Pharmacologic Treatment of Rett Syndrome With Glatiramer Acetate

Pediatric Neurology 61 (2016) 51e57

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Pediatric Neurology journal homepage: www.elsevier.com/locate/pnu

Original Article

Pharmacologic Treatment of Rett Syndrome With Glatiramer Acetate Aleksandra Djukic MD, PhD a, b, c, *, Roee Holtzer PhD a, d, Shlomo Shinnar MD, PhD a, b, c, e, Hiren Muzumdar MD f, Susan A. Rose PhD c, Wenzhu Mowrey PhD e, Aristea S. Galanopoulou MD, PhD a, b, g, Ruth Shinnar RN, MSN a, Jeffrey J. Jankowski PhD h, Judith F. Feldman PhD c, Sophia Pillai MD i, Solomon L. Moshé MD a, b, c, g a

Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, New York Rett Syndrome Center, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York c Department of Pediatrics, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York d Ferkauf Graduate School of Psychology of Yeshiva University, Bronx, New York e Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York f Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh, Pittsburgh, Pennsylvania g Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York h Department of Social Sciences, Queensborough Community College, City University of New York, Bayside, New York i Department of Pediatrics, Weill Cornell Medical College, New York, New York b

abstract BACKGROUND: Rett syndrome (RTT) is a severe neurological disease that primarily affects females. The level of brain

derived neurotropic factor (BDNF) expression directly correlates with the severity of RTT related symptoms. Because Glatiramer acetate (GA) stimulates secretion of BDNF in the brain, we conducted the study with the objective to assess its efficacy in improving gait velocity cognition, respiratory function, electroencephalographic findings, and quality of life in patients with RTT. METHODS: Phase two, open label, single center trial. Inclusion criteria: ambulatory girls with genetically confirmed RTT, 10 years or older. Pre- and post-treatment measures were compared using the non-parametric Wilcoxon signed rank sum test and paired t-tests. RESULTS: Ten patients were enrolled and completed the trial. Gait velocity improved significantly (improvement range 13%-95%, p¼0.03 for both tests) and emerged as an especially valuable outcome measure with excellent test- retest reliability of the 2 trials within sessions (intraclass correlation coefficient¼0.94). Memory, and the breath holding index also improved significantly (p0.03). Epileptiform discharges decreased in all four patients who had them at baseline. There was a trend towards improved quality of life, which did not reach statistical significance. CONCLUSIONS: This prospective open-label trial provides important preliminary information related to the efficacy of GA in improving gait velocity in female patients with RTT who are 10 years or older. The results of this trial justify the need for larger scale controlled trials of GA as well as provide a template for assessing the efficacy of other interventions in RTT. Keywords: Rett syndrome, glatiramer acetate, gait, clinical trial

Pediatr Neurol 2016; 61: 51-57 Ó 2016 Elsevier Inc. All rights reserved.

Article History: Received February 27, 2016; Accepted in final form May 20, 2016 * Communications should be addressed to: Dr. Djukic; Saul R. Korey Department of Neurology; Albert Einstein College of Medicine; 3351 Steuben Avenue; Bronx, NY 10467. E-mail address: adjukic@montefiore.org 0887-8994/$ e see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pediatrneurol.2016.05.010

Introduction

Rett syndrome is a severe neurological disease that affects 1:10000 females. It is caused by loss-of-function mutations in the methyl-CpG-binding gene (MeCP2).1 Patients with Rett syndrome are typically completely dependent,

