Conditions: Fragile X Syndrome
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 29
Healthy volunteers: no

Brief summary:
The aim of this study is to utilize neurophysiologic assessments, behavioral measures and clinical measures to assess how much deficits associated with Fragile X Syndrome from pre-dose to post-dose using pharmacology.

Detailed description:

Arms:
arm 1: Participants received, in random order, a single dose of placebo, acamprosate, lovastatin, minocycline, or baclofen, with a two-week washout period between doses. Midway through the study (n=16) it was determined that acamprosate was undetectable in serum and this intervention was replaced by baclofen. Remaining participants (n=13) received baclofen and 5 participants were re-enrolled to receive baclofen or a second round of placebo, so investigators and participants would remain blinded to drug status during the baclofen visit. The second round of placebo was not analyzed.

Interventions: intervention 1: Acamprosate intervention 2: Lovastatin intervention 3: Minocycline intervention 4: Placebo intervention 5: Baclofen
Intervention descriptions: intervention 1: two 666mg pills intervention 2: two 20mg pills intervention 3: two 135mg pills intervention 4: placebo pill intervention 5: one 30mg pill

Protocol document:
Prot_SAP_001.pdf: 
1 
U54 Project 2 v1_12012015 
 
 
 
 
 
 
 
 
 
 
 
 
 
Title: Evaluating the Neurophysiologic and Clinical Effects of Single-Dose Acamprosate, 
Lovastatin, Minocycline and Placebo in Fragile X Syndrome 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator:  
Craig Erickson, M.D. 
 
 
 
 
Associate Professor of Psychiatry 
Children’s Hospital Medical Center  
3333 Burnet Avenue  
Cincinnati, OH 45229-3039 
Office 513-636-4611 
craig.erickson@cchmc.org 
 
 
 
Co-Investigators 
 
Ernest Pedapati, M.D. 
Logan K. Wink, M.D. 
Ismail Badran, M.D. 
Kelli Dominick, M.D., PhD 
Rebecca Shaffer, PsyD 
Amie Duncan, PhD 


2 
U54 Project 2 v1_12012015 
 
1.0 Introduction  
 
Fragile X Syndrome (FXS) is a family of genetic conditions caused by mutations in the Fmr1 
gene. FXS is the most prevalent cause of inherited intellectual disability (Crawford, Acuna, & 
Sherman, 2001). Approximately 1:4,000 males and 1:8,000 females have FXS (Crawford et al., 
2001). This impairment ranges from learning disabilities to severe intellectual disability. Between 
15% and 60% of individuals with FXS have autism (Budimirovic & Kaufmann, 2011). Other 
symptoms also can include characteristic physical (e.g. elongated face, prominent ears, and 
enlarged testes) and behavioral (e.g. stereotypic movements and social anxiety) features.  
Sensory hypersensitivities are an especially common, clinically distressing feature of FXS.  The 
aim of this project is to determine whether single-dose administration of acamprosate, 
minocycline and lovastatin, which lead to recovery of neuronal function in Fmr1 knockout (KO) 
mice, reduce (i.e., normalize) sensory evoke-related potentials (ERPs) in FXS patients. The 
proposed study will contribute to an understanding of neural hyperexcitability as a substrate of 
sensory hypersensitivities in FXS. 
 
2.0 Specific Aims 
 
Prior research has documented that pharmacological strategies targeting different neural 
mechanisms can reduce neocortical hyperexcitability in Fmr1 KO mouse models. Unlike nearly 
all areas of clinical psychopharmacology, marked to full recovery of function has been shown 
within hours of drug administration in animal models. We propose to leverage that observation 
to investigate single-dose drug effects on event-related potential (ERP) biomarkers and 
behavior in humans with FXS.  
 
Primary Aim 
Our aim is to clinically validate the acute pharmacologic recovery of auditory processing deficits 
seen in Fmr1 KO mouse models, and to assess the relative impact and consistency of effect 
across patients of drugs targeting three different mechanisms on this recovery. If ERP 
normalization is observed in the proposed pharmacologic study, it will not only provide new 
knowledge about underlying mechanisms, but potentially speed drug development by guiding 
selection of mechanisms to target, choice of drugs to investigate, and outcomes to assess in 
next-step early phase clinical trials. 
 
Secondary Aim 
Neurophysiologic assessments will mirror the assessments in “Mechanisms and brain circuits 
underlying fragile X syndrome” IRB # 2015-8425 and include spectral analysis, N1 amplitude 
and habituation ERP, chirps, and talk/listen paradigms. Behavioral measures were chosen 
based on prior validation in FXS populations and an ability to allow brief assessments with 
limited learning effects. The primary clinical measure will be the Clinical Global Impressions 
Improvement (CGI-I) scale (Guy, 1976). The CGI-I requires the clinician to assess how much 
the patient's illness has changed relative to pre-dose, from 1 (very much improved) to 7 (very 
much worse). The recently validated Aberrant Behavior Checklist (ABC) using the 6-factor FXS 
model (Sansone et al., 2012) will be used as a secondary outcome of behavioral status.  
 
To assess cognitive outcomes as a secondary aim, we selected the following tests:  Woodcock 
Johnson (WJ)-III Spatial Relations (visual spatial thinking/5 min (McGrew & Woodcock, 2001); 
Grooved Pegboard Test (sensory motor integration/15 min); RBANS List Learning (immediate 
memory/5 min (Randolph, 1998); and Test of Attentional Performance for Children (KiTAP) 
(attention and inhibition/30 min (Knox et al., 2012). The WJ-III Auditory Attention test will assess 


3 
U54 Project 2 v1_12012015 
 
auditory processing-speech sound discrimination, which aligns well with our focus on auditory 
cortical function. This test was validated in ages 5-81 and requires 5 min to complete (McGrew 
& Woodcock, 2001). 
 
3.0 Background: 
 
Fmr1 gene full mutation leads to a cascade of brain changes, which offer multiple potential 
pharmacologic targets to treat functional deficits associated with FXS (Wijetunge, Chattarji, 
Wyllie, & Kind, 2013). This protocol is designed to investigate clinical homologues in sensory 
processing abnormalities that can provide more sensitive and mechanism-related assessments 
of pharmacologic effects.  We plan to evaluate single-dose drug effects on a core symptom of 
FXS, auditory hypersensitivity, using robust ERP measures of auditory response that translate 
from mouse models to humans and back.  Specifically, our study will evaluate the effects of a 
single-dose approach to test drug effects on putative ERP biomarkers and behavior of persons 
with FXS. In 2009, Berry-Kravis et al. (Berry-Kravis et al., 2009) reported that a single oral dose 
of fenobam, an mGluR5 antagonist (Porter et al., 2005), was well tolerated and resulted in 
response (defined as 20% improvement) in auditory prepulse inhibition (PPI). These PPI effects 
were demonstrated just 60 minutes after oral dosing (Berry-Kravis et al., 2009). This study lays 
a foundation for using acute pharmacologic studies to probe putative etiologic pathways of FXS. 
We will extend this pharmacologic strategy to test the neurophysiologic effects of multiple drugs 
targeting different mechanisms in the same patients. 
 
Among the most promising targets for pharmacologic intervention in FXS are treatments 
targeting mGluR5 and GABA systems and the ERK and MMP-9 pathways (Wijetunge et al., 
2013). Fmr1 mutation results in upregulation of mGluR5 receptors and “downstream” effects of 
increased mGluR5 signaling in extracellular signal related kinase (ERK) and matrix 
metalloproteinase-9 (MMP-9) pathways that lead to abnormal dendritic spines and auditory 
circuit hyperexcitability [Preliminary data from our collaborators at UTSW (Huber & Gibson) and 
UC Riverside (Abdulrazak & Ethell)].  Fortunately, safe and well-tolerated drugs with actions 
relevant to these mechanisms already exist in agents FDA-approved for other conditions:  
acamprosate inhibits mGluR5 (and enhances GABA function (Harris et al., 2002)); lovastatin 
inhibits ERK (Osterweil et al., 2013), and; minocycline inhibits MMP-9 (Bilousova et al., 2009). 
Limited, but growing evidence suggests these agents can counter auditory hyperexcitability 
associated with the Fmr1 mutation. Acute administration of acamprosate results in a significant 
reduction in UP state duration (a measure of cortical hyperexcitability) in somatosensory cortex 
slices of Fmr1 KO mice (Huber & Gibson preliminary data). Acamprosate has also been shown 
to reduce cortical hyperexcitability in ERP and magnetoencephalopathy studies in the 
substance dependence literature (Boeijinga et al., 2004) (Pietrzak & Czarnecka, 2005). 
Lovastatin, an ERK inhibitor, corrects audiogenic seizures in the Fmr1 KO mouse model 
(Osterweil et al., 2013). A related ERK inhibitor, U0126, blocks hyperexcitability in 
somatosensory cortex as shown in reduced UP states in Fmr1 KO mice (UTSW collaborators 
Huber & Gibson preliminary data). Our UC Riverside collaborators (Abdulrazak & Ethell) 
recently found that chronic minocycline treatment partially attenuates the number and severity of 
audiogenic seizures in Fmr1 KO mice (Dansie et al., 2013). A study of mice and rats found that 
minocycline was protective against partial seizure induction in vivo (Wang, Englot, Garcia, 
Lawton, & Young, 2012). Particularly relevant to our proposed pharmacologic study, a single 
oral dose of minocycline exerted acute inhibitory effects on cortical excitability (motor cortex 
response to transcranial magnetic stimulation) in normal humans just 4 hours after dosing 
(Lang, Rothkegel, Terney, Antal, & Paulus, 2013). Chronic treatment with minocycline has 
already been shown to significantly reduce auditory N1 ERP in a double-blind, placebo-
controlled crossover treatment trial in children with FXS (Schneider et al., 2013).   


4 
U54 Project 2 v1_12012015 
 
 
This pharmacologic study is designed to leverage clinical and preclinical preliminary data 
showing robust acute drug effects on neurophysiological parameters. If our strategy of single-
dose rapid testing of drug effects on ERP biomarkers is effective, the approach would inform 
preclinical work on the mechanisms most relevant to auditory hyperexcitability, speed the 
identification of effective pharmacologic treatments, develop an urgently needed biomarker 
strategy for predicting and evaluating drug effects, and ultimately, could allow for rapid 
personalization of treatment in FXS.  
 
4.0. Concise Summary of Project: 
 
4.1 Study Design 
 
This pharmacologic study employs a double –blinded 4-drug single dose, 4-period, 4-sequence 
balanced-crossover design.   36 patients age 15 to 55 years with full mutation fragile X 
syndrome characterized by greater than 200 CGG repeats in the FMR1 gene by Southern Blot 
and PCR genetic testing will be eligible to participate if they previously successfully completed 
baseline testing (behavioral, medical, neurophysiological tests) for our study “Mechanisms and 
brain circuits underlying fragile x syndrome (IRB # 2015-8425)”. In this project, subjects will be 
randomly assigned to 4 different sequences for receiving acamprosate, lovastatin, minocycline 
and placebo, with a 2-week washout between administrations. Patients will be stratified based 
on gender and then randomized in a 1:1 ratio to one of the 4 possible drug sequences (e.g.,1: 
ADBC; 2: BACD; 3: CBDA; and 4: DCAB). The study is expected to enroll 36 subjects who 
randomize, once they are randomized, subjects will not be replaced, Subjects and legally 
authorized representatives will be informed during consent that they will be blinded to treatment 
status throughout the study. Agents of this study will be dosed as a single administration to each 
patient at the following doses:  acamprosate=1,332 mg, lovastatin=40 mg, minocycline 
extended release 270 mg (bioequivalent to 200 mg immediate release minocycline).  The timing 
of evaluations in relation to the single-dose of each agent/placebo is guided primarily by the 
time to maximum plasma concentration (Tmax) of the drugs but is also supported by 
considerable preclinical and clinical literature. The primary outcome ERP will be conducted 4 
hours post-dosing, with post-dose clinical and cognitive evaluations being done immediately 
thereafter. Pharmacokinetic data regarding ½-lives of the drugs support a 2 week washout 
period between drug exposures. 
 
The power analysis supports our plan to enroll subjects until at least 36 subjects have fully 
completed the drug dosing protocol. The study will last approximately 10-11 weeks and there 
will be a total of 5 visits in the study (and 5 follow-up phone calls with a study coordinator). 
 
4.2 Blindness and Breaking the Blind 
 
The study blind will be maintained throughout the duration of the clinical trial. Only the CCHMC 
Investigational Pharmacy will be aware of study drug assignment. The blind will be broken 
only at the discretion of a participating study physician or Medical Monitor in cases necessary 
to assure patient safety. Such significant safety events would include, but are not limited to, 
any circumstance where an adverse effect documented as possibly, probably, or definitely 
related to study treatment and moderate or more severe in intensity results in the need for 
additional medical intervention (hospitalization, emergency/urgent care room visit, additional 
outpatient prescribing/other management etc.). 


5 
U54 Project 2 v1_12012015 
 
5.0 Study Procedures: 
 
5.1 Baseline Visit Procedures 
 
All FXS age 15-55, who successfully complete testing (behavioral, medical, neurophysiological 
tests) for our study “Mechanisms and brain circuits underlying fragile x syndrome (IRB # 2015-
8425)” will be eligible to participate in this pharmacologic study. At the day of recruitment, 
consenting participants will complete a urine drug screen (if possible) and females of 
childbearing age (as determined by report regarding age of menarche or menopause) will be 
required to take a urine or serum pregnancy test before study procedures. A positive test will 
exclude them from participating in the study.  Safety labs including comprehensive metabolic 
panel (CMP), antinucleotide antibody (ANA), and complete blood count with differential (CBC with 
differential) will be collected at baseline.  All subjects will have the option to provide a blood 
sample for Extracellular Signal Related Kinase (ERK) Activation Molecular Biomarker Assay and 
Amyloid Precursor Protein (APP) Molecular Biomarker Assay (described in further detail below).    
Baseline cognitive and clinical measures (described in further detail below) will be assessed.  
Each patient will have a medical examination performed by a study physician who will review 
medical history, and assess height, weight, vitals (Blood pressure, heart rate, respiratory rate, 
and temperature), muscle tone, Tanner staging, head circumference, history of recurrent otitis 
media, dysmorphologies and other comorbid conditions at baseline. 
  
Subjects who successfully complete baseline testing will be randomized to study treatment and 
assigned to 4 different sequences for receiving acamprosate, lovastatin, minocycline and 
placebo. Subjects will return within 7 days to receive the first dose of the first of 3 drugs or 
placebo.   
 
 
5.2 Drug Administration Visit Procedures  
 
 The day of each drug administration day, a medical evaluation/physical exam, vital signs, event 
related potentials (ERP) neurophysiology measures, RBANS, KiTap and CGI-I will be completed 
as “baseline” assessments for the day. Approximately two hours later, study drug (acamprosate 
1,332 mg, lovastatin 40 mg, or minocycline ER 270 mg) or matching placebo will be 
administered orally to the subject with FXS. Optional pharmacokinetic blood samples will be 
drawn 1 to 2 hours post dose.  Approximately, four hours post-dose event related potentials 
(ERP) will be performed, followed immediately thereafter by cognitive, clinical, and side 
effect/safety (including vital sign) assessments. Optional pharmacokinetic blood sampling will be 
after the ERP evaluation approximately 4 to 6 hours following dosing. Optional Molecular 
assays (ERK/APP) will be drawn at the same time as Pk draw.  At the end of single-dose drug 
administration days, Side Effect/Safety (including vital sign) assessments will be repeated. 
These visits will take approximately 8 hours to complete. These four drug administration days 
will occur once every 2 weeks over the course of 8 weeks.  A detailed description of each 
procedure being performed on drug administration days is provided below in Table 2. 
 
 
5.3 Follow Up Phone Call Procedures 
 
We will bolster safety by adding weekly Side Effect/Safety assessments by phone midway 
between planned drug administration visits and the day following drug administration and finally 
1 month after final study drug administration. Additional visits will be provided at any time that 
signs of concern arise.  
 
 


6 
U54 Project 2 v1_12012015 
 
Table 2. Schedule of Assessments 
 
Visit 
1  
Base
line 
2  
Pre-
Dose
2  
Post-
Dose
 
Call
 
Call 
3 
Pre-
Dose
3  
Post-
Dose
 
Call
 
Call 
4  
Pre-
Dose
4 
Post-
Dose
 
Call 
 
Call 
5  
Pre-
Dose
5 
Post-
Dose
 
Call 
 
Call 
Safety 
follow-up
Call 
Day 
Day 
-7to 0
Day 
 1 
Day 
 1 
Day
2 
Day 
8 
Week 
2 
Week 
2 
Week 
2+1
Week 
3 
Week 
4 
Week 
4 
Week 
4+1 
Week 
5 
Week 
6 
Week 
6 
Week 
6+1 
Week 7
Week  
10 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
Urine drug 
screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital Signs  
X 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
 
Urine or serum 
pregnancy test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety Labs 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biomarker 
Molecular 
Assays 
(optional) 
X 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
Pk Assays 
(optional) 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
Neurophysiology 
Measures 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
 
CGI 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
 
ABC 
X 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
WJ Subtests and 
Pegboard Test 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
RBANS and 
KiTap 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
 
Side effects/ 
Safety  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X* 
Randomization 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*The Safety Follow-up visit at week 10 will include phone call with physician regarding participants overall health status and a general 
inquiry about AE’s. Day 0 and Day 1 may occur on the same day. Safety labs include comprehensive metabolic panel, ANA, and 
CBC. 
 


