A Double-Blind, Placebo-Controlled Trial of Metformin in Individuals With Fragile X Syndrome (FXS)
Phase: PHASE2
Conditions: Fragile X Syndrome, Fragile X Mental Retardation Syndrome, Mental Retardation, X-Linked, Genetic Diseases, X-Linked, Trinucleotide Repeat Expansion, Fra(X) Syndrome, Intellectual Disability, FXS, Neurobehavioral Manifestations, Sex Chromosome Disorders
Sponsor: University of Alberta

A Double-Blind, Placebo-Controlled Trial of Metformin in Individuals with Fragile X Syndrome 
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Protocol Title: A Double-Blind, Placebo-
Controlled Trial of  Metformin in Individuals 
with Fragile X Syndrome (FXS) 
  NCT Number:  NCT03862950 
  Protocol Version Date:  1 November 2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1) Protocol Title: A Double-Blind, Placebo-Controlled Trial of  Metformin in Individuals with 
Fragile X Syndrome (FXS) 
 
Nominated Principal Investigator: Dr. Francois Bolduc, MD PhD FRCPC 
Sub Investigators for University of Alberta: Dr. Janette Mailo, MD PhD FRCPC,  Dr. 
Jeffrey Jirsch, MD FRCPC, and Dr. Brenda Clark, MD FRCPC 
Canadian Co-Principal Investigators (Co-PIs): Dr. Sebastien Jacquemont,  MD, Dr. Sarah 
Lippé, Ph.D. 
Study Sponsor: The Governors of the University of Alberta 
Funding Sponsor: Women and Children’s Health Research Institute, CHU Sainte-Justine 
Foundation, and the Azrieli Foundation  
Phase: Clinical Trial Phase 2 
Sites/Facilities enrolling participants: Stollery Children’s Hospital (Edmonton, AB), CHU 
Sainte-Justine (Montreal, QC) 
Study population: 120 children and adults (aged 6-35 years), with FXS and controls 
Study duration: estimated duration of 3 years, starting in Spring 2019 
Participant duration: 4 months   
      Protocol Version Date:  1 November 2022 
Protocol Number: FXSMET-2018 Version 14 
 
2) Objectives 
1. Assess safety and tolerability of metformin in individuals with FXS ages 6 to 35 who are 
treated over a 4-month period (and an additional 2 years if they participate in the open label 
extension). 
2. Assess the benefit of metformin in the treatment of language deficits, behavior problems, 
and obesity/excessive appetite in individuals with FXS over a 4-month period (and an 
additional 2 years if they participate in the open label extension). 
3. Assess the utility of innovative outcome measures, including measures of brain processing 
(event related potential habituation paradigm and social gaze monitoring using the Tobii 
Eye Tracker), language/cognitive measures (NIH Toolbox cognitive battery measures and 
expressive language sampling), a measure of memory (The Memory Game), behavioral 
and quality of life measures [Aberrant Behavioral Checklist–Community (ABC-C) fragile 
X version, Child Sleep Habits Questionnaire (CSHQ), Anxiety Depression and Mood Scale 
(ADAMS) Swanson, Nolan and Pelham questionnaire (SNAP-IV), and Pediatric Quality 
of Life Questionnaire (PedsQL) Parent Proxy], EuroQol-5D (EQ-5D-3L or EQ-5D-Y), 
Sensory Profile-2 (up to 15 years old) or Sensory Profile Adolescents/Adults (11 years and 
older), and Care Related Quality of Life (CarerQoL)], a food questionnaire (Automated 
Self-Administered 24-Hour Dietary Assessment Tool (ASA24)), in addition to standard 
measures of outcome [Clinical Global Impression Scale-Severity (CGI-S), Clinical Global 
Impression Scale-Improvement (CGI-I), Visual Analog Scales (VAS), and Vineland 

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Adaptive Behavior Scales–Third Edition (for English speakers) and Second Edition for 
French speakers)]. 
4. Assess molecular biomarkers that may predict which patients may be likely to benefit from 
metformin treatment. A panel of molecular biomarkers, including FMRP level, MMP9, S6 
Kinase, EIF4E, CYFIP1 mRNA, CYP450 allelic variants, and metabolome variants, 
genomic variants, proteomics and mRNA sequencing will be studied. 
 
3) Background 
FXS is the leading monogenic cause of intellectual disability and autism spectrum disorder. 
Treatment for FXS is currently non-pharmacological or symptomatic interventions. FXS is 
associated with an increase in body mass index (McLennan et al., 2011), and a subgroup of patients 
with FXS develops severe hyperphagia, obesity, and hypogonadism or delayed puberty. This 
presentation has been described as the Prader-Willi phenotype of FXS (PWP) (Nowicki et al., 
2007), as it is similar to those with the Prader-Willi syndrome caused by a deletion at 15q11-q13. 
Unlike Prader-Willi syndrome, the FXS-PWP does not have a deletion of 15q11-q13 or 
chromosome 15 maternal uniparental disomy (McLennan et al., 2011). However, FMR1 protein 
(FMRP) binds to cytoplasmic interacting FMR1 protein (CYFIP1), a protein that affects synaptic 
remodeling, including FMRP protein translation (Schenck et al., 2001); CYFIP1 is localized to 
15q, the critical region in Prader-Willi-syndrome (Chai et al., 2003). The level of CYFIP1 mRNA 
expression has been found to be lower in those with the PWP compared to FXS without the PWP 
(Nowicki et al., 2007), so this appears to be a molecular correlate of the obesity in the PWP. One 
of the biomarkers to be evaluated in this proposal will be CYFIP1 mRNA expression levels that 
may change with metformin treatment in those with or without the PWP of FXS. 
 
Metformin is a type 2 diabetes medication that can also improve obesity and excessive 
appetite. The Food and Drug Administration approved metformin for its effects in lowering blood 
glucose levels in patients with noninsulin-dependent, type 2 diabetes. Some studies report minimal 
to moderate decrease in weight in groups of patients with insulin resistance (Klein et al., 2006). 
Metformin has been shown to be effective and safe in decreasing weight gain associated with 
atypical antipsychotic use and is well tolerated by children and adolescents with ASD aged 6-17 
years old, with up to 500mg twice daily for children aged 6-9 years and 850 mg twice daily for 
those 10-17 years old (Anagnostou et al., 2016). It has also been utilized for the treatment of obese 
children and adults who do not have type 2 diabetes with a short-term reduction in BMI (Park et 
al., 2009; Klein et al., 2006). Metformin is also effective in glycemic control in patients who are 
not obese (Ong et al., 2006). There is little to no risk of hypoglycemic episodes in the use of 
metformin because its mechanism of action does not directly stimulate insulin (Bolen et al., 2007; 
Bodmer et al., 2008).  
 

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Metformin has emerged as a candidate drug for the targeted treatment of FXS based on 
animal studies showing rescue in the FXS model (Monyak et al., 2016; Weisz et al., 2015; Gantois 
et al., 2017). The fly, Drosophila melanogaster, has a homologue of FMR1 called dfmr1. The 
Drosophila FXS model has shown that drosophila insulin-like peptide 2 (Dilp2) in insulin-
producing cells results in elevated insulin signaling via the PI3K/Akt/mTOR pathway. The 
dysregulated insulin signaling in this fly model of FXS leads to defects in circadian rhythm and 
short- and long-term memory deficits. Use of metformin in this study had been able to rescue and 
restore memory deficits (Monyak et al., 2016). Further studies performed in the Sonenberg lab 
(Gantois et al., 2017) showed that adult treatment in the FXS knock-out mouse rescued multiple 
phenotypes, 
including 
social 
novelty, 
grooming, 
dendritic 
spine 
morphology, 
and 
electrophysiology in eCA1 of the hippocampus. Metformin may contribute to normalizing 
signaling pathways in FXS in the central nervous system, which may include activities of ERK, 
mTOR and PI3K, which have shown to be pathogenically overactive in FXS.  
 
In addition, metformin inhibits phosphodiesterase, which would lead to correction of 
cAMP levels. Moreover, metformin inhibits MMP9 production, which is also elevated in FXS 
(Hoeffer et al., 2012; Muzar et al., 2016; Dziembowska et al., 2013), and MMP9 as a biomarker 
will be assessed in this proposal. Looking at the potential signaling pathways, metformin appears 
to be a good candidate for targeting several of the intracellular functions in neurons disrupted in 
FXS and therefore possibly rescuing several types of symptoms in individuals with FXS. 
 
Dr. Hagerman’s team (MIND Institute) has utilized metformin in the clinical treatment of 
over 20 individuals with FXS between the ages of 4 and 58 years and have found benefits not only 
in lowering weight gain and normalizing appetite, but also in language and behavior with doses up 
to 1000 mg twice a day (Dy et al., 2017). Dr. Hagerman’s team has recently reported in Dy et al. 
(2017) the details of the first 7 patients treated clinically for whom pre and post Aberrant Behavior 
Checklist results were obtained and documented improvement in weight, language, and behavior. 
This medication has been well tolerated, even in the 4-year-old patient with FXS, and it is time 
now to carry out a controlled trial.  
 
Considering these promising preliminary results and the established safety of metformin in 
children, we propose to carry out a randomized, placebo-controlled trial of metformin to further 
assess safety and benefits in the areas of language/cognition, eating, and overall behavior, in 
addition to weight loss. The trial will include a US site at the MIND Institute and two Canadian 
centers (University of Alberta and CHU Sainte-Justine).  
 
This study also provides an opportunity to carry out novel outcome measures that are capable 
of documenting improvements in the processing of information in the CNS (event related 
potentials (ERPs) and Tobii eye tracking), cognitive abilities (NIH Toolbox cognitive battery 
measures and expressive language sampling), critical biomarkers of involvement by the FMR1 

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mutation (CGG repeats, degree of methylation, and FMRP expression levels), and neurochemical 
changes in the CNS that can also be measured in blood and fibroblasts (MMP9, S6K, EiF4E, and 
CYFIP1). Finally, the cytochrome P450 (CYP) isozymes, highly polymorphic drug-metabolizing 
enzymes, have been demonstrated to be responsible for the metabolism of metformin in humans, 
mainly via CYP2C11, 2D1, and 3A1/2 (Choi and Lee, 2006). Thus, in this study we will assess if 
the CYP450 allelic variant genotypes involved in the metformin pharmacokinetics determine the 
efficiency of response to metformin. Since metformin absorption is done by active transport via 
the organic cation transport (OCT), we will also assess polymorphism in that gene. 
 
Leveraging the current RCT program and adding an open label extension. New information 
pointing toward the importance of considering a metformin open-label extension to our current 
randomized control trial of metformin in individuals with Fragile X syndrome (FXS) has emerged 
for several reasons. We are therefore adding a 2 year (24 months) optional extension of open-label 
metformin trial to all participants of our approved RCT of metformin in FXS.  
  
Rationale: 1) Based on reports from open-label trial papers from Dr. Hagerman’s group 
suggesting potential improvements in development, testicular size, and IQ in 11 patients with FXS, 
there are important long term tolerance and effects that need to be documented at the end of our 
controlled trial should the patients continue on an open-label treatment of metformin. 2) We know 
that metformin is well tolerated during short term treatments in a majority of individuals with FXS 
(from our RCT participants in Edmonton and at UC Davis). This supports the initial results 
observed in individuals with ASD (Dr. Anagnostou trial). In addition, a recent open label trial in 
Sherbrooke (QC) by Dr. Caku identified no adverse event and pointed toward the need for longer 
treatment (personal communication, paper in preparation). 3) There is a strong demand for 
an extension from families involved so far in our trial at all sites. Previous RCTs in fragile X were 
also followed by open labels in part because there was a strong demand from families who knew 
that they had a 50% chance of not receiving the active molecule (e.g. the NIH supported the 
Neuro NExt trial testing the effect of AFQ056 in a new combined paradigm (PI: Dr. Berry-Kravis) 
which also includes an open-label extension). 4) Importantly, adding the open-label extension to 
the trial allows significant cost-saving compared to a stand-alone project. Indeed, database, server, 
biobank, and pharmacy cost are not duplicated in this design.  
   
Scientific Merit: In addition to allowing all participants to have a chance to assess the effect 
of metformin, the extension would allow us to: 1) Evaluate potential variation in the long-term 
effect of metformin, particularly on cognition, which is the most important issue for the families. 
For instance from visit 1 to visit 3, we will measure potential change on the impact of metformin 
long-term treatment on our primary outcome measure (expressive language sampling), but also 
activity of daily living abilities (as measured by Vineland and IQ). 2) Leverage the baseline 
measures of psychological and biological biomarkers for all participants. This means that every 
participant will have the opportunity to be on metformin at some point in the trial within a 

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monitored framework and all patients will have baseline measures (from visit 1 of the RCT) which 
can be used as internal control. This is very important as most families want to test the effect on 
their children and will do so outside of the investigational framework if it is not made available to 
them. Some families will get it prescribed by their general practitioner for instance. This will lead 
to significant loss of data.  3) Provide additional longitudinal data on cognitive development, 
functional performance, the health economics and quality of life for individuals and families with 
fragile X syndrome with metformin. 4) Provide additional longitudinal biological samples 
(including brain activity) for each given individual with FXS at 1 and 2 years, allowing for a better 
understanding of potential changes in biomarkers over time. This would make the dataset unique 
and very useful in integrating the developmental origin of disease concept to the study of FXS. 
 