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nonverbal with no or minimal purposeful hand use, wheelchair bound, or with abnormal gait, with autonomic instability, seizures, and scoliosis.2,3 Clinical diagnosis is based on a set of clinical criteria, the main of which include gait abnormalities, loss of acquired purposeful hand skills and spoken language, and stereotypic hand movements.3 MeCP2 acts as a transcription regulator of other genes.1 One of the genes that have consistently shown expression changes when MeCP2 is absent is brain-derived neurotrophic factor (BDNF), which functions as a key signaling molecule in brain development and plasticity.4-6 The level of BDNF expression directly correlates with the severity of Rett syndromeerelated symptoms. Overexpression of BDNF in affected mice led to a delayed onset of Rett syndromeelike symptoms, improved quality of life (QOL) and survival rates, and reversed electrophysiological deficits, whereas a decrease in the BDNF level led to earlier development of Rett syndromeelike symptoms and lethality.7 Unfortunately, BDNF cannot be used in human trials of Rett syndrome because it does not cross the bloodebrain barrier8; however, in animal studies, pharmacologic interventions that successfully stimulated BDNF (glutamatergic a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid receptor activatordampakine, tropomyosin receptor kinase B small molecule partial agonist, insulin-like growth factor 1 (IGF1)dmecasermin, sphingosine-1-phosphate receptor 1 agonistdfingolimod) resulted in a marked functional improvement (locomotion, respiration, longevity) and treatment with IGF-1 had a positive effect on dendritic arborization, which is a histologic hallmark of Rett syndrome.9-14 Glatiramer acetate (GA) is a collection of synthetic polypeptides approved by the US Food and Drug Administration for the treatment of relapsingeremitting multiple sclerosis.15 It activates GA-specific suppressor T cells in the periphery that cross the bloodebrain barrier and stimulate secretion of several neurotrophic factors, including BDNF in the brain.16-19 Administration of GA caused elevation of BDNF expression up to normal levels in several cortical areas in an animal model of Rett syndrome.20 Incubation of neurons with GA for 24 hours resulted in about a twofold increase in neuronal expression of BDNF.21 In this open-label pilot study, we determined the effects of GA (Copaxone) administration in 10 girls with Rett syndrome. The primary end point was gait velocity because of its predictable course (highly unlikely to spontaneously improve), and relative stability compared to other Rett syndromeerelated symptoms that can be assessed objectively but are characterized by significant hour-to-hour and day-to-day fluctuations.3,22,23 Furthermore, it is noteworthy that gait velocity has been routinely used as an outcome measure in animal models of Rett syndrome23 and in human studies of other neurological diseases because of its “excellent psychometric properties.”24-27

epilepsy (well controlled in all). No changes were made to the patient medications during the study period. The study was approved by the Institutional Review Board of Montefiore Medical Center and Albert Einstein College of Medicine and registered at Clinicaltrials.org (NCT02153723). Written consent was obtained from all families. Inclusion criteria were: female patients with genetically confirmed Rett syndrome who were 10 years or older and ambulatory (without assistance at the time of their enrollment). Exclusion criteria included: prolonged QT syndrome, presence of comorbid non-Retterelated disease, presence of immunodeficiency requiring immunoglobulin therapy during the three months before enrollment, and allergy/sensitivity to GA, or mannitol (a contraindication as per the manufacturer).15

Materials and Methods

Secondary end points

Participants

Secondary end points included change in respiratory and cognitive functions, QOL, and electroencephalograph (EEG). Respiratory function monitoring was performed by evaluating ambulatory wake respirations over three hours with sleep monitoring equipment (Xltek, Natus Medical Incorporated, Oakville, ON, Canada) during the daytime at the

In this phase 2, open-label trial, 10 girls with genetically confirmed Rett syndrome were recruited from the Rett Syndrome Center at Montefiore Medical Center (MMC), New York, United States. Six girls had

Assessments Neurological evaluations, history updates and interviews with parents were performed by A.D. before drug initiation, before each drug escalation, and after the completion of the trial. Outcomes were assessed at baseline and after 24 weeks of treatment.

Treatment The dose selected for treatment was 20 mg/day, which is the standard dose used and tolerated in this age group of children with multiple sclerosis.28 Parents were trained to administer GA subcutaneously. The first dose was administered at the MMC Clinical Research Center; all subsequent doses were given by the parents, at home, according to the following dose escalating schedule: once a week for 4 weeks, twice a week for four weeks, and daily for the remaining 16 weeks of the trial. Total duration of treatment was 24 weeks. When any persistent intolerance (other than the mild transitory injection site redness) occurred, the dose was reduced to the previous maximally tolerated dose for the duration of the trial. To assess compliance of patients, a drug administration log was kept by parents and unused vials were returned. All patients were compliant, with no patient missing more than five doses during the 24-week trial (one patient missed five doses and another patient missed two doses during ongoing viral illnesses). Patients who showed improvements in gait velocity and whose parents were interested in continuing the treatment continued to receive daily treatment with GA as part of their medical care after the termination of the trial. These patients were followed clinically (A.D.) and underwent gait assessment about one year after the termination of the study.