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U54 Project 2 v1_12012015 
 
5.4 Neurophysiology Measures 
 
For acquisition of electrophysiological) data, we will use up to 128 lead channels placed 
according to the standard 10-20 electroencephalography array (which allows for a brief 5-10 
minute setup) referenced to the CMS-DRL ground.  We will use an EGI NetAmp400 (EGI, 
Eugene, OR) with hydrocel nets.  Participants will view a standard silent movie during 
acquisition for all paradigms except talk-listen, as is common practice to facilitate participation 
with developmentally disabled subjects. Auditory stimuli will be delivered with Etymotic or 
Seinheiser earphones via a programmable sound module (Presentation Software). data will be 
acquired continuously, amplified (12,500x), and digitized (1000 Hz). Eye movements will be 
recorded (EOG) for offline data correction. 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 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.  
  
Spectral analysis: Spectra analysis will be completed on data acquired with eyes open for 5 
minutes. Data will be segmented into 1-sec epochs, detrended, and transformed into time-
frequency space in Matlab with a fast Fourier transform (FFT) algorithm, yielding 1Hz frequency 
steps.  
  
Auditory habituation ERP:  ERPs will be recorded during passive listening (150 sets of four 75 
ms broadband noise bursts) separated by an ISI of 500 ms, with inter-set interval of 4000 ms, 
for ~14 minutes of recording. Data will be filtered from .5-55 Hz (6 and 12 db/octave rolloff, 
respectively, zero-phase).  For each 4 stimulus train, trials will be blocked into 3000ms epochs, 
from 500 ms prior to stimulus onset to 500 ms after stimulus offset, baseline corrected using the 
prestimulus interval, and averaged across trials. Spatial PCA in BESA 5.3 (MEGIS Software, 
Grafelfing, Germany) will be used to define spatial topographies for ERP components in the 
grand average of all subjects. The PCA component capturing the N1 waveform will be identified 
with topography defined as the largest negative deflection between 50-200 ms post-stimulus.  
  
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. Data will be filtered from .5-120 Hz (6 and 12 
db/octave rolloff, respectively, zero-phase).  Trials will be blocked into 3500 ms epochs, from 
500 ms before to 1000 ms after stimulus offset. Representative topographies for the steady-
state response will be computed using spatial PCA in BESA 5.3 (MEGIS Software, Grafelfing, 
Germany) on the grand average of all subjects to determine the sensors used for the final 
analysis. 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.  
  
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 


8 
U54 Project 2 v1_12012015 
 
conditions. Data will be filtered 1- 50Hz and then epoched from -800 to 800 ms with respect to 
the onset of each “ah” and baseline corrected using data from the -800 to -500ms epoch 
preceding vocalization. ERP averages are generated using a robust averaging approach 
included in SPM8. Inspection of the grand average ERP waveform in preliminary studies and 
previous work with this paradigm indicates three components: N1 peak at ~100ms after speech 
onset, P2 peak at ~200ms after speech onset, and a slow negative component before speech 
onset from -300 to 0 ms.  Mean amplitudes of these components will be extracted at the anterior 
locus from which each of these three components are robust (collapsed across 26 electrodes 
surrounding FCz): N1: 80-120ms; P2: 170-210 ms; pre-speech: -300-0 ms. Correlations will be 
computed between amplitude of the pre-speech component and N1 ERP suppression 
(difference between N1 amplitude in the Talk vs. Listen conditions). 
 
5.5 Blood Sampling 
 
All subjects will have the option to provide a blood sample as noted in Table 2 to complete both 
molecular pharmacodynamic and pharmacokinetic studies. Pharmacokinetic data will be limited 
to a spot pK draw following neurophysiology evaluation approximately 4 to 6 hours following 
dosing. Molecular assays will be drawn at the same time as pK draw and at Baseline. 
 
5.5.1 Extracellular Signal Related Kinase (ERK) Activation Molecular Biomarker Assay 
ERK is a central cellular signaling kinase who dysregulation is implicated in the pathophysiology of 
FXS and in autism spectrum disorder. Our group has previously utilized ERK activation as a 
pharmacodynamics marker in FXS drug study (Erickson et al. 2011). For ERK biomarker assays, 
blood will be drawn at Baseline and 1-2 and 4-6 hours post dose at all drug dosing days.  Assays 
will be analyzed in Dr. Craig Erickson’s Molecular Translational Biomarker Lab at CCHMC. All blood 
biomarker assays will be performed blinded to study assignment. Blood will be collected and 
lymphocytes will be isolated, washed, counted, and re-suspended. Samples will be frozen until 
ERK total and phosphorylated ERK levels will be determined in duplicate using commercially 
available ELISA kits (Abcam) per manufacture instructions. All ERK measurements will be 
attempted, but are not required for study participation. 
5.5.2 Amyloid Precursor Protein (APP) Molecular Biomarker Assay 
APP and its derivatives are involved in neuronal growth, maturation, and senescence. APP 
dysregulation has been implicated in the pathophysiology of FXS and autism of unknown cause. 
Our group has published on the use of plasma APP derivative analysis in FXS clinical trials 
(Erickson et al. 2014).  For APP biomarker assays assessing plasma levels of sAPP (total), 
sAPPα, Aβ40, and Aβ42, blood will be drawn at Baseline and 1-2 and 4-6 hours post dose at all 
drug dosing days.  Assays will be analyzed in Dr. Craig Erickson’s Molecular Translational 
Biomarker Lab at CCHMC. 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), sAPP (IBL), and Aβ peptides (Wako Chemical Industries, Japan) in 
duplicate per manufacturer instructions. All APP measurements will be attempted, but are not 
required for study participation.  
5.5.3 Pharmacokinetic (PK) sampling 


9 
U54 Project 2 v1_12012015 
 
Pharmacokinetic evaluation blood samples will be drawn 1 to 2 and 4 to 6 hours post dose (max two 
PK draws per drug dosing day) on each of four drug dosing days. All PK measurements will be 
optional and are not required for study participation.   
Acamprosate, minocycline, and lovastatin concentrations will be determined with a low volume, 
validated liquid chromatography tandem mass spectrometry (LC-MS/MS) assay provided by the 
iC42 Clinical Research and Development Laboratory (Uwe Christians, MD, PhD, director), 
University of Colorado, Aurora, Colorado, USA. 
Concentration data will be analyzed by compartmental and non-compartmental 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. 
Parameter estimates generated will include Cmax, total body clearance, distribution and 
elimination half-lives, volume of distribution and the area under the curve (AUC). 
 
6.0 Study Outcome Measures 
 
 
6.1 Clinical Assessments 
 
 
CGI-I: The Clinical Global Impressions Improvement (CGI-I) will be the primary clinical 
measure completed both before and after the subject receives study medication. The CGI-I 
requires the clinician to assess how much the patient's illness has changed relative to pre-
dose, from 1 (very much improved) to 7 (very much worse).  
 
 
ABC: The recently validated Aberrant Behavior Checklist (ABC) using the 6-factor FXS 
model will be used as a secondary outcome of clinical status. This test will be completed 
only after the subject receives study medication. 
 
6.2 Cognitive Assessments 
 
To assess cognitive outcomes as a secondary aim, we selected tests with minimal practice 
effects, good reliability (weighted kappa scores ≥0.7), and a lack of floor effects in persons with 
FXS with a mental age of 3+ years (Berry-Kravis, Sumis, Kim, Lara, & Wuu, 2008). See Table 1 
below for descriptions of each of the tests. 
 
Table 1. Cognitive and neuropsychological assessment measures 
 
Measure 
Domain 
Task Description 
Estimated Time 


10 
U54 Project 2 v1_12012015 
 
WJ-III Spatial 
Relations (McGrew 
& Woodcock, 2001)
Visual Spatial 
Thinking 
Identify parts needed to form a 
complete shape. Responses can 
be oral or motoric (pointing).
05 minutes 
RBANS: List 
Learning (Randolph, 
1998) 
Immediate Memory 
A 10-item list of unrelated words 
is presented orally to the 
examinee who is then required 
to immediately recall words 
presented. 
05 minutes 
KiTAP: Test of 
Attentional 
Performance for 
Children (Knox et 
al., 2012) 
Attention and 
Inhibition 
Computerized task comprised of 
8 subtests where an examinee is 
required to push a key when a 
target stimulus is presented on 
the screen.  
 30 minutes 
Grooved Pegboard 
Test 
Sensory Motor 
Integration 
Pegs, with a key along one side, 
must be rotated to match the 
holes in the pegboard before 
they can be inserted.  
 15 minutes 
WJ-III Auditory 
Attention (McGrew 
& Woodcock, 2001) 
Auditory Processing-
Speech Sound 
Discrimination 
Identify orally presented words 
amid increasingly intense 
background noise. Examinee 
points to a picture of the word 
presented.  
05 minutes 
 
7.0 Side Effect/Safety Assessments 
 
Safety assessments will include comprehensive metabolic panel (CMP), ANA, complete blood 
count with differential (CBC with differential), urine drug screen, urine or serum pregnancy test in 
female participants of childbearing age, physical exam, medical history, concomitant 
medication review, vital signs at baseline.  
 
At dosing visits, Side Effect/Safety Assessments will include: a general inquire about AE’s, a 
detailed inquiry of common side effects of the 3 study drugs, and vital signs. In an effort to ensure 
subject safety, we plan to schedule all study drug dosing visits at the CTRC.  Phone calls to the 
patient/caregiver assessing for adverse effects will be completed 1 day following each dose. The 
Safety Follow-up visit at week 10 will include phone call with physician regarding participants 
overall health status and a general inquiry about AE’s. 
 
Additional study safety oversight will be provided by Dr. Sergio Delgado, Outpatient Clinical 
Director and Professor with the division of child and adolescent psychiatry at Cincinnati 
Children’s Hospital will serve as independent study medical monitors for the study. The monitors 
will review recruitment and adverse events every 6 months and report assessment to PI.  
 
8.0 Research Methods 
 
  
8.1 Criteria for Inclusion of Subjects: 
 
 
Subjects ages 15-55, with FXS who completed the study entitled “Mechanisms 
and brain circuits underlying fragile X syndrome (IRB # 2015-8425). FXS is 


11 
U54 Project 2 v1_12012015 
 
defined as full FMR1 mutations (>200 CGG repeats) confirmed by genetic 
testing. 
 
 
General good health as determined by physical exam, medical history and 
laboratory work up. 
 
 
 
 
8.2 Criteria for Exclusion of Subjects: 
 
 
Subjects with a history of intolerance to acamprosate, lovastatin, or minocycline 
will be excluded.  
 
Subjects will also be excluded if they have taken any investigational drug within 
3 months, have a history of substance abuse or dependence within 6 months, or 
significant psychiatric or CNS neurological disease unrelated to FXS. 
 
Uncontrolled seizures impact EEG data as do anticonvulsants, barbiturates, 
lithium and benzodiazepines and are exclusions (within 5 half-lives). Those 
taking other psychiatric medications must be on stable doses for 4 weeks before 
any testing.  
 
For female subjects of child bearing potential, a positive urine pregnancy test. 
 
Potential subjects with a creatinine clearance < 50 mL/min will be excluded. 
 
Identified medical issues, inability to tolerate study procedures or study 
drug per the discretion of the Principal Investigator. 
 
 
 
8.3 Vulnerable Populations: 
 
 Inclusion of Children in Research: 
One special class of subjects that will be involved in this study is children.  FXS is a genetic 
disorder that is a cause of intellectual disability and neuropsychiatric disorders.  A central aim of 
this study is to examine the effects to investigate single-dose drug effects on event-related 
potential (ERP) biomarkers and behavior in humans with FXS. This can only be done by 
studying affected individuals across the age span.  We have chosen ages 15-55 to represent a 
broad developmental spectrum. The upper age of 55 is chosen because of variable and 
common age-related changes in behavior, sensory function and brain function beyond that age. 
The age 15 is chosen for the low end of the age range because of the level of cooperation 
required to complete procedures.   
 
Persons with Cognitive Impairment or other limited decision making capacity  
To be inclusive and most generalizable in our study findings, and because some elements of 
our protocol can be completed with minimal cognitive requirements such as ERP monitoring of 
cortical responses to tones, we will recruit any study eligible patient with Fragile X Syndrome 
regardless of level of cognitive disability into as many study components as they can 
complete. We will recruit all FXS patients who we believe can complete study procedures and 
use IQ scores to describe study participants in research reports.  
 
Inclusion of Women and Minorities: 
No person shall be excluded from participation in this research study on the basis of gender 
alone. However, FXS occurs at a higher rate in males than in females.  Therefore, it is 
anticipated that the largest number of proband participants will be male.  Every effort will be 


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U54 Project 2 v1_12012015 
 
made to recruit and include minorities among the participants in this research study.  In regard 
to gender and minority representation, our research cohort will reflect consecutive referrals to 
our clinic meeting the inclusion-exclusion criteria of the study.  Recruitment is expected to be 
30% minority because of the population our clinics serve. 
 
9.0 Sources of Research Material: 
 
In addition to sociodemographic information, we may collect sensitive clinical information 
including information about medical history, drug use, psychiatric symptoms and developmental 
history during individual testing sessions and via questionnaires completed during pre-screening 
or testing sessions. This information can be collected as part of structured and semi-structured 
interviews, self-report and caregiver questionnaires, and clinical tests of behavioral functioning.   
 
Neuropsychological and electrophysiological data may be acquired during individual testing 
sessions. Medical records may be obtained after obtaining a signed permission for release of 
records.   
 
All information collected is for research purposes only. If some assessments were recently done 
in the clinic by our faculty before recruitment, then we will use the existing clinical data to reduce 
patient burden; but we expect this overlap to be rare. All material will be protected by limited 
access and stored under a unique ID number.   
 
10.0 Recruitment Methods and Consenting Process: 
 
10.1 Recruitment 
 
Recruitment for the study will be conducted by the Cincinnati Fragile X Research and Treatment 
Center Subjects ages 15-55, will be recruited from participants who have completed the study 
entitled “Mechanisms and brain circuits underlying fragile X syndrome (IRB #2015-8425). 
 
10.2 Informed Consent Process 
 
Screening and consent procedures for the study will be completed prior to enrollment. 
Participants (if they have decisional capacity) or legally authorized representatives together with 
participants initially will receive written materials and be given an oral explanation of the study. 
Informed consent procedures will be conducted by research staff or investigators. In addition, 
members of the protocol team will be available to address any questions for participants or 
families.   
 
Each participant must give informed consent or assent to participate when possible. Given the 
developmental disability associated with FXS, particularly in males, some subjects themselves 
will not be able to provide assent or consent. Consent also will be provided by the guardian of 
minors and from adult subjects who are not their own legal guardian.  
 
Participants (and if they have one, their legally authorized representative) will be informed 
during their initial consent, prior to the initiation of the pharmacologic study, and on several 
additional occasions throughout their experience in the study that they may elect to not to 
participate at any time.  
 
Consent and assent will be collected using the procedures outlined above. This will take place 
after initial screening but before any formal assessments and experimental procedures begin. 


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U54 Project 2 v1_12012015 
 
Both written and oral information will be provided to participants and legally authorized 
representatives. Refusal to participate in the study will not limit their ability to seek services at 
Cincinnati Children’s Hospital Medical Center. One signed and witnessed copy of the consent 
and assent forms will be stored in secure storage and the participant and legally authorized 
representative will each be given a copy of the signed forms, as is appropriate. Participants will 
receive payment for taking part in this study.  
 
Some individuals may be interested in study participation but live far away from CCHMC.  In 
these circumstances, the consent forms will be mailed to potential participants after they have 
initiated contact with our research program. Our research team will arrange for a conversation 
with the potential participant by phone after the documents have been received. During the 
phone conversation, our research team member will review the consent form (i.e., study 
purpose, voluntary participation, study procedures, risks and benefits, and confidentiality) and 
answer any questions.  Then, the participant will sign the documents and mail the documents 
back to the researcher.  Once the researcher at our Center receives them, he/she will sign the 
consent forms and return copies of the documents to the participant for their records and place 
the originals in a secure area.  
 
 
If a subject turns 18 years-old while enrolled in the study, the parent(s)/legal guardian(s) will be 
asked to provide appropriate documentation indicating that they are actively seeking legal 
guardianship. Copies of documentation will be filed in the subject’s study chart. If parent(s)/legal 
guardian(s) are not pursuing legal guardianship when the subject turns 18 years-old, then the 
subject will need to demonstrate the capacity to provide informed consent and voluntarily sign 
the informed consent document in order to continue participation in the study. 
 
