ARTICLE OPEN ACCESS CLASS OF EVIDENCE

Double-blind randomized placebo-controlledstudy of trofinetide in pediatric Rett syndromeDaniel G Glaze MD Jeffrey L Neul MD PhD Walter E Kaufmann MD Elizabeth Berry-Kravis MD PhD

Sean Condon DPH George Stoms BS Sean Oosterholt MSc Oscar Della Pasqua MD PhD Larry Glass BA

Nancy E Jones PhD and Alan K Percy MD on behalf of the Rett 002 Study Group

Neurologyreg 201992e1-e14 doi101212WNL0000000000007316

Correspondence

Dr Jones

njonesneurenpharmacom

AbstractObjectiveTo determine safety tolerability and pharmacokinetics of trofinetide and evaluate its efficacy infemale childrenadolescents with Rett syndrome (RTT) a debilitating neurodevelopmentalcondition for which no pharmacotherapies directed at core features are available

MethodsThis was a phase 2 multicenter double-blind placebo-controlled parallel-group study inwhich safetytolerability pharmacokinetics and clinical response to trofinetide were charac-terized in 82 childrenadolescents with RTT aged 5 to 15 years Sixty-two participants wererandomized 1111 to receive placebo twice a day (bid) for 14 days followed by placebo 50100 or 200 mgkg bid of trofinetide for 42 days Following blinded safety data review 20additional participants were randomized 11 to the 200 mgkg or placebo bid groups Safetyassessments included adverse events clinical laboratory tests physical examinations andconcomitant medications Clinician- and caregiver-based efficacy measurements assessedclinically relevant phenotypic dimensions of impairment of RTT

ResultsAll dose levels were well tolerated and generally safe Trofinetide at 200 mgkg bid showedstatistically significant and clinically relevant improvements relative to placebo on the RettSyndrome Behaviour Questionnaire RTT-Clinician Domain Specific ConcernsndashVisual AnalogScale and Clinical Global Impression ScalendashImprovement Exploratory analyses suggested thatobserved changes correlated with trofinetide exposure

ConclusionThese results together with those from a previous adolescentadult trial indicate trofinetidersquospotential for treating core RTT symptoms and support further trials

Classification of evidenceThis study provides Class I evidence that for childrenadolescents with RTT trofinetide wassafe well-tolerated and demonstrated improvement over placebo at 200 mgkg bid in func-tionally important dimensions of RTT

RELATED ARTICLE

Turning the tide ontargeted treatments forneurodevelopmentaldisorders

Page 741

MORE ONLINE

Class of EvidenceCriteria for ratingtherapeutic and diagnosticstudies

NPuborgcoe

These authors made equal contributions to the conduct andor analysis of the study described in this manuscript

From the Department of Pediatrics and Neurology (DGG) Baylor College of Medicine Houston TX Department of Neurosciences (JLN) University of California San DiegoGreenwood Genetic Center (WEK) Center for Translational Research Greenwood SC Pediatrics Neurological Sciences and Biochemistry (EBK) Rush University Medical CenterChicago ILVital Systems Inc (SC GS) Rolling Meadows IL Clinical Pharmacology ampTherapeutics Group (SO ODP) University College London UK Neuren Pharmaceuticals Ltd(LG NEJ) Camberwell VIC Australia Department of Pediatrics (AKP) Division of Neurology University of Alabama at Birmingham JLN is currently affiliated with the VanderbiltUniversity Medical Center Vanderbilt Kennedy Center Nashville TN

Go to NeurologyorgN for full disclosures Funding information and disclosures deemed relevant by the authors if any are provided at the end of the article

The Article Processing Charge was funded by ACADIA Pharmaceuticals Inc and Neuren Pharmaceuticals Limited

Coinvestigators are listed in the appendix at the end of the article

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology e1

Published Ahead of Print on March 27 2019 as 101212WNL0000000000007316

Rett syndrome (RTT) is a neurodevelopmental disorder af-fecting 1 in 10000ndash15000 females1ndash4 Most cases are causedby loss of function mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2)56 encoding a protein thatbinds to DNA and regulates transcription6ndash8

RTT is characterized by developmental regression includingloss of expressive language and purposeful hand use impairedor absent ambulation and onset of stereotypical handmovements9 Individuals with RTT have severe motor deficitsand autonomic gastrointestinal and other systemic symp-toms10 Neurobehavioral impairments are prevalent in-cluding anxiety-like behaviors disruptive behavior and mooddysregulation11ndash14 Disease burden is severe for individualsand their families the effects of which are lifelong1516 Noapproved or effective treatment is available for the core orother prevalent symptoms of RTT

Trofinetide (glycyl-L-2-methylprolyl-L-glutamic acid) is an analogof the amino-terminal tripeptide of insulinlike growth factor 1(IGF1 [1ndash3]) RTT mouse models demonstrated that IGF1(1ndash3) treatment improves disease symptoms17 Trofinetide isbelieved to have potential in treating RTT by normalizing aber-rant neuronal and glial function secondary to anti-inflammatoryand trophic effects which inhibit astrogliosis and pathologicmicroglial activation normalizing synaptic protein synthesisdendritic morphology and neuronal signaling and enhancingantioxidant response18ndash20 A phase 2 study in adolescentadultfemales with RTT demonstrated excellent safetytolerability andpreliminary evidence of efficacy at 70mgkg twice a day (bid) for28 days21 Herein we report a phase 2 study in a larger pediatriccohort evaluating higher doses and longer treatment durationand assessing pharmacokinetic-pharmacodynamic (PKPD) cor-relations to support the dose rationale for future studies

MethodsClassification of evidenceThe primary research questions were (1) Is treatment withtrofinetide safe and well-tolerated in the pediatric RTT pop-ulation and (2) What is the pharmacokinetic (PK) profile oftrofinetide in this population The secondary question was (3)Does trofinetide treatment demonstrate improvement of RTTsymptoms over placebo This study provides Class I evidenceshowing that trofinetide at doses of 50 100 and 200 mgkg bidwas well tolerated and generally safe in childrenadolescents withRTT in whom increasing doses were associated with

proportional increase in systemic exposure (linear PKs) and at200 mgkg bid demonstrated improvement over placebo acrossfunctionally important dimensions of RTT The study also pro-vides insight into potential efficacy measures for future studies

Study designStudy design (dose length of treatment and safety and effi-cacy outcome measures) was informed by the previousadolescentadult trial (Rett-001)21 The present study(hereafter Rett-002) was an exploratory phase 2 multicenterdouble-blind placebo-controlled parallel-group trial Thefirst 62 participants were randomized 1111 to placebo or 1of 3 dose levels of trofinetide 50 100 or 200 mgkg bidstratified by age Following review of blinded safety and tolera-bility data the study design wasmodified to enable enrollment of20 additional participants randomized 11 to 200 mgkg bid orplacebo bid The aim was to enrich both the high-dose andplacebo groups to maximize the likelihood of detecting clinicalbenefit in this population for whom there are no alternativepharmacotherapies Total duration of treatment was 56 dayswith participants receiving single-blind placebo treatment for 14days followed by 42 days of double-blind treatment with eitherdrug or placebo Participants had a posttreatment visit approxi-mately 10 days after the end of treatment Study design andsubject disposition are shown in figure 1