Primary end pointdgait velocity Gait velocity (cm/second) was measured by a standardized walking test as used in previous studies by one of the coinvestigators (R.H.). Research assistants conducted quantitative gait evaluations, independent of the clinician’s evaluation, using a computerized mat with embedded pressure sensors (GAITRite System). The walkway measures 8.5  0.9  0.01 m (L  W  H) with an active recording area of 6.1  0.61 m (L  W). Patients were asked to walk on the mat, at their normal walking speed, for two trials in a quiet and well-lit hallway. Start and stop points were marked by white lines on the floor and included 0.9 m (three feet) each for initial acceleration and terminal deceleration. Monitoring devices were not attached to the participants during the test. A parent walked alongside the electronic walkway to provide reassurance and, when indicated, support to maintain balance while walking. Software computed quantitative parameters based on footfalls recorded. Each trial was one walkway in length, and values analyzed were the mean of two trials computed automatically by the software.25,27

A. Djukic et al. / Pediatric Neurology 61 (2016) 51e57 polysomnography laboratory at Children’s Hospital at Montefiore. Respiratory impedance, plethysmography, oronasal airflow, and electromyography were recorded. Because breathing in patients with Rett syndrome improves or normalizes during sleep, the patients also underwent polygraphic recording and video monitoring during the three-hour study period to confirm wakefulness and to eliminate the possibility of improvement in respiratory function caused by the patients falling asleep.22,29 Normal breathing was defined as breathing with a regular, homogenous pattern; breath hold (BH) as a reduction in respiratory amplitude (flattening of the tracing in inspiration) by 90% for two or more breaths. BHs in Rett syndrome are usually sustained inhalations and not apneic events (absence of inspiration after exhalation). Since the threshold for distress is not always clear and can vary with individuals, we noted all measurable events. Shorter but more frequent BHs can be equally distressing to patients as less frequent but prolonged ones. To capture change in both frequency and duration of BH, parameters measured included BH index (BHI ¼ number of BH events/hour) and percentage of BH time (proportion of time with BH events). We also measured 3% desaturations as an index of gas exchange disturbance (oxygen desaturation index). Finally, we measured the duration of breathing that was not regular and not a BH as irregular breathing. Cognitive function was assessed with measures of attention and recognition memory, with eye-tracking data recorded using a Tobii T300 Eye Tracker, which is built-in to a 23-inch LCD monitor (Tobii Technology AB, Danderyd, Sweden). Testing was conducted in a quiet room, in a three-sided black-curtained enclosure, with girls seated on a parent’s lap (which calms them and aids in steadying their head). Verbal instructions, limited to “Look at the TV,” were used at the beginning of the session. To assess recognition memory of faces and patterns, we used the same methodology as employed in our prior study, in which two identical targets are presented for familiarization, and then a novel and familiar are presented on test.30 Recognition is indexed by a novelty score or percentage of time looking to the novel target on test. Attention is indexed by duration and number of fixations. The cognitive basis of eye gaze measures is supported by data showing: (1) normal adults demonstrate significant novelty scores on recognition tasks, whereas those with amnesia (due to hippocampal damage) show chance performance and (2) normal adults show a significant relationship between novelty scores (using a task similar to that noted above) and recognition using a standard forced-choice test.31-34 A 21-channel video EEG was performed over the course of 1 hour at baseline and at the end of the treatment, using the 10-20 system (Xltek, Natus Medical Incorporated, San Carlos, CA). Two electroencephalographers (A.S.G. and S.L.M.) analyzed visually the EEG for background abnormalities and for the presence and hourly frequency of epileptiform discharges (spikes or spike-and-slow-wave complexes). The electroencephalographers were not aware of when the EEGs were obtained (before or after treatment). QOL was assessed using the Child Health Questionnaire-P50, which has been used previously with patients with Rett syndrome.35 This questionnaire includes broad-spectrum areas, which can be divided into two summary categories or scores: (1) physical summary scores, generated based on the physical functioning, social limitationsdphysical, general health, bodily pain, parent impactdtime, and parent impactdemotional domains, and (2) psychosocial summary scores, generated based on the social limitationsdemotional and/or behavioral, self-esteem, mental health, behavior, parent impactdtime, and parent impactdemotional domains.30 A higher score indicates better QOL.

