An Initial Double-Blind, Placebo-Controlled Two-Dose Crossover Study of AZD7325 in Adults with Fragile X Syndrome

Protocol Version 14

1 Version 14.0 Study Sponsor

Craig Erickson, MD Professor of Clinical Psychiatry Cincinnati Children’s Hospital Medical Center 3333 Burnet Avenue Cincinnati, Ohio 45229 Phone: 513.636.4200

Study Principal Investigator

Ernest Pedapati , MD, MS, FAAP Assistant Professor of Clinical Psychiatry Cincinnati Children’s Hospital Medical Center 3333 Burnet Ave, MLC 3014 Cincinnati, OH 45229 513.636.6265

Sub-Investigators:

Craig Erickson, MD Rebecca Shaffer, PsyD Martine Lamy, MD, PhD Kelli C. Dominick, MD, PhD

2 Version 14.0 Introductory Statement and General Investigational Plan

Introductory Statement

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability. FXS is the result of a cysteine-guanine-guanine (CGG) trinucleotide repeat expansion (>200 repeats) within the fragile X mental retardation 1 gene (FMR1) located near the long arm of the X chromosome. Translation of the FMR1 gene leads to the synthesis of fragile X mental retardation protein (FMRP). Mutations in FMR1 lead to a lack of FMRP, resulting in FXS. As a single gene disorder with increasingly well-understood neurobiology, FXS is a model disorder for targeted treatment development. We believe that AZD7325 may be effective for reducing impairments commonly seen in FXS. We propose the first study of AZD7325 in adults with FXS given our promising Preliminary Data describing use of the drug in the FXS mouse model and given the demonstrated safety profile of the molecule.

Drug Product Summary

Name: AZD7325 (4-amino-8-(2-fluoro-6-methoxyphenyl)-N-propylcinnoline-3-carboxamide)

Pharmacological Class: AZD7325 is a gamma-amino butyric acid type A (GABA A) receptor modulator. In vitro AZD7325 is functionally selective for GABAAα2 and GABAAα3 receptor subtypes. This in vitro profile translates to a non-sedative anxiolytic profile in vivo as characterized in multiple rat models of sedation and anxiety.

Structural Formula of the Drug:

Formulation of the Dosage Form: Orange gelatin capsules

Route of Administration: PO

Broad Objectives and Planned Duration of Clinical Investigation:

Hypotheses and Specific Aims:

Primary Aim #1. Determine if AZD7325 shows evidence of efficacy and determine an estimate of effect size for the short-term treatment of peripheral amyloid precursor protein (APP) dysregulation in adults with FXS. Hypothesis: AZD7325 use will be associated with significant correction of elevated plasma APP.

Primary Aim #2. Determine estimates of the short-term safety and tolerability of AZD7325 in adults with FXS. Hypothesis: AZD7325 will not be associated with a significant increase in treatment discontinuation compared to placebo at both doses evaluated. AZD7325 will not be associated with significant laboratory or vital sign abnormalities at both doses studied.

3 Version 14.0 Secondary Aim #1. Determine if AZD7325 shows evidence of efficacy and determine an estimate of effect size for the short term treatment of interfering behavior associated with FXS in adults. Hypothesis: AZD7325 use will be associated with a positive directional change compared to placebo on phenotyping measures commonly utilized in FXS including the Aberrant Behavior Checklist Social Withdrawal subscale, the Pediatric Anxiety Rating Scale, Anxiety Depression and Mood Scale (ADAMS), social gaze eye tracking, and computerize neurocognitive performance.

Secondary Aim #2. Determine if AZD7325 show evidence of efficacy and to determine an estimate of effect size for the short term treatment of electrophysiological abnormalities associated with FXS. Hypothesis: AZD7325 use will be associated with reduction in elevated EEG gamma power and correction of auditory evoked response habituation deficits in adults with FXS compared to placebo.

Exploratory Aim #1. Determine if AZD7325 shows evidence of efficacy and determine an estimate of effect size for the short-term treatment of excessive peripheral lymphocytic extracellular signal related kinase (ERK) activation dysregulation in adults with FXS. Hypothesis: AZD7325 use will be associated with significant correction of excessive lymphocytic ERK activation in adults with FXS.

The proposed Initial Double-Blind, Placebo-Controlled Two-Dose Crossover Study of AZD7325 in Adults with Fragile X Syndrome will involve a randomized double-blind, placebo-controlled, crossover pilot study of two doses 5mg PO BID and 15mg PO BID of AZD7325. Each study treatment period will be two weeks with two week wash out periods between treatments. Study visits will occur in two week intervals at the Clinical and Translational Research Center (CTRC) at Cincinnati Children’s Hospital Medical Center (CCHMC). Total duration of study participation will be approximately 12-16 weeks per subject.

General Investigational Plan

Background and Rationale: FXS is associated with a common genotype and a substantially increased risk, particularly in males, for a particular neurobehavioral phenotype marked by severe interfering behavioral symptoms in addition to cognitive delay. About 2 in 3 (67%) males with FXS are thought to exhibit behavior consistent with an additional diagnosis of autism spectrum disorder (ASD)1. FXS is associated with significant behavioral disturbance which contributes not only to the disability of the affected individual, but also has a significant negative impact on parents and caregivers. In one report, 89.8% of 49 boys with FXS exhibited significant interfering externalizing behaviors most often driven by anxiety which contributed to more familial stress compared to families of children with other chronic illness and controls2. Fifty-eight percent of boys with FXS exhibited self-injurious behavior (SIB) in a survey report including fifty-five families3. Behaviors such as aggression and SIB in FXS are most often driven by treatment-refractory anxiety4,5. Problematic associated behaviors, including aggression to self and others and property destruction, can significantly limit the ability of an individual with FXS to participate in critical early intervention strategies such as speech and language therapy and formal educational programs. Later in life, behaviors commonly seen in FXS contribute to lifelong disability as they can limit vocational opportunities and curtail the individual’s ability to contribute to more typical societal efforts and norms. The costs necessary to care for these individuals throughout their lives are therefore escalating and ever- increasing. Recent pre-clinical findings in FXS knockout animal models have led to targeted treatment development efforts in this field. To date, drug development efforts have focused on metabotropic glutamate receptor type 5 (mGluR5) antagonists and a gamma-aminobutyric acid receptor B (GABA(B)) agonist with results that have not been marked by a robust drug effect. In both mGluR5 and GABA(B) human trials to date, only small subsets of persons with FXS have potentially shown response with treatment6. Increasing evidence has pointed to dysregulation of GABA receptor A (GABA(A)) neurotransmission in the pathophysiology of FXS7. Among potential targets of drug therapy in FXS, modulation of GABA(A) activity, in particular selective agonism, remains largely unexplored in humans with FXS. 4 Version 14.0 Significant pre-clinical data supports the hypothesis that deficient GABA(A) activity contributes to the underlying pathophysiology of FXS. Fragile X Mental Retardation Protein (FMRP) has been shown to transcriptionally regulate GABA(A) receptor subunit RNA expression with reductions in GABA(A) receptor mRNA noted in FXS KO mice lacking FMRP8. GABA(A) receptor expression has been shown to be significantly down regulated in a number of brain regions in FXS KO mice9-13. In animal models of FXS, GABA(A) agonism has shown significant promise as a pharmacotherapy target. The GABA(A) agonist alphaxalone was associated with reductions in anxiety and rescue of audiogenic seizures in FXS KO mice14. Also in FXS KO mice, the GABA(A) agonist gaboxadol restored neuron excitability deficits in the amygdala, reduced hyperactivity, and reduced prepulse inhibition (PPI) deficits15. Improvements in memory acquisition and retention have been noted in FXS KO mice receiving taurine, a GABA(A) agonist16. Non-selective GABA(A) modulators including benzodiazepines are uncommonly used in FXS due to sedating and cognitively dulling features17. Recently the neuroactive steroid allosteric modulator of GABA(A) receptors ganaxolone was not associated with significant clinical improvement on primary outcome measures employed and the drug was associated with lethargy, diarrhea, and sedation (http://www.biospace.com/news_story.aspx?StoryID=424678). As an α2,3 specific agonist, AZD7325 holds promise to breakthrough targeting the GABAergic deficits noted in FXS while not being associated with the significant negative effects noted with use of broader spectrum potent GABA agonists in FXS.

Preliminary Data:

We and our collaborators have elucidated several quantitative aspects of FXS human pathophysiology that hold promise for use in initial first in human FXS trials to show drug-treatment engagement with and potential correction of molecular and electrophysiological dysregulation. Two molecular human blood studies in first-in-human FXS trials conducted by our group include analysis of amyloid precursor protein (APP) derivatives and analysis of extracellular signal-related kinase (ERK) activation.

Amyloid Precursor Protein (APP): The CGG repeat expansion associated with FXS leads to FMR1 gene methylation and silencing and subsequent deficiency in Fragile X Mental Retardation Protein (FMRP) production. FMRP is a known repressor of neuronal mRNA translation 18-22 and thus is important to synaptic plasticity and regulation of local protein synthesis at the synapse 23. Similar to reports in idiopathic autism, children with FXS also exhibit early brain overgrowth 24. Additionally, FMRP has been demonstrated to regulate APP mRNA expression 25,26. Baseline APP levels are elevated in Fmr1 knockout (KO) mouse synaptoneurosomes and primary neurons25. A preliminary study reported a relative elevation of plasma APP in 18 youth with FXS compared to age-matched control subjects 27. Additionally, Westmark and colleagues (2011) have demonstrated abnormal levels of the APP derivative Aβ42 in the plasma of persons with FXS. For reference, we estimate normal population total plasma APP in a range from 20-28 ng/mL28-30. Findings from FXS studies point to enhanced translation of APP. It may be that analyses of plasma APP holds promise as a target of treatment, pharmacodynamic marker of target engagement, and as a potential means to subgroup populations matched to potential treatments of interest in FXS. We conducted the first analysis of potential drug-associated change in plasma APP with treatment in youth with FXS and in youth with idiopathic autism. Fifteen subjects (mean age 11.1 years, range 6-15 years) underwent open label acamprosate treatment with plasma APP sampled pre-treatment and after 10-weeks of treatment28. Acamprosate, a drug FDA approved for the maintenance of abstinence from alcohol use in adults, is a novel agent with pleiotropic effects likely impacting glutamate and gamma-aminobutyric acid (GABA) neurotransmission31. In this report, plasma APP total levels were uniformly reduced with treatment in this pilot patient sample with the mean level changing from 32.6 ± 9.9 ng/mL at baseline to 21.45 ± 8.34 ng/mL at week 10 (p<0.05; effect size= 1.22). Interestingly, in subjects with FXS, reduction in plasma APP correlated significantly with reduction in the Aberrant Behavior Checklist Social Withdrawal Subscale (ABC-SW; p=0.009, Kendall’s Tau Correlation Analysis). This uniform correction of APP molecular dysregulation with drug treatment and correlation of molecular correction with clinical change stands as proof of concept for utilizing plasma APP as a peripheral blood marker sensitive to significant positive treatment-associated change in initial study of novel molecules in FXS. We anticipate that reduction of plasma APP will correlate with positive clinical change in FXS-specific targeted pharmacotherapy study and that these reductions are a sign of engagement of targeted treatment with an aspect of FXS pathophysiology. 5 Version 14.0 Peripheral Lymphocytic Extracellular Signal Related Kinase (ERK) Activation

