype and RTT mice. This resulted in an inverted U-shaped dose response with maximal improvement of AEP features at 3 â€‹mg/kg but with no marked effect on basal EEG power or epileptiform discharges in RTT mice and no significant changes in wildtype mice. These findings suggest that M1 potentiation can improve neural circuit synchrony to auditory stimuli in RTT mice and that neurophysiological features have potential as pharmacodynamic or treatment-responsive biomarkers for preclinical and clinical evaluation of putative therapies in RTT.

A small-molecule TrkB ligand improves dendritic spine phenotypes and atypical behaviors in female Rett syndrome mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in MECP2, which encodes methyl-CpG-binding protein 2, a transcriptional regulator of many genes, including brain-derived neurotrophic factor (BDNF). BDNF levels are lower in multiple brain regions of Mecp2-deficient mice, and experimentally increasing BDNF levels improve atypical phenotypes in Mecp2 mutant mice. Due to the low blood-brain barrier permeability of BDNF itself, we tested the effects of LM22A-4, a brain-penetrant, small-molecule ligand of the BDNF receptor TrkB (encoded by Ntrk2), on dendritic spine density and form in hippocampal pyramidal neurons and on behavioral phenotypes in female Mecp2 heterozygous (HET) mice. A 4-week systemic treatment of Mecp2 HET mice with LM22A-4 restored spine volume in MeCP2-expressing neurons to wild-type (WT) levels, whereas spine volume in MeCP2-lacking neurons remained comparable to that in neurons from female WT mice. Female Mecp2 HET mice engaged in aggressive behaviors more than WT mice, the levels of which were reduced to WT levels by the 4-week LM22A-4 treatment. These data provide additional support to the potential usefulness of novel therapies not only for RTT but also to other BDNF-related disorders.

A meta-analysis of the efficacy and safety of trofinetide in patients with rett syndrome.

BACKGROUND: Rett syndrome (RTT) is an uncommon inherited neurodevelopmental disorder that affects brain development, mostly in females. It results from mutation in MECP2 gene in the long arm (q) of the X chromosome. OBJECTIVE: Trofinetide is a recently developed drug that has a neuroprotective effect on neurons, and it is our aim in this meta-analysis to evaluate its efficacy and safety in treating Rett syndrome patients. METHODS: We searched 5 databases (PubMed, Scopus, Embase, Web of Science, and Cochrane Library databases) to identify randomized controlled trials (RCTs) comparing Trofinetide and placebo in patients with Rett syndrome until August 13, 2023.Our primary outcomes were the Clinical Global Impression-Improvement (CGI) and the Rett syndrome Behavior Questionnaire (RSBQ). We used Risk of Bias Assessment tool-2 (ROB2) to assess the methodological quality of the included randomized controlled trials. RESULTS: Three RCTs with a total of 325 patients were included with a follow-up duration ranging from one month to three months. 186 patients received the intervention drug (Trofinetide) and 138 received the placebo. Trofinetide was found to reduce CGI and RSBQ significantly more than placebo (MD = -0.35, 95% CI [-0.52 to -0.18], P 0.0001), (MD = -3.40, 95% CI [-3.69 to -3.12], P 0.00001) respectively. Most adverse events did not show any statistical difference between Trofinetide and the placebo. CONCLUSION: Trofinetide offers promise as a potential effective and safe therapeutic opportunity for a population without many available treatments, with improvements seen on both CGI and RSBQ assessments and no severe adverse effects reported.

Metformin Induces MeCP2 in the Hippocampus of Male Mice with Sex-Specific and Brain-Region-Dependent Molecular Impact.

