Effects of STX209 (Arbaclofen) on Neurobehavioral Function in Children and
Adults with Fragile X Syndrome: A Randomized, Controlled, Phase 2 Trial
Elizabeth M. Berry-Kravis, David Hessl, Barbara Rathmell, Peter Zarevics, Maryann
Cherubini, Karen Walton-Bowen, Yi Mu, Danh V. Nguyen, Joseph Gonzalez-Heydrich,
Paul P. Wang, Randall L. Carpenter, Mark F. Bear, and Randi J. Hagerman
Research on animal models of fragile X syndrome suggests that STX209, a γ-
aminobutyric acid type B (GABAB) agonist, might improve neurobehavioral function in
affected patients. We evaluated whether STX209 improves behavioral symptoms of
fragile X syndrome in a randomized, double-blind, placebo-controlled crossover study in
63 subjects (55 male), ages 6 to 39 years, with a full mutation in the FMR1 gene (>200
CGG triplet repeats). We found no difference from placebo on the primary endpoint, the
Aberrant Behavior Checklist—Irritability (ABC-I) subscale. In the other analyses
specified in the protocol, improvement was seen on the visual analog scale ratings of
parent-nominated problem behaviors, with positive trends on multiple global measures.
Post hoc analysis with the ABC—Social Avoidance scale, a newly validated scale for the
assessment of fragile X syndrome, showed a significant beneficial treatment effect in the
full study population. A post hoc subgroup of 27 subjects with more severe social
impairment showed improvements on the Vineland II–Socialization raw score, on the
ABC—Social Avoidance scale, and on all global measures. STX209 was well tolerated,
with 8% incidences of sedation and of headache as the most frequent side effects. In this
exploratory study, STX209 did not show a benefit on irritability in fragile X syndrome.
Nonetheless, our results suggest that GABAB agonists have potential to improve social
function and behavior in patients with fragile X syndrome.
Targeted treatments are therapies that address the specific molecular pathophysiology of
a disease. Until very recently, neurobiological knowledge was not adequate to allow the
development of targeted treatments for neurodevelopmental disorders. Instead,
psychopharmacologic agents were developed on the basis of fortuitous observations [for
example, psychostimulants for attention deficit/hyperactivity disorder (1)] or by
extension from their use in distinct adult conditions [for example, antipsychotics for the
treatment of irritability in autism (2)]. Now, science has advanced to the point that drug
development for several neurodevelopmental disorders can proceed within a rational
neurobiological framework (3, 4). Fragile X syndrome (FXS) provides a key example of
this approach (5).
FXS is the most common known inherited cause of autism and of intellectual disability
(6), and social impairment is among its core features (7, 8). It is caused by an expansion
of the CGG triplet repeat in the FMR1 gene on the X chromosome. The discovery that the
FXS phenotype is a result of the transcriptional silencing of the FMR1 gene (9) led to the
creation of animal models in which this gene has been knocked out. Study of these
animals led, in turn, to the mGluR theory of fragile X (10), which posits that the
neurobehavioral abnormalities in FXS result from dysregulation of neuronal signaling
through group 1 metabotropic glutamate receptor–activated (mGluR1 and mGluR5)
pathways and excessive dendritic protein synthesis. Pharmacologic and genetic rescue
experiments show that negative modulators of mGluR5 correct many of the abnormal
phenotypes in animal models of FXS, including disruptions of neural plasticity and
excitability in the hippocampus, neocortex, and amygdala; deficits in learning and
memory; and altered neuronal morphology (11–15).
Deficiencies in γ-aminobutyric acid (GABA)–mediated inhibitory neurotransmission also
have been implicated in FXS. In Fmr1-knockout mice, decreased GABAergic inhibition
occurs in many areas of the brain, including the hippocampus, striatum, somatosensory
cortex, and amygdala (16, 17). Humans with FXS show overactivation of the amygdala
when asked to perform a face-processing task (18). Excessive activation in the amygdala
and elsewhere in the limbic system is hypothesized to be a basis of the social anxiety and
social avoidance that characterize FXS (19–21). GABAergic compounds reverse
phenotypes in the FXS Drosophila model (22), and pharmacological stimulation of
GABA receptors has therefore been suggested as a therapeutic strategy for FXS (17, 23).