Extracellular signal-related kinase (ERK) is a nodal point for several cellular signaling cascades and acts as a central element of intracellular signaling governing neuronal development 32,33, synaptic plasticity 34, and memory formation 35 which are all functions that are likely dysregulated in FXS. ERK is expressed in peripheral blood cells including lymphocytes. Analysis of ERK activation in lymphocytes is well established in the leukemia literature36-38. ERK requires phosphorylation for full activity and employs phosphatases to regulate signal transduction cascades39. In FXS, ERK activation (phosphorylation) has been shown to be excessive in human post-mortem brain samples40, in FMR1 KO mouse brain40, and in human platelets41. Delayed early-phase phosphorylation (activation) kinetics of ERK has been demonstrated in neurons and thymocytes of FMR1 knockout mice 42. We have reported on lymphocytic ERK phosphorylation in humans with ASD compared to neurotypical control subjects (unpublished data). We compared the ratio of lymphocytes staining positive for phosphorylated (activated) ERK to total lymphocytes counted in 71 persons with ASD (mean age 13.8 ± 9.2 years; range 5-52 years) and 45 matched neurotypical control subjects (mean age 14.6 ± 9.4 years; range 5-52 years). The cell count ratio (cytosolic positive/ nuclear negative pERK lymphocytes counted, divided by the total number of cells counted) was significantly higher in the ASD group compared to controls (ratio of 0.064 ± 0.044 versus 0.034 ± 0.031, p =0.002). In a subgroup of samples with sufficient material for additional analysis, whole cell pERK Western Blotting analysis demonstrated an increased phosphorylated to total ERK ratio in the autistic disorder group (n=37) compared to neurotypical controls (n=17; p= 0.004). Overall, the literature has described enhanced ERK activation in FXS in the Fmr1 KO mouse and in humans and our lab has demonstrated the ability to efficiently detect ERK activation in peripheral lymphocytes. We hypothesize that reduction in enhanced ERK activation in lymphocytes from humans with FXS will show engagement with one aspect of the pathophysiology of FXS and will correlate with clinical change with targeted pharmacotherapy.

Dense Array Resting State EEG Assessment

Our U54 Fragile X Center NIH supported EEG group has recently collected resting state dense array data in 21 FXS patients (mean age 25.6 years ± 11.1 years; range 12-57 years; mean IQ= 55.1) and 21 matched neurotypical control subjects (unpublished data). Cluster-permutation analysis showed stronger relative activity in those with FXS in the theta and gamma frequency bands in both occipital and frontal regions. The gamma abnormalities were of the greatest significance including half the FXS participants having more than 1 standard deviation power beyond controls and all FXS participants showing reduced local gamma with local theta activity. Also, increasing gamma power significantly correlated with more elevated scores on the Social Communication Questionnaire (SCQ; r=0.58, p=0.02), a measure of social impairment utilized in developmental disorders (higher score indicates more severe deficits). Given our experience to date, we hypothesize that correction of gamma power deficits in FXS will be associated with positive clinical change and will provide a potential signal of targeted drug treatment engagement with an aspect of the pathophysiology of FXS.

Reduced Habituation of Auditory Evoked Event-Related Potentials (ERP)

Our U54 Fragile X Center NIH supported EEG recently completed an analysis of habituation to auditory evoked potentials in 14 adolescents and adults with FXS (mean age 28.5 +/- 11.7 years; range 14-57 years; mean IQ 54.9) and 15 match neurotypical control subjects43. The subjects with FXS showed a significantly (p=0.014) reduced habituation of the N1 ERP compared to control subjects. Habituation deficits also correlated with clinical measures employed including the Achenbach Adult and Child Behavior Checklists, the Aberrant Behavior Checklist (ABC), and the Social Communication Questionnaire (SCQ). We believe that reduced habituation to auditory stimuli in FXS potentially relates to the clinical over responsiveness to sounds and resultant enhanced anxiety that is in noted in the disorder. We hypothesize that correction of habituation deficits will be associated with clinical improvement in persons with FXS undergoing targeted pharmacotherapy trials.

6 Version 14.0 PRECLINICAL DATA: Improved Memory, Sensory Response Neuroplasticity, and Molecular Markers in Fmr1 KO mice treated with AZD7325

In animal models of FXS, GABA(A) agonism has shown significant promise as a pharmacotherapy target. In Fmr1 KO mice, benzodiazepines have been associated with normalization of morphological features, GABA(A) expression, and behavior9,14,15,44. In wild-type (WT) rodents, benzodiazepines have been shown to increase synaptic phasic inhibition45. Regarding preclinical treatment, study of the GABA(A) agonist in FXS, alphaxalone, a neuroactive steroid and general anesthetic with multiple potential pharmacodynamics effects including modulation of nicotinic acetylcholine receptors, activation of chloride channels, and non-selective extrasynaptic GABA(A) agonism, was associated with reductions in anxiety and rescue of audiogenic seizures in FXS KO mice14. Also in FXS KO mice, the GABA(A) extrasynaptic δ-subunit agonist gaboxadol (THIP) restored neuron excitability deficits in the amygdala, reduced hyperactivity, and reduced prepulse inhibition (PPI) alterations, but did not improve startle reactivity or cued fear behavior15. Importantly, our Preliminary Data show that manipulation at the GABA(A) α2,3 subunits with AZD7325 improves many of these same behaviors and additionally improves memory, startle reactivity, and fear behavior in the Fmr1 KO mice. In addition to significant literature outside of our group justifying selective GABA(A) agonism as an effective treatment target in FXS, our lab has conducted recent key work with AZD7325 in the Fmr1 KO mouse (unpublished data). We studied 1mg/kg AZD7325, 3mg/kg AZD7325 and vehicle in adult Fmr1 KO mice compared to wildtype control littermates. Adult mice were treated for ~4 weeks. No observable AZD7325-associated deficits were noted in cerebellar or striatal dependent motor tasks as measured by open- field activity, pole descent test, adhesive removal task, and rotorod time to fall. In addition to the positive findings below, we have a preliminary finding that both the 1mg/kg and 3mg/kg AZD7325 doses were associated with correction of enhanced freezing behavior following fear conditioning stimuli. All behavior data were analyzed by two-way ANOVA with repeated measures when warranted (2-sided). Data were corrected for multiple comparisons using the False Discovery Rate method.

MEMORY: We demonstrated improved memory in Fmr1 KO mice treated with low dose AZD7325 (Fig. A; human correlate: KiTap testing).

Object memory is impaired in Fmr1 KO mice and was improved with low dose AZD7325 treatment (Fig. A; human correlate: KiTap testing) In an object memory test, FXS mice treated with placebo had significant difficulty remembering a novel object they had previously investigated which is indicated by the lower memory index. In processes requiring higher order functioning such as memory processing and retrieval, modulation of synaptic excitatory/ inhibitory signaling requires fine tuning and balance. This is indicated by the presence of a treatment response in the FXS mice receiving the low dose but not the high dose of AZD7325. This augmented response in the low-dose treated group rather than complete correction is realistic of the type of response an oral treatment can be expected to have on memory with clinical treatment of adults with FXS.

Fig. A AZD7325 treatment improved object memory in Fmr1 KO mice. Treatment with the low dose of AZD7325 attenuated deficits in object memory when assessed in a novel object recognition paradigm (Figure 4 left; main effect of genotype for memory index (P<0.03). Low dose-treated animals showed greater interest in the novel object during the second phase compared to placebo-treated KO mice indicating a greater memory of the familiar object. n=14- 18/group; *p < 0.05 pairwise group comparisons.

Fig. A

BRAIN ERK ACTIVATION: We demonstrated attenuation of elevated ERK activation in FXS mice with improved memory (Fig. B; human correlate: lymphocytic ERK activation assay).

7 Version 14.0 ERK activation is reduced in the hippocampus of FXS mice that exhibited improved memory following low dose AZD7325 treatment (Fig. B; human correlate: lymphocytic ERK activation assay). The hippocampus is a brain region intimately involved in learning and memory in both rodents and humans. ERK activation in the hippocampus is attenuated following low dose AZD7325 and is correlative to our finding of improved memory in this group but not the high dose-treated group Fig. B AZD7325 treatment attenuated increased ERK activation in Fmr1 KO mice. Reductions in hippocampal activated (phosphorylated) ERK were observed in the low dose treated group that also demonstrated an improvement in object memory. There were no changes in total ERK levels with any treatment indicating the observed changes were dependent on differences in the phosphorylated protein. n=6-8/group; *p < 0.05 pairwise group comparisons. Fig. B

SENSORY PROCESSING AND MOTOR GATING: We demonstrated proper gating and response following both weak and intense sensory stimuli following treatment with AZD7325 (Fig. C; human pilot study correlate: auditory evoked ERP habituation).