4) Inclusion and Exclusion Criteria 
Inclusion criteria: A subject will be eligible for study participation if subject meets all following 
criteria: 
1. Subject has Fragile X syndrome with a molecular genetic confirmation of the full FMR1 
mutation (>200 CGG repeats) or the other loss of function mutations of the FMR1 gene 
(SNVs and deletions of the gene).  
2. Subject is a male or non-pregnant, non-lactating female age 6 through 35 years, inclusive. 
3. Subjects who are capable of becoming pregnant must use an acceptable method of birth 
control for the duration of the study. Acceptable forms of birth control include abstinence 
(only for subjects who are not sexually active), intrauterine devices in place for at least 3 
months, oral contraceptives, surgical sterilization, or adequate barrier methods. 
4. Subject must have a caregiver (parent, guardian, or other legally authorized 
representative) who is willing to participate in the whole study.  
5. Subject and caregiver are able to attend the clinic regularly and reliably. 
6. Subject and/or subject’s caregiver is able to understand, read, write and speak English or 
French fluently to complete study-related materials. 
7. For subjects who are not their own legal guardian, subject’s caregiver is able to understand 
and sign an informed consent to participate in the study. 
8. The use of concomitant medication must be stable, in terms of dose and dosing regimen, 
for at least 4 weeks prior to Screening and must remain stable during the period between 
first visit (Screening) and the commencement of the study; every effort should be made 
to maintain stable regimens of allowed concomitant medications from the time of 
commencement of double-blind study medication until the last study assessment. 
9. Behavioral/educational treatments must be stable for 4 weeks prior to first visit 
(Screening) and must remain stable during the period between Screening and the 
commencement of randomized double-blind study medication. 

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10. Overall age equivalent is not higher than 13 and IQ is not higher than 85, as assessed at 
Screening on the Leiter-III, and subject must speak at least occasional 3-word phrases. 
Exclusion criteria: 
1. Families that are not cooperative and will not follow through with the demands of this 
study. 
2. Subject has a life-threatening medical problem or other major systemic illness that 
compromises health or safety and/or would interfere with this study. 
3. Age younger than 6 or older than 35 years. 
4. History of intolerable adverse events with metformin. 
5. Current or recent metformin treatment (within the past 4-months). 
6. BMI inferior to 2 standard deviations below the mean for age using the World Health 
Organization scale.  
7. Serum creatinine > 1.4 mg/dl (female) or > 1.5 mg/dl (male). 
8. History of metabolic acidosis or a condition with lactic acidosis. 
9. Severe Vitamin B12 deficiency. 
10. Pregnancy at screening or unwillingness to use acceptable method of birth control, if 
applicable. 
11. Age equivalent higher than 13 or IQ higher than 85 on the Leiter-III at Screening. 
No additional inclusion criteria will be assessed for the extension phase. Participants can choose 
to enter the extension phase after successfully completing the main study (randomized controlled 
trial - RCT). A washout between the RCT and extension phase will not be required. 
 
Inclusion criteria for the controls providing a stool sample: 
1. Able to provide a stool sample 
2. Able to read and write in English in order to complete a questionnaire (or have a 
parent/caregiver able to do this) 
3. Age 6-35 years old 
 
Exclusion criteria for the controls providing a stool sample: 
1. Diagnosis of Fragile X Syndrome 
2. Unable to provide a stool sample 
3. Younger than 6 or older than 35 years old 
4. Metformin Treatment within the past 4 months 
Consenting Process: 
Subjects will be recruited and consented at each site. The research coordinator will speak 
with both the subject and at least one caregiver to explain the research and associated study 
procedures and assess the subject’s understanding of the protocol. Subjects aged 6 will not provide 

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assent; consent will be obtained from the caregiver (Caregiver (Parent/ Legal Guardian) 
Consenting for Participants Information & Consent Form). For all subjects 7-35, cognitive ability 
will be assessed utilizing the Capacity Assessment Checklist for Informed Consent with 
Cognitively Impaired Subjects, also used at the MIND. Study coordinators will perform the initial 
capacity assessment, which will be reviewed with the study doctor before any other study 
procedures are done and signed by the study doctor. If appropriate based on cognitive age and 
ability, assent will be obtained from subjects aged 7-17; if the assent form is not appropriate for 
the subject’s cognitive age and ability, study staff will instead provide a letter of information that 
will be read to and reviewed with the subject. In the event of changes to the protocol that could 
possibly affect subjects’ safety or decision to participate in the study, current subjects and/or their 
caregiver will be informed of the changes and re-consented for continuing participation in the 
study. 
In addition to the assenting process described above, all subjects under the age of 18 will 
need to have their caregiver sign the consent form. Subjects 18 and older who are cognitively 
capable of providing consent (based on the Capacity Assessment Checklist for Informed Consent 
with Cognitively Impaired Subjects mentioned previously) will sign an informed consent form 
(Adult Participants and Caregiver Information and Consent). For subjects 18 or older at 
screening/baseline, or for those who turn 18 during the course of study participation, who are not 
cognitively capable of providing consent, a caregiver will need to sign the consent form used for 
subject with parents consenting (Caregiver (Parent/ Legal Guardian) Consenting for Participants 
Information & Consent Form). All potential subjects will attend their screening visit with a 
caregiver able to give surrogate consent.  
 
Separate consent forms (at University of Alberta) or separate signatures (at CHU Sainte-
Justine) for either subject or caregiverwill be filled or obtained for the optional biobanking of 
blood,urine (cell culture), and stool as well as for the optional skin biopsy.  
 
Extension Consent: The consent for the extension will be shown to participants/caregivers at 
the beginning of the trial and again at the end of the RCT component. A separate informed consent 
form and signature page for the extension will be signed for participation in the extension phase 
of the trial.  
 
Consent for controls providing a stool sample: There will be a separate consent form and 
signature page for this portion of the study. Adult participants will sign on their own behalf and 
parents/caregivers will sign on behalf of children. If appropriate based on cognitive age and 
ability, assent will be obtained from subjects aged 7-17. We will be asking family members of 
particpants in the trial to complete this portion of the study and we will also recruit controls 
through the Healthy Infants and Children's Clinical Research Program (HICCUP).  
 

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5) Study Timelines 
The study plans to enroll 60 participants over a 3-year period at each site. Approximately 
20 patients per year with FXS will be randomized at each site to receive either metformin or 
placebo for a 4-month period. During the 4-month study period, subjects will attend three visits to 
the recruiting site: screening/baseline, 2-month, and 4-month visits. In addition, routine phone calls 
will be made once per week during the first month of the study, with the week 4 phone call being 
conducted by the study physician, and once at week 12/month 3. 
 
Extension Visit schedule: Informed consent will be obtained either concomitantly to visit 3 
of the original schedule of the RCT portion or later if the participant finished the RCT already, 
but would like to enter the open-label extension. A follow up in-person visit will be done at 1 
year of the open-label trial, or before if needed, and another in-person visit will occur at 2 years. 
Scheduled follow up phone calls will be done weekly for the first month of the extension and 
then every 4 months. If a participant begins the extension study within 1 year of ending the RCT 
portion of the study, Visit 3 of the RCT will be considered to be Visit 1 of the extension and the 
participant would be seen at a 1 year follow up from when they had their Visit 3 of the RCT 
portion.  Those participants previously enrolled in the RCT who have been taking metformin off-
label for more than a year can be enrolled in the extension phase. These participants will have a 
‘first visit’ for the extension phase in addition to the previously-completed third visit for the RCT 
(as that visit occurred more than a year before starting the extension phase). Informed consent 
will be obtained at this ‘first visit’. 
6) Study Endpoints 
 
The primary outcome measure will be the Expressive Language Sampling (ELS) measure 
which is conducted at baseline and at the end of treatment, as described in detail in Section 7. The 
ADOS-2 and Leiter-III will be administered at baseline only for purposes of study population 
characterization and will not be repeated as outcome measures. All other measures will be 
secondary outcome measures and are also described in detail in Section 7. Attempts will be made 
to administer all secondary measures for each participant, to the extent his or her cognitive age and 
ability allows. Safety endpoints will include safety blood draws and physical exams at each study 
visits. 
For the extension, the primary outcome measure will remain the Expressive language 
sampling (ELS) measured at the 1 year and 2 year visits. This will provide a connection with the 
RCT primary outcome.  The extension secondary outcome measures will be measured at the 1 year 
and 2 year visits and will include: Leiter IQ test, Vineland (version 3 for English and version 2 for 
French), Memory Game (online), NIH Toolbox, CarerQol-Quality of life questionnaires (online), 
EQ-5D- Health economics questionnaires (online), Pediatric Quality of Life (PedsQL) Parent 

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Proxy Questionnaire (online), Aberrant Behavior Checklist–Community (ABC-C) (online), Visual 
Analogue Scale (VAS) (online), Swanson, Nolan and Pelham Questionnaire (SNAP-IV) (online), 
Clinical Global Impression Scale–Improvement (CGI-I) (online), Anxiety Depression and Mood 
Scale (ADAMS) (online), Sensory Profile-2 or Sensory Profile Adolescent/Adult, Child Sleep 
Habits Questionnaire (CSHQ) (online), Automated Self-Administered 24-Hour Dietary 
Assessment Tool (ASA24), biological biomarkers (blood, stool, and urine), resting EEG and event 
related potentials (ERPs), and eye tracking. 
 
7) Procedures Involved 
 
At the first visit, baseline testing will include IQ testing with the Leiter-III and assessment 
for autism with the Autism Diagnostic Observation Scale (ADOS-2). Cognitive ability will be 
derived from the Leiter-III, only at baseline. We have shown that full mutation males often perform 
at the floor of standardized intelligence tests, such as the Wechsler Scales of intelligence, which 
severely restricts test sensitivity and variability, and therefore presents major limitations in 
analyses examining genotype-phenotype associations. The primary reason that we have chosen the 
Leiter-III is because it is a non-verbal test, and it can be used even with low-functioning patients 
who have little or no language. The ADOS-2 (Lord et al., 2000) is a semi-structured standardized 
assessment administered directly for the purposes of diagnosing ASD, and will be done only at 
baseline. The ADOS-2 uses developmentally appropriate social and object-based interactions in a 
30–45 minute assessment to elicit symptoms of ASD in four areas: social interaction, 
communication, play, and repetitive, restrictive behaviors. The ADOS-2 consists of different 
modules, each directed at a particular level of language ability, and is thus appropriate to use across 
subjects of varying ages and functioning levels. 
 
The CGI-S, VAS, ABC-C, ADAMS, CSHQ, SNAP-IV, PedsQL, EQ-5D, CarerQoL, 
Sensory Profile-2 or Sensory Profile Adolescents/Adults, ASA24, and the memory game will be 
completed.  
 
Blood will also be drawn for safety laboratory tests, including a hemoglobin A1c 
(HgbA1c), blood glucose, complete blood count (CBC), vitamin B12 level, comprehensive 
metabolic panel (CMP), cholesterol, renal clearance function, blood pregnancy test for female 
participants in childbearing age, along with biomarker studies described below. A small skin 
biopsy (optional procedure) will be obtained from the back of the shoulder, utilizing a small punch 
biopsy after numbing the skin with lidocaine or EMLA cream, so that fibroblasts can be grown on 
each patient at baseline to determine the level of Fragile X Mental Retardation Protein (FMRP), 
the presence of tissue mosaicism, and other studies described below. At the first visit, a detailed 
medical history and physical and neurological examination will be carried out, with all medications 
and medical problems documented. A urine and stool sample will also be collected. 
 

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Patients will be randomized to either metformin or placebo by the Alberta Health Services 
Pharmacy Research Office and by the Service pharmaceutique de support à la recherche (SPSR) 
of CHU Sainte-Justine (University of Montreal) for their participants after the baseline studies are 
carried out, as detailed below. For patients 50 kg and over, the initial dose will be 500 mg once a 
day at dinner by mouth, and then increasing each week by 500 mg until a maximum dose of 2000 
mg daily is reached. For those patients who are less than 50 kg, the initial dose will be 250 mg 
once a day at dinner by mouth, and if this dose is well tolerated, they will increase each week by 
250 mg until a maximum dose of 1000 mg daily is reached. As such, the maximum dose of 
metformin in patients under 50 kg will be 1000 mg daily. We will recommend all patients to take 
a multiple vitamin that has B12 to avoid potential anemia. In the first 4 weeks following visit 1, 
each patient will receive a weekly call to evaluate tolerability of the medication, any adverse events 
(AEs), changes in concomitant medications, and gradual titration of the metformin. If potentially 
related minor AEs such as diarrhea or gastrointestinal pain are experienced, the dose can be 
lowered, or medication held until clinically indicated to resume the trial. 
 