Randomization and treatment allocationThe randomization scheme used 2 age strata (ages 5ndash10 and11ndash15 years) and then assignment to treatment blocks At theend of the 1-week screening period the participant was ran-domized if she met all eligibility criteria and written informedconsent was provided by a legally authorized representativeRandomization was via a web-based randomization systemoperating 24 hours per day 7 days per week Participants wereassigned a unique randomization number Treatmentassignments were not disclosed to the sponsor participantscaregivers investigators or research site personnel

Dosingdose scheduleTrofinetide (also known as NNZ-2566) and placebo wereadministered as a volume-matched strawberry-flavored liquideither orally or via gastrostomy tube Participants were blindlyup-titrated to their assigned dose based on a predefined dosingschedule The 50 mgkg group was up-titrated over 2 days(85 mgkg bid 35 mgkg bid then 50 mgkg bid) the 100mgkg over 3 days (175 mgkg bid 35 mgkg bid 50 mgkgbid then 100 mgkg bid) and the 200 mgkg group over5 days (175 mgkg bid 35mgkg bid 50mgkg bid 100mgkg

GlossaryAE = adverse event AUC = area under the concentration vs time curve bid = twice a day CGI-I = Clinical Global ImpressionScalendashImprovement IGF1 = insulinlike growth factor 1 IRB = institutional review board ITT = intention to treat MBA =Motor Behavioral Assessment mITT = modified intention to treat PK = pharmacokinetic PKPD = pharmacokinetic-pharmacodynamic RSBQ = Rett Syndrome Behaviour Questionnaire RTT = Rett syndrome RTT-DSC = RettsyndromendashClinician Domain Specific Concerns SAE = serious adverse event VAS = Visual Analog Scale

e2 Neurology | Volume 92 Number 16 | April 16 2019 NeurologyorgN

bid 150 mgkg bid then 200 mgkg bid) The 100 and 200 mgkg groups were also down-titrated from their maximum dose afterday 54 (100 mgkg bid on day 55 and 50 mgkg bid on day 56)As such day 54 was the last day when all participants were treatedon their maximally assigned dose level

ParticipantsEighty-two girls between 5 and 15 years of age participated inthe study All participants met the 2010 diagnostic criteria forclassic RTT9 had molecular documentation of a pathogenicMECP2 variant were in the postregression stage and werestable on current pharmacologic and behavioral treatmentsfor at least 4 weeks (seizure profile and antiepileptic drugs hadto be stable for 8 weeks) Caregivers recorded seizure fre-quency medications and behavioral treatments in a paperdiary during the 1-week screening period to confirm stabilitythrough the start of the study medication Data were collectedat 12 RTT research centers in the United States

Standard protocol approvals registrationsand patient consentsThe study was registered on Clinicaltrialsgov (NCT02715115)Enrollment commenced in March 2016 and the study wascompleted in January 2017 after target enrollment was reachedand participants completed the study The studywas approved ateach study site by its institutional review board (IRB) or by thedesignated centralized IRB for the study Written informedconsent was obtained by the parent or legal guardian for allparticipants Oral or written assent was obtained from partic-ipants deemed able by the recruiting physician and per local IRBregulations

Safety assessmentsSafety evaluations included monitoring of adverse events(AEs) clinical laboratory tests (urinalysis hematologychemistry [including hemoglobin A1c electrolytes mineralsprotein lipids and tests of thyroid renal and liver function])

Figure 1 Study design (A) and participant disposition (B)

BID = twice daily dosing ITT = intention to treat mITT = modified intention to treat PK = pharmacokinetic

NeurologyorgN Neurology | Volume 92 Number 16 | April 16 2019 e3

vital signs ECGs physical examinations funduscopy andtonsil size and concomitant medications Seizures weremonitored in the caregiver diary

PK samplingPK samples were collected on day 28 (predose and 2ndash4 hourspost dose) and day 54 (predose 2ndash3 hours and 4ndash6 hourspost dose) Samples of whole blood were collected into 2-mLlithium heparin Vacutainer tubes from a cannula port or viavenipuncture and stored until shipment at minus70degC (or colder)no more than 2 hours after sample collection The date andtimes of collectionstorage in the freezer were recorded ina log The date and time of the most recent dose of studymedication and most recent meal relative to that dose wererecorded in the caregiver diary Samples were shipped atdesignated times on dry ice and using temperature monitor-ing to the laboratory for storage and analysis22

Efficacy outcome measuresEfficacy assessments were performed during the treatmentand posttreatment visits Baseline assessments occurred beforethe first dose of study medication All clinician raters completeda standardized training and completed regular calibration ses-sions during the study Study staff reviewed instructions forcompleting the caregiver measures with the caregiver rater ateach session and reviewed the forms for completeness

Efficacy measurements were categorized into 4 domains (1)clinician-completed syndrome-specific measures (RTT MotorBehavioral Assessment [MBA] RTT Domain SpecificConcernsndashVisual Analog Scale) (2) clinician-completedsyndrome-specific global measures (Clinical Global ImpressionScales Improvement and Severity) (3) caregiver-completedsyndrome-specific measures (Rett Syndrome Behaviour Ques-tionnaire Caregiver Top 3 ConcernsndashVisual Analog Scale RTTCaregiver Burden Inventory) and (4) physiologic measures(heart and respiratory rate)

For statistical analysis the end points were further charac-terized into priority levels of core secondary and exploratoryThe main efficacy analysis was conducted on core end pointswhich were identified in the statistical analysis plan prior tounblinding of treatment codes Core efficacy end points in-cluded 5 measures (both clinician- and caregiver-completed)as described below

Core efficacy end pointsThe RTT MBA is a clinician-completed rating scale that hasbeen used as part of the Rett Syndrome Natural HistoryStudy23ndash25 The MBA has 34 items captured on a 4-pointLikert scale grouped in 3 subscalesmdashBehaviorSocialOrofacialRespiratory and MotorPhysical Signs and a modi-fied scoring rubric used as an outcome variable in Rett-00121

The RTT-Clinician Domain Specific ConcernsndashVisual Ana-log Scale (RTT-DSC-VAS) is a clinician-completed VASassessing the severity of concerns in (1) hand use (2)

ambulation (3) seizures (4) autonomic features (5) be-havior (6) attentiveness (7) social interaction and (8)languagecommunication Concerns are identified on an in-dividual basis at baseline Severity is scored at baseline andfollow-up visits for each concern by measuring the number ofcentimeters on a 10-cm VAS line and reported as a percentageof the line A total VAS score for each participant is calculatedas the sum of the scores for the 8 concerns If a subject had nosymptomsconcerns in an area that domain was not ratedand the score was null for that concern

The Clinical Global Impression ScalendashImprovement (CGI-I)is a clinician-completed assessment of howmuch the individualrsquosillness has improvedworsened relative to a baseline state scoredusing a standardized rubric that is specific to the clinical featuresof RTT26 A 7-point scale is used 1 = very much improved 2 =much improved 3 = minimally improved 4 = no change 5 =minimally worse 6 = much worse 7 = very much worse Day 14assessments (treatment baseline end of placebo run-in) weremade relative to the pretreatment baseline visit For all sub-sequent visits assessments weremade relative to the day-14 visit