Rett Syndrome (RTT) is a progressive X-linked neurodevelopmental disorder with no cure. RTT patients show disease-associated symptoms within 18 months of age that include developmental regression, progressive loss of useful hand movements, and breathing difficulties, along with neurological impairments, seizures, tremor, and mental disability. Rett Syndrome is also associated with metabolic abnormalities, and the anti-diabetic drug metformin is suggested to be a potential drug of choice with low or no side-effects. Previously, we showed that in vitro exposure of metformin in a human brain cell line induces MECP2E1 transcripts, the dominant isoform of the MECP2 gene in the brain, mutations in which causes RTT. Here, we report the molecular impact of metformin in mice. Protein analysis of specific brain regions in the male and female mice by immunoblotting indicated that metformin induces MeCP2 in the hippocampus, in a sex-dependent manner. Additional experiments confirm that the regulatory role of metformin on the MeCP2 target "BDNF" is brain region-dependent and sex-specific. Measurement of the ribosomal protein S6 (in both phosphorylated and unphosphorylated forms) confirms the sex-dependent role of metformin in the liver. Our results can help foster a better understanding of the molecular impact of metformin in different brain regions of male and female adult mice, while providing some insight towards its potential in therapeutic strategies for the treatment of Rett Syndrome.

Safety and efficacy of trofinetide in Rett syndrome: a systematic review and meta-analysis of randomized controlled trials.

INTRODUCTION: Rett syndrome is a rare genetic neurodevelopmental disorder that predominantly impacts females. It presents with loss of acquired skills, impaired communication, and stereotypic hand movements. Given the limited treatment options for Rett syndrome, there is a dire need for effective interventions. OBJECTIVE: To evaluate the safety and efficacy of trofinetide in Randomized Controlled Trials (RCTs) that report on Rett syndrome patients. METHODS: We identified 109 articles from four databases (Scopus, PubMed, Web of Science, and Cochrane CENTRAL). After removing the duplicates, we narrowed them down to 59 articles for further assessment. We included RCTs that evaluated the efficacy and safety of trofinetide in patients with Rett syndrome. Three studies were eligible for inclusion. Two independent reviewers evaluated the identified studies' titles, abstracts, and full texts, extracting pertinent data. We assessed the quality of the studies using the Cochrane Risk of Bias (RoB) 2.0 tool. We then conducted a meta-analysis using the fixed effects model in the case of insignificant heterogeneity; otherwise, we used the random effects model. Based on the nature of the outcome, we analyzed the mean difference or the odds ratio. Analysis was conducted using RevMan version 5.3. RESULTS: Among the analyzed outcomes in 181 patients in the trofinetide group and 134 patients in the placebo group, significant improvement in Rett Syndrome Behavior Questionnaire (RSBQ) scores was observed at 200 mg dosage (overall mean difference: -3.53, p = 0.001). Clinical Global Impression-Improvement (CGI-I) scores improved considerably at 200 mg dosage (overall mean difference: -0.34, p < 0.0001). No substantial changes were observed in Motor Behavioral Assessment (MBA) or Top 3 Caregiver Concerns. We evaluated Treatment Emergent Adverse Events (TEAEs) across the various dosages and noted significant associations with diarrhea (200 mg), vomiting (200 mg), and irritability (200 mg). However, we did not find a significant association between any of the dosages and the incidence of decreased appetite. CONCLUSION: Trofinetide demonstrated potential in improving RSBQ and CGI-I scores at 200 mg dosage. Although no substantial changes were found in MBA and top 3 caregiver concerns. Adverse events were linked to specific dosages.

Exposure-Response Efficacy Modeling to Support Trofinetide Dosing in Individuals with Rett Syndrome.