11.0. Potential Risks: 
 
11.1 Study Drugs 
 
All participants will receive single doses of 3 FDA-approved drugs (acamprosate, lovastatin, 
minocycline) that have proven benign safety profiles during chronic treatment.  Minocycline and 
acamprosate were well-tolerated in studies of humans with FXS (Erickson et al., 2013; Leigh 
et al., 2013).  Lovastatin, not tested in humans with FXS, is one of the most widely prescribed 
medicines worldwide. At the doses proposed for this study the incidence of adverse effects is low. 
Furthermore the administration of a single dose will limit the risk for side effects that might emerge 
with longer term treatment. The study PI (Dr. Erickson) has experience in the use of 
pharmacotherapy in persons with FXS. All participants will have a careful medical examination 
and detailed monitoring of side effects in the Fragile X clinic.  
 
11.1.1 Acamprosate  
 
Common side effects (more than 1 in 100 patients): 
 
headache, abdominal pain, back pain, infection, flu syndrome, chest pain, chills, suicide 
attempt 
 
palpitations (feelings of having rapid, fluttering or pounding heartbeats), fainting 
 
vomiting, upset stomach, constipation, increased appetite 
 
swelling of the legs and feet, weight gain 
 
muscle pain, joint pain  
 
sleepiness,  decreased interest in sex, memory loss, thinking abnormal, shaking, 


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U54 Project 2 v1_12012015 
 
vasodilatation, high blood pressure 
 
stuffy nose, cough increased, shortness of breath, sore throat, bronchitis (lung infection) 
 
rash 
 
abnormal vision, change in sense of taste 
 
difficulty in getting or keeping an erection 
 
Less common side effects (between 1 in 1000 patients and 1 in 100 patients): 
 
fever, intentional overdose, bodily discomfort, allergic reaction, abscess, neck pain, 
hernia (part of an internal organ or tissue that bulges through a weak area of muscle), 
intentional self-injury  
 
low blood pressure, fast or irregular heart rate, bleeding, angina pectoris (chest 
discomfort due to poor blood flow through the blood vessels in the heart), migraine, 
varicose vein, heart attack, phlebitis (vein inflammation),  a drop in blood pressure when 
getting up from a seated position or lying down 
 
abnormal liver function tests, stomach flu, gastritis (inflammation of the stomach lining), 
difficulty with swallowing , burping, bleeding of the stomach or intestines, pancreatitis 
(inflammation of the pancreas), rectal bleeding, liver cirrhosis (scarring of the liver), 
inflammation of the esophagus, vomiting blood, nausea and vomiting, hepatitis 
(inflammation of the liver)  
 
anemia (low red blood cell count), bruising, eosinophilia (higher than normal level of a 
type of white blood cells, which could be a sign of an allergic-type drug reaction), 
lymphocytosis (an increase in a type of white blood cells, which could be a sign of an 
allergic-type drug reaction), thrombocytopenia (abnormally low amount of platelets, 
which can increase risk of bleeding)  
 
weight loss, high blood sugar and diabetes, (increase in chemicals made by the liver (a 
sign of liver injury), , hyperuricemia (high level of uric acid), gout (type of arthritis caused 
by too much uric acid in blood), thirst,  low vitamin levels, bilirubinemia (presence of 
excess bilirubin in the blood, which can be sign of liver dysfunction)  
 
leg cramps 
 
convulsion, confusion, increased interest in sex, dizziness, withdrawal syndrome, 
general lack of interest, suicidal thoughts, neuralgia (nerve pain), hostility, agitation (a 
state of marked excitement or restlessness), unrealistic thoughts, abnormal dreams, 
hallucinations, hypesthesia (a reduced sense of touch or sensation) 
 
asthma, nosebleed, pneumonia;   
 
acne, eczema (skin irritation condition), hair loss, maculopapular rash (flat discolored 
area of the skin covered with small bumps), dry skin, hives, exfoliative dermatitis (severe 
inflammation of the entire skin surface, vesiculobullous rash (a rash like a blister),  
 
ringing in the ears, lazy eye, deafness;  
 
metrorrhagia (bleeding from the uterus between menstrual periods), frequent urination, 
urinary tract infection, sexual dysfunction, loss of bladder control, vaginitis (inflammation 
of the vagina) 
 
Rare side effects (less than 1 in 1000 patients): 
 
ascites (build-up of fluid inside the abdomen), face swelling, photosensitivity reaction 
(sensitivity to sunlight), abdomen enlarged, sudden death 
 
heart failure, closing of an artery of the intestines, cardiomyopathy (weakening of the 
heart muscle), deep thrombophlebitis (a blood clot blocking one or more of your veins), 
shock (dangerously low blood pressure) 
 
melena (black stools from bleeding), stomach ulcer, inflammation of the gallbladder, 
inflammation of the colon, duodenal ulcer (an ulcer in the first part of the small intestine), 


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U54 Project 2 v1_12012015 
 
mouth ulceration, live cancer 
 
goiter (enlargement of the thyroid gland), hypothyroidism (underactive thyroid) 
 
leukopenia (low white blood cell count, which could increase the risk of infection), 
swollen or enlarged lymph nodes, monocytosis (increase in number of particular type of 
white blood cells, which could be a sign of an allergic-type drug reaction). 
 
alkaline phosphatase increased (a possible sign of liver, bone or gall bladder injury), 
creatinine increased (a possible sign of kidney injury), low sodium, lactic dehydrogenase 
increased (a possible sign of tissue injury) 
 
rheumatoid arthritis, muscle injury 
 
alcohol craving, psychosis, excessive abnormal movements, twitching, 
depersonalization (periods of feeling disconnected or detached from one's body and 
thoughts), increased salivation, paranoid reaction, torticollis (a muscle spasm of the neck 
in which the head is tipped to one side)), brain disturbance resulting in confusion, manic 
reaction (elevated, expansive, or irritable mood) 
 
laryngismus (spasm of the throat, which can cause difficulty breathing), blood clot of the 
vessels in the lung 
 
psoriasis (a skin condition that causes skin redness and irritation) 
 
eye inflammation, double vision, sensitivity of eyes to light 
 
kidney stone, abnormal ejaculation, blood in urine, an abnormally heavy and prolonged 
menstrual period, a need to get up at night to urinate, excessive urination, a sudden urge 
to urinate 
 
11.1.2 Lovastatin  
 
Common side effects (more than 1 in 100 patients): 
 
weakness 
 
abdominal pain, constipation, diarrhea, upset stomach, flatulence, nausea 
 
muscle cramps, muscle pain 
 
dizziness, headache 
 
rash 
 
blurred vision 
 
Less common side effects (between 1 in 1000 patients and 1 in 100 patients): 
 
chest pain 
 
acid regurgitation (“heartburn”), dry mouth, vomiting 
 
leg pain, shoulder pain, joint pain 
 
insomnia (difficulty falling or staying asleep), paresthesia (numbness and tingling) 
 
hair loss, itching 
 
eye irritation 
 
Rare side effects (less than 1 in 1000 patients): 
Myopathy/Rhabdomyolysis 
Lovastatin, like other “statin” cholesterol-lowering medicines, can cause myopathy (muscle 
injury) which shows as muscle pain, tenderness or weakness with an increase of creatine 
kinase (“CK” a protein leaked from the muscles when injured) to ten times the normal limit. 
Myopathy sometimes takes the form of rhabdomyolysis (high levels of muscle proteins leaked to 
the bloodstream) with or without acute kidney failure, and rare fatalities have occurred. The risk 
of myopathy is increased by high levels of “statin” drug inhibitory activity in plasma. 


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U54 Project 2 v1_12012015 
 
As with other “statin” cholesterol-lowering medicines, the risk of 
myopathy/rhabdomyolysis is dose related. In a clinical study (EXCEL) in which patients were 
carefully monitored and some interacting drugs were excluded, there was one case of myopathy 
among 4933 patients randomized to lovastatin 20-40 mg daily for 48 weeks, and 4 among 1649 
patients randomized to 80 mg daily. 
There have been rare reports of immune-mediated necrotizing myopathy (IMNM), an 
autoimmune myopathy, associated with statin use. IMNM is characterized by: proximal muscle 
weakness and elevated serum creatine kinase, which persist despite discontinuation of statin 
treatment; muscle biopsy showing necrotizing myopathy without significant inflammation; 
improvement with immunosuppressive agents. 
All patients starting therapy with lovastatin, or whose dose of lovastatin is being 
increased, should be advised of the risk of myopathy and told to report promptly any 
unexplained muscle pain, tenderness or weakness particularly if accompanied by 
malaise or fever or if muscle signs and symptoms persist after discontinuing lovastatin. 
Lovastatin therapy should be discontinued immediately if myopathy is diagnosed or 
suspected. In most cases, muscle symptoms and CK increases resolved when treatment was 
promptly discontinued. Periodic CK determinations may be considered in patients starting 
therapy with lovastatin or whose dose is being increased, but there is no assurance that such 
monitoring will prevent myopathy. 
Many of the patients who have developed rhabdomyolysis (high levels of muscle proteins 
leaked to the bloodstream) on therapy with lovastatin have had complicated medical histories, 
including kidney disease usually as a consequence of long-standing diabetes. Such patients 
should have closer monitoring. Lovastatin therapy should be discontinued if markedly elevated 
CPK levels occur or myopathy is diagnosed or suspected. Lovastatin therapy should also be 
temporarily withheld in any patient experiencing an acute or serious condition predisposing to 
the development of kidney failure secondary to rhabdomyolysis, e.g., sepsis (infection in the 
bloodstream); hypotension (low blood pressure); major surgery; trauma; severe metabolic, 
endocrine, or electrolyte disorders; or uncontrolled epilepsy. 
 
11.1.3 Minocycline ER  
 
Common side effects (more than 1 in 100 patients): 
 
headache 
 
fatigue 
 
dizziness 
 
itching 
 
bodily discomfort 
 
mood change 
 
sleepiness 
 
hives 
 
tinnitus (ringing in the ears) 
 
joint pain 
 
dizziness 
 
dry mouth 
 
muscle pain 
 
Less Common or Rare (occurrence rates not further specified in PI), but potentially serious side 
effects 


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U54 Project 2 v1_12012015 
 
Teratogenic Effects 
Minocycline can cause fetal harm when taken by women during pregnancy.  Minocycline should 
not be used during pregnancy or by individuals of either gender who are attempting to conceive 
a child. 
The use of minocycline during tooth development (last half of pregnancy, infancy and childhood 
to the age of 8 years) may cause permanent discoloration of the teeth (yellow-gray-brown).  
This adverse reaction is more common during long-term use of the drug but has been observed 
following repeated short-term courses. Enamel hypoplasia (a defect of the teeth in which the 
enamel is hard but thin and deficient in amount, which can cause abnormally shaped teeth) has 
also been reported. Minocycline, therefore, should not be used during tooth development.   
 
All tetracyclines (the class of drug that minocycline is in) form a stable calcium complex in any 
bone-forming tissue. A decrease in fibula (a bone in the leg) growth rate has been observed in 
premature human infants given oral tetracycline in doses of 25 mg/kg every 6 hours. This 
reaction was shown to be reversible when the drug was discontinued. 
 
Results of animal studies indicate that tetracyclines (like minocycline) cross the placenta, are 
found in fetal tissues, and can cause retardation of skeletal development on the developing 
fetus. Evidence of embryotoxicity has been noted in animals treated early in pregnancy. 
 
Pseudomembranous Colitis 
Note, the risk of pseudomembranous colitis typically occurs only with repeated dosing of 
antibiotics over days or weeks (just a single dose of minocycline will be taken in this 
study, thus the risk for pseudomembranous colitis is exceedingly low). A description of 
pseudomembranous colitis, as usually only seen with repeated dosing follows.  
Clostridium difficile associated diarrhea (CDAD) has been reported with nearly all antibacterial 
agents, including minocycline, and may range in severity from mild diarrhea to fatal colitis. 
Treatment with antibacterial agents alters the normal flora of the colon leading to overgrowth of 
C. difficile. C. difficile produces toxins A and B which contribute to the development of CDAD. 
Hypertoxin producing strains of C. difficile cause increased morbidity and mortality, as these 
infections can be refractory to antimicrobial therapy and may require colectomy. CDAD must be 
considered in all patients who present with diarrhea following antibiotic use. Careful medical 
history is necessary since CDAD has been reported to occur over two months after the 
administration of antibacterial agents. If CDAD is suspected or confirmed, ongoing antibiotic use 
not directed against C. difficile may need to be discontinued. Appropriate fluid and electrolyte 
management, protein supplementation, antibiotic treatment of C. difficile, and surgical 
evaluation should be instituted as clinically indicated. 
 
Hepatotoxicity 
Post-marketing cases of serious liver injury, including irreversible drug-induced hepatitis and 
fulminant hepatic failure (sometimes fatal) have been reported with minocycline use in the 
treatment of acne. 
 
Metabolic Effects 
The anti-anabolic action of the tetracyclines (like minocycline) may cause an increase in blood 
urea nitrogen (BUN).  A BUN test measures the amount of nitrogen in blood that comes from 
the waste product urea. Urea is made when protein is broken down in the body. While an 
increase in BUN is not a problem in those with normal kidney function, in patients with 
significantly impaired kidney function, higher serum levels of minocycline-class drugs may lead 
to azotemia (high levels of nitrogen-containing waste products in the blood stream), 


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U54 Project 2 v1_12012015 
 
hyperphosphatemia (high levels of phosphate), and acidosis (which can disturb normal 
functioning of the body).  
 
Central Nervous System Effects 
Central nervous system side effects including light-headedness, dizziness or vertigo have been 
reported with minocycline therapy. Patients who experience these symptoms should be 
cautioned about driving vehicles or using hazardous machinery while on minocycline therapy. 
These symptoms may disappear during therapy and usually rapidly disappear when the drug is 
discontinued. 
 
Benign Intracranial Hypertension 
Pseudotumor cerebri (benign intracranial hypertension/pressure) in adults and adolescents has 
been associated with the use of tetracyclines like minocycline. Minocycline has been reported to 
cause or precipitate pseudotumor cerebri, the hallmark of which is papilledema. Clinical 
manifestations include headache and blurred vision. Bulging fontanels have been associated 
with the use of tetracyclines in infants. Although signs and symptoms of pseudotumor cerebri 
resolve after discontinuation of treatment, the possibility for permanent problems such as visual 
loss that may be permanent or severe exists. Patients should be questioned for visual 
disturbances prior to initiation of treatment with tetracyclines. If visual disturbance occurs during 
treatment, patients should be checked for papilledema.  
 
Autoimmune Syndromes 
Tetracyclines like minocycline have been associated with the development of autoimmune 
syndromes (a condition that occurs when the immune system mistakenly attacks and destroys 
healthy body tissue). The long-term use of minocycline in the treatment of acne has been 
associated with drug-induced lupus-like syndrome (condition that mimics the symptoms of 
systemic lupus erythematosus, or SLE, but is not actually lupus).  Lupus-like syndrome can 
cause rash (especially of the face), skin sensitivity to sunlight, fatigue, joint pain and arthritis, 
and overall feeling of sickness. Long-term use of minocycline can also cause autoimmune 
hepatitis (autoimmune reaction to the liver) and vasculitis (autoimmune reaction to the blood 
vessels). Rare cases of serum sickness have presented shortly after minocycline use, including 
symptoms of fever, rash, arthralgia, and malaise.  
 
Photosensitivity 
Photosensitivity manifested by an exaggerated sunburn reaction has been observed in some 
individuals taking tetracyclines. This has been reported rarely with minocycline.  
 
Serious Skin/Hypersensitivity (allergic) Reaction 
Cases of anaphylaxis (severe, whole-body allergic reaction), serious skin reactions, and drug 
rash with eosinophilia (type of white blood cell) and systemic (whole-body) symptoms (DRESS) 
syndrome have been reported post marketing with minocycline use in patients with acne. 
DRESS syndrome consists of skin reaction (which is sometimes severe), eosinophilia, and one 
or more of the following: organ complications such as inflammation of the liver, lungs, kidneys or 
heart.  Fever and swelling of the lymph nodes may be present. In some cases, death has been 
reported.  
 
Tissue Hyperpigmentation 
Note, this side effect is exceedingly unlikely to occur because it tends to occur only with 
prolong taking of minocycline over days, weeks, or months (and just a single dose of 
minocycline will be taken in this study). Tetracycline-class antibiotics like minocycline are 
known to cause hyperpigmentation (darkening pigment). Minocycline may induce 


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U54 Project 2 v1_12012015 
 
hyperpigmentation in many organs, including nails, bone, skin, eyes, thyroid, visceral tissue, 
oral cavity (teeth, linings of the mouth, bone), sclerae (“whites” of the eye) and heart valves. 
Skin and oral pigmentation has been reported to occur independently of time or amount of drug 
administration, whereas other tissue pigmentation has been reported to occur upon prolonged 
administration. Skin pigmentation includes diffuse pigmentation as well as over sites of scars or 
injury. 
 
Superinfection 
As with other antibiotic preparations, use of minocycline may result in overgrowth 
of non-susceptible organisms, including fungi.  
 