The Rett Syndrome Behaviour Questionnaire (RSBQ) isa validated caregiver-completed rating scale assessing a widerange of neurobehavioral symptoms known to be impaired inRTT27ndash31 The RSBQ has been correlated with functioningand quality of life and characterized across a range of ages andgenetic variations in RTT13143032 The scale includes 45items rated on a Likert scale from 0 to 2 39 of them groupedinto 8 subscales (General Mood Breathing Problems HandBehaviors Repetitive Face Movements Body Rocking andExpressionless Face Night-time Behaviors FearAnxiety andWalkingStanding) whose ratings reflect severity and frequencyof symptoms A total representing the sum of the 45 items(maximum score 90) and 8 subscale scores are obtained

The Caregiver Top 3 Concerns VAS is a syndrome-specificmeasure of 3 signs or symptoms identified by caregivers atbaseline on an individual per-participant basis as being pri-ority concerns they would like to see improved as a result oftreatment33 Caregivers were required to choose 3 concernsfrom any symptom domain related to the participantrsquos RTTThe severity of each concern is scored using a 10-cm VASusing the aforementioned methodology for the RTT-DSC

Sample sizeThis was an exploratory study with primary outcomes relatingto assessment of safety and PK and secondary outcomesrelating to efficacy The study design with approximately 82participants enabled randomization of approximately 15 pergroup to the 50 and 100 mgkg groups and approximately 24per group for the 200 mgkg and placebo groups which wasdeemed to be sufficient to detect treatment differences

Statistical analysesSafety analyses were conducted for all participants random-ized according to actual treatment received (intention-to-

e4 Neurology | Volume 92 Number 16 | April 16 2019 NeurologyorgN

treat [ITT] population) AEs and other safety data weresummarized as frequencies and percentages by treatmentgroups AEs were also summarized by system organ class andpreferred term using number and percentages AEs and seriousAEs (SAEs) were listed by severity and by relationship to studytreatment Discontinuations caused by AEs were also listed

Percent and number of participants with abnormal clinicallaboratory or ECG findings were summarized by time pointand treatment group Vital signs ECG thyroid funduscopyand tonsillar findings were summarized by time point andtreatment group Participants taking concomitant medi-cations were summarized by preferred term and treatmentgroup using numbers and percentages

An integrated population PK model was developed to describetrofinetidersquos PK in pediatric patients with RTT using sparsesamples22 In addition to clearance volume of distribution andabsorption rate constant secondary PK parameters includedTmax (time to maximum concentration) Cmax (maximumconcentration) Cmin (trough concentration) Css (steady-stateconcentration) t12 (terminal elimination half-life) and AUC(area under the concentration vs time curve) Derived PKparameters were used to assess the PKPD correlations between12-hour exposure (AUC0-12) and cumulative exposure over thecourse of treatment (AUC0-x) and change in coremeasures ThePK population included participants who received study drug toat least themorning day-28 dose and had a PK sample collectionat the specified postdose time point(s)

The efficacy analysis was conducted for the modified ITT(mITT) population which included all participants randomizedto receive at least one dose of double-blind study medicationand differences between each active treatment group and theplacebo group were assessed Comparisons of mean change oneach core efficacy measure were assessed from treatment base-line (day 14 end of placebo run-in) to day 54 (end of treatmentassessment the last visit before down-titration) For the CGI-Iactual values at the end of treatment were compared as there areno baseline values for CGI-I

Analyses were performed for each core end point usinga generalized linear model Treatment baseline and placeboresponse (change from pretreatment baseline to treatmentbaseline or observed value at treatment baseline for CGI-I)were included as covariates in the model but dropped fromthe final model if not significant at the p le 01 two-sided levelEffect sizes were determined using Cohen d For efficacyanalyses of the mITT population missing data were imputedwith the median value for the participantrsquos assigned dosegroup at that visit The imputation was performed for in-dividual instrument items any subscale subtotals and totalsfor a given instrument were calculated based on the imputedindividual items Nomultiplicity adjustments were performed Ifnormality and homogeneity of variance assumptions were sub-stantially violated an appropriate nonparametric test would beperformed instead Conclusions about the studyrsquos overall

evidence of clinical improvement were based on the totality ofefficacy data for the prespecified core end points rather than fora single primary end point

Data availabilityThe data reported are part of an ongoing sponsor-led clinicaldevelopment program As such complete datasets for thestudy will not be made available with the report

ResultsDemographicsA total of 82 participants from 12 study sites were randomizedin this study 24 in the placebo bid group 15 in the 50 mgkgbid trofinetide group 16 in 100 mgkg bid trofinetide groupand 27 in the 200 mgkg bid trofinetide group All 82 partic-ipants were included in the ITT population (safety analysis) andin the mITT population (efficacy analysis) The PK populationincluded all 58 participants who received treatment with trofi-netide Themean age of the cohort was 97 years (range 51ndash159years) 94 were white and mean weight was 261 kg Overalldemographic characteristics for participants were balancedacross the treatment groups information for the mITT pop-ulation is shown in table 1

SafetySafety and tolerability of trofinetide was very good at all 3 doselevels No deaths occurred in the study Only one participant(200 mgkg bid group) was withdrawn from the study at therequest of her parents because of increased mild gastro-esophageal reflux moderate diarrhea and mild vomitingwhich resolved uneventfully after discontinuation Four SAEsoccurred in 3 participants 1 participant receiving placebo 1participant receiving 100 mgkg bid and 1 participant re-ceiving 200 mgkg bid All the SAEs were deemed not relatedto study medication and resolved by the end of the study

A summary of AEs during the double-blind treatment periodoccurring in at least 2 participants is shown in table 2 Themost common AEs reported during the double-blind periodacross all treatment groups were diarrhea (27) vomiting(15) upper respiratory tract infection (12) and pyrexia(10)Diarrhea was reported in 27 in the 50mgkg bid group13 for the 100mgkg bid group and 56 in the 200mgkg bidgroup Most AEs were mild or moderate in intensity and mostevents were considered not related to study drug

No systematic evidence of withdrawal effects was observedwhen the study drug was discontinued Clinical laboratorytests ECGs vital signs and physical examinations (includingfunduscopy and tonsil size) indicated no time- or dose-dependent patterns

EfficacyTrofinetide demonstrated statistically significant evidence ofclinical improvement (p lt 005) for the 200 mgkg bid doseover placebo in 3 core measures RSBQ (total score core

NeurologyorgN Neurology | Volume 92 Number 16 | April 16 2019 e5

neurobehavioral RTT symptoms p = 0042) CGI-I (overallclinical status p = 0029) and RTT-DSC (most concerningaspects of RTT identified by clinicians p = 0025) Results forthe 5 core efficacy measures across the treatment groups areshown in table 3

Among the 3 outcome measures with p lt 005 the magnitudeof effect ( change of themedian score) for RTT-DSCwas 15(vs 5 for placebo) and themagnitude of effect ( change of themean score) for RSBQwas 16 (vs 6 for placebo) For CGI-Ithe mean was 30 for the 200 mgkg bid group and was 35 forplacebo with more than 20 of participants scoring a 2 ldquomuchimprovedrdquo compared with less than 5 of those on placebo

Cohen d effect sizes were minus0645 for CGI-I minus0247 for the RTT-DSC and minus0487 for the RSBQ total

Pharmacokinetics and pharmacodynamicsTrofinetide showed linear PKs across the dose range testedThese results are consistent with previous data in adult andadolescent patients (Rett-001) From a drug metabolism per-spective no accumulation metabolic inhibition or inductionwas observed during treatment Geometric mean estimates forthe predicted area under the concentration vs time curve(AUC0ndash12 μgmLmiddoth) were 1365 (50 mgkg bid) 3212 (100mgkg bid) and 5306 (200 mgkg bid) Geometric meanestimates of peak concentration (Cmax μgmL) were 187 (50