Statistical analyses For the primary outcome of gait velocity, testeretest reliability was computed as intraclass correlation coefficient (ICC) for the two trials both before and after treatment, separately. The magnitude of change in each end point was computed for each individual as “post-treatment value  pretreatment value.” Percentage of change was computed as (post-treatment value  pretreatment value)/pretreatment  100. Median and interquartile range (IQR) was computed for pretreatment and post-treatment, respectively. Pretreatment and post-treatment

53

measures were compared using the nonparametric Wilcoxon signed rank sum test (primary analysis approach) and paired t tests (secondary analysis approach). We presented results from both tests because a sample size of ten may not be sufficient to test the normality assumption required for paired t tests. For the end points that had demonstrated significant treatment effects, the Pearson correlation was used to assess whether there was an association between the magnitude of change and age, seizure status, and the Rett syndrome severity score. The only data read blind in regards to the timing of the evaluation (before or after treatment) were EEG data.

Results Patients

Ten patients met the inclusion and exclusion criteria and completed the trial (age range 10-21 years, median [IQR]: 14 [12 to 16]). One additional patient was evaluated but not enrolled into the trial because of prolonged QT syndrome. The clinical characteristics of the cohort are described in Table 1. GA was well tolerated in all except one patient (ID ¼ 1), who developed asymptomatic elevation of creatine kinase (up to 600 units) when given daily injections. For this patient, the dosage was reduced to twice weekly injections for the duration of the trial, which lead to normalization of creatine kinase levels. All patients were compliant, with no patient missing more than five doses during the 24-week trial. None of the parents had problems administering injections. Focal redness around the injections sites was reported in three patients on several occasions during the trial. In all cases, the redness was self-limited and lasted from few hours to one day. Seven patients continued the treatment after the study termination. Primary endpointdgait velocity

Comparison of the gait velocities before and after the trial for the entire cohort revealed a significant improvement after treatment with GA (Table 2; median [IQR]; pretreatment: 62.7 [42.3 to 103.2] cm/second, posttreatment: 84.3 [58.1 to 119.3] cm/second; Wilcoxon signed rank sum test, P ¼ 0.03; paired t test, P ¼ 0.03). Comparison of pretreatment and post-treatment velocities in individual patients revealed improvements in seven patients, with the range of improvement 14.7 to 41.4 cm/ second and 13.1% to 95.2% compared with pretreatment gait velocity. One of these patients was able to complete only one of the two pretreatment walks, because of severe TABLE 1. Characteristics of the Cohort

ID

Age (years)

Genetics

1 2 3 4 5 6 7 8 9 10

10 11 12 12 13 15 16 16 18 21

T158M R306C R294X R255X R168X Large deletion R306C P101S R133C R294X

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TABLE 2. Gait VelocitydGroup and Individual Results

ID

T1 mean Walk1 Walk2 T2 mean Walk1 Walk2 T3 mean Walk1 Walk2

Individual Data (cm/second)

Group Data Median (IQR)