INTRODUCTION: Trofinetide was recently approved for the treatment of Rett syndrome (RTT) on the basis of the efficacy and safety findings of the phase 3 LAVENDER study, which used a body weight-based dosing regimen. Exposure-response (E-R) efficacy modeling was used to characterize relationships between trofinetide exposure measures (maximum drug concentration and area under the concentration-time curve for the dosing interval of 0-12 h [AUC0-12]) and efficacy endpoints in RTT clinical studies to support the trofinetide dosing regimen. METHODS: Efficacy endpoints were modeled using trofinetide exposure measures predicted from the population pharmacokinetic model and Bayesian estimates. The analysis population for each E-R model comprised individuals receiving placebo or trofinetide who had available trofinetide exposure measures. Efficacy endpoints were scores from the Rett Syndrome Behaviour Questionnaire (RSBQ), the Clinical Global Impression-Improvement, the Communication and Symbolic Behavior Scales Developmental Profile™ Infant-Toddler Checklist (CSBS-DP-IT) Social Composite, and the Rett Syndrome Clinician Rating of Ability to Communicate Choices (RTT-COMC). RESULTS: Higher trofinetide exposure was associated with improvements in RSBQ, CSBS-DP-IT Social Composite, and RTT-COMC scores. Assuming target trofinetide AUC0-12 values of 800-1200 µg·h/mL, the reductions in RSBQ total scores at week 12 were approximately five- to seven-fold greater with trofinetide (range 3.55-4.94) versus placebo (0.76). Significant E-R relationships were also found for the CSBS-DP-IT Social Composite and RTT-COMC scores. CONCLUSION: E-R efficacy modeling demonstrated significant relationships between trofinetide exposure and RSBQ, CSBS-DP-IT Social Composite, and RTT-COMC scores. Trofinetide is efficacious within the target exposure range, supporting the approved dosing regimen for trofinetide. TRIAL REGISTRATION: NCT01703533, NCT02715115, NCT04181723.

Trofinetide is the first approved treatment for people living with Rett syndrome, a rare genetic condition affecting brain development. This approval was based on the findings of clinical studies in which trofinetide showed significant improvements in the symptoms of Rett syndrome. In this study researchers were looking to see if the level of trofinetide in the blood was related to the level of improvement in symptoms observed in clinical studies. Information on the effectiveness of trofinetide was obtained from the phase 3 LAVENDER study which used doses of trofinetide according to body weight. Trofinetide's effectiveness was assessed on the basis of clinical measurements of key Rett syndrome symptoms. All the information on trofinetide dose, blood levels, and how much symptoms changed (i.e., effectiveness of trofinetide) was then used to develop models to predict symptom responses in the observed population. Researchers found that as the blood levels of trofinetide increased the symptom improvement also increased. When the blood levels were at the recommended level that was achieved in the LAVENDER study, the model predicted that symptom improvement was up to seven times greater with trofinetide than having no treatment (i.e., placebo). This study shows a positive relationship between trofinetide blood levels and improvement in the symptoms of Rett syndrome. Trofinetide was effective within the recommended blood level range in the LAVENDER study using the approved weight-based dosing.

Managing Gastrointestinal Symptoms Resulting from Treatment with Trofinetide for Rett Syndrome: Caregiver and Nurse Perspectives.

Rett syndrome (RTT) is a rare genetic neurodevelopmental disorder mainly affecting female individuals. Trofinetide was recently approved as the first treatment for RTT, largely on the basis of results from the phase 3 LAVENDER trial, in which trofinetide showed improvements in core symptoms of RTT compared with placebo. However, gastrointestinal (GI) symptoms such as diarrhea and vomiting were commonly reported side effects, and taste was also a reported issue. The objective of this article is to describe the perspectives of five caregivers of girls in trofinetide clinical trials as well as those of three nurse trial coordinators, with a focus on management of GI symptoms of trofinetide treatment.Audio Abstract available for this article. Audio Abstract: Jane Lane provides an overview and discusses key findings of the article titled "Managing Gastrointestinal Symptoms Resulting from Treatment with Trofinetide for Rett Syndrome: Caregiver and Nurse Perspectives." (MP4 83274 KB).

Treatment guidelines for rare, early-onset conditions associated with epileptic seizures: a literature review on Rett syndrome and tuberous sclerosis complex.