Table 1 Baseline demographics by treatment group (modified intention to treat)

Placebo(n = 24)

50 mgkg(n = 15)

100 mgkg(n = 16)

200 mgkg(n = 27)

All participants(n = 82)

Age y

Mean (SD) 938 (326) 1006 (318) 1081 (310) 923 (388) 973 (343)

Median 964 954 966 749 941

Heighta cm

Mean (SD) 12269 (1267) 12412 (1170) 12955 (1276) 12155 (1519) 12386 (1350)

Median 11970 12210 13030 11700 12090

Weight kg

Mean (SD) 2420 (687) 2613 (978) 3043 (1216) 2522 (1151) 2610 (1024)

Median 2375 2260 2865 2130 2305

BMIa kgcm2

Mean (SD) 1600 (285) 1650 (361) 1770 (506) 1631 (357) 1652 (370)

Median 1597 1504 1696 1570 1581

Age category n ()

le10 y 15 (63) 10 (67) 10 (63) 17 (63) 52 (63)

gt10 y 9 (38) 5 (33) 6 (38) 10 (37) 30 (37)

Ethnicity n ()

Hispanic 0 (0) 1 (7) 1 (6) 6 (22) 8 (10)

Not Hispanic 24 (100) 14 (93) 14 (88) 21 (78) 73 (89)

Not reported 0 (0) 0 (0) 1 (6) 0 (0) 1 (1)

Race n ()

American Indian or Alaskan Native 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Asian 1 (4) 0 (0) 0 (0) 2 (7) 3 (4)

Black or African American 0 (0) 0 (0) 1 (6) 0 (0) 1 (1)

Native Hawaiian or other Pacific Islander 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

White 22 (92) 15 (100) 15 (94) 25 (93) 77 (94)

Other 1 (4) 0 (0) 0 (0) 0 (0) 1 (1)

Abbreviation BMI = body mass indexa For height and BMI for the placebo group n = 23

e6 Neurology | Volume 92 Number 16 | April 16 2019 NeurologyorgN

mgkg bid) 487 (100 mgkg bid) and 837 (200 mgkg bid)The geometric mean of the apparent terminal elimination half-life(T12) varied from 53 to 61 hours across the 3 dosing groups

As observed previously in adults and adolescent patientsbody weight had a significant effect on clearance and volumeof distribution and consequently on overall systemic exposureto trofinetide Participants with lower body weight had lower

overall systemic exposure to trofinetide despite the use ofa dosing regimen based on body weight (ie mgkg) Dif-ferences in bioavailability were also observed betweenmorning and evening doses which may be explained by dif-ferent factors including circadian variation in absorption ormetabolism or eventually food effect22 which also appears tooccur in preclinical studies (unpublished data BioAnalyticalSystems Inc 2011)

Table 2 Incidence of treatment-emergent adverse events during the double-blind period (days 15ndash56 intention-to-treatpopulation)

System organ class preferred termaPlacebo(n = 24)

50 mgkg(n = 15)

100 mgkg(n = 16)

200 mgkg(n = 27)

Total(n = 82)

Reported at least 1 event 14 (58) 8 (53) 11 (69) 19 (70) 52 (63)

Gastrointestinal disorders

Diarrhea 1 (4) 4 (27) 2 (13) 15 (56) 22 (27)

Vomiting 3 (13) 1 (7) 2 (13) 6 (22) 12 (15)

Constipation 0 (0) 0 (0) 0 (0) 2 (7) 2 (2)

General disorders and administration-site conditions

Pyrexia 2 (8) 0 (0) 3 (19) 3 (11) 8 (10)

Infections and infestations

Upper respiratory tract infection 3 (13) 1 (7) 0 (0) 5 (19) 9 (11)

Nasopharyngitis 2 (8) 1 (7) 0 (0) 1 (4) 4 (5)

Ear infection 2 (8) 0 (0) 0 (0) 1 (4) 3 (4)

Gastroenteritis 0 (0) 0 (0) 1 (6) 1 (4) 2 (2)

Pneumonia 1 (4) 0 (0) 0 (0) 1 (4) 2 (2)

Metabolism and nutrition disorders

Decreased appetite 1 (4) 1 (7) 0 (0) 0 (0) 2 (2)

Nervous system disorders

Somnolence 1 (4) 1 (7) 0 (0) 0 (0) 2 (2)

Seizure 1 (4) 0 (0) 0 (0) 0 (0) 1 (1)

Seizure cluster 0 (0) 0 (0) 0 (0) 1 (4) 1 (1)

Tonic convulsion 0 (0) 0 (0) 1 (6) 0 (0) 1 (1)

Psychiatric disorders

Irritability 0 (0) 0 (0) 1 (6) 1 (4) 2 (2)

Respiratory thoracic and mediastinal disorders

Cough 2 (8) 0 (0) 1 (6) 1 (4) 4 (5)

Sinus congestion 0 (0) 0 (0) 1 (6) 2 (7) 3 (4)

Rhinorrhea 1 (4) 1 (7) 0 (0) 0 (0) 2 (2)

Skin and subcutaneous tissue disorders

Dermatitis diaper 0 (0) 1 (7) 0 (0) 1 (4) 2 (2)

Rash maculopapular 1 (4) 0 (0) 0 (0) 1 (4) 2 (2)

Data represent n () of participantsa Events occurring in at least 2 participants Seizure adverse events are an adverse event of interest in this population so all seizure-related adverse events arelisted

NeurologyorgN Neurology | Volume 92 Number 16 | April 16 2019 e7

At the end of the dosing period data indicate that clinicalbenefit was continuing to accrue as the difference betweenthe placebo and 200 mgkg bid groups appeared to continueto increase (figure 2) Furthermore there is evidence of a dimi-nution of effect following cessation of dosing Moreover asshown in figure 3 an exploratory PKPD analysis suggests a cor-relation between trofinetide exposure (expressed as AUC0ndash12

[dosing interval] and cumulative AUC [over 42 days]) and themagnitude of response on RSBQ RTT-DSC and CGI-I

Additional prespecified analysesAcross the core efficacy measures improvement was seenin clinically important symptom areas core to RTTbreathing problems repetitive movements (includinghand function) mood dysfunction (including nighttime

behaviors) ambulation and seizures On the RSBQ basedon comparison with placebo and Cohen d effect sizes(figure 4A) all of the subscales except one were direc-tionally in favor of the 200 mgkg bid treatment group withnotable improvement in mood dysfunction and disruptivebehavior (General Mood subscale p = 0007) breathingproblems (Breathing subscale p = 0095) and repetitivemovements (Repetitive Face Movement subscale p =0047)

On RTT-DSC improvements were notable in 2 domainsambulation (p = 0040) and seizures (p = 0057) but were alsoobserved to be directionally in favor of 200 mgkg bidtreatment for attentiveness and social interaction based onCohen d effect sizes (figure 4B)

Table 3 Change from treatment baseline (day 14) to end of treatment (day 54) in core efficacy outcomes (modifiedintention to treat)

Outcome measurePrespecifiedcovariates p le 01a

Placebo(n = 24)

50 mgkg(n = 15)

100 mgkg(n = 16)

200 mgkg(n = 27)

RSBQ total

Treatment baseline mean (SD) 395 (1183) 447 (1357) 403 (1126) 422 (1099)