11.2 Cognitive Testing 
 
There are minimal risks anticipated to individuals participating in observational or clinical 
behavioral assessments. In the rare case that an individual becomes overly fatigued or 
distressed during assessment sessions, the session will be discontinued and rescheduled.  
Evaluation procedures, when appropriate, are designed in a way that is compatible with the 
attention span of a person with an intellectual disability, greater activity level, and need for 
access to his or her parents. In addition, specialized procedures for children with FXS (e.g., 
reducing language demands, providing praise for task attention and attempts, using work-
reward routines) will be implemented. Three clinical psychology investigators with extensive 
experience working clinically with developmentally disabled patients are investigators in the 
study. 
 
11.3 Neurophysiology Procedures 
 
There are minimal risks associated with participation in the neurophysiology part of the study. 
The auditory stimuli presented are relatively benign, and have been well tolerated in our pilot 
studies. We are sensitive to monitoring participant distress associated with sensory paradigms, 
which we have found more commonly in participants younger than 12 years of age. The most 
common risk associated with these tasks is boredom. In rare circumstances the electrode gel 
used to place the recording electrodes to the scalp can cause minor irritation to the skin, which 
resolves rapidly with no permanent effects.  
 
11.4 Pregnancy and Protection against Risk 
 
Females of childbearing age (as determined by report regarding age of menarche or 
menopause) enrolling in the pharmacologic study will be required to take a urine pregnancy test 
before study procedures. A positive test will exclude them from participating in this portion of the 
study. The PI or his designee will discuss with the subject in private the results of any drug 
screen and/or pregnancy test and if positive, that they will no longer be able to participate in the 
study. Consent forms will make clear that this information will be shared with parents of minors. 
A subject who tests positive will be referred to appropriate medical care. 
 
11.5 Risk of Discrimination 
 
There is a theoretical risk for discrimination towards individuals who are at risk for a medical 
disorder or have a medical disorder in their family. Potential discrimination may include barriers 
to insurability, employability, or other unidentified adverse effects. Extensive efforts are made to 
protect all research subjects from prejudice, discrimination, or uses of this information that will 
adversely affect them.  


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U54 Project 2 v1_12012015 
 
 
11.6 
Venipuncture Procedures 
 
The risks of venipuncture are modest and include mild discomfort, infection, bleeding, and 
fainting.  Standard methods and precautions will be used to protect the puncture site from 
bleeding and infection.  The Research Coordinator or Research Assistant will be familiar with 
the subjects and will accompany the subject and their parents to the blood drawing setting.  
Parents are encouraged to remain with the subject at all times.  To minimize the subject’s 
anxiety and phobic reactions, we utilize Child Life personnel when needed and available.  We 
also suggest that the parents reassure the subject concerning their safety.  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. 
 
12.0 Procedures for Protecting Against and Minimizing Potential Risks 
 
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. There will be repeated monitoring of behavior 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.  All of the research data is kept in locked files to ensure 
confidentiality. The other procedures to ensure confidentiality follow the regulations and policies 
of the Medical Center. 
 
All SAEs will be reviewed in detail by the PIs and Independent Safety Monitor.  If the stopping 
rule is met, the study will close to enrollment for interim analysis of safety and efficacy data and 
the Safety Monitor would provide a recommendation to the PI regarding continuation of the trial. 
 
13.0. Potential Benefits: 
 
Subjects may or may not benefit directly from study participation. The possibility of contributing 
to the discovery of treatments for FXS may benefit all affected individuals and their families. 
The following knowledge can be gained from this research: a more detailed understanding of 
auditory processing abnormalities in FXS, a better understanding of the pathophysiology of 
FXS, new perspectives on pharmacologic mechanisms in FXS, and insight into possible 
biomarkers that can be used to track drug effects in FXS. 
 
The potential gain in knowledge can change our clinical understanding and treatment of FXS 
and related disorders. This could lead to more effective treatments and possibly earlier 
identification and personalized treatment of at-risk children. Given the severity of impairments 
associated with FXS, this could dramatically impact the lives of affected individuals and their 
families. Given the potential for gain, the relatively modest risks are reasonable for the study 
participants, most of whom stand to receive modest personal gains by participating in the 
research.  
 
13.1 Risk vs Benefit Ratio 
 


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U54 Project 2 v1_12012015 
 
The subjects will be exposed to the risks of blood sampling and the potential side effects of 
acamprosate, lovastatin, or minocycline. 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. 
 
14.0. Subject Safety and Data Monitoring: 
 
Dr. Sergio Delgado, Outpatient Clinical Director with the division of child and adolescent 
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.  
 
All problems (i.e. adverse events, unanticipated events, etc.) will be characterized by the 
following:  
 
 
Severity = Mild, Moderate or Severe  
 
Relatedness = Definite, Probable, Possible, Unlikely or Unrelated  
 
Expectedness = Expected or Not Expected  
 
A stopping rule for excessive toxicity is in place in the event of ≥ 2 SAEs considered possibly, 
probably or definitely related to treatment on study.  All SAEs will be reviewed in detail by the 
PIs and Independent Safety Monitor.  If the stopping rule is met, the study will close to 
enrollment for interim analysis of safety and efficacy data and the Safety Monitor would provide 
a recommendation to the PI regarding continuation of the trial. 
 
Events will be reported consistent with Institutional IRB reporting policies. An aggregate listing 
of all safety events will be reported to the IRB in summary form at the time of continuing review. 
These reports will also be provided to any applicable regulatory agencies per applicable 
regulatory agency requirements. Data (such as lab values, vital signs, and outcome measure 
data) will be entered from source documents to Case Report Forms by the study coordinator. 
The study coordinator will review case report form entries for accuracy by comparison with the 
source documents.  Research records and source documents will be maintained in a research 
chart and stored in the investigator's locked file cabinet. Information will be kept in a secure 
database that is password protected. Records will be kept secure, and individually identifiable 
information will not be included in any reports or data sets.  
 
All subjects will be assured that data will be kept confidential. Data will be identified only by a 
unique identification number and not by the subject’s name. Paper data will be stored in a 


22 
U54 Project 2 v1_12012015 
 
locked office in a locked cabinet to which only staff affiliated with the project will have access.  
Any discrepancies in the maintenance of confidentiality or other irregularities involving the data 
will be reported to the PI. Any such events will be documented and reviewed by the PI and 
reported to the IRB within a timeframe set out by guidelines set forth by the CCHMC IRB.  
 
14.1 Adverse Events 
 
Any adverse events--whether due to assessment, medication or other aspects of the study--will 
be reported to the PI. Adverse events will be documented and reviewed by the PI within 24 
hours of receipt. Serious/unanticipated events will be reported to the IRB within 48 hours by 
phone, email, or fax. All adverse events will be compiled, and reported in summary form to the 
IRB, on an annual basis and at the conclusion of the study.  
 
15.0 Procedures to Maintain Confidentiality: 
 
Unique numeric identifiers that do not include any PHI will be assigned. All data will be 
maintained either in locked storage facilities with limited access or in secure, electronic facilities. 
Data transmitted electronically will be protected by encryption over a very secure network that 
limits access only to CCHMC faculty and staff who are reviewed and specifically cleared for 
access to the password protected, secure network. The data will be stored in protected hard 
drives in a server system that is protected by two firewalls, one at the hospital level and a 
second at the Departmental level. De-identified data will be shared with the national database 
for ASD research (NDAR) as may be required as part of NIH funding via this mechanism, 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 (a more detailed description of data sharing procedures is below). 
 
15.1 Data Management  
 
Data will be collected on paper source documents, CRFs, and/or in the electronic medical record 
and will be entered by study staff in a password protected, encrypted REDCap64 database. 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. 
 
16.0 Biostatistics: 
 
The sample size analysis was based on a primary outcome of N1 amplitude. This is the best 
established ERP abnormality and the only validated ERP drug outcome measure in FXS 
patients. The estimate of effect size was drawn from a double-blind trial of minocycline vs. 
placebo in FXS on N1 amplitudes (Schneider et al., 2013). Assuming that omnibus drug effect 
size of d~0.68, and no carryover or period effects, then 36 patients (9 in each of 4 drug 
sequences) provides power of 0.80 with α of 0.05 (two-tailed) ("Power Analysis and Sample 


23 
U54 Project 2 v1_12012015 
 
Size (PASS)," 2008). Patients will be recruited from the STU 052013-043 population until 36 
patients have completed the pharmacologic study.   
 
A 4-drug, 4-period, 4-sequence balanced-crossover design (Williams, 1989) will be used for the 
proposed pharmacologic study. The primary ERP outcome will be N1 amplitude. Secondary 
outcomes will be N1 habituation, auditory evoked gamma power in the AM modulated chirps, 
and N1 amplitude reduction during the talk/listen task, and clinical and cognitive outcomes.   
 
The analysis will be a linear mixed model analysis of repeated measures. A separate mixed 
model analysis will be conducted on each outcome. If deemed necessary, the outcomes will be 
appropriately transformed to obtain a more normal distribution. The fixed effects of drug, drug 
sequence, and drug period will be examined. Potential carryover effects of drug will be 
estimated in the mixed model and, if significant, the drug effects will be adjusted. Patients will be 
nested within drug sequence and accounted for as a random effect in the mixed model. The 
intercept will be included as a random effect (if it improves model fit). Sex, age, and IQ will be 
considered as covariates in the respective mixed models, as will baseline clinical and cognitive 
data for analyses of those parameters. Restricted maximum likelihood estimation, Type 3 tests 
of fixed effects, and generalized least-squares will be used, with the Kenward-Roger correction 
(Kenward & Roger, 1997) applied to the appropriate and best fitting covariance structure. The 
robust covariance matrix estimator will also be considered. The six pairwise differences of least 
squares means among the drug effects will be estimated, and these comparisons will be carried 
out using the Tukey multiple comparison procedure. Hedges' ĝ will be calculated and 
interpreted as the effect size estimator for the pairwise contrasts between the drug effects.  
Findings will be examined in relation to Fmr1 gene expansion and methylation. Baseline and 
changes in neurophysiologic measures will be examined for relationships to cognitive and 
clinical outcomes as an exploratory evaluation of these variables as potential biomarkers of drug 
effects in FXS. The mixed model procedures of PROC MIXED in SAS software (SAS Institute, 
Inc., Cary, NC) will be used to conduct the mixed model analysis making use of all available 
data from all participants to provide a robust mechanism for handling data that are assumed 
missing at random (Little, 1995) (Mallinckrodt et al., 2003) (Rubin, 1976) (Wolfinger & Chang, 
1995). The level of significance for all tests will be set at α = .05 (two-tailed) and, to address 
multiple testing, p-values will be adjusted using the False Discovery Rate (Benjamini & 
Hochberg, 1995). 
 
17.0 Subject Cost and Payment:  
 
Subjects will be paid $100 on a ClinCard for their time and transportation at the end of each 
fully completed visit.  Other than cost of travel to and from visits and potential time away from 
work for caregivers, we anticipate no other costs for subjects associated with study 
participation. 
 
 
 
 
 
 
 
 
References: 


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Benjamini, Y, & Hochberg, Y. (1995). Controlling the flase discovery rate: a practical and 
powerful approach to multiple testing. Journal of the Royal Statistical Society, 57, 289-
300.  
Berry-Kravis, E., Hessl, D., Coffey, S., Hervey, C., Schneider, A., Yuhas, J., . . . Hagerman, R. 
(2009). A pilot open label, single dose trial of fenobam in adults with fragile X syndrome. 
J Med Genet, 46(4), 266-271. doi: 10.1136/jmg.2008.063701 
Berry-Kravis, E., Sumis, A., Kim, O. K., Lara, R., & Wuu, J. (2008). Characterization of potential 
outcome measures for future clinical trials in fragile X syndrome. J Autism Dev Disord, 
38(9), 1751-1757. doi: 10.1007/s10803-008-0564-8 
Bilousova, T. V., Dansie, L., Ngo, M., Aye, J., Charles, J. R., Ethell, D. W., & Ethell, I. M. (2009). 
Minocycline promotes dendritic spine maturation and improves behavioural performance 
in the fragile X mouse model. J Med Genet, 46(2), 94-102. doi: 
10.1136/jmg.2008.061796 
Boeijinga, P. H., Parot, P., Soufflet, L., Landron, F., Danel, T., Gendre, I., . . . Luthringer, R. 
(2004). Pharmacodynamic effects of acamprosate on markers of cerebral function in 
alcohol-dependent subjects administered as pretreatment and during alcohol 
abstinence. Neuropsychobiology, 50(1), 71-77. doi: 10.1159/000077944 
Budimirovic, D. B., & Kaufmann, W. E. (2011). What can we learn about autism from studying 
fragile X syndrome? Dev Neurosci, 33(5), 379-394. doi: 10.1159/000330213 
Crawford, D. C., Acuna, J. M., & Sherman, S. L. (2001). FMR1 and the fragile X syndrome: 
human genome epidemiology review. Genet Med, 3(5), 359-371. doi: 10.109700125817-
200109000-00006 
Dansie, L. E., Phommahaxay, K., Okusanya, A. G., Uwadia, J., Huang, M., Rotschafer, S. E., . . 
. Ethell, I. M. (2013). Long-lasting effects of minocycline on behavior in young but not 
adult Fragile X mice. Neuroscience, 246, 186-198. doi: 
10.1016/j.neuroscience.2013.04.058 
Erickson, C. A., Wink, L. K., Ray, B., Early, M. C., Stiegelmeyer, E., Mathieu-Frasier, L., . . . 
McDougle, C. J. (2013). Impact of acamprosate on behavior and brain-derived 
neurotrophic factor: an open-label study in youth with fragile X syndrome. 
Psychopharmacology (Berl), 228(1), 75-84. doi: 10.1007/s00213-013-3022-z 
Erickson CA, Weng N, Weiler IJ, Greenough WT, Stigler KA, Wink LK, McDougle 
 
CJ. Open-label riluzole in fragile X syndrome. Brain Res. 2011 Mar 
 
22;1380:264-70. doi: 10.1016/j.brainres.2010.10.108. Epub 2010 Nov 5. PubMed 
 
PMID: 21059347. 
Erickson CA, Ray B, Maloney B, Wink LK, Bowers K, Schaefer TL, McDougle CJ, 
 
Sokol DK, Lahiri DK. Impact of acamprosate on plasma amyloid-β precursor protein  
 
in youth: a pilot analysis in fragile X syndrome-associated and idiopathic autism 
 
spectrum disorder suggests a pharmacodynamic protein marker. J Psychiatr Res. 
 
2014 Dec;59:220-8.  
 
Guy, W. (ed). (1976). ECDEU Assessment for Psychopharmacology, Revised Edition. Rockville, 
MD: NIMH Publication. 
Harris, B. R., Prendergast, M. A., Gibson, D. A., Rogers, D. T., Blanchard, J. A., Holley, R. C., . . 
. Littleton, J. M. (2002). Acamprosate inhibits the binding and neurotoxic effects of trans-
ACPD, suggesting a novel site of action at metabotropic glutamate receptors. Alcohol 
Clin Exp Res, 26(12), 1779-1793. doi: 10.1097/01.ALC.0000042011.99580.98 
Kenward, M. G., & Roger, J. H. (1997). Small sample inference for fixed effects from restricted 
maximum likelihood. Biometrics, 53(3), 983-997.  


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Knox et al.: Feasibility, reliability, and clinical validity of the Test of Attentional Performance for 
 
Children (KiTAP) in Fragile X syndrome (FXS). Journal of Neurodevelopmental 
 
Disorders 2012 4:2. 
 
Lang, N., Rothkegel, H., Terney, D., Antal, A., & Paulus, W. (2013). Minocycline exerts acute 
inhibitory effects on cerebral cortex excitability in humans. Epilepsy Res, 107(3), 302-
305. doi: 10.1016/j.eplepsyres.2013.09.006 
Leigh, M. J., Nguyen, D. V., Mu, Y., Winarni, T. I., Schneider, A., Chechi, T., . . . Hagerman, R. 
J. (2013). A randomized double-blind, placebo-controlled trial of minocycline in children 
and adolescents with fragile x syndrome. J Dev Behav Pediatr, 34(3), 147-155. doi: 
10.1097/DBP.0b013e318287cd17 
Little, RJA. (1995). Modeling the drop-out mechanism in repeated-measures studies. Journal of 
the American Statistical Association, 90, 1112-1121.  
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Tollefson, G. D. (2003). Assessing and interpreting treatment effects in longitudinal 
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Riverside Publishing. 
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M. F. (2013). Lovastatin corrects excess protein synthesis and prevents epileptogenesis 
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10.1016/j.neuron.2012.01.034 
Pietrzak, B., & Czarnecka, E. (2005). Effect of the combined administration of ethanol and 
acamprosate on rabbit EEG. Pharmacol Rep, 57(1), 61-69.  
Porter, R. H., Jaeschke, G., Spooren, W., Ballard, T. M., Buttelmann, B., Kolczewski, S., . . . 
Malherbe, P. (2005). Fenobam: a clinically validated nonbenzodiazepine anxiolytic is a 
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(2013). Electrocortical changes associated with minocycline treatment in fragile X 
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Users Group Conference 

ICF_002.pdf: 
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Informed Consent/Parental Permission Form
For Participation in a Research Study
STUDY TITLE:  Evaluating The Neurophysiologic And Clinical Effects Of Single-Dose Baclofen, 
Lovastatin, Minocycline And Placebo In Fragile X Syndrome 
     
STUDY NUMBER: 2015-8804             
FUNDING ORGANIZATION:  CCHMC Department of Psychiatry
Craig Erickson, M.D.
   