1

2

3

4

5

6*

51.6 43.5 59.7 93 78 108 80.6 92.4 68.8

39.8 36.5 43 58.1 60 56.1 60.6 58.0 62.5

76.5 78.1 74.8 113.1 109.8 116.3 108.7 103.1 114.2

54.3 65 43.6 75.7 86.1 65.2 62.1 79.8 44.4

42.3 32.9 51.7 32.7 38.6 26.8

103.2 103.2 123.1 150.7 95.4

y

7

8

9

10

129.2 132.2 126.1 119.3 112.4 126.2 110.5 128.2 92.7

71.2 78.8 63.5 65.8 68.4 63.2

20.7 18.9 22.4 40.3 33.6 47 48.7 41.0 56.4

112.6 110.1 115 127.3 127.1 127.4

62.7 (42.3-103.2)

84.3 (58.1-119.3)

71.4 (60.6-108.7)

Abbreviations: IQR ¼ Interquartile range T1 ¼ Trial entry testing T2 ¼ Trial exit testing T3 ¼ 1-year follow-up * The patient has severe fluctuating dystonia and was unable to perform the second walk before the treatment. She completed both walks after treatment. y The patient required parental support before treatment, walked without assistance after treatment.

dystonia, requiring the use of a stroller for all daily transportations. After the treatment, she was not only able to easily complete both trials, but her parents also reported almost complete elimination of the need for a stroller for her daily transportations. One patient, who required assistance to walk before treatment, was able to walk with no assistance after GA treatment. Parents’ reports of improvement or absence of improvement in the gait were consistent with the post-trial results of the objective gait assessment. Testeretest reliability of gait velocity was excellent before (ICC ¼ 0.94) and after (ICC ¼ 0.82) treatment (Table 2). There appeared to be an inverse correlation between pretreatment gait speed and the percent improvement, although this did not reach statistical significance (r ¼ 0.53, P ¼ 0.11; Figure). A subset of six patients continued the treatment after the study termination and underwent the third gait assessment at a year after the termination of the trial. The median (IQR) for this subset of patients was 53.0 (39.8 to 76.5) for T1 before treatment and 71.4 (60.6 to 108.7) for T3 at 1-year

follow-up. The Wilcoxon signed rank sum test revealed a trend (P ¼ 0.09), suggesting that treatment gains may be sustained at one-year follow-up compared with baseline. Secondary endpoints Respiratory function

Nine patients completed the evaluations. One patient did not tolerate the entry evaluation due to anxiety, and the change in respiratory function could not be assessed. The median (IQR) of the percentage of time patients were fully awake was 100% (99%, 100%) (range: 87.3% to 100.0%) of the recording time before treatment and 100% (100%, 100%) (range: 99.5% to 100%) after treatment. The BHI and BH time both significantly decreased in the entire group after treatment (Wilcoxon signed rank sum test, P ¼ 0.03 and P ¼ 0.004, respectively; Table 3). When data were analyzed for individual patients, BHI improved in five patients (change range: 2.3 to 14.5 events/hour, 54.1% to 95.9%), worsened in one from 3.8 events/hour to 4.9 events/hour (27.4%), and remained almost unchanged in the remaining three patients. Oxygen desaturation index and duration of irregular breathing did not change after treatment. Cognition

Seven participants completed the cognitive assessment. The remaining three participants were cooperative, but the eye tracker was unable to capture their eye gaze. Treatment with GA resulted in significant improvement in memory scores for faces (paired t test, P ¼ 0.03), but not for patterns, nor for either index of attention-number of fixations or fixation duration (Table 3). Five of seven participants had increased novelty scores, indicating improved recognition (19.3% to 58.2% improvement in comparison with pretreatment novelty scores). Electroencephalography FIGURE. Inverse relationship between pretreatment gait velocity and the magnitude of improvement.

Wakefulness was captured in all studies, allowing comparisons of awake background in all patients before and after treatment. In contrast, drowsiness was captured in six

A. Djukic et al. / Pediatric Neurology 61 (2016) 51e57

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TABLE 3. Group Analysis of Secondary End Points

Statistics Pretreatment, median (IQR) Post-treatment, median (IQR) Post  pre difference, median (IQR) Wilcoxon signed rank sum test, P Paired t test, P

BHI (n ¼ 9)

BH% (n ¼ 9)

Memory Novelty Score % (n ¼ 7)

Attention Number Attention Fixation of Fixations (n ¼ 7) Length(s) (n ¼ 7)