Sensory responses were improved and motor responses were more appropriately gated with AZD7325 treatment (Fig. C; human pilot study correlate: auditory evoked ERP habituation). In an acoustic startle paradigm FXS mice inappropriately gate motor responses to acoustic stimuli, resulting in exaggerated responses to low intensity stimuli and reduced responses to high intensity stimuli. Treatment with AZD7325 both reduces exaggerated responses and normalizes reduced responding in a manner that suggests improved neuroplasticity. This is in contrast to an expected result of treatment with a broad GABAergic positive modulator or neurosteroid that would simply increase tonic GABAergic signaling resulting in diminished response regardless of stimulus intensity. Fig. C Response to acoustic stimuli is corrected and response to intensity is appropriately gated with AZD7325 treatment. In adulthood, Fmr1 KO mice have abnormal motor responses to sensory stimuli compared to WT mice in an acoustic startle paradigm. When presented with a short low level white noise burst (82 db) over 10 trial blocks, Fmr1 KO mice will flinch at a higher amplitude than WT mice (presentation of these startle bursts do not elicit seizure activity). The Left Panel shows In the left panel, there was a main effect of genotype (P<0.001) and drug (P<0.0001) for motor response. Fmr1 KO mice treated chronically with the low dose of AZD7325 have significantly reduced whole body flinching in this low-lintensity acoustic startle paradigm. Interestingly, in a paradigm employing a high level acoustic stimulus (120 dB), adult Fmr1 KO mice will repeatedly respond to the stimulus with a lower amplitude whole body flinch compared to WT mice. The Right Panel shows a significant Gene x Drug interaction (P<0.0001) was found during these high intensity trials and that treatment with both doses of AZD7325 improves this response to WT levels. These data indicate that drug treatment isn’t simply reducing overall whole body flinching in response to sensory stimuli, but is rather mediating responses so that they are becoming more appropriate and reflective of startle intensity. n=14- 18/group; *p < 0.05 pairwise group comparisons. 8 Version 14.0 FEAR/ANXIETY BEHAVIOR: Next, we report on reduction in fear/anxiety behavior with AZD7325 treatment in the Fmr1 KO mouse (Fig. D; human correlate: Aberrant Behavior Checklist Anxiety rating scale)

Treatment with both doses of AZD7325 reduced fear behavior in FXS mice (Fig. D; human correlate: Pediatric Anxiety Rating Scales (PARS) Exaggerated fear and anxiety behavior is a hallmark of the clinical presentation of FXS. Freezing behavior (or the absence of movement other than required for respiration) is a species specific (rodents) behavioral outcome of fear. In placebo-treated FXS mice, freezing behavior is greatly elevated compared to WT mice following a conditioning paradigm where mice learn to associate a stimulus with a mild foot shock. Chronic treatment with both the low and high dose of AZD7325 reduced this fear behavior to WT levels indicating a therapeutic effect.

Fig. D Exaggerated fear response is reduced in Fmr1 mice treated with AZD7325. Conditioned fear responses to a mild foot shock were shown to be exaggerated compared to WT mice during the cued portion of the test with AZD7325 treatment at both dose levels normalizing this behavior in KO mice (Gene x Drug interaction; P<0.003). This type of deficit in the placebo-treated Fmr1 KO mice is reminiscent of the exaggerated anxiety responses people with FXS commonly display. n=5/group Fig. D (preliminary); *p < 0.05 pairwise group comparisons.

NEURONAL HYPEREXCITABILITY: Finally, we report on a classic paradigm, audiogenic seizure threshold in the Fmr1 KO mouse, where AZD7325 protects against the development of seizures and death following an intense and prolonged auditory stimulus.

AZD7325 treatment greatly reduced tonic/clonic seizure from 60% in the placebo group to 0% in the high dose group. Following exposure to a loud siren, juvenile FXS mice will experience a tonic/clonic seizure and cardiac arrest due to increase excitatory signaling whereas WT mice never experience seizure activity in this same auditory paradigm. Both the low dose and high dose of AZD7325 dramatically reduced seizure frequency in juvenile FXS mice from 60% frequency in placebo-treated FXS mice to 11% in the low dose group to 0% in the high dose group. Fig. E Seizure susceptibility is greatly reduced with AZD7325 treatment in a dose dependent manner. Audiogenic seizure susceptibility in Fmr1 KO mice, which is thought to be the result of increased neuronal activity in response to sensory stimuli, peaks around the third week of life and manifests as wild running which is typically followed by tonic-clonic seizures and cardiac arrest. WT mice do not respond to this siren with any noticeable running or seizure behavior. The Top Panel shows that ~ 60% Fmr1 KO mice treated with placebo displayed wild running followed by seizure in response to a loud stimulus (120 dB siren), but when treated acutely (30 min prior to testing) with both the low (11%) and high (0%) dose of AZD7325 seizure activity was significantly reduced indicating that the drug is reducing hyperexcitability in the brain Furthermore, the Bottom Panel shows significant reduction in seizure severity scores in treated FXS mice (Fisher’s exact test p<0.0002). Seizure severity was calculated by using an animal’s most severe response number (0 indicating no altered behavior, followed by 1 indicating wild-running, 2 indicating clonic seizure (rapid limb flexion and extension), 3 indicating tonic seizure (static limb extension), and 4 indicating the most severe response of cardiac arrest. n= ~15/group *p < 0.05 9 Version 14.0 pairwise group comparisons. Please note in the left side of Figure E, Wild Type mice in this experiment do not have seizures under any condition, hence they have seizure severity scores of zero.

Amyloid Precursor Protein Expression We demonstrated reduction of APP in FXS mice (Fig. F; human correlate: plasma APP derivative analysis).

The low dose of AZD7325 reduced APP total expression by ~ 23% in the cortex of adult Fmr1 KO mice following chronic systemic treatment. Amyloid Precursor Protein derivatives have been shown to be dysregulated in Fmr1 KO mouse brain previously. Plasma APP derivatives in humans with FXS have also been reported to be altered with elevations in plasma secreted APP responsive to drug treatment and reductions correlating to improved social behavior. The significant reduction in APP total protein in the brains of Fmr1 KO mice following AZD7235 treatment and improved behavior compliments previously reported data in which genetic reduction in APP in Fmr1 KO mice attenuated many characteristic Fmr1 KO phenotypes46. Therefore, in human FXS clinical study of AZD7325, APP derivative analysis holds great promise as a biomarker for target engagement. Fig. F APP total protein in the cortex of adult FXS mice is reduced with AZD7325 treatment. APP total (C-terminal domain) was assessed by Western blot analysis with ERK total used as the loading control. Data are expressed as the ratio of APP total signal/ERK total signal. n = 6/group; *p < 0.05 pairwise group comparisons

Study DESIGN

Given our preclinical experience with AZD7325 in the Fmr1 KO mouse combined with the significant background information pointing to reduced GABA(A) activity in FXS, we propose an initial in human 15 subject FXS study involving a low (5mg BID) and high dose (15mg BID) of AZD7325, a crossover design, and two week treatment periods. The illustrative figure below shows 3 of a possible 6 orders of low dose-high dose- placebo treatment in the crossover design.

Design (Study Measures will remain the same across Treatment Phases 1, 2 and 3)

We propose to mirror AZD7325 dosing and total AZD7325 exposure limited utilized in Study D114C0014 (see IB version 9) and dose with 2 week treatment periods with placebo, 5mg BID AZD7325 or 15mg BID 10 Version 14.0 AZD7325. Two weeks will allow for drug to reach steady state while balancing an eye towards overall project length and subject burden. We will have two week drug washout periods. We will additionally have a Final Follow Up visit 2 weeks after Treatment Phase 3. Fifteen 18 to 49 year olds with full mutation FXS will be enrolled in this study.

Inclusion Criteria:

 Diagnostic confirmation of full mutation FXS  50 ≥ Age ≥18 years. Males and Females included in study.  General good health as determined by physical exam, medical history and laboratory work up.  FXS genetic reports at screening  IQ less than or equal to 80. Note: IQ cutoff is used as a means to exclude cases of females with FXS who have the full mutation, but may have neurotypical development (ie: do not have the full FXS phenotype despite positive FXS genetic testing) due to variability in X chromosome inactivation patterns.  Male study participants who are sexually active with a female partner of childbearing potential must be surgically sterilized, practicing abstinence, or agree to use highly effective methods of birth control (defined in the list below), and not rely on barrier methods and spermicide alone, from the time of screening until 1 week after final dose of study drug. Male study participants must also not donate sperm from the time of screening until 1 week after final dose of study drug. Given that AZD7325 is not mutagenic, there is no mandatory requirement for condom use, either for avoidance of procreation or in the case of treated males with a pregnant partner.  Women of childbearing potential may be included in the study provided they are practicing abstinence or are established on, and continue to use, highly effective contraceptive methods from the time of screening until 1 week after the final dose of study drug. Highly effective methods of contraception associated with inhibition of ovulation (either oral, intravaginal or transdermal), progestin-only hormonal contraception associated with inhibition of ovulation (either oral [specifically Micronor, Nor-QD or their generic equivalents], injectable or implantable).  Aberrant Behavior Checklist total score of 20 or higher at screening

Exclusion Criteria:

 Concomitant use of modulators of GABA A neurotransmission. (examples)  Use of more than three psychotropic drugs that do not directly impact GABA transmission, and/or unstable dosing of any psychotropic medication in the 4 weeks prior to baseline visit.  Subjects are prohibited from use of strong and moderate modulators of CYP3A and CYP2C19 during the screening (at least 2 weeks before initiation of the study) and treatment periods of the study. Such prohibited drugs are outlined in http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm 292362.pdf  CNS-suppressing agents such as central analgesics, muscle relaxants, benzodiazepines, other sedatives hypnotic drugs, and should also limit alcohol intake to ≤1 alcoholic beverage per day.  Unstable seizure disorder as defined by any seizure in the 6 months prior to baseline visit and/or a change in any anti-convulsant drug dosing in the 60 days prior to study entry.  All patients with abnormal baseline safety lab assessments including, but not limited to ALT or AST greater than 1.5 the upper limit of normal, total bilirubin or creatinine greater than 1 time the upper limit of normal or other clinically relevant lab abnormality or abnormality in ECG, HR or BP at screening as judged by the investigator.  Clinical relevant history or presence of any medical disorder judged by the investigator at potentially interfering with this trial.  History of or current abuse of drugs or alcohol including prescription medication.

11 Version 14.0  For female subjects of child bearing potential (women 50 & under is “amenorrhoeic for 12 months or more (following cessation of exogenous hormonal treatments – if these have been previously taken) and with luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in the post- menopausal range) a positive pregnancy test.

Contraception Guidance: Highly effective methods of contraception also include combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation (either oral, intravaginal or transdermal), progestogen-only hormonal contraception associated with inhibition of ovulation (either oral [specifically Cerazette], injectable or implantable). However, AZD7325 has been shown in vivo to be a weak inducer of CYP3A4 at daily doses >10 mg, which may impair the contraceptive efficacy of these hormonal methods; therefore an additional highly effective method of contraception should be used by female study participants of childbearing potential who are already using a hormonal method of contraception from the time of screening until 1 week after the final dose of study drug. These hormonal methods are, however considered to be highly effective for the female partners of study participants.

Avoidance of procreation can be through use of a highly effective contraceptive method by the study participant or by the partner, as detailed above.

Rescreening may be allowed under circumstances where the subject passed the screening but could not be randomized within the 28-day screening window due to logistical, personal or other unforeseeable reasons.

Study Treatment(s)

Placebo Capsules:

Ingredients: Microcyrstalline Cellulose Powder (PCCA) Orange #0 capsules (PCCA)

Procedure: Fill capsule with Microcyrstalline Cellulose Powder. Securely close capsule. Wipe excess powder. Document on Compounding Worksheet.