Name of Principal Investigator 
513-636-4124 
Telephone Number 
INTRODUCTION
We are asking you to be in a research study so that we can learn new information that may help 
others. If you decide not to be in this study, we will still take good care of you.  If you decide to be in 
this study, you may change your mind at any time during the study and you can stop being in the 
study.  Take all the time you need to make your choice.  Ask us any questions you have. It is also 
okay to ask more questions after you decide to be in the study.  You can ask questions at any time.  
If you are 18 years and older:  This is a consent form. It explains this research study.  If you decide 
that you want to be in this research study, then you will sign this form to show that you agree to be part 
of this study.  If you sign this form, you will receive a signed copy of this form for your records. 
If you are 15 to 17 years old:  This form is also an assent form.  That means that if you decide that 
you want to be in this research study, then you will sign this form to show that you agree to be part of 
this study.  Your parent or guardian will also need to give their permission and sign this form for you to 
join the study.   
 
Parents/Guardians:  You have the option of having your child or teen join this research study. This is 
a parental permission form.  It explains this research study.  If you decide that your child can be in this 
study, you will sign this form to show that you agree. If you sign this form, you will receive a signed 
copy for your records. 
The word “you” or “I” in this form refers to your child/teen.
WHY ARE WE DOING THIS RESEARCH?
In this research study we want to see if medication can treat excessive sensitivity to sounds which is a 
common and distressing symptom of Fragile X Syndrome (FXS). One medication and a placebo
will be tested: baclofen. This drug has been approved by the U. S. Federal Food and Drug 
Administration (FDA) for the treatment of other conditions.


U54 Project 2 Consent_PP v9 28Aug2019
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WHO IS IN CHARGE OF THE RESEARCH? 
Dr. Craig Erickson is the researcher at Cincinnati Children’s Hospital Medical Center (CCHMC) that is 
in charge of this study.  
This study is being paid for by Cincinnati Children’s Hospital Medical Center.
WHO SHOULD NOT BE IN THE STUDY
You cannot be in this study if you have any of the following:
You are unable to take baclofen 
If you have taken any investigational drug within 3 months
If you have a history of drug or alcohol abuse within the last 6 months.
Any significant psychiatric or CNS neurological disease unrelated to FXS.
If you take anticonvulsants, barbiturates, lithium and benzodiazepines 
If you have just started any psychiatric medications  within the last 4 weeks 
Women who have a positive urine pregnancy test.
After screening, you have a creatinine clearance < 50 mL/min
If you have certain medical issues, unable to tolerate study procedures or study 
drug decided by the study doctor.
WHAT WILL HAPPEN IN THE STUDY?
The research staff will explain each visit to you and may give you a handout that explains each visit in 
more detail.  You will be able to ask questions to make sure that you understand what will happen.
If you qualify and decide you want to be in the study, your study participation will last about 11 weeks 
and include 5 study visits. You can choose to stop participating for any reason at any time. However, if 
you decide to stop participating in the study, we encourage you to tell the researchers. You may be 
asked if you are willing to complete some study termination tests.
These are the things that will happen to you while you are in the study:
Baseline Procedures: Visit 1
If you qualify and wish to continue, the following will be done:

You will give a urine sample to be tested for illegal drugs.

Women of child bearing years, you will have a pregnancy test to see if are pregnant

You will be given several tests of your memory and concentration.

You will have an optional blood sample drawn.

You will have an evaluation of your fragile x symptoms.

 You will have a review of your medical history.

You will have a medical evaluation, which includes your height, weight, head measurements, 
physical examination, and vision and hearing tests.

You will have your vital signs (pulse, blood pressure, breathing rate, and temperature) checked.

Due to the large size of the study pill, you will have the option to practice swallowing a placebo 
pill


U54 Project 2 Consent_PP v9 28Aug2019
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
You will be randomized to study treatment (see description below)

Your caregiver will be asked to complete questions about your behavior 
 You will be given a task to test your thinking and motor skills which approximately takes 25 
minutes
This baseline visit will take about 2 hours. You will return to the clinic within 60 days tobegin the single-
dose phase of the study and receive your first dose of study drug
Randomization
If the researchers believe you can take part in this study, you will be assigned randomly (like pulling a 
number from a hat) to 1 of 2 treatment groups. Each treatment group will receive the same dose of 
each of the study drugs – baclofen and placebo, but the order of drugs and placebo will be different for 
each group. You have a 50% chance (1 in 2) of being assigned to each group. Neither you nor the 
researchers will be allowed to choose which group you are assigned to. Neither you nor the 
researchers will know which group you are in and which study drug you are taking. However, the 
information about your assignment can be released to you after completing all phases of the study if 
you wish.  But, if there is an emergency, they are able to get this information.
Study Medication
If you decide to participate in this study you will be assigned to 1 of 2 treatment
groups and take each of the following study drugs in different orders:
•
baclofen (30 milligrams) or
•
placebo
The medication and placebo will be given in 2 capsules.
Study Drug Visits: Visits 2, 3, 4, 5
After you have been randomized to your treatment group, you will begin the weekly single-dose phase. 
You will come to the clinic once every 2 weeks to receive a single-dose of study drug (baclofenor 
placebo). You will complete study procedures both before and after receiving the study drug (see 
description below). These 4 visits will each take about 8 hours to complete.
Pre-dose Procedures
You will be asked to come in to the clinic for each visit. The following procedures will be done:

You will have a medical evaluation, which includes your height, weight, physical examination, 
motor skills, and vision and hearing tests.

 You will have your vital signs (pulse, blood pressure, breathing rate, and temperature) 
checked.

You will have an optional blood sample drawn

 You will participate in an electroencephalogram (EEG) neurophysiological measures.  During the 
EEG measures, you will wear a cap with sensors to measure the electrical activity of your brain. 
You will not feel these sensors working. These tests will look at how the brain functions at rest 
and when you are presented with visual images or sounds such as beeps or human voices. The 
study team will use a program to take a picture of the electrode placement on your head. This 


U54 Project 2 Consent_PP v9 28Aug2019
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helps the study team analyze the EEG data.

You will watch targets and faces on a large screen while we measure and record your eye 
movements

Women of child bearing years, you will have a pregnancy test to see if are pregnant. 

You and your caregiver will be asked to complete questions about your behavior 

You will be given several tasks to test your thinking skills, memory and concentration.

These tasks require that you be seated at a table, listen and follow directions presented by the 
examiner, look at pictures or other testing materials, and respond to questions either verbally or 
by manipulating objects. The total time spent performing such tasks is approximately 35 
minutes.

You will have an evaluation of your FXS symptoms. You will be asked about current side 
effects.
Following the pre-dose procedures, at approximately 2 hours after you arrive, your assigned study 
drug for the day will be administered orally (by mouth) by a research nurse.  
Post-dose Procedures
Optional blood samples will be drawn 1 to 2 hours after post dose.
Four hours after you have received the study drug: 
• You have your vital signs (pulse, blood pressure, breathing rate, and temperature) checked.
• You will participate in EEG neurophysiological and eye tracking measures.  
• Optional blood sampling will be 4 to 6 hours following dosing. 
• You will have an evaluation of your FXS symptoms.
• You will be given several tasks to test your thinking skills, memory and concentration.
These tasks require that you be seated at a table, listen and follow directions presented 
by the examiner, look at pictures or other testing materials, and respond to questions 
either verbally or by manipulating objects. The total time spent performing such tasks is 
approximately 1 hour. Frequent breaks will be offered throughout the testing session.
• You will be questioned about and examined for possible side effects of the study drug.
At the end of each visit, the study doctor will ensure that it is safe for you to leave the clinic by having 
your vital signs checked, asking questions about how you are feeling and by doing a physical 
examination. 
Each of these 2 single-dose phase visits will take about 8 hours to complete. You will return to the 
clinic once every 2 weeks to complete these study visits.
Phone/Email Evaluation 1 Day after Single-dose Visits:
We will call or email you the day after each of your Single-dose Visits and conduct an interview. We 
will ask you about any side effects you may have been experiencing and about any changes in your 
general health. This phone call will take about 20 minutes.
Phone/Email Evaluation 1 Week after Single-dose Visits:
We will call or email you a week after each of your Single-dose Visits and conduct an interview. We 
will ask you about any side effects you may have been experiencing and about any changes in your 
general health. This phone call will take about 20 minutes.


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Phone Evaluation 1 Month after Final Visit:
We will call you 4 weeks after Visit 5 and conduct a phone interview. We will ask you about any side 
effects you may have been experiencing and about any changes in your general health. This phone 
call will take about 20 minutes. 
The procedures in this study are designed for research, not for medical purposes. They are not useful 
for finding problems or diseases. Even though the researchers are not looking at your EEG measures 
and other tests to find or treat a medical problem, you will be told if they notice something unusual. 
You and your regular doctor can decide together whether to follow up with more tests or treatment. 
Because the procedures done in this study are not for medical purposes, the research results will not 
be sent to you or to your regular doctor.
Clinical Report Following Study Completion: 
Following the Week 10 call, the participating family will receive a report of the clinical measure scores 
including changes with drug/placebo treatment during each treatment period. A clinician not involved 
with the individual participant’s clinical ratings during study will sign off on the score report provided to 
the participating family.
WHAT ARE THE GOOD THINGS THAT CAN HAPPEN FROM THIS RESEARCH?
If you agree to take part in this study, there may not be direct benefits to you. The researchers cannot 
guarantee that you will benefit from participation in this research. We hope the information learned 
from this study will benefit others with FXS in the future. Information gained from this research could 
lead to better treatments for FXS.  
You will receive a report of the clinical measures obtained throughout the different treatment periods in 
this study. This may be helpful by providing clinical information associated with specific study drugs. 
WHAT ARE THE BAD THINGS THAT CAN HAPPEN FROM THIS RESEARCH?
Because of your participation in this study, you are at risk for the following side effects. You should 
discuss these with the researchers and your regular health care provider.
There are also risks associated with the medicine. 
Baclofen is FDA approved to treat reversible spasticity associated with multiple sclerosis or spinal 
cord lesions. Common side effects include hypotension (low blood pressure), nausea, constipation, 
diarrhea, vomiting, dryness of mouth, muscle pain, muscular weakness, sedation, drowsiness, 
dizziness, vertigo, headache, insomnia, euphoria (intense joy), depressive state, ataxia (loss of full 
body control), tremor, nightmares, tinnitus (ear ringing), nystagmus (uncontrolled eye movement), 
visual disturbance, rash, pruritus (itching), hyperhidrosis (excessive sweating), dysuria (difficult 
urination), and enuresis (involuntary urination). Less common side effects are arrthymias (abnormal 
heart beat), dyspnea (difficult breathing), palpitations (irregular heart beat), chest pain, ankle edema, 
abdominal pain, anorexia, disorders of hepatic function, paresthesias (tingling), taste disturbance, 
dysarthia (unclear speech), syncope (loss of consciousness), dyskinesia (impaired voluntary 
movement), coma, urine retention, helplessness, inability to ejaculate, hematuria (blood in urine), 
nasal congestion, and weight gain. 


U54 Project 2 Consent_PP v9 28Aug2019
Page 6 of 10
Risks related to EEG Measurements:
A paste or saline is used to attach the sensors during EEG testing which may irritate the skin on the 
scalp or face. Irritation is mild and usually goes away quickly. Paste used on the scalp during one of 
the tests requires that you not use hair products on that day of testing (or it has to be washed out prior 
to testing), which may be inconvenient.
Risks to Fetus:
If you are part of this study while pregnant, it is possible that you may expose the unborn child or infant 
to risks. For that reason, pregnant females cannot participate in the study. If you can become 
pregnant, a urine pregnancy test will be done, and it must be negative before you participate in this 
study. If you do become pregnant during this study, you must tell the researchers immediately
Pregnancy test results will be shared with you and your legal guardian if you are younger than 18 
years old.
Risks associated with blood draw
The risks of blood draw 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.
Psychological Stress
Some of the questions we will ask you as part of this study may make you feel uncomfortable. You 
may refuse to answer any of the questions, take a break or stop your participation in this study at any 
time.
Loss of Confidentiality
Any time information is collected; there is a potential risk for loss of confidentiality.
Every effort will be made to keep your information confidential; however, this cannot be guaranteed.
There may be other risks that we do not know about yet.
WHAT OTHER CHOICES ARE THERE?
You do not have to participate in this research to receive care for your medical problem. Instead of 
being in this study, you have the following options:
 Besides the study drugs, there are other similar medications that you can receive outside the study in 
regular clinical care. Please talk to the researchers or your personal doctor about these options.
HOW WILL INFORMATION ABOUT YOU BE KEPT PRIVATE?
 
Making sure that information about you remains private is important to us.  To protect your privacy in 
this research study we will code records to remove identifiers, limit access to the study records, not 
using any individual identifiers in publications or reports resulting from the study.
Your identity will be kept as confidential as possible as required by law. Except as required by law, you 
will not be identified by name, social security number, address, telephone number, or any other direct 


U54 Project 2 Consent_PP v9 28Aug2019
Page 7 of 10
personal identifier.  Your research records may be disclosed outside of Cincinnati Children’s Hospital 
Medical Center, but in this case, they will be identified only by a unique code number.  Information 
about the code will be kept in a secure location and access limited to research study personnel.  
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by U.S. 
Law. This website will not include information that can identify you. At most, the website will include a 
summary of the study results. You can search this website at any time.
WHAT IF WE LEARN NEW INFORMATION DURING THE RESEARCH?
The study doctor will tell you if they find out about new information from this or other studies that may 
affect your health, safety or willingness to stay in this study.
 WILL IT COST YOU ANYTHING EXTRA TO BE IN THE RESEARCH STUDY? 
Other than cost of travel to and from visits and potential time away from work for caregivers, we 
anticipate no other costs for subjects associated with study participation.
WILL YOU BE PAID TO BE IN THIS RESEARCH STUDY? 
You will be reimbursed for your time, effort and travel while you are in this research study.
You will receive payment for this study in the form of a reloadable debit card (Clincard).  We will give 
you a handout that will explain how to use the card.  Because you are being paid for your participation, 
CCHMC is required by the Internal Revenue Service (IRS) to collect and use your social security 
number (SSN) or taxpayer identification number (TIN) to track the amount of money that we pay you.  
You will need to complete a Federal W-9 form for this income tax reporting.  This form requires your 
Social Security number.  This form will be given to the CCHMC business office.  It will not be kept as 
part of your study chart.  If you move, you will need to complete another W-9 with an updated address.
You will be paid $100 for each study visit as reimbursement for your time and effort.
Tissues or body fluids collected for this research may result in the development of a product that could 
be patented/licensed and sold. You will not be paid if this happens.
WHAT HAPPENS IF YOU ARE INJURED FROM BEING IN THIS STUDY? 
If you believe that you have been injured as a result of this research you should contact the study 
doctor as soon as possible to discuss the concerns.  Treatment for injuries is available at CCHMC.  If 
you go to the Emergency Room or to another hospital or doctor it is important that you tell them that 
you are in a research study.  If possible, you should give them a copy of this consent form.
CCHMC follows a policy of making all decisions about compensation for the medical treatment of 
physical injuries that happened during or were caused by research on an individual basis.
WHO DO YOU CALL IF YOU HAVE QUESTIONS OR PROBLEMS? 
For questions, concerns, or complaints about this research study you can contact the study person 


U54 Project 2 Consent_PP v9 28Aug2019
Page 8 of 10
listed on page 1 of this document. 
If you would like to talk to someone that is not part of the research staff or if you have general 
questions about your research study rights or questions, concerns, or complaints about the research, 
you can call the CCHMC Institutional Review Board at 513-636-8039. 
AUTHORIZATION FOR USE/DISCLOSURE OF HEALTH INFORMATION FOR RESEARCH
To be in this research study you must also give your permission (or authorization) to use and disclose 
(or share) your “protected health information” (called PHI for short). 
What protected health information will be used and shared during this study?
CCHMC will need to use and share your PHI as part of this study.  This PHI will come from:

Your CCHMC medical records

Your research records
The types of information that will be used and shared from these records include:

Laboratory test results, diagnosis, and medications

Reports and notes from clinical and research observations

Imaging (like CT scans, MRI scans, x-rays, etc.) studies and reports

If applicable, information concerning HIV testing or the treatment of AIDS or AIDS-related 
conditions, drug or alcohol abuse, drug-related conditions, alcoholism, and/or 
psychiatric/psychological conditions (but not psychotherapy notes).
Who will share, receive and/or use your protected health information in this study?