At 2 months (Week 8 +/- 7 days, scheduled visit 2), when at their maximum tolerated dose, 
they will be seen in clinic for an examination, fasting safety labs (consisting of the lipid panel 
which includes cholesterol, LDL, and triglycerides; complete blood count; and comprehensive 
metabolic panel),  urine,  biomarkers, assessment of AEs, and limited studies, including a CGI-I, 
VAS, and behavioral and quality of life questionnaires (ABC-C, ADAMS, CSHQ, SNAP-IV, 
PedsQL, EQ-5D, Sensory Profile-2 or Sensory Profile Adolescents/Adults, and CarerQoL). We 
will require 8 hours of fasting prior to the visit 2 blood draw. Participants will only be allowed 
water during the fasting period. 
 
Subjects will remain at the designated dose through Visit 3 (at Week 16/end of treatment 
+/-7 days) and will be contacted at week12 by study staff via phone to assess for possible adverse 
events, changes in concomitant medications, and verification of proper dosing. Study participants 
will be dispensed study medication at Visits 1 and 2 and will discontinue dosing after completing 
Visit 3. Any change in medication or new medication will also be documented. The examination, 
safety labs, biomarkers, urine and stool collection, documentation of AEs, and outcome measures 
will be repeated at the final follow-up visit at 4 months/end of treatment. We will also record any 
seizure activity that occurs throughout the study using a seizure diary (which the 
participant/caregiver will take home) where the date, time, seizure type, duration, whether the 
participant was awake or asleep during the seizure, use of baseline and abortive medication, as 
well as any changes to medication. We will also suggest to families to attempt to video record the 
episodes when possible (on a phone or other camera). They will be asked to show the study doctor 
or their primary care physician the video as part of standard of care for patients. We will not retain 
a copy of the video.  
 

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Remote visits may be utilized for visit 2 if deemed appropriate by the PI. It will save on 
travel cost and burden to families. When this is the case, the urine dipstick and research blood 
work will not be performed. Questionnaires will be sent to the family and the physical exam will 
be performed virtually via tele/videoconference, for example via Zoom. Study medication will be 
mailed to participants. 
 
Metformin Titration Schedule ( < 50 kg) 
 
Week 1 
Week 2 
Week 3 
Weeks 4-16 
Morning Dose 
 
    250mg 
250mg 
500mg 
Evening Dose 
     250mg 
    250mg 
500mg 
500mg 
Total daily dose 
     250mg 
    500mg 
       750mg 
  1,000mg 
 
Metformin Titration Schedule ( ≥ 50 kg) 
Week 1 
Week 2 
Week 3 
Weeks 4-16 
Morning Dose 
 
500mg 
500mg 
1,000mg 
Evening Dose 
500mg 
500mg 
    1,000mg 
1,000mg 
Total daily dose 
500mg 
    1,000mg 
    1,500mg 
2,000mg 
 
Optional open label Metformin extension treatment regimen:  We will use the same 
treatment regimen as for the RCT portion of the trial. The extension component would begin after 
the end of the 4 month RCT. It will include a progressive ramping up of metformin over 4 weeks 
based on weight as for the RCT. Treatment will be based on weight (measured at the time of 
entering the open label phase and can be adjusted during the 2 year period based on change in 
weight) as shown below:  
  
Metformin Titration Schedule ( < 50 kg)  
 
Week 1  
Week 2  
Week 3  
Weeks 4-104  
Morning Dose  
 
250mg  
250mg  
500mg  
Evening Dose  
250mg  
250mg  
500mg  
500mg  
Total daily dose  
250mg  
500mg  
750mg  
1,000mg  
   
   
Metformin Titration Schedule ( ≥ 50 kg)  
 
Week 1  
Week 2  
Week 3  
Weeks 4-104  
Morning Dose  
 
500mg  
500mg  
1,000mg  
Evening Dose  
500mg  
500mg  
1,000mg  
1,000mg  
Total daily dose  
500mg  
1,000mg  
1,500mg  
2,000mg  
   

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To maintain consistency with the trial and our current Health Canada approval, 
metformin 500mg (Sandoz) will be used for all participants. Medication will be dispensed at visit 
1 and then every 3 months either in person or via secure mail service. Participants that have been 
taking off-label metformin after completing the RCT (but before being given the opportunity to 
join the extension phase) will not be required to ramp up the dosage of the drug. 
 
For the controls providing a stool sample: Participants will only be asked to provide a stool 
sample and complete the ASA24 questionnaire as well as provide information about 
medcations/supplements they are on.  
Treatment Measures:  
Expressive Language Sampling (ELS) – Expressive language samples will be collected 
from each participant twice, at study baseline and at the end of treatment/week 16. At each 
assessment, samples will be collected in two different contexts: conversation and narration. In 
conversation, the examiner engages the participant in talk on a variety of topics (e.g., school) 
according to guidelines that specify the order of topics and the ways in which topics are introduced 
and maintained. In narration, the participant tells the story in a wordless picture book. The 
examiner prompts and responses are scripted. In both contexts, the examiner follows a script that 
minimizes his/her participation, maximizes the participant’s contribution, and avoids the use of 
examiner language that would constrain the participant’s talk. In both conversation and narration, 
all talk is digitally recorded and transcribed by highly experienced transcriptionists following 
procedures that yield high intertranscriber agreement (Abbeduto et al., 1995). Computerized 
analyses of the transcripts characterize multiple dimensions of the talk produced by the participant 
(e.g., vocabulary diversity, syntactic complexity). The mean Number of Different Words (NDW) 
in conversation and in narration will be computed (and statistically adjusted through analysis of 
covariance for differences in talkativeness as suggested by Conners et al., 2018) and serve as the 
primary outcome measure. This measure has been shown to have excellent test-retest reliability, 
display negligible practice effects, be free of floor and ceiling effects, have good construct validity 
for individuals with FXS, and improve with treatment (Berry-Kravis et al., 2013; Channell et al., 
2018; McDuffie et al., 2016). For French speaking participants, we will use a French translation 
of the ELS. 
Clinical Global Impression Scales of Severity (CGI-S) and Improvement (CGI-I) – These 
scales are standard assessment for medication studies because it allows the clinician to utilize the 
history from the parent or caretaker and incorporate it into a clinical rating for the clinical follow 
up of the patient through the treatment trial. In the initial evaluation of the patient, we will use the 
CGI-S (severity) to judge the severity of the symptoms with a scale of normal, not at all ill; 
borderline ill; mildly ill; moderately ill; markedly ill; severely ill; or among the most extremely ill. 
The CGI-I scale will be utilized at the week 8 and end of treatment/week 16 follow-up visits. We 

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will use with CGI-I to look at improvement or worsening of symptoms with a scale of very much 
improved; much improved; minimal improvement; no improvement; minimally worse; or very 
much worse (Guy, 1976; Psychopharmacology, 1985). 
Eye Tracking Measures: Social Gaze and Pupillometry – For individuals with FXS, we 
have demonstrated that the social gaze measure shows decreased visual fixations on the eye region 
while viewing human faces (with greater fixation to the nose region), and these individuals show 
abnormal pupillary dilation, an indication of sympathetic nervous system reactivity, compared 
with controls (Farzin et al., 2009). While these measures do not appear to map directly onto 
caregiver-reported anxiety of the individuals level of anxiety, the results are consistent with high 
rates of social anxiety (Cordeiro et al., 2011) and sympathetic reactivity in FXS (Miller et al., 
1999). In a subsequent replication study, we showed that the gaze abnormalities (fixation to eye 
region, pupillary reactivity) are stable in FXS across a 9-10 day period, with intraclass correlations 
of 0.70–0.91 for pupillary response to faces and 0.94 for proportion of looking time to the eye 
region. All stimuli are presented on a Tobii T120 binocular eye-tracker monitor (Tobii Technology 
AB, Sweden). This eye-tracking system consists of a high-resolution camera embedded in a 17-
inch TFT monitor (1280 X 1024 pixels resolution, 120 Hz sampling rate, average precision of 0.5 
degrees of visual angle). The Tobii system has several benefits that make it conducive to testing 
individuals with developmental disorders, including approximately 30 cm of tolerance to head-
motion in any direction without requiring any head apparatus or restraints. Stimuli consist of sixty 
colored photographs of adult human faces (equal numbers of males and females, different races 
and ethnicities) from the NimStim Face Stimulus Set (Tottenham et al., 2009), each showing a 
calm, happy, or fearful expression, and sixty scrambled versions of the face images. Since it is 
critical that pupil responses following the onset of the face stimulus be independent of a pupillary 
light reflex, each face and its scramble are matched on mean luminance confirmed using a 
photometer (Minolta, LS-100, Osaka, Japan). Face images subtend a 12.12 degree by 17.19 degree 
region (the size of an actual human face) when viewed from a distance of 60 cm, and are presented 
on a standard 50% gray background. Percent duration of fixation to the eye, nose, mouth, and other 
regions are calculated for each participant, as well as mean pupil size calculated for each 250 ms 
interval. 
 
This assessment will be administered twice, at study baseline and at the end of 
treatment/week 16. 
 
Event Related Potential (ERP) Measures – The ERP measures described below will be 
carried out twice, at study baseline and at the end of treatment/week 16. We will use the 3000-
0751 this is a 128 channel GES300 system. This is already approved by Health Canada 
(https://www.egi.com/images/stories/company/documents/Health_Canada_GES_300_issued_20
14_03_11.pdf) 
 

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In a recently published study (Schneider et al., 2013), researchers were able to obtain EEG 
recordings and ERP responses during a passive auditory oddball paradigm from 12 patients with 
FXS enrolled in a controlled trial of minocycline (4 females, 8 males, mean age 10.5 years, SD 
3.7) at baseline, 3 months, and after 6 months at the end of the trial. Current source density (CSD) 
and ERP analysis at baseline showed high amplitude, long latency components in the temporal 
regions. After 3 months of treatment with minocycline, the temporal N100 and P200 amplitudes 
were significantly reduced. There was a significant amplitude increase at the Cz electrode position 
on minocycline treatment. Electrocortical habituation to the auditory stimuli was improved with 
minocycline treatment (Figure 1). This preliminary study demonstrated the potential feasibility 
and sensitivity of ERPs as an indicator of cortical processing changes in a targeted treatment trial. 
It appears to provide a biomarker and measure for the human equivalent of cortical 
hyperexcitability. 
 
 
 
 
 
The ERP method to be followed in this study is as follows. Subjects are presented with an 
auditory oddball paradigm using Presentation software (Neurobehavioral Systems, Albany, CA). 
The auditory stimuli are sinusoidal tones with frequencies of 1000 Hz (standard tone) and 2000 
Hz (target/oddball). The tones have a 10ms rise/fall, 50ms plateau, and a sound pressure intensity 
of 70 dB. The randomized order of both auditory stimuli consists of first six 1000 Hz standard 
tones, then one target tone (2000 Hz) either at 7th, 8th, 9th, or 10th position, with standard tones 
presented in the remaining positions. The tones are presented with a consistent interstimulus 
interval (ISI) of 1000ms over stereo speakers. Standard stimuli are presented 80% of the time and 
deviant stimuli are presented 20% of the time. The auditory oddball task includes a total of 240 
standard and 60 deviant stimuli, which are presented in three blocks, each containing 80 standards 
and 20 deviants. After a 3-minute resting phase, the passive auditory oddball paradigm will be 
administered, and then following the experimental compliance and depending on the functioning 
level of the individual, the auditory oddball paradigm will be administered as an active paradigm, 
requiring a button press for the deviant stimuli. This behavioral data can be used for an analysis of 
Cz
-100 0
100 200 300 400 500
-10
-5
0
5
10
Baseline
Cz
-100 0
100 200 300 400 500
-10
-5
0
5
10
Placebo
Cz
-100 0
100 200 300 400 500
-10
-5
0
5
10
first 45 
last 45
Minocycline 
N1
N1
N1
P2
P2
P2
p .012
0
2
4
6
8
Baseline
Placebo
MINO
-8
-6
-4
-2
0
Cz
p .002
N1
P2
a)
b)
mV
first 45
last 45
first 45
last 45
Cz
mV
mV
mV
mV
ms
ms
ms
Figure 1. (a) Grand average 
waveforms at Cz, highlighted in 
yellow the N1 component, the P2 
component in blue. Black line 
signifies first 45 stimuli, the red 
line last 45 stimuli potentials. 
Negative plotted upwards. (b) 
Significantly 
improved 
attenuation of N1 and P2 on 
minocycline 
condition, 
not 
significant for baseline and 
placebo. 