Treatment with a GABA agonist could work directly, by augmenting the deficiencies in
inhibitory neurotransmission, or indirectly as a negative modulator of the mGluR
pathway, by decreasing the synaptic release of glutamate, a known effect of presynaptic
GABA type B (GABAB) receptor stimulation (24).
STX209 (arbaclofen, R-baclofen) is a GABAB receptor agonist and is the active
enantiomer of racemic baclofen. In Fmr1-knockout mice, STX209 rescues the increased
susceptibility to seizures and corrects the excessive dendritic spine density and protein
synthesis believed to be pathogenic in FXS (25). In healthy human subjects, STX209
shows pharmacokinetic properties similar to those of racemic baclofen, with high
bioavailability and a terminal half-life of 4 to 5 hours. STX209 undergoes renal
elimination with no significant metabolism (26). Here, we have examined the safety and
efficacy of STX209 in the treatment of neurobehavioral symptoms in humans with FXS.
Subjects, disposition, and dosing
Sixty-three subjects were randomized into the study. Their demographic and baseline
characteristics are shown in Table 1. Fifty-six (89%) subjects completed the entire study.
Subject disposition and the composition of the primary analysis populations are shown in
There were no withdrawals related to drug tolerability. Each subject’s optimal titrated
dose (OTD) was defined as either the highest tolerated dose or the lowest dose at which a
subject was judged to be “very much improved” on the CGI-I, the Clinical Global
Impression of Improvement. This instrument is a seven-point Likert scale that requires
the clinician to evaluate overall improvement (or worsening), considering the entirety of
the data available on the subject. Among subjects for whom an OTD was reported, the
OTD was the maximum allowed dose for 91% of subjects aged 6 to 11 years [10 mg,
twice daily (BID)] and 63% of subjects aged 12 to 40 years [10 mg, thrice daily (TID)].
Pharmacokinetic analyses confirmed correct treatment assignment for all subjects.
Total 63 27
6–11 24 11
12–17 22 9
18–40 17 7
Sex (M/F) 55/8 24/3
Race (>1 response permitted)
Caucasian 55 25
African-American 3 0
Other 5 2
Ethnicity: Non-Hispanic/Hispanic 58/5 25/2
Comorbid autism (DSM-IV and ADI-R criteria) 37 19
Concomitant psychoactive medication (any) 54 27
Antipsychotics 30 16
Psychostimulants 18 9
SSRI/SNRI 25 12
Antiepileptics 12 10
IQ (mean ± SD) 46 ± 8 47 ± 11
Baseline characteristics of study subjects. Included are the ITT population and those assessed in the
secondary exploratory analyses. ADI-R, Autism Diagnostic Interview-Revised; SSRI/SNRI, selective
serotonin reuptake inhibitor/serotonin-norepinephrine reuptake inhibitor.
Prespecified efficacy analyses
The primary outcome measure was the Irritability subscale of the Aberrant Behavior
Checklist (ABC-I), which measures irritable mood, aggression, and self-injury. No
treatment effect was found on the ABC-I in the intent-to-treat (ITT) study population. A
possible period effect was seen, with greater improvement in period 1 (−5.5 ± 0.84) than
in period 2 (−3.2 ± 0.87, P < 0.10). No explanation for a period effect was evident in the
data, and the treatment × period interaction was not significant, failing to support a
possible carryover effect. In the analyses of the secondary outcome measures, STX209
was associated with significant improvement on the visual analog scale (VAS) problem
behavior ratings (Table 2), in which each parent or guardian provided severity ratings for
the three most problematic behaviors for their child. Parents or guardians nominated a
variety of problems for VAS rating, including both externalizing (for example,
aggression and outbursts) and internalizing (for example, anxiety, self-injurious behavior,
and stereotypies) behaviors. Consistent but not statistically significant trends toward
STX209 effects were found on several global measures, including the CGI-I and the
blinded treatment preference (Table 2 and Fig. 2A). No significant treatment effects or
period effects were found on the summary scores for other prespecified analyses. No
significant age-related effects were found on any of the prespecified analyses.