AZD7325 Capsules:

Ingredients: AZD7325 hydrogen sulfate powder (AstraZeneca)

Orange #0 capsules (PCCA)

Procedure: 1.28mg AZD7325 hydrogen sulfate powder is equivalent to 1mg AZD7325 free base.

5mg capsule Weigh 6.4mg (+/-10% range i.e. 5.8mg to 7.0mg) AZD7325 hydrogen sulfate powder. Place in #0 capsule.

Securely close capsule. Wipe excess powder. Document on Compounding Worksheet The bulk supply of drug powder is weighed and the appropriate amount is placed in the individual capsule. This is repeated until each capsule is filled.

15mg capsule Weigh 19.2 mg AZD7325 (+/-10% range i.e. 17.3mg to 21.1mg) hydrogen sulfate powder. Place in #0 capsule.

12 Version 14.0

Securely close capsule. Wipe excess powder. Document on Compounding Worksheet The bulk supply of drug powder is weighed and the appropriate amount is placed in the individual capsule. This is repeated until each capsule is filled.

Container Closure System: The drug product is packed in high density polyethylene (HDPE) bottles.

Administration of Study Treatments

AZD7325 will be supplied as a 5 or 15 mg capsule, and matching placebo, in bottles. The caregiver or independent adult will receive a sufficient number of capsules in in separate bottles for “Morning” and “Evening” at the phase 1, phase 2 and phase 3 visits. All bottles will be clearly labeled. All subjects will receive a morning dose of AZD7325 or placebo and an evening dose of AZD7325 or placebo during the entire duration of the treatment phase.

For the morning dose, it is recommended that the study drug be given either before or after breakfast.

For the evening dose, it is recommended that study drug be given within 30 minutes of scheduled bedtime

Labelling

Cincinnati Children's Hospital Medical Center 3333 Burnet Avenue Cincinnati, Ohio 45229 (513) 636-3016 Caution : Federal Law prohibits the transfer of this product to any other person than the patient for whom it was prescribed. Caution: New Drug - Limited by Federal Law to Investigational Use

Date: Dr: Patient: Directions: Storage: Expiration: Drug: Amount : RPh: Study:

Storage

All study medication is dispensed Study treatments are to be dispensed on a specific order/prescription from the principle investigator or his designee. The prescription is filled based on the instructions are requirements of the protocol. Receipt of the prescription is confirmation that eligibility workup and consent are complete. Each time study drug is dispensed; it is logged on the IDS log. The investigator or designee is responsible for keeping accurate records of study treatment supplies received from CCHMC investigational pharmacy and the amount dispensed to and returned by the subjects, and the amount remaining at the conclusion of the study. Study treatment should be stored at ≤ 86°F.

13 Version 14.0 Study Drug Dosing:

Study drug or placebo will provided to subject and their guardian distributed from the CCHMC investigational Drug Pharmacy. At the beginning of each Treatment Period the subject will receive their first dose of study drug or placebo at the Schubert Research Clinic (SRC)at Cincinnati Children’s Hospital Medical Center CCHMC) and stay at the SRC for a minimum of 4 hours post- first dose. All subsequent visits will occur 14 days apart at an interval of 14 +/- 2 days between visits (goal 14 days). A final follow up visit will occur approximately 2 weeks following the final treatment period. .A dose will be given during final visit. The active treatment with AZD7325 will be 5mg BID or 15mg BID daily given in orange gelatin capsules by mouth. Our chosen dosing is based on an extensive review of the AZD7235 safety literature, our pre-clinical experience with the drug in the FMR1 KO mouse, and closely aligns with AZD7325 dosing and drug exposure in Study D1140C00014 (safety data described below under Risk/Benefit Discussion), a large Phase II 28 day study of AZD7325 5mg or 15mg BID versus lorazepam or placebo.

After taking the first dose of compound (AZD7325), it is strongly recommended that study subjects be advised not to drive upon discharge from the visit. You should consider wording such as: Following the first dose, the subject will continue taking the medication at home. It is strongly recommended that subjects be advised not to drive or perform duties that require dexterity (e.g. operating machinery) until the subject becomes familiar with the effects of the investigational product and individual susceptibility to the potential CNS effects of AZD7325 is known. As a general guidance, subjects should be advised not to drive or perform duties that require dexterity for at least 3 hours after taking investigational product during the first 7 days.

Subject Compliance

Study drug compliance will be assessed by the investigator and/or study center staff by recording capsule counts of study treatments from the previously dispensed capsules, separately for “Morning” and “Evening” bottles. The investigator and/or study center staff will also assess whether the subject has been given the capsules in the prescribed order. The total number of doses administered to each subject will be derived from dosing information recorded in the source document. The participants or their caregivers must bring the bottles back at each study center visit. Any remaining capsules will be counted and recorded to assess compliance. Participants or caregivers shall be questioned as to the reason why remaining capsules have not been administered (e.g. forgot because not part of routine, subject refused, subject had side effects so parent decided to give a drug holiday, couldn’t open the bottle, etc.) and the approximate time of any missed doses.

Study Drug Accountability

The CCHMC Investigational Pharmacy shall be responsible for study drug accountability records. These records shall include dates, quantities, batch numbers, expiry dates and the unique code numbers assigned to the study drug and to the study subjects.

Blindness and Breaking the Blind: This is a double-blind, placebo-controlled study. Investigators, study staff, and study subjects will be blinded to the randomized study treatment assignments. Both randomization and blinding techniques will be used in this study to minimize bias. The study blind will be maintained throughout the duration of the clinical trial for all study staff interacting with participants, including research coordinators and study physicians assessing participants during the treatment period. Only the CCHMC Investigational Pharmacy will be aware of study drug assignment. The blind may be broken after the participant has completed or withdrawn from study.

Participant or parent/guardian may receive randomization unblinded information after the study results are reported in the public domain-poster, talk and/or on paper.

14 Version 14.0 Procedure for Breaking the Blind: . In a medical emergency where knowledge of the subject’s treatment assignment may influence the subject’s clinical care, the investigator, may access the subject’s treatment assignment. The investigator will contact the CCHMC Investigational Pharmacy to be authorized to access the emergency unblinding. Emergency unblinding can thus be made for any subject without affecting the double-blind nature of the study. Subject treatment information may only be accessed in the event of an emergency and out of necessity to know the identity of the allocated study drug to institute appropriate therapeutic management. The investigator should make every effort to discuss the rationale for emergency unblinding with the Medical Monitor prior to unblinding the individual subject. Emergency unblinding should only be considered in situations where the knowledge of the treatment code has an impact on the planned treatment of the emergency. Once the randomization code is broken for a subject, he/she must be withdrawn from the study. The investigator must record the reason for the emergency unblinding in the source documents and recorded in the electronic database.

Withdrawal of Subjects: A subject may voluntarily withdraw at any time and for any reason. If a subject withdraws, at his or her request or at the request of his or her caregiver or legal representative, the reason(s) must be recorded on the relevant page of the subject’s source documentation. Subjects who withdraw from the study prematurely should undergo all end-of-study assessments where possible. It is vital to obtain follow-up data on any subject withdrawn because of an AE. In any case, every effort must be made to undertake protocol- specified safety follow-up procedures. If a subject refuses to continue with study procedures, the reason for refusal should be fully documented in the subject’s source document and subjects who withdraw from the study prior to completing the final follow-up visit may be replaced at the discretion of investigator. Subjects who discontinue the study due to an AE considered related to study drug should be followed until the event is resolved, considered stable, or the investigator determines the event is no longer clinically significant. Study drug discontinuation due to AEs considered not related to study drug will be followed until end of study.

STUDY PROCEDURES:

Primary and Secondary Outcome Measures:

Primary Outcome Measure: Blood Product Plasma Amyloid Precursor Protein Blood Marker:

Plasma total APP level (sAPP) will be the primary outcome measure of this pilot trial. Levels of sAPP (total), sAPPα, Aβ40, and Aβ42 will be analyzed from plasma specimens of subjects drawn as noted on the Schedule of Measures. APP assays will be processed in Dr. Craig Erickson’s Neurobehavioral Research Lab at CCHMC. APP data results will be analyzed by an independent statistician, Paul Horn. All blood biomarker assays will be performed blinded to study assignment. Plasma will be isolated and platelets will be removed prior to platelet-free plasma storage at -80ºC. Aliquots will be thawed and pre-coated ELISA plates will be used to determine the amount of sAPPα (IBL-America), sAPP (IBL- America), and Aβ peptides (IBL-America) in duplicate per manufacturer instructions. Western blot analysis may be used to confirm ELISA results. Methodology follows work previously published by our group

As a first in disorder study, there is inherently a dearth of available data upon which to base power calculations. Among quantitative measures associated with FXS-pathophysiology, the APP peripheral plasma marker been utilized in small first in human FXS trials and shown the potential for consistent change with targeted drug treatment. We will power this project focused on the plasma APP primary molecular marker for several reasons 1) the assay 15 Version 14.0 is consistently linked to FXS pathophysiology in human and preclinical models, 2) the assay has been utilized effectively in small scale FXS trials, 3) positive change (reduction) in APP levels has been clinically correlated with improvements in FXS-associated interfering behavior. Given our prior experience estimating a potential 1.22 effect size drug-associated change in plasma APP levels, a total of 15 patients enrolled in this crossover trial design will have 84% power to detect a drug associated difference in plasma APP at a 2-sided 0.05 significance level.

Secondary Outcome Measures:

Blood Product Extracellular Signal Related Kinase (ERK) Marker:

Lymphocytic ERK analysis specimens will be gathered as noted in the Schedule of Measures. ERK Assays will be a secondary outcome measure and will be processed in Dr. Craig Erickson’s Neurobehavioral Research Lab at CCHMC. All ERK blood biomarker assays will be performed blinded to study assignment. Blood will be collected and lymphocytes and/or platelets will be isolated, washed, lysed, and resuspended. Samples will be frozen until assayed. ERK measurements will be quantified by immunocytochemical staining; ELISA , flow cytometry and/or Western blot analysis as previously published by our group.

Eye Tracking and Pupillometry:

Videos, static faces, and inanimate images/marks will be shown on a Tobii X300 eye tracking apparatus to all subjects throughout the trial as noted in the Schedule of Measures. In FXS using Tobii eye tracking systems gaze abnormalities including reduced eye gaze to eye regions has been noted in humans with FXS47,48. Additionally, aberrant pupil response to viewing emotional faces has been noted in FXS thus pointing to aberrant autonomic reactions in this disorder47,48. Eye tracking and pupillometry evaluations have been employed in a number of FXS-specific targeted treatment trials including studies of minocycline49,50 and mavoglurant51 where early reports indicated potential normalization of eye tracking parameters with treatment. Eye tracking and pupillometry will be a secondary quantitative outcome measure in this study.