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attentional performance (Van der Molen et al., 2012). Following the experimental compliance, 
auditory steady-state of a 1000Hz tone at 6 and 40 Hz (3 min task) and visual steady state of 
abstract shapes at 6, 15 Hz (2.5 min tasks) will be administered.  Before the experiment, the 
subjects pick a favorite movie, which is shown during the preparation and the oddball task. The 
movie is required to provide a comforting environment for the patients and provide a fixation point 
for their eyes. During the oddball paradigm, the movie is muted. Before the experiment, we collect 
2 minutes of resting EEG, and in compliant subjects, we include an Alpha-block paradigm with 
four 30-second blocks of alternating eyes-open and eyes-closed continuous EEG recording.  
In addition to the oddball paradigm, we will administer a recently developed task looking 
at EEG habituation to pseudowords to assess word learning with EEG (Knoth et al., 2018). Fifty 
pseudowords will be successively repeated 6 times each with an inter-stimulus interval of 1000ms. 
Each sequence of pseudowords appears in randomized order. 
 
Throughout the protocol, positive reinforcement through age appropriate gifts (stickers 
and/or reward sheet) will be provided. 
 
EEG data is acquired using a 72-channel (or 128-chanel modified) Brainproducts 
Quickamp system with an Acticap 32-channel Ag+/Ag+Cl- active EEG electrode array 
[International 10-20 system, positions (Fp1, Fp2, F7, F3, Fz, F4, F8, FC5, FC1, FC2, FC6, T7, C3, 
Cz, C4, T8, TP9, CP5, CP1, CP2, CP6, TP10, P7, P3, Pz, P4, P8, PO9, O1, Oz, O2, PO10] using 
a common average reference and a ground electrode positioned between Fz and Pz sites. Electrode 
impedances are maintained below 10 kΩ and electrical activities amplified and recorded with Brain 
Vision Recorder and Quickamp® amplifier (Brain Products, Germany). During the recording, 
bandpass filters are set at 0.3–100 Hz, and data are digitized continuously at 250-500 Hz. Raw 
data is than imported into Brainvision Analyzer software (Version 2.01) for analysis. 
 
The continuous data is segmented according to the event type (standard or target tone with 
a 1000ms time window, -100ms before the event until 900ms after the event) and filtered 
(Butterworth Zero Phase Filters with low cutoff 0.5 Hz, time constant 0.3, 12 dB/oct, high cutoff: 
80 Hz, 12 dB/oct, a notch filter is not included because of the active shield technology). 
 
For artifact rejection, we define the maximal allowed voltage step in a segment to 50 
μV/ms, with a maximal allowed difference of values in intervals of 1000 μV, minimal allowed 
amplitude -500 μV, maximal allowed amplitude: 500 μV, lowest allowed activity in intervals 0.5 
μV. For the detection and correction of blinks we use the electrode sites Fp1 and Fp2 as source for 
an independent component analysis (ICA) Infomax restricted slope algorithm. The components 
relevant for vertical activity are selected by computing the global field power. The number of ICA 
steps and convergence bound will be selected individually according to the quality of the data; in 
general, the ocular correction ICA converged between 90–120 steps, with the last step’s matrix 
modification usually smaller than 9.575E-08. In general, there was a loss of 6% of all trials in both 

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control and FXS participants. We exclude participants without a sufficient number of artifact free 
trials (>30). ERPs are baseline corrected using the 100 ms prestimulus interval and averaged for 
standards and target tones separately. Peak amplitude and latency of the N100, P200, N200, and 
P300 components are determined at the Fz, F3, F4, Cz, C3, C4, Pz, P3, and P4 electrode positions 
by the largest voltage deflection within the 1000ms time window relative to stimulus onset, 
depending on the specific latency range for each component (N100 = 50-150ms, P200 = 150-
250ms, N200 = 150-250ms, P300 = 250-400ms). The peak detection is performed 
semiautomatically, and a large voltage deflection also determined as a peak if it is outside the 
predefined latency range. 
 
We also analyze the EEG data at resting state for the oddball paradigm according to 
frequency distribution and the occurrence of spontaneous oscillations, and pseudoword habituation 
with ERP (similar to oddball), energy and time-frequency analyses (Rigoulot et al, 2017; Knoth et 
al., 2018).  
NIH Toolbox Measures, Cognitive Battery – The following set of measures will be 
administered at the first visit and end of treatment/week 16: 
 
Picture Sequence Memory [PSM; Episodic Memory; (Bauer et al., 2013)] – This 
test involves recalling increasingly lengthy series of illustrated objects and activities around 
different themes (e.g., “playing at the park,” “working on the farm”) that are presented in 
a particular order on the screen. For each trial, pictures appear in the center of the computer 
screen and then are moved one at a time into a fixed spatial order, as an audio file 
simultaneously describes the content of each (e.g., “Plant the tomatoes”) and until the entire 
sequence is displayed on the screen. Then the pictures return to the center of the screen in 
a random display and the participant moves them into the sequence that was shown. The 
score is derived from the cumulative number of adjacent pairs of pictures remembered 
correctly over 2-3 learning trials. Level of task difficulty is adjusted for the various age 
groups. Administration time is about 10 minutes. Encoding, storage, and retrieval of 
episodic memories depend on a neural network including the temporal lobe (especially the 
hippocampus), the prefrontal cortex, and limbic/temporal association areas (Eichenbaum, 
2001; Zola and Squire, 2000). Patients with FXS perform approximately two standard 
deviations below normal on PSM, which demonstrates good feasibility, test-retest 
reliability [ICC= 0.76 in both healthy children and those with ID; (Hessl et al., 2016)] and 
moderate correlation with FSIQ. Although this is not a major relative weakness, compared 
to their executive function (EF) deficits, we note that Fmr1 knock-out mice demonstrate 
memory deficits and hippocampal dependent long term depression (LTD) that are rescued 
by metformin in the Sonenberg studies and by the CB1 receptor antagonist/inverse agonist 
rimonabant. We therefore included PSM in the current battery. 

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Flanker Inhibitory Control and Attention Test (Zelazo et al., 2013) – Flanker is a 
measure of inhibition and visual attention. On each trial, a central directional target (fish 
for mental age younger than 8, arrows for ages 8 and older) is flanked by similar stimuli 
on the left and right. The participant chooses the direction of the central stimulus. On 
congruent trials, the flankers face the same direction as the target. On incongruent trials, 
they face the opposite direction. A scoring algorithm integrates accuracy, a suitable 
measure in early childhood/low mental ages, and reaction time, a measure more relevant 
to adult performance on this task, yielding computed scores from 0 to 10. There are 40 
trials, and the test duration is about 4 minutes. Patients with FXS demonstrate profound 
deficits on Flanker, performing about 7 standard deviations below normal, and significantly 
worse than IQ-matched controls with Down syndrome (Hessl et al., 2016). This test 
demonstrates excellent test-retest reliability (ICC=0.94) and correlates significantly with 
FSIQ and dialing functioning in children and adolescents with ID. Like the Dimensional 
Change Card Sort test describe below, Flanker is an EF task dependent on prefrontal cortex 
activity. Because EF deficits are consistently observed in patients with FXS, significantly 
affecting their daily functioning, we predicted that metformin, if it normalizes aspects of 
brain function in the disorder, would improve EF as demonstrated in the KO mice in the 
Sonenberg lab. 
 
Dimensional Change Card Sort Test (Zelazo et al., 2013) – Dimensional Change 
Card Sort Test is a measure of cognitive flexibility. Two target pictures are presented that 
vary along two dimensions (i.e., shape and color). Participants are asked to match a series 
of bivalent test pictures (e.g., yellow balls and blue trucks) to the target pictures, first 
according to one dimension (e.g., color) and then, after a number of trials, according to the 
other dimension (e.g., shape). “Switch” trials are also employed, in which the participant 
must change the dimension being matched. For example, after four trials matching on 
shape, the participant is asked to match on color in the next trial and then switch back to 
matching by shape. Scoring is based on a combination of accuracy and reaction time 
(computed score, ranging from 0–10), and the test duration is about 4 minutes. Dimensional 
Change Card Sort Test demonstrates good test-retest reliability (ICC=0.74) and correlates 
well with FSIQ (r=0.66) in children and adolescents with ID, including FXS (Hessl et al., 
2016). Aside from having general EF deficits, individuals with FXS have notable 
impairments in cognitive flexibility and show perseveration in response patterns (Abbeduto 
and Hagerman, 1997; Hooper et al., 2008). 
 
List Sorting Working Memory Test (Zelazo et al., 2013) – This test requires 
immediate recall and sequencing of different visually and orally presented stimuli. Pictures 
of different foods and animals are displayed with accompanying audio recording and 
written text (e.g., “elephant”), and the participant is asked to state the items in size order 
from smallest to largest, first within a single dimension (either animals or foods, called 1-

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List) and then on 2 dimensions (foods, then animals, called 2-List). The raw score is the 
number of items recalled and sequenced correctly, and the test takes approximately 7 
minutes to administer. 
 
Pattern Comparison Processing Speed Test (Zelazo et al., 2013) – This test 
measures speed of processing by asking participants to discern whether two side-by-side 
pictures are the same or not the same by touching “yes” or “no” (or a happy or frowning 
face for lower mental age). Participants’ raw score is the number of items correct in a 90-
second period. The items are designed to be simple to distinguish. The test takes 
approximately 3 minutes to administer. 
 
Oral Reading Recognition Test (Zelazo et al., 2013) – The participant is asked to 
read and pronounce letters and words as accurately as possible. The test administrator 
scores them as right or wrong. The items are administered by computer adaptive testing 
(CAT; continuously adapted depending on performance), and participant responses are 
scored by the examiner. For the youngest children, the initial items require identification 
of letters (as opposed to symbols) and identification of a specific letter in an array of 4 
symbols. The test requires approximately 3 minutes. A theta score is calculated for this 
test. 
Picture Vocabulary Test (Zelazo et al., 2013) – This measure of receptive 
vocabulary is administered in a CAT format. The respondent is presented with an audio 
recording of a word and four photographs on the screen and is asked to select the picture 
that most closely matches the meaning of the word. The test takes approximately 4 minutes 
to administer. A theta score is calculated for this test. 
 
Speeded Matching Test (introduced in 2018) – This test measures speed of 
processing by asking participants to discern whether four side-by-side pictures match the 
target image at the top of the screen. The items are presented one group at a time on the 
iPad screen, and the participant is given 119.999 seconds of actual presentation time 
(excluding transitions between items) to respond to as many items as possible (up to a 
maximum of 130). The items are designed to be simple so as to most purely measure 
processing speed. Speeded Matching gives a raw score, which is the number of items the 
participant correctly responds to (up to 130) in 119.999 seconds. Overall, the test takes 
approximately 3 minutes to administer. 
 
Because the NIH toolbox has not been validated in French, we will use the original English 
version and only administer the non-verbal tasks : Flanker inhibitory control, Dimensional card 
change sort task, and Pattern comparison processing speed. We will say the instructions verbally 
to the participants, in French.  

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Visual Analogue Scale (VAS) – Parents will mark on a visual line measuring 10 cm with 
one side marked “worst behavior” and the other side marked “best behavior.” They will mark three 
key behaviors that we are targeting with this study: behavior problems (parents can note which 
problems are most severe and track on them throughout the study), language abilities, and eating 
behavior. For each behavior, they will mark their impression of the behavior at baseline and during 
the follow-up visits at week 8 and end of treatment/week 16. The horizontal marks are measure in 
centimeter distance where they fall from the worst behavior side so that we can see improvements 
or worsening of behavior over this time period. 
 
