A Single-Center, Open Label Study to Assess the Safety and Tolerability of Metformin in Subjects With C9orf72 Amyotrophic Lateral Sclerosis Over 24 Weeks of Treatment
Phase: PHASE2
Conditions: C9orf72 Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia
Sponsor: University of Florida

 
 
 
 
The Foundation for The Gator Nation 
An Equal Opportunity Institution 
College of Medicine 
PO Box 103610 
Department of Molecular Genetics & Microbiology 
Gainesville, FL 32610-0266 
Center for NeuroGenetics 
PH:  352-273-6177 
 
FX:  352-273-8284 
 
 
 
 
 
Study Title: Safety and Therapeutic Potential of the FDA-approved Drug Metformin for 
C9orf72 ALS/FTD 
 
NCT04220021 
 
Document approval date: 4/6/2023 
Document signature date: 8/28/2023 
 
 
 
 
 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
CONFIDENTIAL INFORMATION 
The information in this study protocol is confidential. Any disclosure, copying or distribution of the information contained within is strictly prohibited 
without written consent from University of Florida. 
Page 1 of 61 
 
FULL STUDY TITLE 
 
A Single-Center, Open Label Study to Assess the Safety and Tolerability of Metformin in Subjects with 
C9orf72 Amyotrophic Lateral Sclerosis over 24 Weeks of Treatment   
 
SHORT STUDY TITLE 
Safety and therapeutic potential of the FDA-approved drug Metformin for C9orf72 ALS 
 
 
Protocol number: UF2019-001 
 
Protocol Version: 0.9 
Protocol Version Date: December 16, 2023 
 
Sponsor: 
University of Florida 
 
Phase 2 
 
IND Number: IND144443 
 
Confidentiality Statement 
 
The information contained herein is confidential and proprietary in nature and will not be disclosed to any third 
party without written approval of authorized designee. This document may be disclosed to the appropriate 
institutional review boards or to duly authorized representatives of the US Food and Drug Administration or a 
national regulatory authority under the condition that they maintain confidentiality. 
 
 

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
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PROTOCOL APPROVAL SIGNATORY PAGE 
Study Title: A Single-Center, Open Label Study to Assess the Safety and Tolerability of Metformin in 
Subjects with C9orf72 Amyotrophic Lateral Sclerosis over 24 Weeks of Treatment  
SAFETY APPROVAL
I have read this protocol and agree that the design of the protocol adequately protects the safety of the 
patients.
Date:

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
CONFIDENTIAL INFORMATION
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without written consent from University of Florida.
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INVESTIGATORS AND FACILITIES
Principal Investigators
Laura Ranum, PhD
Director, Center for NeuroGenetics
Department of Molecular Genetics and Microbiology
College of Medicine
University of Florida
Gainesville, Florida 32610
Email: ranum@ufl.edu
James Wymer, MD, PhD
Department of Neurology, College of Medicine
University of Florida
Gainesville, Florida 32610
Email: james.wymer@neurology.ufl.edu
David Vaillancourt, MD
Department of Applied Physiology & Kinesiology
College of Health and Human Performance
University of Florida
Gainesville, Florida 32610
Email: vcourt@ufl.edu
Timothy J. Garrett, PhD
Department of Pathology, Immunology and Laboratory 
Medicine
College of Medicine
University of Florida
Gainesville, Florida 32610
Email: tgarrett@ufl.edu
Collaborator
Sponsor:
Emily Plowman, PhD, CCC-SLP
Department of Otolaryngology
Ohio State University
Columbus, Ohio 43210
Email: emily.plowman@osumc.edu
University of Florida

