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