 
Vineland Adaptive Behavior Scales–Third Edition (VABS-III) – The Vineland, which is a 
gold standard test for assessing adaptive behavior that is widely used in clinical trials, will be 
administered to the parent/caregiver at baseline and end of treatment/week 16. Subtests include 
Communication, Daily Living Skills, Socialization, Motor Skills, and Adaptive Behavior 
Composite. The Vineland has been normed for individuals with intellectual disability and ASD. 
The third edition includes updated item content to streamline similar items and reduce redundancy, 
to reflect changes in daily living (e.g., technology) and in conceptions of developmental disabilities 
(e.g., ASD), and to allow for potential cultural differences by using more generalized wording of 
certain items. French participants will perform the Vineland II  Échelles de comportement adaptatif 
as version III is not available in French.  
The Aberrant Behavior Checklist – Community Edition (ABC-C), scored using the FXS-
specific factoring system [ABC-FX; (Sansone et al., 2012)] – This measure will be completed by 
the parent/caregiver at the first visit (baseline), week 8, and end of treatment/week 16. This 
parent/caregiver report measure is the gold standard measure of problem and interfering behaviors 
in clinical trials in developmental disabilities (Aman et al., 1995; Kerr et al., 2015). The ABC is 
actively used in over 70 countries and has been translated into over 30 languages, including 
Spanish and French. The ABC asks responders to rate behaviors from 0 “not a problem at all” to 
3 “the problem is severe in degree” across 58 questions. Its use has been validated in a variety of 
clinical populations, including in ASD and FXS, has been used extensively in clinical trials, and 
is a Health Canada and FDA-vetted endpoint used in the Health Canada and FDA approvals for 
use of risperidone and aripiprazole targeting irritability in youth with ASD (Owen et al., 2009). It 
has been subjected to utility analysis in FXS and linked to caregiver stress in families (Kerr et al., 
2015; Bailey et al., 2012). Scores will be analyzed using the FXS-specific factor structure such 
that 54 of the items resolve into 6 subscales (irritability, lethargy, social avoidance, stereotypic 
behavior, hyperactivity, and inappropriate speech) (Sansone et al., 2012). For French speaking 
participants we will use Questionnaire sur les comportements anormaux. Version 28 Jan 1997. 
 
Child Sleep Habits Questionnaire (CSHQ) – This measure consists of a series of questions 
relating to the sleep habits of children. It will be completed by caregivers of all subjects, regardless 

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of age, at baseline, week 8, and end of treatment/week 16. For French speaking participants we 
will use Questionnaire sur les habitudes de sommeil de l’enfant (enfants d’âge préscolaire et 
scolaire) Non validé. 
 
The Anxiety Depression and Mood Screen (ADAMS) – This measure is an informant 
report, 28-item questionnaire, that serves as a screening instrument to determine anxiety, mood, 
and depression among individuals with intellectual disability. The respondent is asked to rate the 
extent to which their child displays target symptoms  (mild/moderate/severe/profound structure). 
The ADAMS yields 5 subscale scores: General Anxiety, Social Avoidance, Depression, 
Manic/Hyperactive and Obsessive/Compulsive Behavior.  It was psychometrically evaluated and 
normed using 265 individuals and validated with 129 psychiatric patients with ID (Esbensen et al. 
2003). It will be completed at baseline, week 8, and end of treatment/week 16. For French speaking 
participants we will use Anxiété Dépression et troubles de l’humeur Non validé. 
 
Swanson, Nolan and Pelham Questionnaire (SNAP-IV) – This measure, based on DSM-V 
criteria for ADHD, is a caregiver-rated questionnaire that effectively identifies those with and 
without ADHD and accurately predicts presentation specifier (inattention, hyperactivity/ 
impulsivity, and combined). Its psychometric properties and clinical utility have been 
demonstrated in multiple studies since its introduction in 2001, and it has been found to be reliable 
and well validated with normative data from both parents and teachers. It will be completed by 
caregivers of all subjects at baseline, week 8, and end of treatment/week 16. For French speaking 
participants we will use SNAP-IV version Fr April 2008. 
 
Pediatric Quality of Life Questionnaire (PedsQL) Parent Proxy – This measure consists of 
a series of questions relating to a child’s quality of life and is administered to the caregiver of the 
child.  The parent proxy module designed for children 8-12 years of age will be administered to 
the caregivers of all subjects, regardless of age, because the questions therein are most appropriate 
for the overall study population’s cognitive age and ability. It will be completed at baseline, week 
8, and end of treatment/week 16. For any subjects not in school, questions pertaining to “school” 
will be replaced with references to “work” or other activities in their life. For French speaking 
participants we will use PedsQL : inventaire de la qualité de vie pédiatrique. 
 
EuroQol-5D (EQ-5D) – This is a standardized instrument developed by the EuroQol 
Group as a measure of health-related quality of life that can be used in a wide range of health 
conditions and treatments. The EQ-5D consists of a descriptive system and the EQ VAS. We will 
use the EQ-5D-Y, EQ-5D-Y parent proxy, EQ-5D-3L, and EQ-5D-3L parent proxy depending on 
the participants age and ability. 
The descriptive system comprises five dimensions: mobility, self-care, usual activities, 
pain/discomfort and anxiety/depression. The EQ VAS records the patient’s self-rated health on a 

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vertical visual analogue scale. This can be used as a quantitative measure of health outcome that 
reflects the patient’s own judgement. The scores on these five dimensions can be presented as a 
health profile or can be converted to a single summary index number (utility) reflecting 
preferability compared to other health profiles.  
Care Related Quality of Life (CarerQoL) – The CarerQol was designed to measure and 
value the impact of providing informal care on carers. It combines a subjective burden measure 
that provides a comprehensive description of the caregiving situation (CarerQol-7D) with a 
valuation of informal care in terms of well-being (CarerQol-VAS).  
 
Automated Self-Administered 24-Hour Dietary Assessment Tool (ASA24)  – This is a free, 
web-based tool that enables multiple, automatically coded, self-administered 24-hour diet recalls 
and/or single or multi-day food records, also known as food diaries. The ASA24 system consists 
of a respondent website used to collect dietary intake data and a researcher website used to manage 
study logistics and obtain nutrient and food group data files. We will collect information related 
to food intake for the 24 hours prior to when the participant provides a stool sample.  
 
Sensory Profile-2 or Sensory Profile Adolescents/Adults – The Sensory Profile 2 family of 
assessments evaluates a child’s sensory processing patterns in the context of everyday life. The 
forms are completed by caregivers, who are in the strongest position to observe the child’s response 
to sensory interactions that occur throughout the day. The Adolescent/Adult Sensory Profile was 
developed to assess sensory processing abilities in individuals beyond childhood. Applicable in a 
variety of typical settings, such as schools, clinics, and programs that use client-centered practices, 
it is designed to promote self-evaluation of behavioural responses to everyday sensory experiences. 
The profile is most appropriate for individuals 11 to 65+ years of age and is intended as a trait 
measure of sensory processing. Questions are answered in a way how an individual generally 
responds to sensations, as opposed to how he or she responds at any given time. This enables the 
instrument to capture the more stable and enduring sensory processing preferences of an 
individual. The assessment tool includes 60 items, covering the sensory processing categories of 
Taste/Smell, Movement, Visual, Touch, Activity Level, and Auditory. The Sensory Profile 
Adolescents/Adult is a self report, however, if the participant is unable to complete it themself, we 
will ask their caregiver to complete it and we will make note of who completed it. The French 
versions are the Profil sensoriel 2 and the Profil sensoriel—adolescent/adulte. 
The Memory Game- This is an online test developed in the Bolduc Laboratory done on a 
tablet. Participants will have access to French and English versions.  Participants are asked to 
remember association between sets of pictures. The test has been done in typical development and 
developmental delay for participants 4 years and up. The test has a short demonstration video and 
a practice session. The participants are then shown pairs of pictures they must remember. Testing 
is performed right after the presentation and again approximately 24 hours later. For the 24 hours 

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testing, the participant will perform the association test this time without being shown the 
demonstration video or doing the practice session. The testing takes about 20 minutes the first day 
and 7 minutes the second day. It will be completed before treatment starts (at visit 1) and after 
treatment ends (at visit 3). 
 
Questionnaires will be done on a tablet device directly into REDCap when possible. Paper 
copies will be available when needed.  
 
 
Laboratory Evaluations: 
Safety Lab Tests –  At baseline and at the end of treatment/week 16 follow-up visit, 
comprehensive metabolic panel, glucose, vitamin B12, folate, lactate, pregnancy testing (for those 
female participants who are both capable of becoming pregnant and sexually active in a way that 
could lead to pregnancy), complete blood count, HgbA1c, and urinalysis (if able to provide a 
sample) will be done. A total of 3 tubes (6mL of blood) are drawn for the safety labs at each visit. 
 
At the second visit complete metabolic panel, fasting glucose, folate, lactate, pregnancy 
testing, complete blood count, lipid panel (including cholesterol, LDL, and triglycerides), and 
urine analysis will be done. 
 
Extension Safety labs: The same safety labs included in RCT visit 3 will be measured at 1 
year  and 2 years (after the beginning of the open-label extension phase).   
 
A summary of all tests is provided below. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Safety Lab Tests 
Chemistry 
Hematology 
Urinalysis 
Comprehensive Metabolic Panel: 
Light green top (3mL minimum) 
Complete Blood Count: 
Purple top (2mL minimum) 
Urine Dipstick Analysis: 
Sterile Urine Container 
Sodium 
Hemoglobin 
pH 
Potassium 
Hematocrit 
Color 
Chloride 
WBC 
Clarity 
Carbon Dioxide 
RBC 
Specific Gravity 
BUN 
MCV 
Occult Blood 
Creatinine 
MCH 
Urobilinogen 
Glucose 
MCHC 
Ketones 
Calcium 
RDW 
Protein 
Total Protein 
Platelet Count 
Glucose 
Albumin 
Neutrophils 
 
ALP 
Lymphocytes 
 
AST 
Monocytes 
 
ALT 
Eosinophils 
 
Total Bilirubin 
Basophils 
 
Lipid Panel: 
 
 
Cholesterol 
HgbA1c 
 
LDL cholesterol 
 
 
Triglyceride 
 
 
Fasting glucose 
 
 
Vitamin B12 
 
 
Folate  
 
 
Pregnancy test 
(bHCG)  
 
 
Lactate  Gray top (1mL minimum)  
 
 
Molecular Biomarkers – In addition to the blood for safety labs, we will collect a minimum 
of 24.5 mL of blood at each visit for biomarker analysis including FMRP level, MMP9, S6 Kinase, 
EIF4E, CYFIP1 mRNA, CYP450 allelic variants, proteomics, mRNA sequencing and metabolome 
variants. The Edmonton site will collect four EDTA tubes (2mL, 2 x 3mL, and 6mL), one 
PAXgene tube (2.5mL), and one CPT tubes (8mL). Due to differences in laboratory processing 
protocols, the Montreal site will collect 4 EDTA tubes (2mL, 3mL, and 2 x 5mL) and one PAXgene 
tube (2.5mL). 
 
 
 

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Protein biomarkers 
 
Plasma, DNA, and peripheral blood mononuclear cells (PBMCs) will be used for the 
proposed experiments. We consider testing various target proteins including the following.  
 
FMRP level. The level of FMRP has been correlated to the IQ in individuals with Fragile 
X. Significant variation in FMRP levels can be observed in FXS individuals based on sex, type of 
tissue examined, degree of FMR1 methylation and presence of mosaicism. FMRP measurements 
will be performed on lymphocytes, fibroblasts, and epithelial cells (obtained from urine sample) 
at baseline.  
 
Tissue type: The cells will be obtained from lymphocytes derived from blood, cells from 
urine, or skin fibroblast. 1) The optional skin biopsy will be carried out after lidocaine or EMLA 
cream is applied topically and approximately 2 cc of lidocaine is injected so that the procedure is 
painless. A small punch biopsy is done and then covered with a steristrip and a 4X4 gauze. This 
will only be done at baseline. However, the aforementioned blood sample and the urine sample 
(described below) will be obtained at baseline, week 8, and end of treatment/week 16. 2) The urine 
sample, a non-invasive method, will provide epithelial cell cultures that are alternatives to 
fibroblast cultures and can also later be derived in stem cells. Methods involve fresh urine samples 
(< 5h) being centrifuged and the cell pellets being re-suspended and then plated into 24-well tissue 
culture plates. Urine derived cells are isolated and characterized as previously described (Zhou, T 
et al. 2011). Numbers of cell clones are counted from each urine sample after they are cultured for 
2 weeks. Cell colonies from fresh urine samples often appeared as a cluster of 5–12 cells within 
5–7 days. 3) If done with blood, peripheral blood mononuclear cells (PBMC) are used as described 
later. 
Method: FMRP measurements will be done using the Cisbio Human FMRP assay 
(63ADK038PEC0). The assay uses HTRF (homogeneous time-resolved fluorescence) technology 
in which fluorescence resonance energy transfer (FRET) occurs between antibody-bound donor 
and acceptor groups located on the same FMRP molecule via the two fluorophores-conjugated 
antibodies (Degorce et al., 2009). Operation in a time-resolved fashion eliminates any background 
fluorescence. The donor is labeled with europium cryptate (Eu3+-Cryptate) and the acceptor is 
“d2”. Total protein concentration is determined using the Thermo Fisher MicroBCA Assay 
(23235). Protein levels will be determined in triplicate in a 384 well format following the 
instructions on the assay protocol. Samples are read on the PerkinElmer VictorX5. 
 