Research Electrophysiology (EEG):

Our Research EEG work will be conducting using the same protocols currently executed by our team in the NIH supported study U54HD082008 “Neurophysiological and acute pharmacological studies in FXS patients.” We have two identical EEG set ups maximized for use in persons with FXS of all ages. Our team utilizes picture schedules, PECS, and social stories to facilitate EEG testing in this population.

Our EEG protocols will be as follows and will occur when noted in our Schedule of Measures:

Data Acquisition:

We will use up to 128 lead channels placed according to the standard 10-20 electroencephalography array. We will use an EGI NetAmp400 (EGI, Eugene, OR) with Hydrocel nets. Data will be acquired continuously, amplified, and digitized. Eye movements will be recorded (EOG) for offline data correction. In order to facilitate accurate EEG electrode locations on the scalp during analysis any subject that receives an EEG may have a 5-10 minute procedure in which the EEG net and electrode positions are captured using photogrammetry software and a stereoscopic camera. The procedure will be performed on all participants but inability to complete the procedure will not be considered a deviation. Data will be used to generate a numerical coordinates file. Participants will view a silent movie during recording for all paradigms except talk-listen, as is common practice to facilitate participation with developmentally disabled subjects. For auditory paradigms, auditory stimuli will be delivered with earphones via a programmable sound module (i.e. Presentation Software).

Resting Paradigm: Resting data will be recorded with eyes open for 5 minutes. Auditory Habituation: ERPs will be recorded during passive listening (150 sets of four 75 ms broadband noise bursts) separated by an ISI of 16 Version 14.0 500 ms, with inter-set interval of 4000 ms, for ~14 minutes of recording. Chirp modulated sweep: Subjects will passively listen to auditory stimuli consisting of a 1000 Hz tone amplitude modulated (AM) by a chirp sinusoid that linearly increases in frequency from 0-100 Hz over a period of 2 sec. Two hundred of these stimuli will be separated by a 1.5-2 sec inter-trial interval, for ~12.5 minutes of testing. Talk/Listen Paradigm: In the Talk condition, participants pronounce short (<300ms), sharp vocalizations of the phoneme “ah” in a self-paced manner about every 1-2s, for 180s. The speech is recorded and transmitted back to participants through headphones in real time (zero delay). In the Listen condition, participants listen to the recording from the Talk condition played back. Participants are coached to produce “ah” vocalizations >75dB and < 85dB by monitoring intensity with a dB meter. Sound intensity is kept the same in Talk and Listen conditions for each participant by ensuring that a 1000Hz tone (generated by a Quest QC calibrator) produces equivalent dB intensities when delivered through earphones during the Talk and Listen conditions. Some participants may be unable to complete the talk/listen paradigm due to inability to follow the directions and/or high anxiety. The data collected during these instances is unusable and therefore can cause undue stress to on the participant. The paradigm will be attempted with all participants. Visits where the paradigm cannot be completed due to participant behavior or distress will not be considered a deviation.

EEG analysis: For all neurophysiology studies, data will be average referenced and artifacts related to muscular, cardiac and ocular activity will be corrected using the ICA toolbox in EEGLAB [67] in Matlab. Source estimates of ERP components and power spectra in frequency bands of interest are computed for time epochs of interest implemented on the canonical mesh using multiple sparse priors under group constraints. Data will be segmented into 0.5 - 2 second epochs, de-trended, and transformed for time frequency analysis in EEG software (i.e. Matlab or BESA). For auditory habituation, the N1 waveform will be identified with topography defined as the largest negative deflection between 50-200 ms post-stimulus as a primary outcome. For chirp, data will be averaged over the representative sensors and Morlet wavelets and/or Gabor transform will be utilized to compute time-frequency plots, single trial power and phase locking across trials. For talk/listen, the N1 and P2 components will be isolated following previously published methods.

Clinical Global Impressions Improvement (CGI-I) and Severity (CGI-S) Subscales: The clinician-rated CGI Improvement scale (CGI-I) will serve as a secondary outcome measure. Treatment response with the CGI-I will be defined as a score of 1 “very much” or 2 “much improved” on the CGI-I. The CGI-I is a 7-point scale designed to measure symptomatic change at a specific time as compared to CGI-S at screen. The CGI-I will be focused on the core symptoms of FXS. CGI-I is a gold standard measure of potential change with treatment in placebo-52controlled pharmacotherapy trials in FXS 51. The CGI-I has been proven to be sensitive to treatment-associated change in a number of controlled trials in FXS. The CGI-S will be administered as an index of general FXS severity. The CGI-S, like the CGI-I, is rated on a 7 point Likert scale from minimally to the most severely affected.

Aberrant Behavior Checklist (ABC):

The ABC will be utilized as a secondary outcome measure in this study as a measure of interfering behavior commonly seen in persons with FXS. The ABC is a caregiver-rated questionnaire with demonstrated reliability and validity that has been used in multiple studies of individuals with developmental disabilities53. Items on the ABC are rated on a scale of 0 to 3 (“behavior not a problem” to “behavior is a severe problem”). The ABC factored specifically for persons with FXS has subscales including irritability, social withdrawal, stereotypy, hyperactivity, inappropriate speech, and social avoidance54.

Social Responsiveness Scale 2nd Edition (SRS):

The SRS is a continuous parent/caregiver reported measure of social impairment commonly utilize in populations of persons with autism and related developmental disorders. The SRS has been commonly utilized in FXS-specific targeted treatment trials as a continuous measure of social impairment55-59. The SRS will be utilized as a secondary behavioral outcome measure in this study.

17 Version 14.0 Pediatric Quality of Life Inventory (PedsQL):

The PedsQL is a modular approach to measuring health-related quality of life that has investigated in FXS specifically60. The adult report from caregiver forms will be utilized and all available modules including family impact, cognition, and the core batteries will be utilized as secondary outcomes. Self-reports may be completed for participants that are cognitively capable of recording answers or responding verbally. The PedsQL self-report is optional.

Vineland Adaptive Behavior Scale 3rd Editions (VABS 3):

The VABS is the gold standard assessment of adaptive functioning in persons with developmental disability including specific work done in FXS61. The VABS has a long history of use in FXS-specific clinical trials. Recently the new 3rd edition has been published. The VABS 3 will be utilized as a structured caregiver interview secondary outcome measure in this study.

Pediatric Anxiety Rating Scale (PARS):

The PARS is the gold standard structured interview based assessment of anxiety in FXS. The measure shows good test-retest reliability and consistency in FXS62. Despite being originally designed for use in pediatrics, in FXS the PARS is commonly also used in and considered appropriate for adult use. The PARS will be a secondary behavioral outcome measure in this study.

Anxiety Depression and Moods Scale (ADAMS):

The ADAMS was developed as a tool for screening for anxiety and mood symptoms in persons with intellectual disability. The ADAMS has demonstrated face validity in FXS63 and in adults with developmental disability the measure has adequate internal consistency and convergent and discriminant validity64. The ADAMS will be utilized as a secondary symptom/behavior outcome measure in our study.

Visual Analog Scales (VAS):

The VAS will be utilized in two ways: 1) as a caregiver rating of top three causes for concern and 2) as a clinician rated Fragile X domain specific concern rated anchored to the following domains commonly seen in FXS: anxiety, repetitive behaviors, communication, sensory over sensitivity, repetitive behaviors, social withdrawal, and cognition. The VAS has been successfully utilized in several FXS-specific targeted treatment trials and will be a secondary behavioral outcome in this study.

Expressive Language Sampling Task (ELS)

An Expressive Language Sampling Task (ELS) will be administered to all subjects65 This task assesses expressive language in a real-world, functional context by allowing participants to create a narrative of their own while viewing a picture book. Narratives will be recorded and later analyzed. Expressive language sampling has been found to be an attractive alternative to standardized language tests when testing individuals with language impairments and intellectual disabilities. The task will be completed at final visits and at screen. Screen ELS data may be used for baseline comparison to avoid further burden on the participants during the baseline visit. Expressive language sampling audio files may be deidentified and sent with the subject’s unique study number to Leonard Abbeduto’s lab at UC Davis who developed the measure in developmental disabilities and is an expert in analysis.

18 Version 14.0 Fragile X Syndrome Rating Scale (FXSRS):

The FXRS covers a wide range of FXS-behaviors, ASD-associated features seen in FXS, and other associated features66. This Likert scale clinician completed measures has been utilized in FXS placebo-controlled targeted treatment trials66 and will serve as a secondary clinician reported measure in this study.

Neuropsychological Tests

Test of Attentional Performance for Children (KiTap):

The KiTap is an automated computer based assessment of attentional performance developed and normed for the pediatric population. Despite its development in pediatrics, the KiTap is well suited for use in FXS across all age ranges and has been normed specifically in FXS in adults and youth67. The task presents an enchanted castle animation and investigates performance in a number of areas including Alertness, Vigilance, Distractability, Flexibility, and Go/No Go.

Repeatable Battery for Assessment of Neuropsychological Status (RBANS):

The RBANS is a neuropsychological battery for adults with neurological disorders. The RBANS covers five domains including Immediate Memory, Language, Attention, Visuospatial/Constructional, and Delayed Memory. The test is designed to control for content practice effects when repeat evaluations are desired and can be used to track neurocognitive status. Specifically in FXS, List Learning, Story Memory, List Recognition and Digit Span tests show high feasibility and good test-retest reliability in adults with FXS in a placebo-controlled trial setting52. Full RBANS will be completed at Screen and final visits with list learning only at all baseline visits pre and post dose. The screen RBANS may be used as a baseline comparison to reduce burden on the participant. The RBANS will be utilized as secondary outcome measure of neuropsychological status.

Woodcock Johnson (WJ)-III

Woodcock-Johnson III Tests of Cognitive Abilities (WJ-III) measures the ability to analyze, synthesize, and discriminate auditory stimuli, including processing and discriminating under distorted conditions. The test was validated in ages 5-8168,69. Participants will receive the Auditory Attention and Spatial Relationships subtests as written in the schedule of procedures.

Stop Signal task (SST):

The Stop-signal (SST) is one of the most commonly used behavioral measures of Prepotent Response Inhibition. This test has been developed and used by study investigators in previous studies examining neurodevelopmental and psychiatric populations as well as pharmacological interventions70,71. The tasks have been adapted for the current study population by revising parameters such as longer allotted reaction times, increased positive feedback, and increased number and duration of rest blocks. All participants will be administered practice in which they must achieve 50% accuracy to proceed to task administration. Error rate and reaction time measures will be calculated and serve as secondary outcome measure of neurocognitive functioning. SST will be completed at screen and final visits. SST completed at screen will be used as a baseline measure to decrease burden on the participant during the baseline visit.