Staff at all the research study sites (including CCHMC)

Personnel who provide services to you as part of this study

Other individuals and organizations that need to use your PHI in connection with the research, 
including people at the sponsor and organizations that the sponsor may use to oversee or conduct 
the study.

The members of the CCHMC Institutional Review Board and staff of the Office of Research 
Compliance and Regulatory Affairs.
How will you know that your PHI is not misused?
People that receive your PHI as part of the research are generally limited in how they can use your 
PHI.  In addition, most people who receive your PHI are also required by federal privacy laws to 
protect your PHI.  However, some people that may receive your PHI may not be required to protect it 
and may share the information with others without your permission, if permitted by the laws that apply 
to them.  
Can you change your mind?
You may choose to withdraw your permission at any time.  A withdrawal of your permission to use and 
share your PHI would also include a withdrawal from participation in the research study.  If you wish to 
withdraw your permission to use and share PHI you need to notify the study doctor, listed on the first 
page of this document, in writing.  Your request will be effective immediately and no new PHI about 
you will be used or shared.  The only exceptions are (1) any use or sharing of PHI that has already 
occurred or was in process prior to you withdrawing your permission and (2) any use or sharing that is 
needed to maintain the integrity of the research.


U54 Project 2 Consent_PP v9 28Aug2019
Page 9 of 10
Will this permission expire?
Your permission will expire at the end of the study.  If the study involves the creation or maintenance 
of a research database repository, this authorization will not expire. 
Will your other medical care be impacted?
By signing this document you are agree to participate in this research study and give permission to 
CCHMC to use and share your PHI for the purpose of this research study.  If you refuse to sign this 
document you will not be able to participate in the study.  However, your rights concerning treatment 
not related to this study, payment for services, enrollment in a health plan or eligibility of benefits will 
not be affected.
Optional Blood Sampling
Pharmacokinetics, or PK sampling, Extracellular Signal Related Kinase (ERK) and Amyloid Precursor 
Protein (APP) are tests to measure how much study medication is in the blood (referred to as 
pharmacokinetics or PK sampling) and ERK/APP are molecular biomarkers found in blood.  FMRP 
assay will be conducted to quantify the level of the fragile X mental retardation protein in cells.  Blood 
samples may be drawn 3 times, at pre-dose, 1-2 hours and 4-6 hours after on each day that study 
drug is given and once at baseline for ERK/APP, and FMRP.  The blood tests are optional and not 
required for participation.  
______ Yes, I agree to have Pk ERK/APP, and FMRP blood samples drawn  
______  No, I do NOT agree to have Pk ERK/APP, and FMRP samples drawn
SIGNATURES
The research team has discussed this study with you and answered all of your questions.  Like any 
research, the researchers cannot predict exactly what will happen.  Once you have had enough time 
to consider whether you should participate in this research you will document your consent by 
signature below.  
You will receive a copy of this signed document for your records. 
_____________________________________________            
 
Printed Name of Research Participant
_
____________________________________________            
____________ 
If research participant is 18 years and older
Date
Signature of Research Participant Indicating Consent       
         
_____________________________________________            
____________ 
If research participant is 15-17 years and older
Date
Signature of Research Participant Indicating Assent      
*********************************
If research participant is younger than 18 years or caregiver:


U54 Project 2 Consent_PP v9 28Aug2019
Page 10 of 10
_____________________________________________            
 
Printed Name of Legally Authorized Representative (Parent or Guardian)
 
_____________________________________________            
____________ 
Signature of Legally Authorized Representative 
Date
_____________________________________________________________
If signed by a Legally Authorized Representative, a description of such representative’s authority must 
be provided
_____________________________________________            
____________ 
Signature of Individual Obtaining Consent
Date

Prot_SAP_000.pdf: 
1
U54 Project 2 v16 27Aug2019
Title: Evaluating the Neurophysiologic and Clinical Effects of Single-Dose Baclofen and 
Placebo in Fragile X Syndrome
Principal Investigator:
Craig Erickson, M.D.
Associate Professor of Psychiatry
Children’s Hospital Medical Center 
3333 Burnet Avenue 
Cincinnati, OH 45229-3039
Office 513-636-4611
craig.erickson@cchmc.org
Co-Investigators
Ernest Pedapati, M.D.
Kelli Dominick, M.D., PhD
Rebecca Shaffer, PsyD


2
U54 Project 2 v16 27Aug2019
1.0 Introduction 
Fragile X Syndrome (FXS) is a family of genetic conditions caused by mutations in the Fmr1 
gene. FXS is the most prevalent cause of inherited intellectual disability (Crawford, Acuna, & 
Sherman, 2001). Approximately 1:4,000 males and 1:8,000 females have FXS (Crawford et al., 
2001). This impairment ranges from learning disabilities to severe intellectual disability. Between 
15% and 60% of individuals with FXS have autism (Budimirovic & Kaufmann, 2011). Other 
symptoms also can include characteristic physical (e.g. elongated face, prominent ears, and 
enlarged testes) and behavioral (e.g. stereotypic movements and social anxiety) features.  
Sensory hypersensitivities are an especially common, clinically distressing feature of FXS.  The 
aim of this project is to determine whether single-dose administration of racemic baclofen 
(subsequently referred to as “baclofen”) reduce (i.e., normalize) sensory evoke-related 
potentials (ERPs) in FXS patients. The proposed study will contribute to an understanding of 
neural hyperexcitability as a substrate of sensory hypersensitivities in FXS.
2.0 Specific Aims
Prior research has documented that pharmacological strategies targeting different neural 
mechanisms can reduce neocortical hyperexcitability in Fmr1 KO mouse models. Unlike nearly 
all areas of clinical psychopharmacology, marked to full recovery of function has been shown 
within hours of drug administration in animal models. We propose to leverage that observation 
to investigate single-dose drug effects on event-related potential (ERP) biomarkers and 
behavior in humans with FXS. 
Primary Aim
Our aim is to clinically validate the acute pharmacologic recovery of auditory processing deficits 
seen in Fmr1 KO mouse models, and to assess the relative impact and consistency of effect 
across patients of drugs targeting three different mechanisms on this recovery. If ERP 
normalization is observed in the proposed pharmacologic study, it will not only provide new 
knowledge about underlying mechanisms, but potentially speed drug development by guiding 
selection of mechanisms to target, choice of drugs to investigate, and outcomes to assess in 
next-step early phase clinical trials.
Secondary Aim
Neurophysiologic assessments will mirror the assessments in “Mechanisms and brain circuits 
underlying fragile X syndrome” IRB # 2015-8425 and include spectral analysis, N1 amplitude 
and habituation ERP, chirps, and talk/listen paradigms. Behavioral measures were chosen 
based on prior validation in FXS populations and an ability to allow brief assessments with 
limited learning effects. The primary clinical measure will be the Clinical Global Impressions 
Improvement (CGI-I) scale (Guy, 1976). The CGI-I requires the clinician to assess how much 
the patient's illness has changed relative to pre-dose, from 1 (very much improved) to 7 (very 
much worse). The recently validated Aberrant Behavior Checklist (ABC) using the 6-factor FXS 
model (Sansone et al., 2012) will be used as a secondary outcome of behavioral status. 
To assess cognitive outcomes as a secondary aim, we selected the following tests:  Woodcock 
Johnson (WJ)-III Spatial Relations (visual spatial thinking/5 min (McGrew & Woodcock, 2001); 
Grooved Pegboard Test (sensory motor integration/15 min); RBANS List Learning (immediate 
memory/5 min (Randolph, 1998); and Test of Attentional Performance for Children (KiTAP) 
(attention and inhibition/30 min (Knox et al., 2012). The WJ-III Auditory Attention test will assess 
auditory processing-speech sound discrimination, which aligns well with our focus on auditory 


3
U54 Project 2 v16 27Aug2019
cortical function. This test was validated in ages 5-81 and requires 5 min to complete (McGrew 
& Woodcock, 2001).
3.0 Background:
Fmr1 gene full mutation leads to a cascade of brain changes, which offer multiple potential 
pharmacologic targets to treat functional deficits associated with FXS (Wijetunge, Chattarji, 
Wyllie, & Kind, 2013). This protocol is designed to investigate clinical homologues in sensory 
processing abnormalities that can provide more sensitive and mechanism-related assessments 
of pharmacologic effects.  We plan to evaluate single-dose drug effects on a core symptom of 
FXS, auditory hypersensitivity, using robust ERP measures of auditory response that translate 
from mouse models to humans and back.  Specifically, our study will evaluate the effects of a 
single-dose approach to test drug effects on putative ERP biomarkers and behavior of persons 
with FXS. In 2009, Berry-Kravis et al. (Berry-Kravis et al., 2009) reported that a single oral dose 
of fenobam, an mGluR5 antagonist (Porter et al., 2005), was well tolerated and resulted in 
response (defined as 20% improvement) in auditory prepulse inhibition (PPI). These PPI effects 
were demonstrated just 60 minutes after oral dosing (Berry-Kravis et al., 2009). This study lays 
a foundation for using acute pharmacologic studies to probe putative etiologic pathways of FXS. 
We will extend this pharmacologic strategy to test the neurophysiologic effects of multiple drugs 
targeting different mechanisms in the same patients.
Among the most promising targets for pharmacologic intervention in FXS are treatments 
targeting mGluR5 and GABA systems and the ERK and MMP-9 pathways (Wijetunge et al., 
2013). Fmr1 mutation results in upregulation of mGluR5 receptors and “downstream” effects of 
increased mGluR5 signaling in extracellular signal related kinase (ERK) and matrix 
metalloproteinase-9 (MMP-9) pathways that lead to abnormal dendritic spines and auditory 
circuit hyperexcitability [Preliminary data from our collaborators at UTSW (Huber & Gibson) and 
UC Riverside (Abdulrazak & Ethell)].  Fortunately, safe and well-tolerated drugs with actions 
relevant to these mechanisms already exist in agents FDA-approved for other conditions:  
baclofen potentiates gamma hydroxybutric acid (GABA) type B (GABA(B)) neurotransmission 
Sinclair et al. 2017). Limited, but growing evidence suggests these agents can counter auditory 
hyperexcitability associated with the Fmr1 mutation. In the Fmr1 KO mouse, baclofen treatment 
has been associated with rescue of abnormal auditory-evoked gamma oscillations, working 
memory deficits, and anxiety (Sinclair et al. 2017). The active enantiomer in baclofen, 
arbaclofen has been associated with rescue of dendritic spine deficits and excessive neuronal 
protein synthesis in the Fmr1 KO mouse (Henderson et al. 2012).  
This pharmacologic study is designed to leverage clinical and preclinical preliminary data 
showing robust acute drug effects on neurophysiological parameters. If our strategy of single-
dose rapid testing of drug effects on ERP biomarkers is effective, the approach would inform 
preclinical work on the mechanisms most relevant to auditory hyperexcitability, speed the 
identification of effective pharmacologic treatments, develop an urgently needed biomarker 
strategy for predicting and evaluating drug effects, and ultimately, could allow for rapid 
personalization of treatment in FXS. 
4.0. Concise Summary of Project:
4.1 Study Design
This pharmacologic study employs a double –blinded 1-drug single dose, 2-period, 2-sequence 
balanced-crossover design. 36 patients age 15 to 55 years with full mutation fragile X syndrome 


4
U54 Project 2 v16 27Aug2019
characterized by greater than 200 CGG repeats in the FMR1 gene by Southern Blot and PCR 
genetic testing will be eligible to participate if they previously successfully completed baseline 
testing (behavioral, medical, neurophysiological tests) for our study “Mechanisms and brain 
circuits underlying fragile x syndrome (IRB # 2015-8425)”. In this project, subjects will be 
randomly assigned to 2 different sequences for receiving baclofen and placebo, with a 2-week 
washout (±2 days) between administrations. Patients will be stratified based on gender and then 
randomized in a 1:1 ratio to one of the 2 possible drug sequences (e.g.,1: AB; 2: BA). The study 
is expected to enroll 36 subjects who randomize, once they are randomized, subjects will not be 
replaced, Subjects and legally authorized representatives will be informed during consent that 
they will be blinded to treatment status throughout the study. Agents of this study will be dosed 
as a single administration to each patient at the following dose:  baclofen=30 mg)The timing of 
evaluations in relation to the single-dose of each agent/placebo is guided primarily by the time 
to maximum plasma concentration (Tmax) of the drugs but is also supported by considerable 
preclinical and clinical literature. The Tmax of each drug studied in this project is not significantly 
impacted by a fasting versus fed state. This is important because we cannot reliably keep 
subjects in a specifically fasting or specifically fed state post-dosing prior to post-dose 
evaluations. The primary outcome ERP will be conducted 4 hours post-dosing, with post-dose 
clinical and cognitive evaluations being done immediately thereafter. Pharmacokinetic data 
regarding ½-lives of the drugs support a 2 week washout period between drug exposures.
The power analysis supports our plan to enroll subjects until at least 36 subjects have fully 
completed the drug dosing protocol. The study will last approximately 6 weeks and there will be 
a total of 3 visits in the study (and 5 follow-up phone calls or emails with a study coordinator).
4.2 Blindness and Breaking the Blind
The study blind will be maintained throughout the duration of the clinical trial for all study staff 
interacting with the 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 will be broken only at the discretion of a participating 
study physician or Medical Monitor in cases necessary to assure patient safety. Such significant 
safety events would include, but are not limited to, any circumstance where an adverse effect 
documented as possibly, probably, or definitely related to study treatment and moderate or 
more severe in intensity results in the need for additional medical intervention (hospitalization, 
emergency/urgent care room visit, additional outpatient prescribing/other management etc.).
Following the Week 6 study call, the participating family will receive a report of the CGI and 
clinical measure scores including changes with drug/placebo treatment during each treatment 
period. This will require unblinding of treatment assignment for each participant following the 
Week 6 call. A clinician not involved with the individual subject's clinical ratings during study 
will sign off on the CGI and clinical score report provided to the participating family. The 
clinicians involved in this study who will be designated the responsibility of providing the 
report is Dr. Erickson. 
5.0 Study Procedures:
5.1 Baseline Visit Procedures
All FXS age 15-55, who successfully complete testing (behavioral, medical, neurophysiological 
tests) for our study “Mechanisms and brain circuits underlying fragile x syndrome (IRB # 2015-


5
U54 Project 2 v16 27Aug2019
8425)” will be eligible to participate in this pharmacologic study. At the day of recruitment, 
consenting participants will complete a urine drug screen (if possible) and females of 
childbearing age (as determined by report regarding age of menarche or menopause) will be 
required to take a urine or serum pregnancy test before study procedures. A positive test will 
exclude them from participating in the study.  Safety labs including comprehensive metabolic 
panel (CMP), antinucleotide antibody (ANA), and complete blood count with differential (CBC with 
differential) will be collected at baseline.  All subjects will have the option to provide a blood 
sample for Extracellular Signal Related Kinase (ERK) Activation Molecular Biomarker Assay and 
Amyloid Precursor Protein (APP) Molecular Biomarker Assay (described in further detail below).    
Baseline cognitive and clinical measures (described in further detail below) will be assessed.  
Each patient will have a medical examination performed by a study physician who will review 
medical history, and assess height, weight, vitals (Blood pressure, heart rate, respiratory rate, 
and temperature), muscle tone, Tanner staging, head circumference, history of recurrent otitis 
media, dysmorphologies and other comorbid conditions at baseline.  Since the study drug pill is 
large, in some cases subjects may be given placebo pills to determine whether they are able to 
swallow the capsule or bring home to practice taking.
 
Subjects who successfully complete baseline testing will be randomized to study treatment and 
assigned to 2 different sequences for receiving baclofen and placebo. Subjects will return within 
60 days to receive the first dose of baclofen or placebo.  
Subjects 01-014, who initially received acamprosate in study participation have the option to re-
consent to the updated baclofen and placebo protocol. If subjects re-consent to the study, they 
will repeat their baseline visit to confirm safety for dosing and tolerance for study procedures. 
Re-consented participants will still return within 60 days to receive the first dose of baclofen or 
placebo. 
5.2 Drug Administration Visit Procedures 
 The day of each drug administration day, a medical evaluation/physical exam, vital signs 
(blood pressure, heart rate, respiratory rate, weight and temperature), event related 
potentials (ERP) neurophysiology measures, urine pregnancy test, RBANS, KiTap and CGI-
S will be completed as “baseline” assessments for the day.  All subjects will have the option 
to provide a pre-dose blood sample for pharmokinetics, Extracellular Signal Related Kinase 
(ERK) Activation Molecular Biomarker Assay and Amyloid Precursor Protein (APP) Molecular 
Biomarker Assay and Fragile X Mental Retardation Protein (FMRP) assay. PIV placement or 
venipuncture for blood sampling will be offered as an option to participants and is not 
required for participation in this study.  Approximately two hours later, study drug (baclofen 
30 mg) or matching placebo will be administered orally to the subject with FXS.  The study 
drug or placebo will be self-administered by the participant (or administered with the help of a 
caregiver). Optional pharmacokinetic blood samples will be drawn approximately 90 minutes ± 
5 minutes post dose.  Approximately 4 hours following dosing ± 5 minutes, optional 
pharmacokinetic blood sampling and molecular assays (ERK/APP) will be drawn.   Then, 
event related potentials (ERP) will be performed, followed immediately thereafter by 
cognitive, clinical, and side effect/safety (including vital signs-(Blood pressure, heart rate, 
respiratory rate, and temperature ± 5 minutes) assessments.  At the end of single-dose drug 
administration days, Side Effect/Safety (including vital sign) assessments will be repeated. 
These visits will take approximately 8 hours to complete. These two drug administration 
days will occur once every 2 weeks over the course of 4 weeks.  A detailed description of 
each procedure being performed on drug administration days is provided below in Table 2.