Global protein synthesis rate. Fmr1 KO mouse model of FXS show that protein synthesis 
rates measured in vivo are increased in many regions of the brain (Qin et al., 2005). Both genetic 
manipulation and pharmacological treatment of Fmr1 KO mice with drugs that normalize rates of 
protein synthesis have been shown to correct some molecular and behavioral phenotypes 
(Bhattacharya and Klann, 2012; Henderson et al., 2012; Liu et al., 2012; Michalon et al., 2012; 

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Osterweil et al., 2013). Preliminary data shows that altered protein synthesis can be reliably 
measured in human fibroblast cultures of patients with Fragile X syndrome and data suggest that 
it may be correlated to clinical outcomes. Protein synthesis rates will be measured using cell culture 
and SuNSET assay. During this trial, we aim to measure the critical “molecular phenotype” to 
understand how it may contribute or co-vary with clinical manifestations and respond to treatment.  
 
We propose to perform Data-independent acquisition mass spectrometry (DIA-MS) 
analysis on a subset of lymphocytes and fibroblasts to determine the influence of metformin 
treatment on the cell proteome. DIA-MS is a recently developed MS approach in which all ions 
within a selected m/z range are fragmented and analyzed in a second stage of tandem mass 
spectrometry (Doerr et al., 2015). Mass spectra are acquired either by fragmenting all ions that 
enter the mass spectrometer at a given time (called broadband DIA) or by sequentially isolating 
and fragmenting ranges of m/z. Metformin is known to alter the translation of numerous proteins, 
and therefore that part of its molecular response should be gauged by the nature and extent of the 
proteomic response. The analysis requires approximately 20-40 ug of total protein, which would 
be obtainable from approximately one-third of blood from a CPT tube. For lymphocytes, the DIA-
MS analysis would be performed both before (baseline) and immediately following metformin 
treatment. For fibroblasts, cells would be subjected to DIA-MS prior to, and following addition of 
metformin. In this instance, we would be looking for differences in the basic proteomic response 
to metformin, and whether the nature of this response would correlate with the magnitude of the 
clinical response. 
 
In addition, we will use mass spectroscopy to study specific metabolites. Indeed, we will 
establish metabolome variants integrating data from three different platforms into a coherent view 
on regulation of metabolites in participants. In all three methods ((1) Complex lipids by CSH-
QTOF accurate mass / high resolution mass spectrometry, (2) Primary metabolites by the cold 
injection GC-TOF mass spectrometry, and (3) Exogenous metabolites and biogenic amines by 
HILIC-QTOF mass spectrometry) we will use ‘metabolite targets’ for arrays of identified 
metabolites, with known quantification ions and retention times and specialized internal standards, 
in addition to detecting ‘novel metabolite signals’ which may be important to innovate biomarkers 
and mechanistic understanding of diseases.  
 
Gene expression biomarkers (RNA). Loss of FMRP has been shown to affect gene 
expression in FMR1KO mice and human. We will therefore obtain RNA from participants at each 
visit to assess levels of candidate targets.  
 
The loss of FMRP is believed to cause dysregulated translation of its target mRNAs 
(including MMP9), many of which are critical for synaptic plasticity, maintaining neuronal 
function, and regulatory control of protein synthesis (Darnell and Klann, 2013). We will perform 
RNA sequencing on RNA isolated from leukocytes isolated in the blood of participants on either 

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placebo or metformin at each visit. mRNA sequencing (RNAseq) – As with DIA-MS, RNAseq 
would be performed before/after treatment of subjects with metformin, and pre- and post-
metformin treatment from a subset of participants. mRNA would be purified in the participating 
laboratories and submitted for RNAseq analysis sequencing/expression core. As with DIA-MS, 
the mRNA would be isolated from one aliquot of pelleted lymphocytes, either the same pellet used 
for the DIA-MS experiment or from a portion of the purified RNA. 
 
Genomic biomarkers 
 
Molecular measures at the FMR1 locus will include CGG sizing (using a combination of 
PCR and Southern blot analysis), methylation status, and FMR1 and CYFIP1 mRNA expression 
using procedures outlined and detailed in our previous studies (Tassone et al., 2000; Tassone et 
al., 2008; Filipovic-Sadic et al., 2010). 
 
To determine whether the efficiency of response to metformin is dependent on the type of 
CYP450 allelic variants, we will determine the genotype in DNA samples obtained from 
lymphocytes of participants and correlate them to the clinical response measures outlined above. 
Analysis of Cytochrome P450s polymorphisms will be obtained by the xTAGv3 Kits (Luminex 
Corporation, Austin, TX) on a Luminex 100/200 instrument. Expression levels of targets in the 
mTOR/p70S6 kinase signaling pathways (Renard et al., 2016) (total and phosphorylated forms) 
will be measured by western blot analysis, as described in Hoeffer et al. (2012). MMP9 activity 
will be measured using a Milliplex assay (EMD-Millipore, Billerica, MA) in plasma samples 
isolated from whole blood collected in EDTA-containing tubes, followed by centrifugation for 10 
minutes at 1000 x g within 30 minutes of blood collection. Samples will be diluted 100-fold with 
assay buffer. Overnight incubation will be carried out for 17 hours at 4°C with shaking. Samples 
will be measured within one hour of finishing protocol using Luminex bead reader. MMP9 activity 
will be correlated to clinical improvement measures, as described above. 
 
In addition, several variations in genes involved directly with metformin pharmacokinetics 
have been identified and will be assessed using next-generation sequencing and Sanger sequencing 
confirmation. Indeed, transport of metformin into the body is determined by several transporters. 
We will analyze the sequence for the transporter OCT1 and OCT2 (coded by the genes SLC22A1 
and SLC22A2) and Multi-drug and toxic extrusion 1 (MATE1). A recent genomic analysis of 
response to metformin also identified other candidates including Ataxia telangiectasia mutated 
(ATM) and other candidate genes who did not reach statistical significance in the study (Zhou, K 
et al, 2011).  
 
Furthermore, other genes involved in pathways related to FXS, metformin, metabolism or 
other genes not directly related could also be associated with variation in the response to metformin 
will be assessed using unbiased exome or genome next-generation sequencing.  

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Gut Microbiome 
Stool collected from participants will be stored in the biobank and used by researchers in 
the future to, for example, look at the differences in the gut microbiome before and after treatment. 
The OMNIgene GUT OMR-200 stool collection kit will be utilized by the participant (with 
assistance from the caregiver if needed) to collect a sample at visits 1 and 3 (and the extensions 
visits after 1 and 2 years) and once from controls. The kit allows for transport and storage at room 
temperature for 60 days.  
Other, Non-Outcome Measure Procedures: 
Demographics – Basic demographic information will be collected on all study subjects at 
the screening/baseline visit. This information includes, but is not limited to, date of birth, race, 
ethnicity, socioeconomic status, education of parents, parental occupation, and parental salary 
range. 
Medical Examination – This will be carried out by the Co-PI and the nursing team at each 
site and may include growth percentiles, vitals, a detailed medical history, general physical and 
neurological examination, and review of medical records (paper, electronic), laboratory testing, 
and educational record, as applicable. In addition, a detailed family history may be obtained if 
medically necessary. A medical history and exam will be conducted at the screening/baseline visit. 
A medical exam and a review of recent history will take place at each subsequent visit. 
The complete schedule of all study procedures is also listed in a table format below:  

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Main Study 
Open-label Extension 
Assessment 
Visit 1 
Week 1, 
Day 0 
Visit 2 
Week 8 
±7 days 
Visit 3 
Week 16 
± 7 days 
Visit 1* 
Week 1 
Visit 2 
Week 52  
±7 days 
Visit 3  
Week 104  
 ±7 days  
Informed Consent 
X 
  
 
X** 
 
 
Inclusion/Exclusion 
Criteria*** 
X 
 
 
 
 
 
Medical History 
X 
  
  
 
X 
X 
Physical and Neurological 
Exam 
X 
X 
X 
X 
X 
X 
Vital Signs 
X 
X 
X 
X 
X 
X 
Adverse Eventsa 
X 
X 
X 
X 
X 
X 
Concomitant Medicationsa 
X 
X 
X 
X 
X 
X 
Autism Diagnostic 
Observation Schedule 
(ADOS-2) 
X 
 
 
 
 
 
Leiter-III 
X 
 
 
 
X 
X 
Clinical Global Impression 
Scale – Severity (CGI-S) 
X 
 
 
 
 
 
Clinical Global Impression 
Scale – Improvement 
(CGI-I) 
 
X 
X 
X 
X 
X 
Anxiety Depression and 
Mood Scale (ADAMS) 
X 
X 
X 
X 
X 
X 
Expressive Language 
Sampling (ELS) 
X 
 
X 
X 
X 
X 
Eye Tracking Measuresb 
X 
 
X 
X 
X 
X 
Event Related Potential 
(ERP) Measuresb 
X 
 
X 
X 
X 
X 
NIH Toolbox Cognitive 
Battery Measuresb 
X 
 
X 
X 
X 
X 
Visual Analogue Scale 
(VAS) 
X 
X 
X 
X 
X 
X 
Vineland Adaptive 
Behavior Scales–Third 
Edition (VABS-III) 
X 
 
X 
X 
X 
X 
Aberrant Behavior 
Checklist–Community 
(ABC-C) 
X 
X 
X 
X 
X 
X 

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Child Sleep Habits 
Questionnaire (CSHQ) 
X 
X 
X 
X 
X 
X 
Swanson, Nolan and 
Pelham Questionnaire 
(SNAP-IV) 
X 
X 
X 
X 
X 
X 
Pediatric Quality of Life 
(PedsQL) Parent Proxy 
Questionnaire 
X 
X 
X 
X 
X 
X 
EuroQol-5D (EQ-5D) 
X 
X 
X 
X 
X 
X 
Care Related Quality of 
Life (CarerQoL) 
X 
X 
X 
X 
X 
X 
Sensory Profile-2/Sensory 
Profile Adolescents/Adults 
X 
X 
X 
X 
X 
X 
Automated Self-
Administered 24-Hour 
Dietary Assessment Tool 
(ASA24)   
X 
 
X 
X 
X 
X 
The Memory Game 
(online)b 
X 
 
X 
X 
X 
X 
Blood Draw 
X 
X 
X 
X 
X 
X 
Urine Collection 
X 
X 
X 
X 
X 
X 
Stool Collection 
X 
 
X 
X 
X 
X 
Skin Biopsy (optional) 
X 
 
 
 
 
 
Dispense Study Drug 
X 
X 
 
 
X 
 
Collect Study Drug 
 
X 
X 
 
X 
X 
a : Also assessed at phone calls at Weeks 1, 2, 3, 4, and 12 and during the optional  
open label extension at weeks 1, 2, 3, 4, 20, 36, 52, 68, 84, 100 
b : If subject is capable of completing assessment 
* If a participant begins the extension study within 1 year of ending the RCT portion 
of the study, Visit 3 of the RCT will be considered to be Visit 1 of the extension and the 
participant would be seen at a 1 year follow up from when they had their Visit 3 of the 
RCT portion.  So assessments, safety laboratory and biological sample collections 
(blood, urine, stool) will not be duplicated if no change in medical condition has 
occured.    
** Informed consent for the open label extension will be done at Visit 3 of the RCT 
portion if possible, otherwise if a participant in the RCT portion decides to participate 
in the open label extension later, consent will be done then. 
*** Inclusion criteria, which are met at the beginning of the RCT, will be used to 
participate in the open label extension as well. If a participant is over 35 by the start of 
the extension or any time during the extension they will still be included.  

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8) Data and Specimen Management and Confidentiality 
Recruitment: 
Subjects will be recruited through the FXS clinics at the University of Alberta and CHU 
Sainte-Justine as well as by referral from other centers in Canada. The University of Alberta site 
will also recruit patients from other centers across Canada (including in Alberta, Manitoba, 
Saskatchewan, and British Columbia) referred to the study. The Universite de Montreal site will 
also recruit patients from the Maritimes, Quebec, and Ontario referred to their center. This way, 
any FXS patient from Canada will be able to participate in the study if they want.  Approved study 
information material will be posted on intellectual disability, autism, and FXS support group 
websites and social media feeds, and in clinical spaces.  
 
Educational and scientific conferences by the Co-I to health professionals, education 
systems, and other individuals interacting with FXS patients and families will also be used to 
disseminate knowledge about the study and recruitment. A website for the study has been created 
with videos and testimonial from clinicians, researchers, patients, and family members to generate 
interest and facilitate recruitment.  
 