19 Version 14.0 Additional Subject Characterization Measures:

Social Communication Questionnaire (SCQ):

The SCQ, originally called the Autism Screening Questionnaire, is a continuous measure of ASD behaviors and symptoms based on the Autism Diagnostic Interview- Revised (ADI-R) 72,73. The SCQ will be used at screen to help characterize the subject sample. Specifically in persons with developmental disorders, the PedsQL has been utilized in youth with ASD74-78, general developmental disability79-81, and in clinical trials in youth with developmental disorders82,83.

Autism Diagnostic Observation Schedule (ADOS):

The ADOS is the gold standard research interactive diagnostic assessment for autism spectrum disorder (ASD). The ADOS is frequently utilized to characterize ASD comorbidity in persons of all ages with FXS1,84. The ADOS will be used at screen to thoroughly characterize the subject sample. ADOS will be completed at screen if an ADOS completed by a research reliable examiner is not available in the past 5 years.

Stanford Binet 5th Edition (SB 5):

The SB 5 is a commonly utilized IQ test in persons with FXS. The SB 5 has specific scoring algorithms derived in persons with FXS that avoid floor effects85. The SB 5 will be utilized in this project to characterize subject baseline cognitive status at screen. Prior SB 5 intellectual testing results completed during previous 2 years will be used if available.

Other Study Procedures:

Pharmacokinetic (pK) Sampling:

Pharmacokinetic evaluation blood samples to evaluate AZD7325 concentration in plasma will be drawn at all study visits with the exception of the screening visit. Plasma concentrations of AZD7325 will be determined using a validated LC-MS/MS method after solid phase extraction. The range of quantification, accuracy, within- run and between-run precision for AZD7325 are expected to be between 0.05-40 ng ml-1, -2.7% ≤ %bias ≤ 14.0%, 11.0% and ≤ 7.4%, respectively. A pre-dose steady state pK will be drawn at the final visit of Treatment Periods 1, 2 and 3. Additionally, a tmax sample will be drawn 60 +/- 15 minutes post dose on Day 1 of each Treatment Period. This equates to 2 pK samples per subject per Treatment Period or 6 total pK samples (4 of which should contain active AZD7325) per subject enrolled.

Concentration data will be analyzed by compartmental and noncompartmental pharmacokinetic analysis with the software package WinNonlin (Version 4.0.1, Pharsight Corporation, Palo Alto, CA) using a weighed least- squares algorithm. Population PK analysis will be conducted using NONMEM version 7.2.0 (ICON, Ellicott City, MD) on a 64-bit Linux Operation System with an Intel Fortran Compiler (v 12.0). PDx-Pop (version 5, ICON, Ellicott City, MD) will be used as the graphical user interface for running NONMEM and for processing NONMEM output. Visualization of NONMEM output was implemented by Xpose 4 package in R (v 2.15.0.). First order conditional estimation with interaction (FOCE-I) will be employed throughout to simultaneously estimate the typical population PK parameters, random effect of inter-individual variability and residual errors. Model structure selection will be based on goodness-of-fit criteria, including convergence with at least 3 significant digits, diagnostic plots, physiological plausibility of the parameter estimates and Akaike Information Criterion (AIC). Inter-individual variability (IIV) will be modeled using an exponential model which assumes a normally distributed inter-individual variable with a mean of zero and a variance of ω2.

20 Version 14.0 Parameter estimates generated will include Cmax, total body clearance, distribution and elimination half- lives, volume of distribution and the area under the curve (AUC). Blood Samples, Analyses, and Genetic Testing:

At CCHMC, phlebotomy-trained personnel will draw all blood samples, which will be identified with the subject’s unique study number before being processed and stored on site or sent to a collaborator’s lab. Participants and/or their guardians will have the option to have an IV catheter placement when multiple draws occur in one visit. For the primary and secondary outcome measures, blood will be processed for plasma and peripheral blood mononuclear cells (PBMCs; lymphocytes) and analyzed for APP and ERK derivatives, respectively, as well as other potential markers of cell signaling, development, or disease.

Additional blood tubes, including whole blood for dried blood spots and whole blood for genetic testing, may be drawn.. Not collecting these samples, however, will not be considered a protocol deviation. Dried blood spots will be collected and stored for the quantification of FMRP levels in the blood, as well as other potential analyses in the future. Whole blood may also be used for extraction of DNA and RNA for genetic testing. Subjects will also have the opportunity to consent to having their unused or remaining samples stored indefinitely as part of the Developmental Disabilities Clinical Repository.

Genetic studies will be done by our collaborator, Dr. Elizabeth Berry-Kravis at Rush University in Chicago, who maintains a laboratory that does Fragile X genetic testing. She is a world expert in this area and has conducted active FXS collaborations with our group for the past 10 years. All samples will be deidentified before being sent to her lab. Genetic studies may include but are not limited to tests for CGG expansion of the FMR1 gene using FMR1 repeat-primed (PCR). Subjects with >200 repeats will be considered to have full mutations and FXS. Methylation of this region will also be assessed in full mutation Fragile X patients using Southern blot analysis of methylation or methylation PCR. The choice of method will depend on assay developments in the Berry-Kravis lab when this assay is run on batch samples as we prepare for statistical analyses of group data. Genetic testing by the Berry-Kravis lab will be optional.

Participants and/or their parents have the option to receive a report of repeat count if they want that information. Reports of methylation and repeat counts from the Berry-Kravis lab will be signed by the principal investigator and sent out by mail to families. No genetic counseling will be provided or necessary as all families that participate in AZD are already aware of their diagnosis before participating in this study. We expect to bank samples in Chicago for methylation studies that will be conducted using the best approach available when we are close to having a sufficiently large sample for statistical group analyses. If a blood sample cannot be collected, prior repeat and methylation analysis may be used from clinical tests.

Banked Blood Samples:

Unused and/or remaining blood samples (i.e. plasma, PBMCs, dried blood spots, whole blood) will be stored for future use from all subjects for whom the participant or caregiver provides consent for the storage and future analysis of blood samples and derivatives thereof (e.g., DNA, RNA, proteins, peptides). These samples will become part of the Developmental Disabilities Clinical Repository (DDCR).. Only research study personnel will have access to the locked freezer in which blood is stored at CCHMC.

SAFETY MEASURES:

Safety monitoring provisions will include comprehensive metabolic panel (CMP), complete blood count with differential (CBC with differential), Lipids, Urinalysis, serum pregnancy test in female participants of childbearing age, physical exam, medical history, concomitant medication review, vital signs (height, weight, heart rate, temperature, respirations, blood pressure), and ECG at screen. Triplicate digital ECG recordings should be

21 Version 14.0 done in a supine position after a 5-minute rest. All safety measure results will be reviewed by a study physician prior to initial drug dosing and prior to starting each subsequent dosing phase. Safety labs and ECGs will be repeated throughout study as noted in the Schedule of Measures. Vital signs will occur at all in person study visits. Medication compliance review will occur at the end of each study treatment phase. Concomitant medication review and an adverse effects review will occur at all study in person visits and all study mid treatment phase investigator phone calls. Adverse events that are ongoing at the end of study will continue to be monitored by a physician until the adverse event is resolved or at the discretion of the PI. The adverse event will be assessed by a physician over the phone with the participant or LAR. In the case that the family cannot be reached, or the family is lost to follow up, the outcome of the AE will be recorded appropriately based on the physician’s last assessment of the AE (ex. Not recovered, recovering or unknown). For safety monitoring purposes, the investigator or designee will review the ECG to determine if normal or abnormal. If ECG is abnormal, the investigator or designee will determine clinical significance. If not clinically significant, this will be noted on the ECG. If clinically significant, ECG will be transmitted to cardiology for reading. Dr. Sergio Delgado, Outpatient Clinical Director with the Division of Psychiatry at Cincinnati Children’s Hospital Medical Center (CCHMC), will serve an independent study monitor for the study. The PI and Co- investigators at CCHMC will be primarily responsible for monitoring data quality and adverse events. A physician will monitor adverse effects at each visit. In addition, he or she will review vital signs and laboratory data, as they become available. All of these values are reviewed continuously by a physician. The monitor will review recruitment and adverse events every 6 months and report their assessment to the PI. The independent monitor will also review any SAEs and significant unanticipated events as they occur.

COVID-19 Safety Measures: Washout periods may be extended at the discretion of the PI and/or the participants as a result of COVID-19. The time required for each extension is dependent on the coronavirus outbreak and the limitations set in place as a result of the pandemic. One call may occur after each phase to review AEs, CGI, and ABC-I as outlined in the study schedule. All other calls and visits may be postponed. All study events and dosing will occur as outlined in the study timeline when study visits are resumed. The change in the visit schedule may impact the caregiver’s ability to physically attend visits when visits are resumed. Parents/caregivers may complete measures over the phone or at home if they are unable to attend the visits with the participant. Measures will be completed the same day as the study visit when possible. Any measures completed outside of the two-day study window will be considered a deviation.

Adverse Event Definitions and Classifications:

An adverse event for the purpose of this protocol is the appearance or worsening of any undesirable sign, symptom, or medical condition occurring after starting the study drug even if the event is not considered to be related to study drug. Medical conditions/diseases present before starting study drug (but after signing informed consent) are only considered adverse events if they worsen after starting study drug. Abnormal laboratory values or test results constitute adverse events only if they induce clinical signs or symptoms, are considered clinically significant, or require therapy.

The occurrence of adverse events will be sought by non-directive questioning of the subject. Adverse events also may be detected when they are volunteered by the subject during or between visits or through physical examination, laboratory test, or other assessments. Each adverse event should be evaluated to determine: 1. Severity - mild, moderate, severe, life threatening, death (grade 1-5) 2. Attribution to the study drug(s) - definite, probable, possible, unlikely, unrelated 3. Duration - start and end dates or if continuing at final assessment 4. Whether it constitutes a serious adverse event (SAE). See sections below regarding SAE definition and

22 Version 14.0 reporting. 5. Action taken

Severity Descriptors: Adverse events will be graded as follows:

Severity Numerical Description

Value

Mild 1 Aware of sign, symptom, or event, but easily tolerated; does not interfere with daily routine

Moderate 2 Discomfort enough to interfere with daily routine and may require some therapeutic intervention

Severe 3 Incapacitating, significantly affects clinical status; requires therapeutic intervention

Life 4 Life-Threatening; immediate intervention required Threatening

Death 5 Adverse event causes death.

Attribution Definitions: The investigator is responsible for adverse event attribution to determine the relatedness of the event to study drug or study procedures. Attribution will be determined as follows:

Unrelated The event is unrelated.

Unlikely The event is unlikely to be related

Possibly Related The event or severity of event is not usually associated, but cannot rule out link to the event to link the event.