Change D14ndashD54 LSmean (SE) PR minus23 (154) minus30 (195) minus15 (196) minus67 (146)

p Value vs placebo NA 0768 0749 0042

RTT-DSC total

Exact median testb

Treatment baseline median 4733 4500 44535 5166

Change D14ndashD54 median UnAdj minus2585 minus3250 minus1210 minus7600

p Value vs placebo NA 0999 0748 0025

CGI-I

Treatment baseline mdashc mdashc mdashc mdashc

Change D14ndashD54 LSmean (SE) UnAdj 35 (014) 33 (017) 34 (017) 30 (013)

p Value vs placebo NA 0391 0703 0029

Top 3 caregiver concerns

Treatment baseline mean (SD) 22387 (5451) 23769 (6397) 21155 (4260) 24590 (4912)

Change D14ndashD54 LSmean (SE) UnAdj minus1252 (878) minus1656 (1110) minus209 (1075) minus1854 (828)

p Value vs placebo NA 0776 0455 0619

MBA total

Treatment baseline mean (SD) 488 (799) 466 (877) 486 (882) 466 (1310)

Change D14ndashD54 LSmean (SE) TBL PR minus26 (099) minus28 (125) minus24 (123) minus29 (094)

p Value vs placebo NA 0872 0925 0840

Abbreviations CGI-I = Clinical Global Impression ScalendashImprovement D = day LSmean = least-squaresmean MBA =Motor Behavioral Assessment NA = notapplicable PR = placebo response RSBQ = Rett Syndrome Behaviour Questionnaire RTT-DSC = Rett syndromendashClinician Domain Specific Concerns TBL =treatment baseline UnAdj = unadjusteda Prespecifiedmodel covariates if p le 01 TBL andor PR UnAdj is noted if neither covariate was included in the final model Nomultiplicity adjustments wereperformedb The distribution of data in the RTT-DSC was nonnormal Consequently groupmedians were used in the analysis of this end point and statistical significancewas determined by the exact median testc CGI-I has no pretreatment baseline value The CGI-I values at day 14 are ratings of change from day 0 (the pre-treatment baseline) to day 14 (end of placeborun-in) CGI-I assessments done after day 14 are referenced to participantrsquos status at day 14Mean of actual scores was assessed at day 54 (end of treatment)

e8 Neurology | Volume 92 Number 16 | April 16 2019 NeurologyorgN

On RTT-DSC concerns are identified on an individual per-participant basis The types of symptoms that improved forambulation included being able to walk unassisted or im-proving on a certain motor milestone (eg walking a certaindistance walking on stairs improved stability) For seizuresconcerns included both frequency and severity but symptomsthat improved appear to be primarily the frequency ofseizures

DiscussionThis pediatric phase 2 study confirmed previous findingsand expanded the safety tolerability and efficacy profileobserved in the initial adolescentadult phase 2 trofinetidetrial in RTT21 All dose levels of trofinetide were generallysafe and well-tolerated in individuals with RTT aged 5 to15 years No sentinel safety events occurred Few SAEsand only one discontinuation because of AEs occurredand no overt time-dependent issues related to tolerability

were noted Diarrhea occurred more frequently in theactive treatment groups but did not affect overall tolera-bility It is important to note that chronic constipation andcorresponding pharmacologic treatment are highly prev-alent in RTT which may be a confounding factor Ob-jective assessments (eg laboratory assessments vitalsigns) did not reveal any systematic pattern of clinicaldetriment

Efficacy measures indicate clinically relevant improve-ments in a range of core symptoms of RTT at the highestdose of 200 mgkg bid as shown by statistically significantimprovement in 3 of the 5 core efficacy end points Themagnitude of this positive response was at a clinicallymeaningful level as evidenced by the effect size of thedifferences in the RSBQ and RTT-DSC total and subscalescores and a face validity assessment of the range ofsymptoms The findings on the 2 rating scales are sup-ported by improvements on overall functioning as

Figure 2 Core efficacy measures demonstrating improvement in the 200 mgkg group compared to placebo

(A) Change from treatment baseline of the 200 mgkg group compared to placebo in the mITT population for the RSBQ total score EOTmeasured at day 54Posttreatment follow-up at day 66 Improvement is a decrease in score LSmeans placebo response included as covariate in themodel (B) Absolute values ofthe 200mgkg group compared to placebo in themITT population for the CGI-I EOTmeasured at day 54 Posttreatment follow-up at day 66 Improvement islower score LSmeans (C) Change from treatment baseline of the 200 mgkg group compared to placebo in the mITT population for the RTT-DSC EOTmeasured at day 54 Posttreatment follow-up at day 66 Improvement is a decrease in score Exact median test Treatment baseline = day 14 CGI-I = ClinicalGlobal Impression ScalendashImprovement EOT = end of treatment LSmean = least-squares mean mITT = modified intention to treat RSBQ = Rett SyndromeBehaviour Questionnaire RTT-DSC = Rett syndromendashClinician Domain Specific Concerns VAS = Visual Analog Scale

NeurologyorgN Neurology | Volume 92 Number 16 | April 16 2019 e9

measured by the CGI-I Both the CGI-I and RSBQ totalhad medium effect sizes and 4 of the RSBQ subscales and 2of the RTT-DSC subscales showed Cohen d effect sizesge03 (small to medium) Effect sizes of this range arereported in trials of other CNS disorders using behavioral-based rating scales and are comparable to those reportedfor Food and Drug Administrationndashapproved treatments

for other disorders such as major depressive disorder(mean 03 range 017ndash042)34

Of note the RSBQ and RTT-DSC data suggest improve-ments across a range of symptom domains and individualsymptoms which included repetitive behaviors breathingproblemsmood abnormalitiesdisruptive behavior ambulation

Figure 3 Relationship between change from treatment baseline in RBSQ CGI-I and RTT-DSC and trofinetide exposure

Relationship between change from treatment baseline in RBSQ CGI-I and RTT-DSC and AUC0ndash12 at different visits and over the active treatment period (A)Relationship between percentage change from treatment baseline in RSBQ-total score and AUC0ndash12 at day 28 (B) Relationship between percentage changefrom treatment baseline in RSBQ-total score and cumulative AUCduring the active dosing period (C) Relationship between CGI-I scores andAUC0ndash12 at day 28(D) Relationship between CGI-I score and cumulative AUC during the active dosing period (E) Relationship between percentage change from treatmentbaseline in RTT-DSC score and AUC0ndash12 at day 28 (F) Relationship between percentage change from treatment baseline in RTT-DSC score and cumulative AUCduring the active dosing period Visit 3 (day 14 end of placebo run-in) visit 4 (day 21) visit 5 (day 28) visit 6 (day 42) visit 7 (day 54 end of treatment) visit 8 (day66 posttreatment) Solid lines are obtained by linear regression Where applicable placebo data (AUC = 0) from different visits are pooled together AUC =area under the concentration vs time curve CGI-I = Clinical Global Impression ScalendashImprovement RSBQ = Rett Syndrome Behaviour Questionnaire RTT-DSC = Rett syndromendashClinician Domain Specific Concerns

e10 Neurology | Volume 92 Number 16 | April 16 2019 NeurologyorgN

impairment and seizures This multiple-domain effect isconsistent with the adolescentadult Rett-001 trial21 whichshowed improvement in measures covering a wide range ofmanifestations of the disease (eg MBA CGI-I) and con-sistent with the generalized mechanisms of action oftrofinetide1735 As in the present study in Rett-001 appar-ent clinical benefits were also exhibited in both clinician andcaregiver assessments and in a number of core RTTsymptoms21 In addition as in the present study the clinicalimprovement was still increasing at the end of treatmentand declined after cessation of treatment The relativeconsistency of effects (albeit captured by a partially differentset of measures and in an older population with shorterexposure to trofinetide) gives additional confidence thatthe results of the previous study were not simply attribut-able to chance

