Neurophysiological and Acute Pharmacological Studies in FXS Patients
Phase: EARLY_PHASE1
Conditions: Fragile X Syndrome
Sponsor: Children's Hospital Medical Center, Cincinnati

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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 

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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).   

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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.). 

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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.  
 
 

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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 

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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 

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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 

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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 

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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. 

13 
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. 

16 
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 

17 
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), 

18 
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 

19 
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 

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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. 
 
 
 
 
 
 
 
 
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