Probably Related The event or severity of event is such that it can likely be correlated.

Definitely Related There is a strong correlation with the event.

23 Version 14.0 Expected Adverse Events: Expected adverse events are those that are a known symptom or associated condition related to AZD7325 or study procedures as described in the protocol, consent and/or IB Version 9.

Unexpected Adverse Events: Unexpected adverse events are defined as any adverse event whose nature, frequency, or severity is inconsistent with the underlying disease, disorder or condition of the subject or is not identified in the Informed Consent, Protocol, or IB Version 9 for AZD7325.

Serious Adverse Events:

Serious adverse event or serious suspected adverse reaction: An adverse event or suspected adverse reaction is considered "serious" if, in the view of either the investigator or sponsor, it results in any of the following outcomes: Death, a life-threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions, or a congenital anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.

When possible, all serious adverse events will be followed until resolution of the event or there is return to baseline status. The duration of the event will be recorded (start and end dates or if continuing at final assessment). Any action taken to address the event will also be recorded.

Adverse Event Reporting:

SAEs require expedited reporting when meeting the following criteria:  Serious  Unexpected  At least possibly related to the study agent or other protocol specific activity

SAEs meeting the above criteria are required to be reported to the FDA as follows:  If characterized as fatal or life-threatening, within 7 calendar days of the sponsor’s initial receipt of the information  If non-fatal or non-life threatening, within 15 calendar days

The Medical Monitor will review SAEs within 48 hours after initial receipt of the information by the investigator(s) to review the PIs assignment of SAE as related or unrelated to treatment, to confirm the grading of toxicity, and assure that the study may continue.

24 Version 14.0 SAEs requiring expedited reporting will be reported to the IRB concurrently. Significant unanticipated events will also be reported as per CCHMC SOP. All other serious adverse events and non-serious adverse events will be reported at the time of submission of annual reports to the FDA and IRB.

Monitoring will be performed by an independent study monitoring (trained staff from the Cincinnati Children’s Hospital Medical Center Office of Clinical and Translational Research) to ensure the study is conducted, documented, and reported in accordance with the IRB approved protocol, the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) Guidelines, and applicable FDA regulatory requirements.

Targeted monitoring will occur at least annually, with the schedule for monitoring to be determined by rate of enrollment, the overall risk level of the study, identified site-level risks, and issues that may arise during the course of the study.

Monitoring will include source data verification and source data review as specified in the Monitoring Plan. The study monitor may require access to the subject medical records and other source documents needed to verify the entries on the CRF/eCRFs. The study monitor will perform verification and/or review of critical site processes as indicated, including:  Eligibility of enrolled research participants  Protocol adherence and appropriate reporting of any protocol deviations  Safety information and safety reporting  Consent process and documentation  Drug accountability, storage and reconciliation  Record keeping of essential documents  Completeness and accuracy of data entry and data query resolution  Investigator oversight.

Data Management Redcap:

The REDCap database will be built upon a developmental disability-focus clinical trial REDCap database. REDCap data collection projects rely on a thorough study-specific data dictionary defined in an iterative self- documenting process by the research team. REDCap provides a secure, web-based application that is flexible and provides 1) an intuitive interface for users to enter data and have real time validation rules (with automated data type and range checks) at the time of entry; 2) HIPAA-compliant and 21 CFR Part 11-ready audit trails for tracking page views, data manipulation and export procedures; 3) record locking and electronic signature functions; 4) fine grained control of user rights to view and manipulate data; 5) a report builder for reporting, monitoring and querying patient records; 6) automated export procedures for seamless data downloads.

Data Monitoring:

A rigorous and systematic approach to data management is critical for the quality of any study. The substantial effort and resources that will be devoted to collecting data in this project will be matched by an equally substantial commitment of effort and resources to edit, verify, correct, update, and assemble the resulting data files. Our data management system incorporates quality control at every juncture from data collection through analysis. The PI and primary research coordinator are responsible for data collection and accuracy of record keeping and the researchers will convey an attitude that the data management procedures be treated with unwavering gravity, therefore maintaining a high level of quality for this project.

25 Version 14.0 Data will be collected on hard-copy forms and then verified by data entry personnel. Data that is collected directly on an electronic device using redcap will not require data verification. Data personnel will be trained to search for potential errors and any questionable or illegible entries will be brought to the attention of the study team member responsible for completion of the form. All questionable or illegible entries will be addressed promptly. Corrections to paper records will be made in a way that allows the original entry to be understood. Corrections to electronic records will be recorded with an audit trail that allows the original entry to be retrieved. The hard copy research data is kept in locked file cabinets at the CCHMC. Only the PI and members of the research team will have access to these files, ensuring the security of the records. All procedures to ensure confidentiality will follow the regulations and policies of CCHMC.

Safety labs will be processed in a CCHMC core laboratory and results made available in our electronic medical record system (EPIC). Any lab abnormalities will be promptly brought to the attention of the study physician. All lab results will be reviewed by the study physician and a certified copy, signed and dated by the principal investigator, will be placed in the subject’s chart with the hard copy data.

Source data (such as lab values, vital signs, and outcome measure data) will be entered from source documents and/or CRFs into our secure electronic database by the study coordinator or delegated study team member. The PI and/or other members of the study team will review case report forms and database entries for accuracy by comparison with the source documents. Original research records and source documents will be maintained in a research chart and stored in a locked file cabinet. Records will be kept secure, and individually identifiable information will not be included in any reports or data sets.

Each subject will be given a unique alphanumeric code and this will serve as the connection between the hard-copy forms and the secure electronic database. The electronic database used to house the data will be password protected and only members of the study team will be given access to the database. This will protect the electronic data against any unauthorized persons from entering the dataset and jeopardizing the integrity of the data. De-identified data may be shared with the national database for ASD research (NDAR) in the future using a Global Unique Identifier (GUID) and the Data Dictionary technology developed by NIH. We have successfully used these methods during the past 5 years to share behavioral phenotyping and genetic information.

Optional Data Sharing Agreement:

Commercial entities and/or investors may access data for participants that have been consented to the supplemental authorization to share health information. Commercial entities/investors are more able to take AZD and design large trials to take steps to make this form of treatment an approved therapy for Fragile X. This is a standard method academic institutions use to channel new discoveries made by their physicians and researchers to commercial entities with the capacity, funding and capability to get the new therapies approved. For this to occur, potential investors need to review the study records to determine if the investment is worthwhile.

Cincinnati Children’s would benefit financially from such a partnership. In the course of developing such a relationship, a potential partner may want to have access to information about participant research data and health information. Although Cincinnati Children’s will remove the participants name and other similar types of 26 Version 14.0 information that would directly identify the participant, there are very few participants in this study. Therefore, it is not possible to de-identify health information fully.

This Supplemental Authorization allows Cincinnati Children’s to share the participants protected health information (PHI) with potential investors and sponsors interested in developing AZD7325 or providing funds to Cincinnati Children’s to research and develop AZD7325. This PHI will come from medical and research records. It may include, but is not limited to, medical history, laboratory test results, diagnosis, medications, reports and notes from clinical and research observations, and imaging studies and results. If Cincinnati Children’s shares PHI with a potential commercial partner, it will not provide name or direct contact information such as address or telephone number. The companies and individuals who receive PHI, if the participant signs this Supplemental Authorization, will be asked to keep this information confidential through an agreement between CCHMC and the sponsor or investor. However, absolute confidentiality cannot be promised. Participants and/or their parent/caregiver/LAR will be asked to consent to have personal health information shared with research sponsors or investors who want to develop and bring to market AZD7325 treatment.

Statistical Analysis and Power Calculation:

As a first in disorder study, there is inherently a dearth of available data upon which to base power calculations. Among quantitative measures associated with FXS-pathophysiology, the APP peripheral plasma marker been utilized in small first in human FXS trials and shown the potential for consistent change with targeted drug treatment. We will power this project focused on the plasma APP primary molecular marker for several reasons 1) the assay is consistently linked to FXS pathophysiology in human and preclinical models, 2) the assay has been utilized effectively in small scale FXS trials, 3) positive change (reduction) in APP levels has been clinically correlated with improvements in FXS-associated interfering behavior. Given our prior experience estimating a potential 1.22 effect size drug-associated change in plasma APP levels, a total of 15 patients enrolled in this crossover trial design will have 84% power to detect a drug associated difference in plasma APP at a 2-sided 0.05 significance level86. Independent sample T tests will be used in the primary APP plasma analysis. An additional power calculation was completed using our groups published auditory ERP data which shows a FXS effect of diagnosis of 0.97 compared to typically developing controls43. Should treatment have a 0.97 effect on our auditory ERP testing compared to placebo, our study as designed will have 67% power to detect this change.

Independent sample t tests will be applied for analysis of continuous variables including the subscales of the ABC, RBANS, VABS 3, ADAMS, KiTap, and eye tracking measures. We will not make adjustments for multiplicity in the statistical analysis given the pilot nature of the project. For the dichotomous outcome we will use the Fisher's Exact version of the Mainland-Gart test for binary outcomes in a 2X2 crossover86. The placebo change from baseline will be compared at each of the active dose levels using a paired analysis. Fisher’s Exact Test will also be used to analyze adverse effect occurrence in the two groups.

Correlation analysis between ERK activation, plasma APP derivatives, clinical response, adverse events and PK AUC will be modeled using regression analysis in SPSS. Comparison of plasma levels of sAPP-total, sAPPα, Aβ40, Aβ42, as well as the ratios of sAPPα/sAPP and Aβ42/Aβ40 pre- and post-AZD7325 treatment will be primary outcomes analyzed for the APP biomarker. For the ERK activation marker, primary outcomes will include baseline lymphocytic p-ERK levels and time to half maximum ERK phosphorylation following lymphocyte activation. All blood biomarker data will be coded into IBM SPSS Statistics or SAS for analysis. The differences between pre- and post- assay values for APP and ERK will be compared by paired t-tests in the patient sample, by bootstrap resampling of the mean difference in the individual treatment groups and 95% confidence intervals and Hedge’s g calculated. An exploratory Kendall’s tau or other appropriate

27 Version 14.0 correlation analysis will be conducted to assess for any relationship between change in primary APP and/or ERK activation outcomes above and change in behavioral outcome measures that may show change during the course of the clinical trial.

The EEG data will be processed using QUASAR’s QStates software. QStates software provides workload data using two different statistical models: multivariate normal probability density function (MVNPDF), and a linear model. The workload model consists of an algorithm that identifies characteristics and features of an EEG that differ between high and low mental workload states. The use of the model automates the data reduction from raw EEG (240 Hz) to workload values (0.5 Hz). A repeated measures analysis will be used on the resulting data across visits. Parameter tests were conducted across subject, trial, and workload conditions.