Flow diagram of subject disposition. The ITT population consisted of all subjects who received at least
one dose of study medication and had at least one post-baseline assessment on the ABC-I scale. The per-
protocol (PP) population consists of all subjects in the ITT population who did not have a major protocol
deviation, as determined before unblinding.
Newly validated ABC scoring algorithm
We performed post hoc analyses with the recently validated, FXS-specific scoring
algorithm for the ABC (27). This scoring algorithm was developed through factor
analysis on data from 630 individuals with FXS, resulting in six reconstituted subscales
instead of the five in the standard Aberrant Behavior Checklist—Community (ABC-C)
scoring algorithm. A sixth subscale emerged primarily because the original ABC—
Lethargy/Social Withdrawal (LSW) split along two separate dimensions of physical
lethargy/social unresponsiveness and active social avoidance. On the new ABC—Social
Avoidance (ABC-SA) scale, STX209 was associated with an overall beneficial effect in
the ITT study population (P = 0.01), with no period effect observed (Table 2). No
significant effects were found on the other validated FXS-specific factors.
Subjects with ABC-LSW greater than or equal to 8
Additional analyses were conducted in the subgroup with more severe social impairment
at baseline as measured by the original ABC-LSW scale (Table 1). These analyses were
motivated by the post hoc ABC-SA result and spontaneous investigator reports of social
and communicative improvement in many subjects. We reasoned that benefits to social
function might not be apparent in subjects whose baseline social impairments were rated
as less severe. The criterion for this post hoc subgroup (ABC-LSW ≥8) was chosen
because it represents the upper half of severity on this subscale among the general
population of males with FXS (27). In this subgroup, we noted significant improvement
on the validated ABC-SA scale (P = 0.04), as well as significant treatment effects on the
Vineland-Socialization measure of adaptive function, and on multiple global assessments,
with a trend on the ABC-LSW itself (Table 3 and Fig. 2B). A responder analysis showed
that significantly more subjects improved on STX209 than on placebo.
STX209 (least squares
mean ± SEM), n = 60*
Placebo (least squares
mean ± SEM), n = 62*
Baseline End of
Baseline End of
ABC-I 21.0 ±
16.4 ± 0.95 21.8 ±
16.2 ± 0.95 0.89
CGI-I — 3.1 ± 0.16 — 3.5 ± 0.16 0.15
CGI-S 5.1 ± 0.13 4.5 ± 0.12 5.1 ± 0.14 4.8 ± 0.12 0.09
— 26 (57%) — 13 (28%) 0.05
— 27 (59%) — 15 (33%) 0.09
VAS problem behaviors 2.2 ± 0.22 4.2 ± 0.32 1.9 ± 0.20 3.1 ± 0.33 0.04
Post hoc analysis
ABC—Social Avoidance 4.5 ± 0.46 3.3 ± 0.44 3.9 ± 0.43 3.6 ± 0.41 0.008
Prespecified and post hoc efficacy analyses. *Numbers reflect n for analysis of ABC-I. n differs slightly
for other variables because of missing data.
The safety and tolerability of STX209 was good. Three subjects (two placebo, one
STX209) discontinued from the study because of adverse events. All three of these cases
were reported to have “increased irritability” during the planned drug taper at the end of
the first treatment period (Fig. 1). The STX209 case was the only serious adverse event in
the study, requiring hospitalization for behavioral management in a subject with two
previous similar hospitalizations. Most other adverse events in this study were mild or
moderate in severity. All adverse events occurring in ≥5% of subjects on either STX209
or placebo are listed in Table 4. There were no differences between the STX209- and
placebo-treated groups on clinical laboratory assessments or physical examination.
Distribution of CGI-I scores. (A) PP population (P = 0.15). (B) Subjects with ABC-LSW ≥8 at screening
and baseline (P = 0.03).
This double-blind, placebo-controlled study evaluated STX209, a GABAB agonist, in
patients with FXS. The STX209-treated group was not different from the placebo group
on the study’s primary endpoint, the ABC measure of irritability. Improvement was seen,
however, on the prespecified analysis of VAS-rated problem behaviors, and multiple
global assessments of neurobehavioral function showed trends in favor of STX209. Our
post hoc analyses were designed to identify the nature of the possible beneficial drug
effects that contributed to the trend for global functional improvement but would not be
accounted for by a change in irritability and aggression as measured by the ABC-I.