There are no validated biomarkers available to assess drug-induced physiologic cognitive and behavioral effects whichcan ultimately be linked to clinical improvement and efficacySuch a limitation may be partly overcome by the evaluation ofexposure-response relationships3637 In this pediatric studydespite the variability of the relatively small cohort a correla-tion was found between drug exposure and magnitude ofchanges in response to treatment While an integrated analysisof all available data needs to be performed (including anevaluation of the response over a longer treatment period toestablish the dose-exposure-response relationship) the effi-cacy data from this study suggest that the group separationduring the dosing period was due to beneficial treatment ef-fect and not to other differences between the 2 arms thatoccurred simply by chance Taken together with the obser-vation of sustained improvement at the end of treatment and

Figure 4 Cohen d effect sizes for the RSBQ and RTT-DSC-VAS (200 mgkg vs placebo at day 54)

Cohend effect sizes for the RSBQ (A) andRTT-DSC-VAS (B) Cohen d values are unadjustedthat is do not incorporate covariate in-formation from treatment baseline or pla-cebo response bid = twice a day RSBQ = RettSyndrome Behaviour Questionnaire RTT-DSC = Rett syndromendashClinician DomainSpecific Concerns VAS = Visual Analog Scale

NeurologyorgN Neurology | Volume 92 Number 16 | April 16 2019 e11

the diminution of effect post treatment these observationsalso suggest that clinical benefit is likely to increase furtherwith a longer treatment period than was studied in this trialOverall the observed clinical improvement in the presentpediatric trial was more manifest than in the previous trialwith younger age (ie greater neuroplasticity) higher doses(ie higher drug exposure) and longer drug treatment du-ration (ie 28 days in Rett-001 vs 42 days in Rett-002) aspotential contributors

The PK findings replicate findings from the adult trialshowing that weight is an influential moderator of systemicexposure a feature that is common to many drugs used inchildren3839 While mgkg dosing was used dose levels werenot banded by weight in this protocol as such systemic ex-posure was not comparable across all participants receivinga given dose of trofinetide Consequently dose per se was notas important as drug exposure as a determinant factor for themagnitude of the response to trofinetide In addition as ex-posure will vary not only with the dose but also with bodyweight identifying dosing regimens that provide optimal ex-posure will likely reduce variability in response Thereforeweight-banded dosing or similar algorithms should be con-sidered in future trials to adjust dosing in this population3940

For this phase 2 study in a pediatric population the RSBQwasadded to the assessment battery The RSBQ is the mostwidely used behavioral instrument in RTT in part because ofits disorder-specificity and its reliability and validity in par-ticular for the RTT pediatric population12ndash1432 More re-cently the RSBQ has also shown sensitivity to interventionsand correlations with functioning and quality of life inRTT142831 The RSBQ could more properly be labeled asa ldquoneurobehavioralrdquo measure since it includes RTT featuresthat are modulated rather than triggered by behavior (egbreathing problems) Thus the RSBQ is an instrument suit-able for assessing multiple core RTT features similar to theMBA The RSBQ subscale analyses in this study also suggestthat despite its generalized effect trofinetide may have greatereffects on some neurobehavioral symptoms (eg moodabnormalitiesdisruptive behavior) Future studies aiming atreplicating the results of this pediatric trial would benefit fromincluding the RSBQ as a primary end point and should alsoconsider evaluating RSBQ subscales such as the GeneralMood

While supportive of trofinetidersquos potential as a treatment formultiple RTT core symptoms the present study had severallimitations As a phase 2 study the 2 most obvious are smallcohort size and short duration In addition no biomarker orclinical characteristic allowed identification of potential res-ponders to trofinetide Also as noted above since weight wasa factor moderating exposure some participants may not haveexperienced the maximal potential exposure for the dose levelthey received Lastly as this was an exploratory study we havedeemed it appropriate not to include adjustment for multi-plicity for the statistical comparisons Consideration of the

above-mentioned factors and the known shortcomings ofavailable outcomemeasures for RTT2941 in the design of futuretrials will be important for their success RTT is a debilitatingand life-threatening neurodevelopmental disorder for which notherapies are available that address its core features The resultspresented here provide evidence that trofinetide is a potentiallyviable treatment for the core signs and symptoms of RTT andsupport further trials in this population

AcknowledgmentThe authors thank Rettsyndromeorg for their support of thestudy and gratefully acknowledge the contribution of patientswho participated in the study and their families

Study fundingThe clinical trial was sponsored by Neuren Pharmaceuticals andfunded by Neuren Pharmaceuticals and Rettsyndromeorg

DisclosureD Glaze was an investigator in the Neuren trofinetide trialreported in this article and the Rett-001 trial of trofinetide inadolescents and adults He is a consultant to and participatesin trials sponsored by Newron Pharmaceuticals SpA J Neulwas an investigator in the Neuren trofinetide trial reported inthis article and the Rett-001 trial of trofinetide in adolescentsand adults He is a consultant to Ovid AveXis Eloxx Phar-maceuticals Biohaven Teva and Takeda PharmaceuticalsHe participates in clinical trials sponsored by Newron Phar-maceuticals SpA W Kaufmann was part of the study per-sonnel for the Neuren trofinetide trial reported in this articleHe is a consultant to Anavex AveXis Biohaven Cydan EchoEdison EryDel GW Pharmaceuticals Marinus NewronPharmaceuticals SpA Ovid Therapeutics Stalicla andZynerba He has been a consultant and participating principalinvestigator (PI) in sponsor-led trials for Roche Novartis andSeaside Therapeutics Walter Kaufmann was also PI of 2previous IGF1 (mecasermin) trials in Rett syndrome He alsoreceived research support from Ipsen Pharmaceuticals andEloxx Pharmaceuticals E Berry-Kravis was an investigator inthe Neuren trofinetide trial reported in this article and a PI ona Neuren-sponsored trial in fragile X She is a consultant toCydan Fulcrum GW Marinus BioMarin Zynerba Yamoand Neurotrope Pharmaceuticals She is a consultant and hasbeen a participating PI in sponsor-led trials for Ovid Thera-peutics Alcobra Roche Novartis and Seaside TherapeuticsShe receives research support and has been a co-PI of a reg-istration trial for VTS-270 in Niemann-Pick type C l forVtesseSucampoMallinckrodt She receives research sup-port from Asuragen Inc for work on validation of FXS testingmethods S Condon is a biostatistician for Vital Systems Inca contract research organization utilized by Neuren for thetrial described in this article G Stoms is president of VitalSystems Inc a contract research organization utilized byNeuren for the conduct and analysis of this clinical trial theRett-001 trial of trofinetide in adolescents and adults andtrials in fragile X and traumatic brain injury sponsored byNeuren S Oosterholt is a clinical pharmacologist at UCL

e12 Neurology | Volume 92 Number 16 | April 16 2019 NeurologyorgN

supporting PKPD data analysis for this trial the Rett-001 trial oftrofinetide in adolescents and adults and trials in fragile X andtraumatic brain injury sponsored by Neuren PharmaceuticalsLtd O Della Pasqua is chair of Clinical Pharmacology ampTherapeutics Group at the University College London He isalso senior director of Clinical Pharmacology Life Cycle Man-agement at GlaxoSmithKline RampD which was not involved withthe study L Glass is an executive of Neuren Pharmaceuticals NJones is an executive of Neuren Pharmaceuticals A Percy was aninvestigator in the Neuren trofinetide clinical trial reported inthis article and Rett-001 trial of trofinetide in adolescents andadults He is a consultant to Anavex AveXis and Teva andparticipates in trials sponsored byNewron Pharmaceuticals SpAGo to NeurologyorgN for full disclosures