QOL Physical (n ¼ 10)

QOL Psychosocial (n ¼ 10)

3.8 (2.3-7.3)

1.8 (0.8-2.3)

42.4 (39.5-61.0)

19.4 (8.0-20.8)

0.46 (0.26-0.64)

19.7 (15.5-38.8)

40.3 (34.7-47.7)

1.6 (0.3-2.0)

0.4 (0.1-0.8)

62.4 (55.7-65.8)

16.2 (11.0-27.4)

0.35 (0.18-0.46)

38.9 (16.7-45.3)

50.1 (37.4-54.5)

2.0 0.1 15.9 (2.0 to 23.0) (5.6 to 0.3) (1.7 to 0.3)

0.8 (7.1 to 8.4)

0.00 (0.34 to 0.09)

4.1 (0.2 to 19.0)

3.5 (0.5 to 11.2)

0.03

0.004

0.08

0.86

0.44

0.13

0.11

0.06

0.007

0.03

0.54

0.27

0.07

0.09

Abbreviations: BH% ¼ Percentage of time with breath hold events during the 3-hour study time BHI ¼ BH index (number of breath hold events per hour) IQR ¼ interquartile range QOL ¼ Quality of life

EEGs from five patients and sleep in four EEGs from three patients. EEGs were abnormal in all cases. There was no clear effect of GA on background. However, in the four patients with epileptiform discharges present at baseline (Patients 2, 4, 8, and 9), the hourly ED rates were lower after treatment compared with baseline in all four patients. Seizure frequency was clinically unchanged. Quality of life

There was a nonsignificant trend toward improved QOL (Table 3). Correlation of covariates with each end point

No correlation was found between the age, presence or absence of seizures, and the magnitude of change after treatment on any of the end points. The cohort is too small to allow assessment of phenotypeegenotype correlation. Discussion

Our pilot study provides class III evidence about the potential of GA to improve some or Rett syndromeerelated symptoms in females with Rett syndrome who are ten years and older. The study was planned as exploratory and aimed to determine if GA administration causes any meaningful change in patients’ symptoms. It was conducted within a relatively short study period (ten months), to avoid commitment of large population of patients who suffer from this rare disease to a large-scale longer trial at this time when several therapeutic options may be available in the near future (May 2016, ClinicalTrials.gov36). In this population of nonverbal patients, it is, unfortunately, impossible to rely on the optimal, patient-reported outcomes, where reports of the status of a patient’s health condition come directly from the patient, without interpretation of the patient’s response by a clinician or caregiver.37 Instead, surrogate measures including behavioral scales, questionnaires, and a limited number of objective and quantifiable assessment tools (growth, sleep, hand function and respiratory function assessment) were used to assess treatment effect (ClinicalTiral.gov, May 2016). Our

preference was to objectively assess physiological performance or disease-related limitations and to evaluate the post-treatment versus pretreatment change in each end point. Gait abnormalities are one of the four necessary clinical requirements for clinical diagnosis of Rett syndrome.3 A recent study used movement kinematics and surface electromyographic activity to assess gait initiation in a group of 11 girls with Rett syndrome (mean age 9  3 years). They documented a distinct impairment in temporal organization of anticipatory postural adjustment, disruption of synergistic activity of muscles, the lack of appropriate temporal scaling, and diminished impulse to move forward.38 Each of the reported motor impairments affects gait velocity. As expected, variability existed between walking trials in this study, but the data presented, including the high ICC between the first and second trials, indicate that gait assessment was reliable despite the challenges. A trend suggesting that treatment gains may be sustained at oneyear follow-up compared with baseline in patients who continued the treatment after the study termination is encouraging, notably given the small number of participants. This finding further suggests that, similar to other patient populations, gait speed maybe an adequate outcome measure for intervention studies in children with RTT. Indeed, a search on www.clinicaltrials.gov (May 2016) revealed over 200 trials with gait velocity as either the primary or secondary outcome in a range of health and disease states and in different age groups. Timed walking is also included in the National Institutes of Health toolbox for use in clinical trials and epidemiologic studies. The feasibility of objective assessment of gait in girls with Rett syndrome has been previously reported, but, to date, it has not been used as an outcome in treatment trials38 (ClinicalTiral. gov, May 2016). The natural history of Rett syndrome, at this stage of the disease we studied, is characterized by worsening of gross motor difficulties caused by increasing tone, development of bradykinesias (decrease in gait velocity), worsening movement disorder, and loss of ambulation in some patients.2,39-42 All participants in this study were in their postregression stage of disease, with limited likelihood of