An effect of treatment on social function was suggested by several post hoc analyses.
Most notably, improvement was found in the full study population on the ABC-SA score,
a measure recently validated for FXS (27). Furthermore, subjects with more severe
impairments in social function showed improvement on two independent measures in this
domain (the ABC-SA and the Vineland-Socialization) and on multiple global
assessments. Analysis of those who responded to treatment suggested that global
improvement was closely associated with improvement in social function. These
mutually reinforcing results are consistent with the hypothesis that social impairments in
FXS may be related to GABA deficiencies or exaggerated glutamatergic signaling. It has
been suggested that the ABC-SA scale may reflect anxiety-driven behaviors in FXS,
which would be consistent with the observations of GABA deficiency and amygdala
overactivation in FXS. However, the scale itself measures manifest social behavior (for
example, “withdrawn; prefers solitary activity”), as does the Vineland-Socialization
scale, and no drug effect was found on the Child and Adolescent Symptom Inventory
(CASI) anxiety scale. Development and validation of other outcome measures that
accurately define the FXS phenotype, including the anxiety phenotype in FXS, will
facilitate discovery of effective treatments for specific domains of function, as indicated
by the results of this study.
STX209 (least squares
mean ± SEM),
n = 27
Placebo (least squares
mean ± SEM),
n = 27
Baseline End of
Baseline End of
ABC-LSW 16.2 ± 1.21 12.4 ± 1.43 16.0 ± 1.30 15.9 ± 1.45 0.07
6.8 ± 0.60 4.6 ± 0.54 5.9 ± 0.62 6.4 ± 0.53 0.04
80.1 ± 8.10 99.6 ± 3.38 83.1 ± 8.65 87.8 ± 3.19 0.03
CGI-I — 2.5 ± 0.24 — 3.6 ± 0.25 0.02
CGI-S 5.4 ± 0.22 4.4 ± 0.21 5.3 ± 0.22 5.2 ± 0.22 0.009
— 16/20 (80%) — 4/20 (20%) 0.01
— 16/20 (80%) — 4/20 (20%) 0.01
Responders‡ — 10/21 (47.6%) — 2/23 (8.7%) 0.04
Efficacy analyses in subjects with ABC-LSW ≥8 at baseline. *Vineland-‐Socialization?? raw?? score?? only??
had?? data?? at?? the?? end?? of?? treatment?? and?? baseline;?? there?? were?? no?? data?? in?? the?? middle?? of?? period.?? †This
table reports on a subgroup of 27 subjects; only 20 subjects had data on these particular variables. Thus,
seven subjects had missing data on these variables. ‡Responders were defined by a rating of “very much”
or “much improved” on the CGI-I and improvement of at least 25% on the ABC-LSW. Not all 27
subjects had ABC-LSW raw score and CGI-I at the end of each treatment.
Translating from animal research to human pharmacotherapy is inherently difficult, and
the effects of STX209 in FXS animal models have not pointed clearly to any specific
endpoint for a clinical trial focused on neurobehavioral function. In particular, no robust
phenotype related to social behavior has been established in FXS animal models, and
thus, STX209 has not been tested on such an endpoint in animals. Moreover, although the
evidence strongly suggests that the core pathophysiology of FXS is evolutionarily
conserved (28), it should not be assumed that FXS pathophysiology will manifest as
similar behavioral abnormalities in animals and humans, as there are profound
differences in brain complexity. Thus, in this exploratory study, we cast a broad net to
find behavioral responses to treatment in humans. The designation of the ABC-I as the
primary endpoint here was driven by a U.S. Food and Drug Administration (FDA)
precedent. Our study’s failure on this primary endpoint could be a result of either a
mistaken choice of endpoint or a true lack of drug effect.
Concomitant use of antipsychotic medications in the study also may have blunted the
effects of STX209 on irritability, the primary endpoint, because antipsychotics are known
to diminish such symptoms in autism (6, 29). Possible effects on social function were less
likely to be confounded by concomitant medications, because no medication is known to
enhance social function.