Potential subjects/caregivers who are interested in participating in the study will be able to 
contact a study team member to schedule their visit 1. This will also allow potential 
subjects/caregivers to ask study-related questions and discuss the study in depth with the research 
team. The research team will use a pre-screen form where questions will be asked to the 
participant/caregiver over the phone to determine if the participant will be eligible to continue to 
visit 1. Informed consent will be signed at the first visit. Informed consent includes authorization 
to access and use their health information for the research study. For participants followed at CHU 
Ste-Justine, the study team is authorized to access medical records of CHU Ste-Justine patients to 
verify eligibility prior to getting their consent for the trial.  
Confidentiality: 
Participant confidentiality and privacy are strictly held in trust by the participating 
investigators, their staff, and the sponsor(s) and their interventions. This confidentiality is extended 
to cover to the clinical information relating to participants. Therefore, the study protocol, 
documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the study or the data will be released to any unauthorized third party 
without the prior written approval of the sponsor. Study files will be kept in a locked cabinet in a 
locked room with access restricted to research staff.  
All research activities will be conducted in the Stollery Children’s Hospital and the 
Biological Sciences building at the University of Alberta in as private a setting as possible. 
The study monitor board, other authorized representatives of the study sponsor (Women 
and Children’s Health Research Institute, Fondation CHU Ste-Justine), representatives of the 

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Research Ethics Board (REB) or regulatory agencies may inspect all documents and records 
required to be maintained by the investigator, including but not limited to, medical records (office, 
clinic, or hospital) and pharmacy records for the participants in this study. The clinical study site 
will permit access to such records. 
The study participant’s contact information will be securely stored at each clinical site for 
internal use during the study. At the end of the study, all records will continue to be kept in a secure 
location for the period dictated by Health Canada regulation (25 years). 
De-identified study participant research data, which is for purposes of statistical analysis 
and scientific reporting, will be stored in the REDCap electronic data capture system at the Women 
and Children’s Health Research Institute (WCHRI) Data Coordinating Centre (DCC) at the 
University of Alberta. This data will not include the participant’s contact or identifying 
information. Rather, individual participants and their research data will be identified by a unique 
study identification number. Permission to store data at the WCHRI DCC will be included in the 
informed consent. 
WCHRI’s REDCap installation is housed in a secure data centre at the University of 
Alberta Hospital and is behind the Faculty of Medicine & Dentistry’s firewall. Data is entered 
through a web based interface using 128 bit SSL encryption. Login is via a username/password 
pair with additional two factor authentication (2FA). Additional information is available in 
WCHRI’s privacy document (https://redcap.ualberta.ca/privacy.pdf). 
Coordinators and investigators at each study site will only have access to data relating to 
their own study participants. Study management staff (PI and research coordinator) at the lead site 
will have access to data for all study participants. Staff at the WCHRI DCC will have access to 
data for all study participants. This access is required to perform system management functions, 
data cleaning, and analysis. Once the study database is “locked” and data has been extracted for 
analysis, read only access to the study database will be granted to the PI and/or their designate if 
requested. 
We will have a material transfer agreement between the Canadian sites and a research 
institute in the United States (UC Davis MIND Institute) that plans to collect similar data, with the 
hopes that it may contribute to the research endpoints. This collaboration may include additional 
biomarker studies. With a material transfer agreement, each site may have access to blood 
epithelial cells cultured from urine and fibroblasts of patients at other sites for future research, if 
the patient consents to this use, in which case all such specimens would be de-identified upon 
transfer. 
Analysis will be based on the intention-to-treat principle. The team at the University of 
Alberta, Ste-Justine, and MIND Institute will collaborate on data analysis. The data will be merged 
into one dataset shared and analyzed across the different sites. De-identified data will be shared 
across sites as stated in the consent form including the UC Davis and Ste-Justine forms. Data will 

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also be analyzed in the lab of Dr. Arnaud Droit using machine learnig approaches and advanced 
statistical analysis, as well as proteomics on urine samples.   
The analysis of treatment efficacy will be based on the analysis of covariance (ANCOVA) 
with outcomes (receptive and expression language at 4-month follow-up with corresponding 
baseline measure as a covariate). The chosen endpoint at 4-month, based on preliminary data, 
provides a reasonable treatment time period to assess changes in the response. The proposed 
method adjusts for potential baseline differences between treatment arms despite randomization, 
if any. The sample size/power for the ANCOVA is based on sample size needed for a t-test by a 
variance inflation factor 1-2 where there is the correlation between baseline and follow-up 
measures. From our preliminary data, the receptive and expressive scores have ranges from 
approximately 0.2 to 0.5. Thus, we predictthe power for the t-test and power for the ANCOVA 
will be higher depending on this factor. We estimate clinically relevant effects sizes, based on our 
preliminary data, at follow-up visit as follows (metformin/placebo): receptive language score mean 
18.1/23.8, SD=7.0; expressive language score mean 13.6/20.7, SD=8.4. For these effect sizes of 
the 2 primary measures, the ANCOVA will have power of at least 80% (or have between 0.2-0.5) 
at level alpha=0.016 with the proposed total sample size n = 60 for each group (placebo and 
treatment). 
Data Safety and Monitoring Board (DSMB): 
 
A DSMB  for the Canadian centers located at the University of Alberta will collaborate 
and exchange information with the DSMB of the US site at the MIND Institute. The Canadian 
DSMB will include a minimum of two physicians who are independent of this study but who have 
significant expertise in carrying out psychopharmacological interventions in individuals with FXS. 
In addition, an independent statistician not involved with the study will be a member of each 
DSMB. The DSMBs will examine unblinded adverse events and clinical data on a semi-annual 
basis to review study progress and to advise whether the trial can be safely continued. The 
investigators and study staff will remain blinded throughout the trial. Any serious adverse events 
(SAEs) will be reported to the  REB of each site and Health Canada in accordance with their 
respective reporting guidelines. 
 
9)  Data and/or Specimen Banking 
Retention of Records: 
Data will remain in REDCap system at the WCHRI DCC until all data management and 
statistical analysis activity has been completed. Following study completion and publication, the 
data will be deleted from the REDCap system.  
The WCHRI DCC will facilitate the deposit of de-identified data in a publicly accessible, 
secure and curated repository for discovery and reuse by others in accordance with the Tri Agency 
Statement of Principles on Digital Data Management (2017). This will include creating a public 

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metadata in an open source system, such as Dataverse or Dryad. This could then be used by other 
investigators as a starting point to contact you if they want to collaborate and/or access de-
identified data sets. 
The PI will be responsible for storing copies of the data and other study materials in a 
secure archival facility in compliance with Health Canada and local institutional research data 
retention policy. At the end of this retention period these materials will be destroyed. 
Specimen Banking: 
All collected samples are de-identified and will only contain the subject’s study ID number 
and date/time of collection. Samples will be used by our team and could be shared with other 
investigators with appropriate ethics approval for investigation.  
 
Biological samples (blood, urine, skin, and stool) will be processed, stored, and destroyed 
in accordance with protocols in place for biological samples. Biological samples for participants 
enrolled through the University of Alberta will be stored in the Canadian Biosample Repository at 
the University of Alberta; biological samples for participants enrolled in Quebec will be stored at  
the CHU Sainte Justine in the Institutional biobank under the name “Biobanque sur les troubles 
neuro-développementaux et les conditions associées”. 
Blood: 
Blood will be drawn at three points throughout the study with a total minimum blood draw 
volume of 30.5 mL at each draw. Serum samples will be frozen immediately and stored at the 
respective biobanks. 
 
Samples may be used in the future for purposes related to this research. On a separate 
consent form, the participant/caregiver may indicate permission to allow for samples to be kept 
and stored indefinitely after the end of the study for use in future research. Alternatively, samples 
may be destroyed upon completion of the study. 
 
The samples are stored in the respective biobank facilities described above. A part of the 
sample will be used for analysis locally or shipped for analysis to other scientists approved by the 
study investigators. Samples collected for safety analyses will not be used for any other purpose. 
Safety labs drawn at each visit will have a minimum volume of 6mL and will be processed through 
the Alberta Health Services Clinical Trial Lab. Safety labs from the Universite de Montreal will 
be assessed locally as well.  Banked samples will be stored and will be used in biomarker studies.  
Urine:  
Urine samples for safety labs, biomarker analysis, and cell culture will be collected at three 
points throughout the main study (and at extension visits). Urine derived cell lines will be obtained 
locally at Ste-Justine and/or at the Universite Laval. We will only collect urine for cell 
culture/biomarker studies until we have a viable sample and then urine will only be collected for 

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safety labs. Samples will be stored in one of the biobank facilities described above. Banked 
samples will be stored and used in biomarker studies.  
Skin:  
Skin sample will be collected at the baseline visit only if the participant/caregiver chooses 
to participate in this optional part of the study (separate consent form). As described above with 
regards to blood specimens, the participant/caregiver may indicate permission to allow for their 
skin sample to be kept and stored indefinitely at the biobank facility for use in future research; 
otherwise, samples will be destroyed upon completion of the study.   
 
Stool:  
 
Stool samples will be collected at visits 1 and 3 of the main study (and at extension visits) 
and once from control participants at any point to be stored in the biobank facility for use in 
biomarker/other gut microbiome research if there is consent from the participant/caregiver to do 
so.  
 
10)  Provisions to Monitor the Data to Ensure the Safety of Subjects 
 
It is the responsibility of the investigators to oversee the safety of the study for subjects 
seen at each site. This safety monitoring will include careful assessment and appropriate reporting 
of physical exam and lab abnormalities, adverse events, and SAEs as noted above. Adverse events, 
SAEs, and laboratory results will be reviewed by the DSMB after each SAE. The WCHRI DCC 
can do the ongoing data monitoring for both sites and prepare reports for the DSMB. Unanticipated 
problems posing risks to subjects or others and serious, unexpected adverse events associated with 
the research will be brought to the attention of the DSMB as soon as possible, accompanied by the 
evaluation of the event conducted by the site investigator. 
 
The role of the DSMB will be to evaluate the participant risk versus benefit, consider factors 
external to the study when relevant information becomes available, such as scientific or therapeutic 
developments that may have an impact on the safety of the participants or the ethics of the trial, 
and make recommendations concerning continuation, termination, or other modifications of the 
trial based on the observed beneficial or adverse effects of the treatment under study, particularly 
in regards to a maximum tolerated dose in this study population. The PI will abide by the 
recommendation of the DSMB regarding stopping the study.  
 
11)  Withdrawal of Subjects 
 
All subjects and their caregiver will be advised that they are free to withdraw from 
participation in this study at any time, for any reason, and without prejudice. Every reasonable 
attempt should be made by the investigator to keep subjects in the study; however, subjects must 
be withdrawn from the study if they/their caregivers withdraw consent to participate. The 

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investigator and/or the Research Coordinator must attempt to contact subjects who fail to attend 
scheduled visits by telephone or other means to exclude the possibility of an AE being the cause 
of withdrawal. Should this be the cause, the AE must be documented, reported, and followed as 
described in Section 10. 
The site investigator also has the right to withdraw subjects from the study at any time for 
lack of therapeutic effect that is intolerable or otherwise unacceptable to the subject, for intolerable 
or unacceptable AEs, inter-current illness, noncompliance with study procedures, administrative 
reasons, or in the investigator’s opinion, to protect the subject’s best interest. If a subject is 
withdrawn before completing the study, the reason for withdrawal and the date of discontinuation 
will be recorded on the appropriate case report form (CRF). Whenever possible and reasonable, 
the evaluations that were to be conducted at the completion of the study should be performed at 
the time of premature discontinuation. Information gathered about a subject who has terminated 
the study early will be kept for analysis unless the subject/subject’s caregivers specifically ask for 
this information to be removed from the analysis. Any unused biological samples will be 
destroyed. 
 Participants/caregivers will be informed of this as a part of the consent process and also 
reminded of this in the event of an early termination. 
 
In addition, the study staff will explain to the participant/caregiver that, although it happens 
rarely, the sponsor may stop the trial early. This could be due to a number of reasons and would 
involve discontinuation of the participant’s and caregiver’s involvement in the study. 
 
12)  Risks to Subjects 
The most common anticipated risks due to participation in the study include anxiety, 
frustration, fatigue, or embarrassment during the answering of questionnaires, study assessments 
and testing, as well as during the medical history and exam. Breaks will be offered to subjects as 
needed.  
 
Risks associated with blood draws include bruising, soreness, and slight risk of infection 
at the needle entry site for the blood draw. This site will be carefully cleaned prior to the draw and 
an appropriate dressing will be applied to the area. Risks associated with skin biopsies include 
bleeding, bruising, scarring, and slight risk of infection at the biopsy site. This site will be carefully 
cleaned prior to the biopsy and lidocaine or EMLA cream will be applied to numb the area. 
 
Metformin is a Health Canada and FDA-approved medication with an established adverse 
events profile explained in the package insert. The clinical data suggest that metformin is safe and 
well-tolerated in diverse populations. The more common observed side effects during treatment 
with metformin are: diarrhea, nausea/vomiting, flatulence, asthenia, and abdominal or stomach 

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discomfort. The less common observed side effects during treatment with metformin are: 
headache, blurred vision, chest discomfort, cold sweats, coma, confusion, difficult or labored 
breathing, dizziness, feeling of warmth, or a heartbeat or pulse that is fast, irregular, pounding, or 
racing. Adverse events and toleration are similar between the liquid and tablet formulations of 
metformin. Safety labs will help rule out candidate at risk of serious side effects such as lactic 
acidosis and anemia. Patients and parents will be instructed about the symptoms of hypoglycemia 
and advised to consult urgently in case of such symptoms.  
 