Publication historyReceived by Neurology July 28 2018 Accepted in final form December19 2018

Appendix 1 Authors

Name Location Role Contribution

Daniel GGlaze MD

Baylor College ofMedicineDepartment ofPediatrics andNeurology

Author Assisted with theconceptualization ofthe study enrolledparticipants andcollected study datainterpreted the dataand criticallyreviewed and revisedthe manuscript

Jeffrey LNeul MDPhD

University ofCalifornia San DiegoDepartment ofNeurosciencesCurrent affiliationVanderbilt UniversityMedical CenterVanderbilt KennedyCenter

Author Assisted with theconceptualization ofthe study enrolledparticipants andcollected study dataand criticallyreviewed and revisedthe manuscript

Walter EKaufmannMD

Greenwood GeneticCenter Center forTranslationalResearch

Author Assisted with thedesign of the studyand interpretation ofoutcome measuresinterpreted the dataand criticallyreviewed and revisedthe manuscript

ElizabethBerry-Kravis MDPhD

Rush UniversityMedical CenterPediatricsNeurologicalSciences andBiochemistry

Author Enrolled participantsand collected studydata interpreted thedata and criticallyreviewed and revisedthe manuscript

SeanCondonDPH

Vital Systems Inc Author Provided statisticaladvice conducted theanalysis of the safetyand efficacy data andreviewed themanuscript

GeorgeStoms BS

Vital Systems Inc Author Provided statisticaladvice conducted theanalysis of the safetyand efficacy data andreviewed themanuscript

Appendix 1 (continued)

Name Location Role Contribution

SeanOosterholtMSc

Clinical Pharmacologyamp TherapeuticsGroup UniversityCollege London

Author Conducted thepharmacokinetic (PK)and pharmacokinetic-pharmacodynamic(PKPD) analysisinterpreted the PKand PKPD data andreviewed themanuscript

Oscar DellaPasquaMD PhD

Clinical Pharmacologyamp TherapeuticsGroup UniversityCollege London

Author Conducted thepharmacokinetic (PK)and pharmacokinetic-pharmacodynamic(PKPD) analysisinterpreted the PKand PKPD data andreviewed themanuscript

Larry GlassBA

NeurenPharmaceuticals Ltd

Author Participated in theconceptualizationand design of thestudy participated intheanalysis interpretedthe data and criticallyreviewed and revisedthe manuscript

Nancy EJones PhD

NeurenPharmaceuticals Ltd

Author Participated in theconceptualizationand design of thestudy assisted withthe coordination andimplementation ofthe studyparticipated in theanalysis interpretedthe data and draftedthe manuscript NJtakes responsibilityfor the article asa whole

Alan PercyMD

University of Alabamaat BirminghamDepartment ofPediatrics Division ofNeurology

Author AlanPercy assistedwiththe conceptualizationdesign andimplementation of thestudy enrolledparticipants andcollected study datainterpreted the dataand critically reviewedand revised themanuscript

Appendix 2 Coinvestigators

Name Location Role Contribution

ArthurBeisang MD

Gillette ChildrenrsquosSpecialtyHealthcare

Siteinvestigator

Enrolledparticipants andcollected studydata Providedadvice onassessments

TimothyBenke MDPhD

Childrenrsquos HospitalColorado

Siteinvestigator

Enrolledparticipants andcollected studydata Providedadvice onassessments

Continued

NeurologyorgN Neurology | Volume 92 Number 16 | April 16 2019 e13

References1 Bienvenu T Philippe C De Roux N et al The incidence of Rett syndrome in France

Pediatr Neurol 200634372ndash3752 Laurvick CL de Klerk N Bower C et al Rett syndrome in Australia a review of the

epidemiology J Pediatr 2006148347ndash3523 Burd L Vesley B Martsolf JT Kerbeshian J Prevalence study of Rett syndrome in

North Dakota children Am J Med Genet 199138565ndash5684 Hagberg B Aicardi J Dias K Ramos O A progressive syndrome of autism dementia

ataxia and loss of purposeful hand use in girls Rettrsquos syndrome report of 35 casesAnn Neurol 198314471ndash479

5 Neul JL Fang P Barrish J et al Specific mutations in methyl-CpG-binding protein 2confer different severity in Rett syndrome Neurology 2008701313ndash1321

6 Cuddapah VA Pillai RB Shekar KV et al Methyl-CpG-binding protein 2 (MECP2)mutation type is associated with disease severity in Rett syndrome JMed Genet 201451152ndash158

7 Kriaucionis S Bird A DNA methylation and Rett syndrome Hum Mol Genet 200312R221ndashR227

8 Hite KC Adams VH Hansen JC Recent advances in MeCP2 structure and functionBiochem Cell Biol 200987219ndash227

9 Neul JL Kaufmann WE Glaze DG et al for the RettSearch Consortium Rett syndromerevised diagnostic criteria and nomenclature Ann Neurol 201068946ndash951

10 Percy A Rett syndrome coming to terms with treatment Adv Neurosci 201420141ndash20

11 Urbanowicz A Downs J Girdler S Ciccone N Leonard H Aspects of speech-language abilities are influenced by MECP2 mutation type in girls with Rett syn-drome Am J Med Genet A 2015167354ndash362

12 Kaufmann WE Tierney E Rohde CA et al Social impairments in Rett syndromecharacteristics and relationship to clinical severity J Intellect Disabil Res 201256233ndash247

13 Cianfaglione R Clarke A KerrM et al National survey of Rett syndrome behaviouralcharacteristics J Neurodev Disord 2015711ndash15

14 Barnes K Coughlin FR OrsquoLeary HM et al Anxiety-like behaviour in Rett syndromecharacteristics and assessment by anxiety scales J Neurodev Disord 2015730

15 Kirby RS Percy AK Lane JB et al Longevity in Rett syndrome probing the NorthAmerican Database J Pediatr 2010159135ndash138

16 Tarquinio DC Hou W Neul JL et al The changing face of survival in Rett syndromeand MECP2-related disorders Pediatr Neurol 201553402ndash411

17 Tropea D Giacometti E Wilson NR et al Partial reversal of Rett syndrome-likesymptoms in MeCP2 mutant mice Proc Natl Acad Sci USA 20091062029ndash2034

18 Derecki NC Cronk JC Lu Z et al Wild-type microglia arrest pathology in a mousemodel of Rett syndrome Nature 2012484105ndash109

19 Lu XC Si Y Williams AJ Hartings JA Gryder D Tortella FC NNZ-2566 a glypro-mate analog attenuates brain ischemia-induced non-convulsive seizures in ratsJ Cereb Blood Flow Metab 2009291924ndash1932