Adverse event STX209, n (%) Placebo, n (%)
Upper respiratory tract infection 8 (13) 6 (10)
Headache 5 (8) 1 (2)
Sedation 5 (8) 1 (2)
Fatigue 4 (6) 1 (2)
Irritability 4 (6) 4 (6)
Diarrhea 3 (5) 5 (8)
Increased appetite 4 (6) 2 (3)
Vomiting 4 (6) 1 (2)
Aggression 3 (5) 4 (6)
Nasopharyngitis 2 (3) 6 (10)
Treatment-emergent adverse events. Only those events reported in ≥5% of subjects in either group are
The safety and tolerability of STX209 was congruent with the known profile of racemic
baclofen. Sedation was reported at a relatively low rate of 8% and in all cases resolved
without dose adjustment. In comparison, the FDA-approved label for racemic baclofen
reports drowsiness in up to 63% of subjects. Increased irritability and aggression were
reported to be equally common in patients given drug and placebo, suggesting that these
were related to underlying pathology, rather than drug treatment effects.
The key limitation of this study is the post hoc nature of most of the positive results. This
issue is mitigated by the cohesive results on multiple endpoints for the subjects with
ABC-LSW ≥8 and by the positive result on the ABC-SA, which has been validated in
FXS (27). Prospective replication of these results is essential, and the results speak to the
importance of developing and using assessments that are validated in the populations
Our findings can be compared with a recent double-blind, placebo-controlled crossover
study that examined the effects of 3 weeks of treatment with AFQ056, an mGluR5-
negative allosteric modulator, in 30 young adults with FXS. In the full study population,
AFQ056 was not associated with improvement on the ABC or on global measures of
behavior, although repetitive behaviors did show improvement (P = 0.05). Post hoc
analyses on the subgroup of seven individuals with full methylation of the FMR1 gene
promoter showed treatment-related improvement on several assessments, including the
ABC subscales of Stereotypy and Hyperactivity, and the CGI-I, but not the ABC-LSW or
ABC-I scales (30). The seven subjects with full methylation were also more severely
affected behaviorally in comparison to the 23 subjects with partial methylation, which
may have been a factor in the positive response of this group, similar to the larger
response in our more socially impaired subgroup.
The effort to develop targeted treatments for neurodevelopmental disorders is just
beginning and its promise is large. Although existing agents can provide some relief of
secondary symptoms, new targeted treatments may be able to ameliorate core
impairments in cognition, language, and social function. Given our limited understanding
of how to anticipate and measure treatment outcomes in these domains, the path to
targeted treatments will require careful interpretation of all potentially informative data
so that future study design and study endpoints can be optimized to find real and valuable
Materials and Methods
Individuals with a DNA-confirmed FMR1 full mutation, aged 6 to 40 years, either male
or female, were eligible for the study. Up to three concomitant psychoactive medications
(including antiepileptic drugs) were permitted, but use of vigabatrin, tiagabine, riluzole,
or racemic baclofen was prohibited because of their GABAergic mechanisms.
Medication regimens were required to be stable for 1 month, and educational, behavioral,
and other treatments for 2 months, before and for the duration of the study. Subjects with
any previous seizure were required to be on anticonvulsant medication and seizure-free
for 6 months. Minimum scores of 9 (if age 12 to 40 years) or 12 (age <12 years) on the
Irritability subscale of the ABC-C and of 4 (moderate) on the CGI-S (Clinical Global
Impression of Severity, a seven-point Likert scale that requires the clinician to evaluate
overall symptom severity, considering the entirety of the data available on the subject.)
were required at the screening visit and at the beginning of treatment period 1. Female
subjects of childbearing potential were tested and excluded if they were pregnant.
All subjects or guardians provided voluntary informed consent or assent, as appropriate.
This study (clinicaltrials.gov identifier NCT00788073) was approved by the institutional
review boards governing each site.
This was an exploratory, first-in-patient trial, using a randomized, double-blind, placebo-
controlled, multisite, two-period crossover design, at 12 sites in the United States,
between December 2008 and March 2010. Study drug was flexibly titrated every 3 to 4
days, starting at 1 mg BID, and then 2 mg BID, 3 mg BID, 5 mg BID, 10 mg BID
(maximum for age <12 years), and 10 mg TID, until the optimal tolerated dose was
established. Regular phone contact was maintained throughout the titration period.