BACKGROUND: Rett syndrome (RTT) and tuberous sclerosis complex (TSC) are two rare disorders presenting with a range of different epileptic seizures. Seizure management requires careful therapy selection, thereby necessitating development of high-quality treatment guidelines. This targeted literature review (TLR) aimed to characterise country-specific and international treatment guidelines available for pharmacological management of seizures in RTT and TSC. METHODS: A TLR was performed between 25-Jan and 11-Mar 2021. Manual searches of online rare disease and guideline databases, and websites of national heath technology assessment bodies were conducted for the following countries: Australia, Canada, France, Germany, Israel, Italy, Japan, Spain, Switzerland, UK, and US as defined by pre-specified eligibility criteria. Search terms were developed for each condition and translated into local languages where appropriate. Eligible publications were defined as guidelines/guidance reporting pharmacological management of seizures in patients with RTT and TSC. Guideline development methodology, geographical focus, author information and treatment recommendations were extracted from guidelines. An author map was generated using R version 3.5.1 to visualise extent of collaboration between authors. RESULTS: 24 total guidelines were included, of which three and six contained only recommendations for RTT and TSC, respectively (some provided recommendations for ≥ 1 condition). Guideline development processes were poorly described (50% [12 guidelines] had unclear/absent literature review methodologies); reported methodologies were variable, including systematic literature reviews (SLRs)/TLRs and varying levels of expert consultation. Most (83% [20/24]) were country-specific, with guideline authors predominantly publishing in contained national groups; four guidelines were classified as 'International,' linking author groups in the US, UK, Italy and France. High levels of heterogeneity were observed in the availability of treatment recommendations across indications, with 13 and 67 recommendations found for RTT and TSC, respectively. For RTT, all treatment recommendations were positive and sodium valproate had the highest number of positive recommendations (Khwaja, Sahin (2011) Curr Opin Pediatr 23(6):633-9). All TSC treatments (21 medications) received either exclusively negative (National Organization for Rare Disorders (2019)) or positive (Chu-Shore et al. (2010) Epilepsia 51(7):1236-41) recommendations; vigabatrin received the highest number of positive recommendations (Kaur, Christodoulou (2019)). CONCLUSIONS: This review highlights the need for the development of international high-quality and comprehensive consensus-based guidance for the management of seizures with pharmacological therapy in RTT and TSC. TRIAL REGISTRATION: Not applicable.

Trofinetide Treatment Demonstrates a Benefit Over Placebo for the Ability to Communicate in Rett Syndrome.

BACKGROUND: Trofinetide was approved by the US Food and Drug Administration for the treatment of Rett syndrome (RTT) in March 2023. Benefiting the ability to communicate in RTT is often identified as the most important caregiver goal for new therapies. This analysis reports the communication-related end points from the phase 3 LAVENDER study of trofinetide in RTT. METHODS: Females with RTT, aged five to 20 years, were randomized 1:1 to trofinetide or placebo for 12 weeks. Secondary efficacy end points related to communication were based on change from baseline to week 12 and included the caregiver-rated Communication and Symbolic Behavior Scales Developmental Profile™ Infant-Toddler Checklist (CSBS-DP-IT) Social Composite score (key secondary end point; scores ranged from 0 to 26 [higher scores indicated better communication]) and novel clinician rating scales (0 [normal] to 7 [severe impairment]) measuring the ability to communicate choices nonverbally (RTT-COMC) and verbally (RTT-VCOM). RESULTS: Trofinetide demonstrated a statistically significant difference versus placebo for the CSBS-DP-IT Social Composite score (least squares mean [LSM] difference = 1.0; 95% confidence interval [CI], 0.3 to 1.7; P = 0.0064; Cohen's d effect size = 0.43) and a nominally significant difference for the RTT-COMC (LSM difference: -0.3; 95% CI, -0.6 to -0.0; P = 0.0257; Cohen's d effect size = 0.36). As expected, there was no difference for the RTT-VCOM. CONCLUSIONS: Significant treatment benefit for trofinetide versus placebo was observed in scales measuring the ability to communicate. These scales may be appropriate for future clinical studies in RTT and other neurodevelopmental disorders.

Efficacy and tolerance of cannabidiol in the treatment of epilepsy in patients with Rett syndrome.