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spontaneous improvement. All participants had been immersed in physical therapies for years before enrollment, but these had not resulted in any improvement in their gross motor functions, limiting the possible role of practice effect as the mechanism of the observed improvement. Yet GA treatment was associated with improved gait velocity. Continuation of therapy after the trial did not lead to additional improvement. It could be argued, however, that the function of GA at this stage of treatment was to maintain the robust gains observed after 24 weeks of treatment. Since we did not study BDNF levels in cerebrospinal fluid in this pilot trial, we can only assume that increase in BDNF levels induced by GA administration may be the mechanism of the observed improvement. The GA treatment also resulted in a decrease of the number of BH per hour (BHI) and percentage of time spent breath holding. Improvement of respiratory function has been reported in the IGF-1 trial (improvement in the apnea index in a group of five patients with clinically significant apneas, longer than 10 seconds) and in individual case reports.8,43,44 Respiratory dysfunction affects 65% to 93% of patients with Rett syndrome and is not only a major challenge and a lifethreatening symptom in Rett syndrome but also a cause of significant morbidity, manifested by oxygen desaturations and loss of consciousness when severe, increased risk of aspiration, and exaggerated and dangerous heart rate changes.22,45-48 BHs are characterized by a pause after inspiration, when lungs remain inflated while overactive expiratory muscles are attempting to push the air out, against the closed glottis.44 They are disruptive for patients not only when prolonged (reflected in BH time) but also when mild but frequent (reflected by BHI). To address the facts that breathing disturbances are exquisitely sensitive to behavioral state and that sleepiness during respiratory function assessment can lead to significant skewing of the findings, we propose inclusion of behavioral state monitoring during respiratory function assessment for any trial in Rett syndrome.22,29,46 Here, we used eye tracking to assess memory and attention for the first time in treatment trials in Rett syndrome. Although eye tracking could be assessed in only about two thirds of the participants, due to various technical reasons (as previously reported by our group),30 it was sensitive to change. Those who were testable showed marked improvement in memory for faces. Such improvement is unlikely to be due to habituation or learning, since neither was found in an earlier work where there was repeated testing at ages five, seven, and 12 months.49 As expected, the EEG was abnormal in all the children with Rett syndrome. The background was unchanged after GA treatment. However, the rate of epileptiform discharges markedly decreased after treatment in the four subjects where they were present at baseline. These were routine EEG rather than prolonged ones. Further, as epileptiform EEG or clinical seizures were not an entry criterion for this study, only four subjects had these discharges present, so the very small sample prevents any definitive conclusions. However, this is of particular interest as a possible biomarker of response to drug therapy and deserves further study in a larger sample with more comprehensive EEG data that control for random or age-dependent variability in spike rates across time.

We did not encounter any patients with evidence of transitory immediate postinjection autonomic reactions, described by the manufacturer (14%) and observed in an Israeli Rett syndrome cohort (personal communication BenZeev), most likely due to small sample size.15 Limitations of this study include the small sample size, lack of placebo control, and lack of blinded assessments for study outcomes, except for the EEG data. The cohort was too small to allow assessment of phenotypeegenotype correlation. Since we conducted the evaluations only before drug initiation and after the trial, and not also before each dose escalation, we cannot comment on a possible doseeresponse relationship. However, the results of this exploratory trial emphasize the need for a larger scale placebo-controlled study of GA in Rett syndrome as the next step and provide a template for assessing efficacy of other interventions in Rett syndrome. The study was funded by Rett Syndrome Research Trust.

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