Subjects returned for evaluations 2 and 4 weeks after starting treatment. Study drug was
then tapered down over 1 to 2 weeks. After a minimum 7-day washout, subjects entered
the second treatment period. An end-of-study safety evaluation was performed 14 days
after the end of the second treatment period, with telephone follow-up 28 days later.
Study drug and matching placebo were provided as 1- and 5-mg capsules. Subjects were
assigned to treatment by the local (eight sites) or a central (four sites) pharmacy,
according to a centrally generated randomization list. Randomization was stratified by
age (6 to 11, 12 to 17, and 18 to 40 years). Treatment compliance was monitored with a
dosing form, which guardians completed on a daily basis.
Baseline assessments included the Stanford-Binet Intelligence Scales, Fifth Edition (SB-
5), the Autism Diagnostic Interview-Revised, and a review of autism spectrum disorder
criteria from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition
Global outcome measures included the CGI-I and CGI-S assessments, both rated on a
seven-point Likert scale, and blinded treatment period preference. Focused assessments
included the ABC-C (31), a 58-item, parent-rated questionnaire yielding five-factor
scores, including Irritability, Lethargy/Social Withdrawal, Hyperactivity, Inappropriate
Speech, and Stereotypy. Other measures included VAS of the three most problematic
behaviors for each child, the Vineland Adaptive Behavior Scales, Second Edition
(Vineland II) (32), the Social Responsiveness Scale (33), the Repetitive Behavior Scale-
Revised (RBS-R) (34), the CASI anxiety scale (35), the ADHD Rating Scale-IV (36), and
measures of vocabulary and short-term and working memory.
Safety assessments included physical examination, standard hematology and clinical
chemistry assessments, urinalysis, electrocardiograms, and spontaneously reported
The ABC-I was designated the primary endpoint because of regulatory precedents (2,
37). It was analyzed with a mixed-effect model repeated-measures approach in the ITT
population. Unstructured within-subject covariance was used. The model included terms
for treatment, time, treatment-by-time interaction, period, treatment-by-period
interaction, age group, and period-specific baseline score as a covariate. The number of
female subjects was too small to support the inclusion of sex as a covariate. Other
appropriate variables were analyzed with a similar approach in the per-protocol
population. Categorical outcomes were analyzed by sign test. For all comparisons, a
nominal P value of 0.05 or less was required to declare significance, and no adjustments
for multiplicity were made.
The study was designed to have 90% power to detect a treatment effect of size 0.6, with a
P level of 0.05 in a crossover design. The planned sample size was 60 subjects.
Post hoc analyses
A post hoc analysis on the entire ITT population was performed with a newly validated
scoring algorithm for the ABC-C recently developed in a separate study (26). Other post
hoc analyses were conducted on the study subgroup with ABC-LSW scores ≥8 at
screening and at baseline visits for both period 1 and period 2. One outlier subject was
excluded from these analyses. Outcomes in this subgroup were analyzed similarly to the
above efficacy analysis.
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We thank the families who participated in this study, and the FRAXA Research
Foundation, the National Fragile X Foundation, and the Elwyn Institute, who assisted
with study recruitment through Web site postings and other mechanisms.
This study was sponsored by Seaside Therapeutics Inc.
E.M.B.-K., B.R., P.Z., M.C., J.G.-H., R.L.C., and R.J.H. designed the study. B.R., P.Z.,
M.C., K.W.-B., P.P.W., and R.L.C. oversaw the execution of the study. E.M.B.-K., D.H.,
and R.J.H. led the subject testing and data collection. K.W.-B., Y.M., and D.V.N. led the
statistical analysis. R.L.C. and M.F.B. provided the original insights and motivation for
the study. All authors contributed to writing and revision of the manuscript.
P.Z.,M.C.,K.W.-B.,P.P.W., and R.L.C. are full-time employees of Seaside Therapeutics.
R.L.C. and M.F.B. are co-founders of Seaside Therapeutics. J.G.-H. has consulted for
Seaside Therapeutics. E.M.B.-K., D.H., and R.J.H. were compensated for their data
collection efforts in this study.
Data and materials availability
This trial is registered at www.clinicaltrials.gov