20 Wei HH Lu XC Shear DA et al NNZ-2566 treatment inhibits neuroinflammationand pro-inflammatory cytokine expression induced by experimental penetratingballistic-like brain injury in rats J Neuroinflammation 2009619

21 Glaze DG Neul JL Percy A et al A double-blind randomized placebo-controlledclinical study of trofinetide in the treatment of Rett syndrome Pediatr Neurol 20177637ndash46

22 Oosterholt SP Horrigan J Jones N Glass L Della PO Population pharmacokineticsof NNZ-2566 in healthy subjects Eur J Pharm Sci 2017109S98ndashS107

23 Percy A Neul J Glaze D et al Rett syndrome diagnostic criteria lessons from theNatural History Study Ann Neurol 201068951ndash955

24 Neul JL Lane JB Lee HS et al Developmental delay in Rett syndrome data from theNatural History Study J Neurodev Dis 201462ndash9

25 FitzGerald PM Jankovic J Percy AK Rett syndrome and associated movementdisorders Mov Disord 19905195ndash202

26 Neul JL Glaze DG Percy A et al Improving treatment trial outcomes for Rettsyndrome the development of Rett-specific anchors for the Clinical Global Im-pression Scale J Child Neurol 2015301743ndash1748

27 Mount RH Charman T Hastings RP Reilly S Cass H The Rett Syndrome BehaviourQuestionnaire (RSBQ) refining the behavioural phenotype of Rett syndrome J ChildPsychol Psych 2002431099ndash1110

28 Khwaja OS Ho E Barnes KV et al Safety pharmacokinetics and preliminary as-sessment of efficacy of mecasermin (recombinant human IGF-1) for the treatment ofRett syndrome Proc Natl Acad Sci USA 20141114596ndash4601

29 Katz DM Bird A Coenraads M Gray SJ Menon DU Philpot BD Rett syndromecrossing the threshold to clinical translation Trends Neurosci 201639100ndash113

30 Cianfaglione R Clarke A Kerr M Hastings RP Oliver C Felce D Ageing in Rettsyndrome J Intellect Disabil Res 201660182ndash190

31 OrsquoLeary HM Kaufmann WE Barnes KV et al Placebo controlled crossover efficacyassessment of mecasermin for the treatment of Rett syndrome Ann Clin TranslNeurol 20185323ndash332

32 Robertson L Hall SE Jacoby P Ellaway C de Klerk N Leonard H The associationbetween behaviour and genotype in Rett syndrome using the Australian Rett Syn-drome Database Am J Med Genet 2006141177ndash183

33 Arnold LE Vitiello B McDougle C et al Parent-defined target symptoms respond torisperidone in RUPP Autism Study customer approach to clinical trial J Am AcadChild Adolesc Psychiatry 2003421443ndash1450

34 Turner EH Matthews AM Linardatos E Tell RA Rosenthal R Selective publicationof antidepressant trials and its influence on apparent efficacy N Engl J Med 2008358252ndash260

35 Corvin AP Molinos I Little G et al Insulin-like growth factor 1 (IGF1) and its activepeptide (1-3)IGF1 enhance the expression of synaptic markers in neuronal circuitsthrough different cellular mechanisms Neurosci Lett 201252051ndash56

36 Taneja A Oosterholt SP Danhof M Della Pasqua O Biomarker exposure-responserelationships as the basis for rational dose selection lessons from a simulation exerciseusing a selective COX-2 inhibitor J Clin Pharmacol 201656609ndash621

37 Danhof M Alvan G Dahl SG Kuhlmann J Paintaud G Mechanism-basedpharmacokinetic-pharmacodynamic modeling a new classification of biomarkersPharm Res 2005221432ndash1437

38 Piana C ZhaoW Adkison K et al Covariate effects and population pharmacokineticsof lamivudine in HIV-infected children Br J Clin Pharmacol 201477861ndash872

39 Cella M Knibbe C DanhofM Della Pasqua OWhat is the right dose for children BrJ Clin Pharmacol 201070597ndash603

40 Cella M Danhof M Della Pasqua O Adaptive trials in paediatric developmentdealing with heterogeneity and uncertainty in pharmacokinetic differences in childrenBr J Clin Pharmacol 201274346ndash353

41 Kaufmann WE Stallworth JL Everman DB Skinner SA Neurobiologically-basedtreatments in Rett syndrome opportunities and challenges Expert Opin OrphanDrugs 201641043ndash1055

Appendix 2 (continued)

Name Location Role Contribution

TimothyFeyma MD

Gillette ChildrenrsquosSpecialtyHealthcare

Siteinvestigator

Enrolledparticipants andcollected studydata

PeterHeydemannMD

Rush UniversityMedical Center

Siteinvestigator

Enrolledparticipants andcollected studydata

Mary JonesMD

UCSF BenioffChildrenrsquos HospitalOakland

Siteinvestigator

Enrolledparticipants andcollected studydata

Eric DMarsh MDPhD

Childrenrsquos Hospitalof Philadelphia

Siteinvestigator

Enrolledparticipants andcollected studydata

SarikaPeters PhD

VanderbiltUniversity MedicalCenter

Siteinvestigator

Enrolledparticipants andcollected studydata

Myron ldquoSkiprdquoPetersonMD PhD

Cato Research Medicalmonitor

Monitored andreviewed safetydata

MustafaSahin MDPhD

Boston ChildrenrsquosHospital

Siteinvestigator

Enrolledparticipants andcollected studydata

SteveSkinner MD

Greenwood GeneticCenter

Siteinvestigator

Enrolledparticipants andcollected studydata

ShannonStandridgeDO MPH

Department ofPediatricsUniversity ofCincinnati Collegeof Medicine OHDivision ofNeurologyCincinnatiChildrenrsquos HospitalMedical Center OH

Siteinvestigator

Enrolledparticipants andcollected studydata

e14 Neurology | Volume 92 Number 16 | April 16 2019 NeurologyorgN

DOI 101212WNL0000000000007316 published online March 27 2019Neurology

Daniel G Glaze Jeffrey L Neul Walter E Kaufmann et al syndrome

Double-blind randomized placebo-controlled study of trofinetide in pediatric Rett

This information is current as of March 27 2019

ServicesUpdated Information amp

316fullhttpnneurologyorgcontentearly20190327WNL0000000000007including high resolution figures can be found at

Citations

316fullotherarticleshttpnneurologyorgcontentearly20190327WNL0000000000007This article has been cited by 2 HighWire-hosted articles

Subspecialty Collections

httpnneurologyorgcgicollectionrett_syndromeRett Syndrome

lled_consort_agreementhttpnneurologyorgcgicollectionclinical_trials_randomized_controClinical trials Randomized controlled (CONSORT agreement)

httpnneurologyorgcgicollectionall_pediatricAll Pediatric

httpnneurologyorgcgicollectionall_clinical_trialsAll Clinical trials

httpnneurologyorgcgicollectionall_clinical_neurologyAll Clinical Neurologyfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpwwwneurologyorgaboutabout_the_journalpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnneurologyorgsubscribersadvertiseInformation about ordering reprints can be found online

ISSN 0028-3878 Online ISSN 1526-632XWolters Kluwer Health Inc on behalf of the American Academy of Neurology All rights reserved Print1951 it is now a weekly with 48 issues per year Copyright Copyright copy 2019 The Author(s) Published by

reg is the official journal of the American Academy of Neurology Published continuously sinceNeurology