We aim to assess the efficacy and tolerance of cannabidiol as adjunctive therapy for Rett syndrome (RTT) patients with epilepsy. We conducted a longitudinal observational study through a monocentric cohort of 46 patients with RTT. Patients were recruited from March 2020 to October 2022 and were treated with Epidyolex® (cannabidiol, CBD, 100 mg/mL oral solution). In our cohort, 26 patients had associated epilepsy (26/46 [56%]), and 10/26 (38%) were treated with CBD, in combination with clobazam in 50% of cases. The median dose at their last follow-up was 15 mg/kg/day. The median treatment duration was 13 months (range: 1-32 months). CBD reduced the incidence of seizures in seven out of 10 patients (70%) with one seizure-free patient, two patients with a reduction of seizures of more than 75%, and four patients with a decrease of more than 50%. No aggravation of symptoms or adverse effects were observed. Only one patient experienced a transitory drooling and somnolence episode at the CBD initiation. Half of the patients showed a reduction in agitation and/or anxiety attacks, and an improvement in spasticity was reported in 4/10 (40%) of patients. CBD appears to have potential therapeutic value for the treatment of drug-resistant epilepsy in Rett syndrome. CBD is well tolerated and, when used in combination with clobazam, may increase the effectiveness of clobazam alone.

Mitochondrial modulation with leriglitazone as a potential treatment for Rett syndrome.

BACKGROUND: Rett syndrome is a neuropediatric disease occurring due to mutations in MECP2 and characterized by a regression in the neuronal development following a normal postnatal growth, which results in the loss of acquired capabilities such as speech or purposeful usage of hands. While altered neurotransmission and brain development are the center of its pathophysiology, alterations in mitochondrial performance have been previously outlined, shaping it as an attractive target for the disease treatment. METHODS: We have thoroughly described mitochondrial performance in two Rett models, patients' primary fibroblasts and female Mecp2tm1.1Bird-/+ mice brain, discriminating between different brain areas. The characterization was made according to their bioenergetics function, oxidative stress, network dynamics or ultrastructure. Building on that, we have studied the effect of leriglitazone, a PPARγ agonist, in the modulation of mitochondrial performance. For that, we treated Rett female mice with 75 mg/kg/day leriglitazone from weaning until sacrifice at 7 months, studying both the mitochondrial performance changes and their consequences on the mice phenotype. Finally, we studied its effect on neuroinflammation based on the presence of reactive glia by immunohistochemistry and through a cytokine panel. RESULTS: We have described mitochondrial alterations in Rett fibroblasts regarding both shape and bioenergetic functions, as they displayed less interconnected and shorter mitochondria and reduced ATP production along with increased oxidative stress. The bioenergetic alterations were recalled in Rett mice models, being especially significant in cerebellum, already detectable in pre-symptomatic stages. Treatment with leriglitazone recovered the bioenergetic alterations both in Rett fibroblasts and female mice and exerted an anti-inflammatory effect in the latest, resulting in the amelioration of the mice phenotype both in general condition and exploratory activity. CONCLUSIONS: Our studies confirm the mitochondrial dysfunction in Rett syndrome, setting the differences through brain areas and disease stages. Its modulation through leriglitazone is a potential treatment for this disorder, along with other diseases with mitochondrial involvement. This work constitutes the preclinical necessary evidence to lead to a clinical trial.

Evaluation of Novel Enhancer Compounds in Gentamicin-Mediated Readthrough of Nonsense Mutations in Rett Syndrome.

Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder, is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). Over 35% RTT patients carry nonsense mutation in MECP2, making it a suitable candidate disease for nonsense suppression therapy. In our previous study, gentamicin was found to induce readthrough of MECP2 nonsense mutations with modest efficiency. Given the recent discovery of readthrough enhancers, CDX compounds, we herein evaluated the potentiation effect of CDX5-1, CDX5-288, and CDX6-180 on gentamicin-mediated readthrough efficiency in transfected HeLa cell lines bearing the four most common MECP2 nonsense mutations. We showed that all three CDX compounds potentiated gentamicin-mediated readthrough and increased full-length MeCP2 protein levels in cells expressing the R168X, R255X, R270X, and R294X nonsense mutations. Among all three CDX compounds, CDX5-288 was the most potent enhancer and enabled the use of reduced doses of gentamicin, thus mitigating the toxicity. Furthermore, we successfully demonstrated the upregulation of full-length Mecp2 protein expression in fibroblasts derived from Mecp2R255X/Y mice through combinatorial treatment. Taken together, findings demonstrate the feasibility of this combinatorial approach to nonsense suppression therapy for a subset of RTT patients.