Adverse events  
 
Serious Adverse Reactions associated with metformin include: 
Lactic acidosis (rare in absence of renal insufficiency)  
Anemia, megaloblastic 
Hepatotoxicity 
Hypoglycemia (rare in absence of concomitant use of sulfonylurea) 
 
Common Reactions include: 
Diarrhea 
Nausea/vomiting 
Flatulence 
Asthenia (fatigue) 
Indigestion 
Abdominal discomfort 
Anorexia 
Headache 
Metallic taste 
Rash 
Ovulation induction 
 
Seeing symptoms of lactic acidosis (eg, weakness, fatigue, drowsiness, unusual muscle 
soreness, difficulty breathing, stomach pain with nausea, vomiting or diarrhea, feeling cold, 
dizziness, lightheadedness, slow or irregular heartbeat), requires discontinuation of metformin and 
immediate medical care. In case of suspicion of lactic acidosis, participants will be instructed to 
proceed to their local emergency room and contact the study doctor. Similarly, for signs of 
hypoglycemia such as shakiness, dizziness, sweating, and hunger, participants will be instructed 
to proceed to their local emergency room and contact the study doctor.  
 
To monitor for any adverse events between visits, weekly phone calls during the first month 
will be made by study personnel to evaluate the presence of side effects. One additional phone call 

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will be made at Week 12 (Month 3), midway between visits 2 and 3. Phone calls will be made 
during the optional extension at weeks 1, 2, 3, 4, 20, 36, 52, 68, 84, and 100. 
 
In case of serious adverse event, the blind will be removed by contacting the study doctor 
who will then open the envelope containing the subject identifier treatment status (placebo or 
metformin). Individualized sealed envelope will be given to the site study doctor from the Alberta 
Health Services Pharmacy Research Office (University of Alberta) and SPSR (CHU Sainte-
Justine, University of Montreal).   The Pharmacy Research Office and the SPSR will retain a copy 
of the randomization codes. The randomization of participants will be done directly in the REDCap 
shared database of the trial.  
 
During the course of a clinical trial, Health Canada and the REB shall be informed of any 
serious unexpected adverse drug reaction: 
a. if it is neither fatal nor life threatening, within 15 days after becoming aware of the 
information; and 
b. if it is fatal or life threatening, within seven days after becoming aware of the information. 
Other unanticipated risks:  
 
Drug-related risk: Prediction of drug effects or side-effects in any individual cannot be 
done with certainty, and unexpected potentially harmful effects could possibly occur. The close 
clinical and laboratory monitoring of subjects is intended to detect any such unanticipated side-
effects so that appropriate corrective measures can be implemented in a timely manner. 
 
Genetic testing risk: Since genetic analysis will be performed to identify the basis of 
clinical variation and differential response to metformin in both a targeted and unbiased manner, 
it may be possible to discover some "incidental findings" i.e. information that we were not looking 
for. This information related to the participant’s health could have a direct impact on their clinical 
care in some cases. We will follow the guidelines from the American College of Medical Genetics 
reported previously and widely adopted including in Canada (Genet Med. 2013 Jul; 15(7): 565–
574). 
In the event that a potential significant and actionable result is found and that preventive 
measures or treatments are available for the participants, they will be notified through the study 
doctor. In this situation, the study doctor will recommend to repeat these tests to confirm the 
research results. A meeting with a geneticist and/or a genetic counsellor will be organized to 
discuss the pros and cons of further testing in the clinical set up If the clinical testing confirms the 
research results, the medical geneticist will proceed as he/she would do normally in the clinical 
setting and the health information will be recorded in the participant’s medical chart. 
 
Genetic test results for children will be communicated to the family if there is actionable 

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conduct. As suggested by the American College of Medical Genetics, results for which an adult 
onset or for which there is no interventions will not be disclosed as this is a research study. In 
addition, results related to non-paternity will not be disclosed in the context of this research testing 
for genes affecting the clinical presentation and drug response.   
 
Possible ineffectiveness of treatment: Some patients may not benefit from the treatment 
provided. If their condition deteriorates to the point that requires immediate effective treatment, 
they will be discontinued from the study and referred for such treatment.  
 
Duty to report:  
If the investigators or study staff learn that a patient is in immediate danger to themselves or 
others as a result of a mental disorder or for any other reason, the investigator is obligated to contact 
the appropriate facility (i.e. mental health facility) for immediate referral, which may include 
involuntary hospitalization. 
 
13) Potential Benefits to Subjects 
The potential benefits of study participation are that subjects with FXS: 
1. may experience an improvement in physical health, behavioral symptoms, and/or cognitive 
abilities as a result of treatment with metformin. 
2. will undergo neuropsychological assessments, the results of which may be made available 
to the family of participants on request. 
3. will receive medical exams offered through the study. Additionally, a complete blood count 
will be conducted as a part of this study. Participants/caregivers will be informed of clinically 
significant findings from either the medical exam or CBC as appropriate. 
4. may understand that they are contributing to the scientific knowledge that may lead to    
expansion of the targeted treatment options for subjects with FXS. 
5. will have a direct access to health professionals and will be followed very closely during 
the study. 
No other benefits of participation are anticipated. 
 
14)  Multi-Site Research 
 
This trial has a multi-site design. The study sponsor/PI has obtained a material transfer 
agreement with an additional international research institution (UC Davis MIND Institute) that 
will collect similar data to contribute to the research endpoints. With the material transfer 
agreement, de-identified data as well as de-identified biological samples including blood, urine 
endothelial cells, and fibroblast lines would be shared to compare biomarkers that are outlined in 
this study (see Section 7). All results and data will be de-identified to protect the confidentiality of 

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the research subjects. Allocation tables generated by the Data Coordinating Centre statistician have 
been uploaded to REDCap and sent to the Alberta Health Services Pharmacy Research Office 
(University of Alberta) and SPSR (CHU Sainte-Justine, University of Montreal). Once the study 
coordinator has randomized a participant in REDCap (done separately for each site), the 
randomization number that is generated will be sent to the respective pharmacy, which will then 
prepare the drug or placebo based on the number they are given and the table that has been provided 
to them by the DCC.  
  
15)  Sharing of Results with Subjects 
 
At the conclusion of the study, the possible medical benefit from the study will be  reviewed 
with the subject and his or her caregiver. Participants/caregivers will not be unblinded at any point 
during enrollment. This is to prevent unconscious biases that may present as the trial progresses 
and to ensure quality data. Participants/caregivers will be unblinded once all study procedures and 
all statistical analyses for all subjects have been completed. Unblinding will be conducted by a 
study physician. If a subject/caregiver has to be unblinded for emergency purposes, then the 
subject/caregiver will be notified by the PI or site investigator.  
 
The results of the study will also be published in a peer reviewed scientific journal that will 
be made publicly available through PubMed Central. In addition, after publication the results will 
be posted on the MIND Institute website under clinical trials.  
 
Preliminary data may be presented annually at scientific meetings. Preliminary data may 
also be shared with the network of 22 USA National Fragile X Foundation (NFXF), Fragile X 
Clinical and Research Consortium clinics, and with the 20 countries involved with the NFXF 
International Consortium. Permission from Drs. Bolduc and Jacquemont will be required prior to 
the presentation of Canadian derived data.  
 
16)  Prior Approvals 
 
Initial submission was approved by Health Canada on 24-OCT-2018 and the REB on 24-
DEC-2018. Amendments were approved by Health Canada on 6-MAY-2019, 15-JAN-2021, and 
6-JUN-2021 and the REB on 7-MAY-2019, 13-JUN-2019, 26-JUN-2019, 25-JUL-2019, 20-FEB-
2020, 7-MAY-2020, 6-OCT-2020, 19-NOV-2020, 24-DEC-2020, 11-FEB-2021, 15-MAR-2021, 

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12-APR-2021, 26-APR-2021, 8-JUN-2021, 17-JUN-2021, 16JUL-2021, 27-OCT-2021, 11-JAN-
2022, 22-MAR-2022, 6-APR-2022, and 5-JUL-2022. 
 
17)  Economic Burden to Subjects 
 
There is no charge for the subject to participate in this study. Neither the subject nor his or 
her caregiver will be charged for taking part in the research. All costs associated with the study 
will be paid by the sponsor/department. 
 
Possible expenses to the subject’s family include time spent at the clinic as well as expenses 
for travelling. Subjects will not be paid just for taking part in this study (i.e., compensated). 
However, all eligible research subjects, who would not otherwise be able to participate due to the 
cost of travelling to the University of Alberta (e.g., flights, gas, lodging), will be offered 
reimbursement per visit for actual costs incurred.  
 
18)  Drugs or Devices 
 
We will treat children with Fragile X syndrome aged 6 to 35 years old in a randomized, 
double-blind, placebo-controlled trial of metformin for 4 months. Randomization to metformin or 
placebo will be carried out by the Alberta Health Services Pharmacy Research Office (University 
of Alberta) and SPSR at CHU Sainte-Justine (University of Montreal) after they are given the 
randomization number that is generated in REDCap by their respective site, and dosing will be 
determined based on patient weight (see below). Metformin is available in a 500 mg tablet, which 
Strathcona Pharmacy staff will split into 250 mg halves and over-encapsulate to create both 250 
mg and 500 mg capsules. Capusules will be white for a dose of 250mg and pink for a dose of 
500mg. Strathcona Pharmacy staff will also develop identical placebo capsules. Metformin pills 
will be purchased, split in half, and encapsulated by each Canadian site (Strathcona pharmacy for 
University of Alberta site and the central pharmacy at CHU Sainte-Justine for Universite de 
Montreal site). Matching placebo capsules will be manufactured by the Strathcona Pharmacy for 
the University of Alberta site and the central pharmacy at CHU Sainte-Justine for the Universite 
de Montreal site.  
 
 
Subjects under 50 kg will start at an initial dose of 250 mg once a day at dinner by mouth 
and increase to 250 mg twice a day after the first week. If well tolerated, after the second week the 
dose will be increased to 250 mg at breakfast and 500 mg at dinner, and then to 500 mg twice a 
day after the third week for a maximum dose of 1000 mg per day. 
 
 
Subjects at and over 50 kg will start at an initial dose of 500 mg once a day at dinner by 
mouth and increase to 500 mg twice a day after the first week. If well tolerated, after the second 

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week the dose will be increased to 500 mg at breakfast and 1000 mg at dinner, and then to 1000 
mg twice a day after the third week for a maximum dose of 2000 mg per day.  
 
Since the maximum tolerated dose may vary between individuals, dose modifications may 
be made at the discretion of the study doctor as well during the treatment dose titration period for 
the first 4 weeks of the study; for instance, if a subject experienced adverse events after increasing 
from 500 mg to 1000 mg, decreasing the dose back to 500 mg would be allowed per protocol. The 
dose may then increase again at the discretion of the study doctor. After the dose titration period, 
for the remaining 12 weeks of the study, dose adjustments may be performed considering 
intercurrent illnesses associated with gastrointestinal symptoms.  
 
The Strathcona pharmacy will buy, store, and dispense the study drugs for the Edmonton 
site. Drug will be dispensed to the Alberta Health Services Research Pharmacy, where the study 
staff can pick it up to dispense to each participant. The SPSR at CHU Sainte-Justine (Universite 
de Montreal) will buy, store, and dispense the study drugs for the Montreal site. 
Randomization: 
Allocation tables generated by the Data Coordinating Centre statistician have been 
uploaded to REDCap and sent to the Alberta Health Services Pharmacy Research Office 
(University of Alberta) and SPSR (CHU Sainte-Justine, University of Montreal). Once the study 
coordinator has randomized a participant in REDCap (done separately for each site), the 
randomization number that is generated will be sent to the respective pharmacy, which will then 
prepare the drug or placebo based on the number they are given and the table that has been provided 
to them by the DCC.  
Participants will be randomized to treatment or control arm with a 1:1 allocation ratio, 
stratified by sex and using permuted blocks of randomly varied sizes 2 and 4. Participant 
randomization will be performed in REDCap, based on allocation tables that have been generated 
and uploaded by the Data Coordinating Centre statistician. REDCap maintains an automated audit 
trail which includes the assigned study identification number, treatment allocation, and date and 
time of the transaction. 
The randomization process will be performed the same way at both sites, i.e.  randomization 
to treatment or control arm with a 1:1 allocation ratio stratified by sex and using permuted blocks 
of randomly varied sizes 2 and 4.  
 
19)  ClinicalTrials.gov Registration 
 
A description of this clinical trial is available on Clinicaltrials.gov (NCT03862950). This 
website does not include information that can identify the participants/caregivers.  At most, the 
website will include a summary of the results of the research. 

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