6
U54 Project 2 v16 27Aug2019
5.3 Follow Up Phone Call/Email Procedures
We will bolster safety by adding weekly Side Effect/Safety assessments by phone or email 
midway between planned drug administration visits and the day following drug administration 
and finally 1 month after final study drug administration. Additional visits will be provided at any 
time that signs of concern arise. 


7
U54 Project 2 v16 27Aug2019
Table 2. Schedule of Assessments
*The Safety Follow-up visit at week 6 will 
include phone call with study coordinator 
regarding participants overall health status 
and a general inquiry about AE’s. Day 0 and 
Day 1 may occur on the same day. Safety 
labs include comprehensive metabolic 
panel, ANA, and CBC.
Visit
1 
Base
line
2 
Pre-
Dose
2 
Post-
Dose
Call
Call
3
Pre-
Dose
3 
Post-
Dose
Call
Call
Safety 
follow-up
Call
Day
Day 
-7to 0
Day
 1
Day
 1
Day
2
Day
8
Week 
2
Week 
2
Week 
2+1
Week 
3
Week 
6
Informed 
Consent
X
Physical Exam
X
X
X
Urine drug 
screen
X
Medical History
X
Vital Signs 
X
X
X
X
X
Urine pregnancy 
test
X
X
X
Safety Labs
X
Biomarker 
Molecular 
Assays 
(optional)
X
X
X
X
X
Pk Assays 
(optional)
X
X
X
X
Neurophysiology
/Eye tracking
X
X
X
X
CGI
X
X
X
X
ABC
X
X
X
X
WJ Subtests and 
Pegboard Test
X
X
X
RBANS and 
KiTap
X
X
X
X
Side effects/
Safety 
X
X
X
X
X
X
X
X
X*
Randomization
X


8
U54 Project 2 v16 27Aug2019
5.4 Neurophysiology Measures
For acquisition of electrophysiological data, we will use up to 128 lead channels placed 
according to the standard 10-20 electroencephalography array (which allows for a brief 5-10 
minute setup) referenced to the CMS-DRL ground.  We will use an EGI NetAmp400 (EGI, 
Eugene, OR) with hydrocel nets.  Participants will view a standard silent movie during 
acquisition for all paradigms except talk-listen, as is common practice to facilitate participation 
with developmentally disabled subjects. Auditory stimuli will be delivered with Etymotic or 
Seinheiser earphones via a programmable sound module (Presentation Software). Data will be 
acquired continuously, amplified (12,500x), and digitized (1000 Hz). Eye movements will be 
recorded (EOG) for offline data correction. 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 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. 
 
Spectral analysis: Spectra analysis will be completed on data acquired with eyes open for 5 
minutes. Data will be segmented into 1-sec epochs, detrended, and transformed into time-
frequency space in Matlab with a fast Fourier transform (FFT) algorithm, yielding 1Hz frequency 
steps. 
 
Auditory habituation ERP:  ERPs will be recorded during passive listening (150 sets of four 75 
ms broadband noise bursts) separated by an ISI of 500 ms, with inter-set interval of 4000 ms, 
for ~14 minutes of recording. Data will be filtered from .5-55 Hz (6 and 12 db/octave rolloff, 
respectively, zero-phase).  For each 4 stimulus train, trials will be blocked into 3000ms epochs, 
from 500 ms prior to stimulus onset to 500 ms after stimulus offset, baseline corrected using the 
prestimulus interval, and averaged across trials. Spatial PCA in BESA 5.3 (MEGIS Software, 
Grafelfing, Germany) will be used to define spatial topographies for ERP components in the 
grand average of all subjects. The PCA component capturing the N1 waveform will be identified 
with topography defined as the largest negative deflection between 50-200 ms post-stimulus. 
 
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. Data will be filtered from .5-120 Hz (6 and 12 
db/octave rolloff, respectively, zero-phase).  Trials will be blocked into 3500 ms epochs, from 
500 ms before to 1000 ms after stimulus offset. Representative topographies for the steady-
state response will be computed using spatial PCA in BESA 5.3 (MEGIS Software, Grafelfing, 
Germany) on the grand average of all subjects to determine the sensors used for the final 
analysis. 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. 
 
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 


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conditions. Data will be filtered 1- 50Hz and then epoched from -800 to 800 ms with respect to 
the onset of each “ah” and baseline corrected using data from the -800 to -500ms epoch 
preceding vocalization. ERP averages are generated using a robust averaging approach 
included in SPM8. Inspection of the grand average ERP waveform in preliminary studies and 
previous work with this paradigm indicates three components: N1 peak at ~100ms after speech 
onset, P2 peak at ~200ms after speech onset, and a slow negative component before speech 
onset from -300 to 0 ms.  Mean amplitudes of these components will be extracted at the anterior 
locus from which each of these three components are robust (collapsed across 26 electrodes 
surrounding FCz): N1: 80-120ms; P2: 170-210 ms; pre-speech: -300-0 ms. Correlations will be 
computed between amplitude of the pre-speech component and N1 ERP suppression 
(difference between N1 amplitude in the Talk vs. Listen conditions).
In order to facilitate accurate EEG electrode locations on the scalp during analysis any subject 
that receives an EEG will have a 5-10 minute procedure in which the EEG net and electrode 
positions are captured using photogrammetry software and a stereoscopic camera. Data will be 
used to generate a numerical coordinates file.
All samples will be deidentified and sent with the subject’s unique study number to Lauren 
Ethridge’s lab at University of Oklahoma Health Sciences Center and Jun Wang’s lab at 
Zhejiang Normal University whom have extensive experience analyzing EEG and ERP.
5.5 Eye Tracking
Persons with full mutation FXS clinically show gaze avoidance. This phenomena has been 
described during application of eye tracking procedures utilizing the presentation of faces from 
the NimStim face set to persons with full mutation. Using this method, what has been reported 
in the literature is reduced gaze towards eye regions in full mutation probands (Farzin et al. 
2009; Farzin et al. 2011). All study subjects will undergo eye tracking evaluation utilizing a 
hands-free Tobii eye tracking unit presenting paradigms (maximum duration 10 minutes) that 
will assess gaze preference. We will utilize this data to correlate eye gaze finding to other 
metrics employed in this cross-sectional study. 
5.6 Blood Sampling
All subjects will have the option to provide a blood sample as noted in Table 2 to complete both 
molecular pharmacodynamic and pharmacokinetic studies. Pharmacokinetic data will be 
collected pre-dose, approximately 90 minutes post-dose and approximately 4 hours following 
dosing. Molecular assays will be drawn at the same time as pK draw and at Baseline.
5.6.1 Extracellular Signal Related Kinase (ERK) Activation Molecular Biomarker Assay
ERK is a central cellular signaling kinase who dysregulation is implicated in the pathophysiology of 
FXS and in autism spectrum disorder. Our group has previously utilized ERK activation as a 
pharmacodynamics marker in FXS drug study (Erickson et al. 2011). For ERK biomarker assays, 
blood will be drawn at Baseline, pre-dose, approximately 90 minutes and 4hours post dose at all 
drug dosing days.  Assays will be analyzed in Dr. Craig Erickson’s Molecular Translational 


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Biomarker Lab at CCHMC. All blood biomarker assays will be performed blinded to study 
assignment. Blood will be collected and lymphocytes will be isolated, washed, counted, and re-
suspended. Samples will be frozen until ERK total and phosphorylated ERK levels will be 
determined in duplicate using commercially available ELISA kits (Abcam) per manufacture 
instructions. All ERK measurements will be attempted, but are not required for study participation.
5.6.2 Amyloid Precursor Protein (APP) Molecular Biomarker Assay
APP and its derivatives are involved in neuronal growth, maturation, and senescence. APP 
dysregulation has been implicated in the pathophysiology of FXS and autism of unknown cause. 
Our group has published on the use of plasma APP derivative analysis in FXS clinical trials 
(Erickson et al. 2014).  For APP biomarker assays assessing plasma levels of sAPP (total), 
sAPPα, Aβ40, and Aβ42, blood will be drawn at Baseline, pre-dose 90 minutes and 4 hours post 
dose at all drug dosing days.  Assays will be analyzed in Dr. Craig Erickson’s Molecular 
Translational Biomarker Lab at CCHMC. 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), sAPP (IBL), and Aβ peptides (Wako Chemical Industries, 
Japan) in duplicate per manufacturer instructions. All APP measurements will be attempted, but 
are not required for study participation. 
5.6.3 Fragile X Mental Retardation Protein (PK) Assay
FMRP assay will be conducted to quantify of the level of the fragile X mental retardation protein 
in cells. For FMRP assays, blood will be drawn at Baseline, pre-dose, approximately 90 minutes 
and 4 hours post dose at all drug dosing days.  Assays will be analyzed in Dr. Craig Erickson’s 
Molecular Translational Biomarker Lab at CCHMC. All blood biomarker assays will be 
performed blinded to study assignment. Blood will be collected and spotted onto Whatman 
Bloodstain Cards. The samples will be allowed to dry between 4 and 24 hours before being 
stored in a gas-permeable bag with desiccant and frozen until analysis. Upon analysis, three 6-
mm disk punches will be collected from separate blood spots within the same card. The proteins 
from the disks will be eluted and analyzed in triplicate using a custom bead-based Luminex 
assay to obtain quantitative measures of FMRP.  All FMRP draws will be attempted, but are not 
required for study participation
5.6.4 Pharmacokinetic (PK) sampling
Pharmacokinetic evaluation blood samples will be drawn pre-dose, 90 minutes, and 4 hours post 
dose on each of four drug dosing days. All PK measurements will be optional and are not required 
for study participation.  
Baclofen concentrations will be determined with a low volume, validated liquid chromatography 
tandem mass spectrometry (LC-MS/MS) assay provided by the iC42 Clinical Research and 
Development Laboratory (Uwe Christians, MD, PhD, director), University of Colorado, Aurora, 
Colorado, USA.
Concentration data will be analyzed by compartmental and non-compartmental 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 


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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.
Parameter estimates generated will include Cmax, total body clearance, distribution and 
elimination half-lives, volume of distribution and the area under the curve (AUC).
6.0 Study Outcome Measures
6.1 Clinical Assessments

CGI-I: The Clinical Global Impressions Improvement (CGI-I) will be the primary clinical 
measure completed both before and after the subject receives study medication. The CGI-I 
requires the clinician to assess how much the patient's illness has changed relative to pre-
dose, from 1 (very much improved) to 7 (very much worse).

CGI-S: The Clinical Global Impressions Severity scale (CGI-S) will be a secondary outcome 
measure completed before the subject receives study medication.  The CGI-S is a 7-point 
scale designed to measure global severity of illness over the previous seven days.

ABC: The recently validated Aberrant Behavior Checklist (ABC) using the 6-factor FXS 
model will be used as a secondary outcome of clinical status. This test will be completed 
only after the subject receives study medication.
6.2 Cognitive Assessments
To assess cognitive outcomes as a secondary aim, we selected tests with minimal practice 
effects, good reliability (weighted kappa scores ≥0.7), and a lack of floor effects in persons with 
FXS with a mental age of 3+ years (Berry-Kravis, Sumis, Kim, Lara, & Wuu, 2008). See Table 1 
below for descriptions of each of the tests.
Table 1. Cognitive and neuropsychological assessment measures
Measure
Domain
Task Description
Estimated Time
WJ-III Spatial 
Relations (McGrew 
& Woodcock, 2001)
Visual Spatial 
Thinking
Identify parts needed to form a 
complete shape. Responses can 
be oral or motoric (pointing).
05 minutes
RBANS: List 
Learning (Randolph, 
1998)
Immediate Memory
A 10-item list of unrelated words 
is presented orally to the 
examinee who is then required 
to immediately recall words 
presented.
05 minutes
KiTAP: Test of 
Attentional 
Attention and 
Inhibition
Computerized task comprised of 
8 subtests where an examinee is 
 30 minutes


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Performance for 
Children (Knox et 
al., 2012)
required to push a key when a 
target stimulus is presented on 
the screen. 
Grooved Pegboard 
Test
Sensory Motor 
Integration
Pegs, with a key along one side, 
must be rotated to match the 
holes in the pegboard before 
they can be inserted. 
 15 minutes
WJ-III Auditory 
Attention (McGrew 
& Woodcock, 2001)
Auditory Processing-
Speech Sound 
Discrimination
Identify orally presented words 
amid increasingly intense 
background noise. Examinee 
points to a picture of the word 
presented. 
05 minutes
7.0 Side Effect/Safety Assessments
Safety assessments will include comprehensive metabolic panel (CMP), ANA, complete blood 
count with differential (CBC with differential), urine drug screen, urine  pregnancy test in female 
participants of childbearing age, physical exam, medical history, concomitant medication 
review, vital signs at baseline. 
At dosing visits, Side Effect/Safety Assessments will include: a general inquire about AE’s, a 
detailed inquiry of common side effects of the 3 study drugs, and vital signs. In an effort to ensure 
subject safety, we plan to schedule all study drug dosing visits at the CTRC. Coordinators will call 
or email the family to ask if they have seen any changes in subject symptomology. If the 
parent/guardian reports no change, the follow up exchange will be considered completed. If the 
parent/guardian reports change in symptomology, a call will be scheduled with a study physician 
to review reported changes and asses safety. Once the study physician speaks with the 
parent/guardian and records their assessment, the follow up will be considered completed. This 
applies to all during study follow up calls as well as the Week 6 call. The Safety Follow-up call at 
week 6 will follow the same follow up call structure as theduring study follow up calls. Following 
the Week 6 call, the participating family will receive a report of the CGI and clinical measure 
scores including changes with drug/placebo treatment during each treatment period. This will 
require unblinding of treatment assignment for each participant following the Week 6 call. A 
clinician not involved with the individual subject's clinical ratings during study will sign off on the 
CGI and clinical measures score report provided to the participating family.
Additional study safety oversight will be provided by Dr. Sergio Delgado, Outpatient Clinical 
Director and Professor with the division of child and adolescent psychiatry at Cincinnati 
Children’s Hospital will serve as independent study medical monitors for the study. The monitors 
will review recruitment and adverse events every 6 months and report assessment to PI. 
8.0 Research Methods
 
8.1 Criteria for Inclusion of Subjects:

Subjects ages 15-55, with FXS who completed the study entitled “Mechanisms 
and brain circuits underlying fragile X syndrome (IRB # 2015-8425). FXS is 
defined as full FMR1 mutations (>200 CGG repeats) confirmed by genetic 
testing.


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
General good health as determined by physical exam, medical history and 
laboratory work up.
8.2 Criteria for Exclusion of Subjects:

Subjects with a history of intolerance to baclofen will be excluded. 

Subjects will also be excluded if they have taken any investigational drug within 
3 months, have a history of substance abuse or dependence within 6 months, or 
significant psychiatric or CNS neurological disease unrelated to FXS.

Uncontrolled seizures impact EEG data as do anticonvulsants, barbiturates, 
lithium and benzodiazepines and are exclusions (within 5 half-lives). Those 
taking other psychiatric medications must be on stable doses for 4 weeks before 
any testing. 

For female subjects of child bearing potential, a positive urine pregnancy test.

Potential subjects with a creatinine clearance < 50 mL/min will be excluded.