Human Subjects: A total of 15 individuals 50 ≥ Age ≥18 years with FXS will participate in the AZD7325 treatment trial. There are no restrictions on gender, ethnicity or social background. We plan to include when available females and members of minority groups and their subpopulations in this research.

Informed Consent: There are no restrictions to study enrollment based on gender, ethnicity or social background. Since all males and some females with FXS have intellectual disability, we anticipate that a small minority of subjects will have the cognitive ability and understanding to give their assent to participate in the project. For every subject, their legal guardian(s) will be required to give voluntary written informed consent. The nature of the project, the risks, the benefits, and the alternatives to participation in the project are discussed with the subject (when possible) and the subject’s parent or legal guardian by a member of the study team. If following these discussions the subject and family continue to be interested in the project, assent will be obtained from the subject when possible, and formal written consent will be obtained from the parent(s)/legal guardian(s) on the consent form approved by the Institutional Review Board. Clinical responsibility for the care of the subject is then assumed by the Principal Investigator and the other members of the research team. All potential subjects and their legal guardians will be encouraged to ask questions about any aspect of the study that is unclear. All questions will be answered and uncertainties clarified. All legal guardians will be provided with copies of the consent form for future reference. Appropriate clinical evaluation and treatment of the referring problem will be offered regardless of the subject’s/legal guardian’s decisions regarding participation in the study.

If the adult subject is able to demonstrate understanding of the informed consent document as determined by a qualified member of the research team then they will be allowed to consent. This will be based on direct observation and clinic judgment. However, if the individual cannot demonstrate an understanding and has a parent or legal guardian who can legally consent for them then the subject’s parent or legal guardian will go through a parental permission document with the researcher. The subject will also be given the opportunity to read through an assent document and ask any questions they might have relating to the study.

The subject or the subject’s parent or legal guardian will also be asked to consent to allow staff to share health information with potential investors or sponsors. This consent process can be completed in person or over the phone to accommodate families that have already completed study. In the case that a phone consent is required the authorization form will be sent for review prior to the phone consent process with study staff. Study staff will review the authorization form over the phone and answer any questions. If subject and/or parent/LAR agree to participate in this portion of the study then they will be asked to sign indicating consent and scan, email or fax the consent form. The study staff will sign the copy. The wet ink document will be mailed to study staff for documentation. Consent to this part of the study is not required to participate. The participant can still enroll in the study if they decline to release health information. 28 Version 14.0 The consent process will also be completed with the subject’s parent, guardian or caregiver. The caregiver must have, at minimum, weekly contact with the participant to consent to complete caregiver questionnaires.

Recruitment of Subjects: Recruitment of subjects will be conducted via IRB approved electronic and paper ads distributed to individuals with FXS, their families, treating clinicians and agencies throughout the referral base of CCHMC and those within our existing clinical services, residential facilities, schools and group homes for the developmentally disabled. Subjects will also be recruited via flyers, brochures, other newsletters, speaking engagements made by members of the research team, and other available advertising including websites, social media, newspaper, radio and television advertisements (only once such ads are individually IRB approved).

Many subjects will be recruited from the clinical patient population base at Cincinnati Children’s Hospital Medical Center Fragile X Center. Members of the research team may also contact other healthcare providers to provide information about this research study. Additionally, an electronic medical record review preparatory to research may be conducted by members of the study team to identify potentially eligible patients from the clinic. Potentially eligible subjects’ parents/legal guardian may be contacted by a member of the study team to assess interest in participation via phone or IRB approved letter. Interested subjects’ parents/legal guardian will undergo a non- invasive pre-screen in which the study purpose, procedures and the inclusion/exclusion criteria will be explained. If it appears that the subject would satisfy the criteria for the study and there is still an interest in participating, subjects and their parent or caregiver will be scheduled for a consent and screening visit.

Risk/Benefit Discussion

AZD7325 Treatment Safety Review and Dosing Discussion

AZD7325 has been evaluated in many human studies including a total of 722 male and female adult subjects exposed to the drug. These human subjects have included to date both healthy volunteers and persons affected by generalize anxiety disorder (GAD). In summary, the PK profile of AZD7325 exhibits linear kinetics with rapid absorption and t ½ of 12 to 17 hours. The rate, but not the extent of absorption, of AZD7325 is impacted by a high fat meal. Accumulation of AZD7325 at steady is minimal for 10, 20, or 50mg daily dosing. Renal elimination of AZD7325 is minimal. The drug is a weak inducer of CYP3A4 in vivo. In a PET study in healthy volunteers 50% GABA A receptor occupancy was achieved with blood level of 4 ng/mL and the dose at which 50% occupancy was achieved is 1.3 mg. At doses above 5 mg, there was <70% receptor occupancy and maximum apparent occupancy was noted at 20mg per day or above. In health Caucasian adult subjects, single doses of up to 100mg and multiple doses up to 50mg per day for 7 days have been generally well tolerated with only one severe event of stupor after a single 50mg dose of AZD7325 noted that resolved within 1 hour. Healthy male Japanese subjects have received single doses up to 75mg which have been well tolerated. In single-dose study in healthy volunteers, no statistically significant effects on sedation, cognition, balance, or coordination was observed. The most common adverse effects in both patients with GAD and healthy volunteers were CNS related and included dizziness, somnolence, headache, and euphoric mood. The abuse potential of AZD7325 10mg daily is considered minimal compared to lorazepam. Treatment discontinuation effects have not been observed in patients following AZD7325 discontinuation other than a case of one patient with GAD with an SAE of anxiety during a follow-up period which was attributed to treatment withdrawal. 29 Version 14.0 Our proposed Phase I FXS study will provide drug exposure and dosing with AZD7325 most closely related to exposure and dosing in the completed Study D1140C00014 a Phase II study with lorazepam arm in subjects with GAD. In this study 368 patients with GAD were administered AZD7325 at doses of 5mg BID (n=93), 15mg BID (n=92), lorazepam 1mg BID for 4 days followed by 2mg BID thereafter (n=92), or placebo BID for up to 28 days. No deaths or SAEs were reported in this study. Overall, 288 (78.3%) of subjects reported an AE during the study period (treatment and follow-up). Most AEs (75.6%) occurred during the treatment period and were mild (39.7%) or moderate (34.2%) in intensity. The incidence of AEs that started during the treatment period were higher in the AZD7325 15mg BID group (82.6%) than in the 5 mg BID (72.0%), lorazepam (78.3%) or placebo groups (69.2%). Among AEs, a total of 237 subjects (64.4%) experienced AEs that the investigator felt were related to the study drug. A total of 32 patients (8.7%) discontinued the study drug due to an AE. A higher number of patients receiving 5mg BID AZD7325 (12.9%) and 15 mg BID (13.0%) discontinued treatment due to an AE than in the lorazepam (5.4%) or placebo (3.3%) group. Most AEs were CNS related including most commonly dizziness (22.6%) noted. Dizziness was higher in the AZD7325 groups (5 mg BID 29% and 15mg BID 33.7%) than in the placebo (12.1%) or lorazepam (15.3%) groups. Somnolence was the second most common AE and was seen in higher percentages in the AZD7325 groups (5mg BID 17.2%, 15mg BID 18.5%) compared to the placebo group (12.1%). Somnolence was higher in the lorazepam group (31.5%) than in the both the AZD7325 groups or the placebo group. No suicidal events were reported in the study. The incidence of euphoric mood was both higher in the AZD7325 groups (5mg BID 7.5%, 15mg BID 8.7%) than in the lorazepam (4.3%) or placebo (1.1%) groups.

Venipuncture and Peripheral IV (PIV) Placement Safety:

The risks of venipuncture and PIV placement include mild discomfort, hematoma, infection, bleeding, and fainting. Standard methods and precautions will be used to protect the puncture site from bleeding and infection. To minimize the subject’s anxiety and phobic reactions, we utilize Child Life personnel when needed and available. At the discretion of the nurse or the investigator, to help reduce pain at the site of the venipuncture, we will offer the use of a topical anesthetic cream or spray.

PIV placement for blood sampling will be offered as an option to participants and is not required for participation on study.

Procedures for Protecting Against and Minimizing Risk:

Effective screening will be used to eliminate subjects who are at greatest risk because of concurrent medical conditions. The subjects will be evaluated and cared for in an advanced well-staffed pediatric neuropsychiatric research environment. Thus, the direct observation by nursing staff and research psychiatrists will allow for careful monitoring of potential adverse effects including drug side effects. If adverse reactions become excessive, the subject will be treated and removed from the study. Psychiatric hospitalization will be facilitated by the PI and Co-I for any subject whose symptoms become difficult to manage or dangerous (hospitalization expenses covered by subject’s family/their insurance provider). There will be repeated monitoring of behavior, safety lab measurements and vital signs that will allow the treatment team to assess the status of the subject and alter or terminate the study if this is warranted.

Potential Benefits: The potential benefits to subjects entering this project are several. 15 adults with FXS will receive a very carefully controlled drug treatment trial with AZD7325 that may be effective for improving cores symptoms 30 Version 14.0 and behaviors of FXS. Subjects will also receive an extensive psychiatric medical/neurological evaluation, which is provided free-of- charge. A standard research report template will be given to parents and/or participant who request a testing report. This report is also available for upload into the participant’s medical records with the appropriate consent. There is not a requirement to have the report uploaded into the medical record.

Risk/Benefit Ratio: The subjects will be exposed to the risks of blood sampling and the potential side effects of AZD7325. For the patients, the benefits potentially offsetting this will be a more intensive and thorough psychiatric and medical evaluation, a documented objective treatment trial, and the possibility of more accurate prescription of treatment designed to meet the individual subject’s needs. Since some of the subjects will have had previous drug trials with poor response or intolerable or dangerous side effects, the opportunity for a more thorough evaluation and clinical trial may be beneficial. Thus, with the risk of drug treatment minimized, the more intensive evaluation and treatment may compensate for the negative risks. The overall benefit to family members and society is considerable.

Subject Payment: o Screen: $100 o Baseline Visit Treatment Phase I: $50 o Final Visit Treatment Phase I: $50 o Baseline Visit Treatment Phase 2: $50 o Final Visit Treatment Phase 2: $50 o Baseline Visit Treatment Phase 3: $50 o Final Visit Treatment Phase 3: $50 o Final Follow up visit: $50

Given the rare nature of FXS and the national recruitment base of our Cincinnati Fragile X Center, we will additionally offer for families traveling more than 100 miles to Cincinnati, cost reimbursable travel reimbursement (mileage, hotel, flights if applicable) to participating families not to exceed $7,500 per subject enrolled over the entire course of the study. Exceptions for travel reimbursement may be made at the discretion of the PI.

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