Identified medical issues, inability to tolerate study procedures or study 
drug per the discretion of the Principal Investigator.
8.3 Vulnerable Populations:
 Inclusion of Children in Research:
One special class of subjects that will be involved in this study is children.  FXS is a genetic 
disorder that is a cause of intellectual disability and neuropsychiatric disorders.  A central aim of 
this study is to examine the effects to investigate single-dose drug effects on event-related 
potential (ERP) biomarkers and behavior in humans with FXS. This can only be done by 
studying affected individuals across the age span.  We have chosen ages 15-55 to represent a 
broad developmental spectrum. The upper age of 55 is chosen because of variable and 
common age-related changes in behavior, sensory function and brain function beyond that age. 
The age 15 is chosen for the low end of the age range because of the level of cooperation 
required to complete procedures.  
Persons with Cognitive Impairment or other limited decision making capacity 
To be inclusive and most generalizable in our study findings, and because some elements of 
our protocol can be completed with minimal cognitive requirements such as ERP monitoring of 
cortical responses to tones, we will recruit any study eligible patient with Fragile X Syndrome 
regardless of level of cognitive disability into as many study components as they can 
complete. We will recruit all FXS patients who we believe can complete study procedures and 
use IQ scores to describe study participants in research reports. 
Inclusion of Women and Minorities:
No person shall be excluded from participation in this research study on the basis of gender 
alone. However, FXS occurs at a higher rate in males than in females.  Therefore, it is 
anticipated that the largest number of proband participants will be male.  Every effort will be 
made to recruit and include minorities among the participants in this research study.  In regard 
to gender and minority representation, our research cohort will reflect consecutive referrals to 


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our clinic meeting the inclusion-exclusion criteria of the study.  Recruitment is expected to be 
30% minority because of the population our clinics serve.
9.0 Sources of Research Material:
In addition to sociodemographic information, we may collect sensitive clinical information 
including information about medical history, drug use, psychiatric symptoms and developmental 
history during individual testing sessions and via questionnaires completed during pre-screening 
or testing sessions. This information can be collected as part of structured and semi-structured 
interviews, self-report and caregiver questionnaires, and clinical tests of behavioral functioning.  
Neuropsychological and electrophysiological data may be acquired during individual testing 
sessions. Medical records may be obtained after obtaining a signed permission for release of 
records.  
All information collected is for research purposes only. If some assessments were recently done 
in the clinic by our faculty before recruitment, then we will use the existing clinical data to reduce 
patient burden; but we expect this overlap to be rare. All material will be protected by limited 
access and stored under a unique ID number.  
10.0 Recruitment Methods and Consenting Process:
10.1 Recruitment
Recruitment for the study will be conducted by the Cincinnati Fragile X Research and Treatment 
Center Subjects ages 15-55, will be recruited from participants who have completed the study 
entitled “Mechanisms and brain circuits underlying fragile X syndrome (IRB #2015-8425).
10.2 Informed Consent Process
Screening and consent procedures for the study will be completed prior to enrollment. 
Participants (if they have decisional capacity) or legally authorized representatives together with 
participants initially will receive written materials and be given an oral explanation of the study. 
Informed consent procedures will be conducted by research staff or investigators. In addition, 
members of the protocol team will be available to address any questions for participants or 
families.  
Each participant must give informed consent or assent to participate when possible. Given the 
developmental disability associated with FXS, particularly in males, some subjects themselves 
will not be able to provide assent or consent. Consent also will be provided by the guardian of 
minors and from adult subjects who are not their own legal guardian. 
Participants (and if they have one, their legally authorized representative) will be informed 
during their initial consent, prior to the initiation of the pharmacologic study, and on several 
additional occasions throughout their experience in the study that they may elect to not to 
participate at any time. 
Consent and assent will be collected using the procedures outlined above. This will take place 
after initial screening but before any formal assessments and experimental procedures begin. 
Both written and oral information will be provided to participants and legally authorized 
representatives. Refusal to participate in the study will not limit their ability to seek services at 


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Cincinnati Children’s Hospital Medical Center. One signed and witnessed copy of the consent 
and assent forms will be stored in secure storage and the participant and legally authorized 
representative will each be given a copy of the signed forms, as is appropriate. Participants will 
receive payment for taking part in this study. 
Some individuals may be interested in study participation but live far away from CCHMC.  In 
these circumstances, the consent forms will be mailed to potential participants after they have 
initiated contact with our research program. Our research team will arrange for a conversation 
with the potential participant by phone after the documents have been received. During the 
phone conversation, our research team member will review the consent form (i.e., study 
purpose, voluntary participation, study procedures, risks and benefits, and confidentiality) and 
answer any questions.  Then, the participant will sign the documents and mail the documents 
back to the researcher.  Once the researcher at our Center receives them, he/she will sign the 
consent forms and return copies of the documents to the participant for their records and place 
the originals in a secure area and a screening visit will be set up with the participant at CCHMC
The study team will attempt to obtain consent/assent from all participants potentially eligible for 
the study. All participants who are cognitively unable (due to limited cognitive functioning and/or 
to provide assent or consent will require a parent or legal guardian to consent on study. Since 
about 3 in 4 persons with autism spectrum disorder have intellectual disability, we anticipate that 
a majority of subjects will not have the cognitive ability and understanding to give their consent 
or assent to participate in the project.
If a subject turns 18 years-old while enrolled in the study, the parent(s)/legal guardian(s) will be 
asked to provide appropriate documentation indicating that they are actively seeking legal 
guardianship. Copies of documentation will be filed in the subject’s study chart. If parent(s)/legal 
guardian(s) are not pursuing legal guardianship when the subject turns 18 years-old, then the 
subject will need to demonstrate the capacity to provide informed consent and voluntarily sign 
the informed consent document in order to continue participation in the study.
11.0. Potential Risks:
11.1 Study Drugs
All participants will receive a single dose of an FDA-approved drug (baclofen) that have proven 
benign safety profiles during chronic treatment.  Arbaclofen (the active enantiomer in racemic 
baclofen) was well-tolerated in studies of humans with FXS (Erickson et al., 2013; Leigh et 
al., 2013At the doses proposed for this study the incidence of adverse effects is low. Furthermore 
the administration of a single dose will limit the risk for side effects that might emerge with longer 
term treatment. The study PI (Dr. Erickson) has experience in the use of pharmacotherapy in 
persons with FXS. All participants will be able to leave the clinic during breaks, however must 
remain on the CCHMC campus.  Participants will be given the PI’s contact information if any 
issues or side effects arise.  All participants will have a careful medical examination and detailed 
monitoring of side effects in the Clinical Translational Research Center. 
11.1.1 Baclofen
Common side effects (more than 1 in 100 patients):

hypotension, nausea, constipation, diarrhea, emesis, dryness of mouth, myalgias, 
muscular weakness


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
sedation, drowsiness, dizziness, vertigo, headache, insomnia, euphoria, depressive 
state, ataxia, tremor, nightmares

tinnitus, nystagmus, visual disturbance, rash, pruritus, hyperhidrosis

fysuria, enuresis
Less common side effects (between 1 in 1000 patients and 1 in 100 patients):

arrthymias, dyspnea, palpitations, chest pain, ankle edema

abdominal pain, anorexia, disorders of hepatic function

paresthesias, taste disturbance, dysarthia, syncope, dyskinesia, coma
urine retention, impotence, inability to ejaculate, nocturia, hematuria

nasal congestion, weight gain
11.2 Cognitive Testing
There are minimal risks anticipated to individuals participating in observational or clinical 
behavioral assessments. In the rare case that an individual becomes overly fatigued or 
distressed during assessment sessions, the session will be discontinued and rescheduled.  
Evaluation procedures, when appropriate, are designed in a way that is compatible with the 
attention span of a person with an intellectual disability, greater activity level, and need for 
access to his or her parents. In addition, specialized procedures for children with FXS (e.g., 
reducing language demands, providing praise for task attention and attempts, using work-
reward routines) will be implemented. Three clinical psychology investigators with extensive 
experience working clinically with developmentally disabled patients are investigators in the 
study.
11.3 Neurophysiology Procedures
There are minimal risks associated with participation in the neurophysiology part of the study. 
The auditory stimuli presented are relatively benign, and have been well tolerated in our pilot 
studies. We are sensitive to monitoring participant distress associated with sensory paradigms, 
which we have found more commonly in participants younger than 12 years of age. The most 
common risk associated with these tasks is boredom. In rare circumstances the electrode gel 
used to place the recording electrodes to the scalp can cause minor irritation to the skin, which 
resolves rapidly with no permanent effects. 
11.4 Pregnancy and Protection against Risk
Females of childbearing age (as determined by report regarding age of menarche or 
menopause) enrolling in the pharmacologic study will be required to take a urine pregnancy test 
before study procedures and prior to study drug at each visit. A positive test will exclude them 
from participating in this portion of the study. The PI or his designee will discuss with the subject 
in private the results of any drug screen and/or pregnancy test and if positive, that they will no 
longer be able to participate in the study. Consent forms will make clear that this information will 
be shared with parents of minors. A subject who tests positive will be referred to appropriate 
medical care.


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11.5 Risk of Discrimination
There is a theoretical risk for discrimination towards individuals who are at risk for a medical 
disorder or have a medical disorder in their family. Potential discrimination may include barriers 
to insurability, employability, or other unidentified adverse effects. Extensive efforts are made to 
protect all research subjects from prejudice, discrimination, or uses of this information that will 
adversely affect them.
11.6 Venipuncture and Peripheral IV Placement Safety
The risks of venipuncture PIV placement are modest and include mild discomfort, infection, 
bleeding, and fainting.  Standard methods and precautions will be used to protect the puncture 
site from bleeding and infection.  The Research Coordinator or Research Assistant will be 
familiar with the subjects and will accompany the subject and their parents to the blood drawing 
setting.  Parents are encouraged to remain with the subject at all times.  To minimize the 
subject’s anxiety and phobic reactions, we utilize Child Life personnel when needed and 
available.  We also suggest that the parents reassure the subject concerning their safety.  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.
12.0 Procedures for Protecting Against and Minimizing Potential Risks
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.  During study visits, subjects will be allowed to leave the 
research clinic, but will be asked to remain on the hospital campus.  If adverse reactions 
become excessive, the subject will be treated and removed from the study. There will be 
repeated monitoring of behavior 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.  All of the research data 
is kept in locked files to ensure confidentiality. The other procedures to ensure confidentiality 
follow the regulations and policies of the Medical Center.
All SAEs will be reviewed in detail by the PIs and Independent Safety Monitor.  If the stopping 
rule is met, the study will close to enrollment for interim analysis of safety and efficacy data and 
the Safety Monitor would provide a recommendation to the PI regarding continuation of the trial.
13.0. Potential Benefits:
Subjects may or may not benefit directly from study participation. The possibility of contributing 
to the discovery of treatments for FXS may benefit all affected individuals and their families.
The following knowledge can be gained from this research: a more detailed understanding of 
auditory processing abnormalities in FXS, a better understanding of the pathophysiology of 
FXS, new perspectives on pharmacologic mechanisms in FXS, and insight into possible 
biomarkers that can be used to track drug effects in FXS.
The potential gain in knowledge can change our clinical understanding and treatment of FXS 
and related disorders. This could lead to more effective treatments and possibly earlier 
identification and personalized treatment of at-risk children. Given the severity of impairments 


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associated with FXS, this could dramatically impact the lives of affected individuals and their 
families. Given the potential for gain, the relatively modest risks are reasonable for the study 
participants, most of whom stand to receive modest personal gains by participating in the 
research. 
13.1 Risk vs Benefit Ratio
The subjects will be exposed to the risks of blood sampling and the potential side effects of 
baclofen. 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.
14.0. Subject Safety and Data Monitoring:
Dr. Sergio Delgado, Outpatient Clinical Director with the division of child and adolescent 
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. 
All problems (i.e. adverse events, unanticipated events, etc.) will be characterized by the 
following: 

Severity = Mild, Moderate or Severe 

Relatedness = Definite, Probable, Possible, Unlikely or Unrelated 

Expectedness = Expected or Not Expected 
A stopping rule for excessive toxicity is in place in the event of ≥ 2 SAEs considered possibly, 
probably or definitely related to treatment on study.  All SAEs will be reviewed in detail by the 
PIs and Independent Safety Monitor.  If the stopping rule is met, the study will close to 
enrollment for interim analysis of safety and efficacy data and the Safety Monitor would provide 
a recommendation to the PI regarding continuation of the trial.
Events will be reported consistent with Institutional IRB reporting policies. An aggregate listing 
of all safety events will be reported to the IRB in summary form at the time of continuing review. 
These reports will also be provided to any applicable regulatory agencies per applicable 
regulatory agency requirements. Data (such as lab values, vital signs, and outcome measure 
data) will be entered from source documents to Case Report Forms by the study coordinator. 
The study coordinator will review case report form entries for accuracy by comparison with the 
source documents.  Research records and source documents will be maintained in a research 
chart and stored in the investigator's locked file cabinet. Information will be kept in a secure 


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database that is password protected. Records will be kept secure, and individually identifiable 
information will not be included in any reports or data sets. 
All subjects will be assured that data will be kept confidential. Data will be identified only by a 
unique identification number and not by the subject’s name. Paper data will be stored in a 
locked office in a locked cabinet to which only staff affiliated with the project will have access.  
Any discrepancies in the maintenance of confidentiality or other irregularities involving the data 
will be reported to the PI. Any such events will be documented and reviewed by the PI and 
reported to the IRB within a timeframe set out by guidelines set forth by the CCHMC IRB. 
14.1 Adverse Events
Any adverse events--whether due to assessment, medication or other aspects of the study--will 
be reported to the PI. Adverse events will be documented and reviewed by the PI within 24 
hours of receipt. Serious/unanticipated events will be reported to the IRB within 48 hours by 
phone, email, or fax. All adverse events will be compiled, and reported in summary form to the 
IRB, on an annual basis and at the conclusion of the study. 
15.0 Procedures to Maintain Confidentiality:
Unique numeric identifiers that do not include any PHI will be assigned. All data will be 
maintained either in locked storage facilities with limited access or in secure, electronic facilities. 
Data transmitted electronically will be protected by encryption over a very secure network that 
limits access only to CCHMC faculty and staff who are reviewed and specifically cleared for 
access to the password protected, secure network. The data will be stored in protected hard 
drives in a server system that is protected by two firewalls, one at the hospital level and a 
second at the Departmental level. De-identified data will be shared with the national database 
for ASD research (NDAR) as may be required as part of NIH funding via this mechanism, 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 (a more detailed description of data sharing procedures is below).
15.1 Data Management 
Data will be collected on paper source documents, CRFs, and/or in the electronic medical record 
and will be entered by study staff in a password protected, encrypted REDCap(Harris, Taylor et al. 
2009) database. 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.
16.0 Biostatistics:
The sample size analysis was based on a primary outcome of N1 amplitude. This is the best 
established ERP abnormality and the only validated ERP drug outcome measure in FXS 


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patients. The estimate of effect size was drawn from a double-blind trial of minocycline vs. 
placebo in FXS on N1 amplitudes (Schneider et al., 2013). Assuming that omnibus drug effect 
size of d~0.68, and no carryover or period effects, then 36 patients (9 in each of 4 drug 
sequences) provides power of 0.80 with α of 0.05 (two-tailed) ("Power Analysis and Sample 
Size (PASS)," 2008). Patients will be recruited from the STU 052013-043 population until 36 
patients have completed the pharmacologic study.  
A 4-drug, 4-period, 4-sequence balanced-crossover design (Williams, 1989) will be used for the 
proposed pharmacologic study. The primary ERP outcome will be N1 amplitude. Secondary 
outcomes will be N1 habituation, auditory evoked gamma power in the AM modulated chirps, 
and N1 amplitude reduction during the talk/listen task, and clinical and cognitive outcomes.  
The analysis will be a linear mixed model analysis of repeated measures. A separate mixed 
model analysis will be conducted on each outcome. If deemed necessary, the outcomes will be 
appropriately transformed to obtain a more normal distribution. The fixed effects of drug, drug 
sequence, and drug period will be examined. Potential carryover effects of drug will be 
estimated in the mixed model and, if significant, the drug effects will be adjusted. Patients will be 
nested within drug sequence and accounted for as a random effect in the mixed model. The 
intercept will be included as a random effect (if it improves model fit). Sex, age, and IQ will be 
considered as covariates in the respective mixed models, as will baseline clinical and cognitive 
data for analyses of those parameters. Restricted maximum likelihood estimation, Type 3 tests 
of fixed effects, and generalized least-squares will be used, with the Kenward-Roger correction 
(Kenward & Roger, 1997) applied to the appropriate and best fitting covariance structure. The 
robust covariance matrix estimator will also be considered. The six pairwise differences of least 
squares means among the drug effects will be estimated, and these comparisons will be carried 
out using the Tukey multiple comparison procedure. Hedges' ĝ will be calculated and 
interpreted as the effect size estimator for the pairwise contrasts between the drug effects.  
Findings will be examined in relation to Fmr1 gene expansion and methylation. Baseline and 
changes in neurophysiologic measures will be examined for relationships to cognitive and 
clinical outcomes as an exploratory evaluation of these variables as potential biomarkers of drug 
effects in FXS. The mixed model procedures of PROC MIXED in SAS software (SAS Institute, 
Inc., Cary, NC) will be used to conduct the mixed model analysis making use of all available 
data from all participants to provide a robust mechanism for handling data that are assumed 
missing at random (Little, 1995) (Mallinckrodt et al., 2003) (Rubin, 1976) (Wolfinger & Chang, 
1995). The level of significance for all tests will be set at α = .05 (two-tailed) and, to address 
multiple testing, p-values will be adjusted using the False Discovery Rate (Benjamini & 
Hochberg, 1995).
17.0 Subject Cost and Payment: 
Subjects will be paid $100 on a ClinCard for their time and transportation at the end of each 
fully completed visit.  Other than cost of travel to and from visits and potential time away from 
work for caregivers, we anticipate no other costs for subjects associated with study 
participation. Travel expenses up to $7500 may be reimbursed for subjects that come from 
distances far from the study site.


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