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
CONFIDENTIAL INFORMATION
The information in this study protocol is confidential. Any disclosure, copying or distribution of the information contained within is strictly prohibited 
without written consent from University of Florida.
Page 4 of 61
Clinical Research Organization:
N/A
Clinical Laboratory:
University of Florida
Pharmacokinetic/Statistical Facility:
University of Florida
CRO Safety (Pharmacovigilance) 
Reporting:
University of Florida
SYNOPSIS
Title
A Single-Center, Open Label Study to Assess the Safety and 
Tolerability of Metformin in Subjects with C9orf72 Amyotrophic 
Lateral Sclerosis over 24 Weeks of Treatment. 
Objectives
The primary objective is to assess the safety and tolerability of 
Metformin in subjects with C9orf72 amyotrophic lateral sclerosis 
administered for 24 weeks. Preclinical studies conducted in the 
Ranum lab show that Metformin improves molecular, behavioral and 
neuropathological phenotypes in a mouse model of C9orf72 ALS.  
These effects are thought to occur because Metformin prevents the 
activation of the protein kinase R (PKR) pathway and reduces the 
production of mutant proteins made from the C9orf72 expansion 
mutation. The overall objective is to determine if Metformin is safe in 
C9orf72 ALS patients and is a potentially viable therapeutic treatment 
for C9-ALS that reduces repeat-associated non-ATG (RAN) proteins 
that are produced by the C9orf72 repeat expansion mutation.   

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
CONFIDENTIAL INFORMATION 
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Page 5 of 61 
 
Experimental Design  
This is a single-center, open label safety and tolerability trial. 
Approximately sixteen (16) individuals with C9orf72 amyotrophic 
lateral sclerosis (ALS) will be enrolled.  Subjects will be screened to 
determine eligibility according to the specified inclusion/exclusion 
criteria.  Following informed consent, screening, and enrollment, 
during study visit 1, subjects will undergo clinical examinations and 
questionnaires including the validated amyotrophic lateral sclerosis 
functional rating scale-revised (ALSFRS-R). Subjects will have blood 
work performed to monitor clinical safety, RAN proteins and 
biomarker analyses.  Lumbar punctures will be performed to allow 
measurements of RAN protein levels in the cerebrospinal fluid (CSF) 
and for molecular and biomarker analyses. MRI/DTI imaging will be 
obtained at the beginning of the study and at the end of the drug 
treatment phase.  Subjects will return on Day 1 for swallowing and 
respiratory evaluation.  The subjects will then be given a first dose of 
Metformin (with escalating dose of the drug. The initial dose during week 
one will begin at 500mg/day, in the following weeks this will increase to 
1000 mg/day (week two), 1500 mg/day (week three) and 2000mg per day 
(weeks 4 to 24). The subjects will begin by taking the first dose in the 
morning during week one and then morning and evening doses in 
weeks 2-24   with an interim assessment at days 42, 84 and final 
evaluation at 168 days (+/- ~7) to assess the safety and tolerability of 
Metformin. 
Interventions  
Metformin is a widely used, well-tolerated drug that has been used for 
decades as a first-line defense for treating type 2 diabetes. Its safety in 
the general population has been well established.  Subjects will begin 
treatment with Metformin at a dosage of 500 mg with an escalation of 
dosage by 500 mg every week to a maximal dosage of 2000 mg. 
Dosing will be once daily during week one and twice daily from 
weeks 2-24.  Due to COVID-19 the Metformin dosing may be altered 
with an additional dispensing to allow for continuation of the drug to 
week 36 (and discontinued at Week 36). This will allow for additional 
time to coordinate lumbar puncture at a remote location and the 
collection of CSF and blood sample while the participant is still 
taking Metformin. For participants continuing on study drug from 
Week 24-36, safety labs will be collected at an alternative site located 
near the participants home area. 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
CONFIDENTIAL INFORMATION 
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Page 6 of 61 
 
Study Duration 
 
Participant Duration: 
The anticipated time from study enrollment until completion of data 
collection and analyses is approximately 10-18 months. 
Total study participant time is expected to be approximately 26 weeks 
from time of screening to completion of study trial phase and then an 
additional 26 weeks of follow-up phase (total 52 weeks). 
 
Study Population and 
Sample Size 
Approximately sixteen (16) subjects with C9orf72 ALS will be 
enrolled. The inclusion criteria are intended to provide a cohort of 
subjects with ALS who are appropriate candidates for treatment with 
Metformin.  Subjects must be suitable candidates in the investigator’s 
judgment and fulfill Inclusion/Exclusion criteria.  Inclusion will 
require a diagnosis of probable, or definite ALS in accordance with 
the Revisited El-Escorial Criteria (Cedarbaum, 1999), positive testing 
for the C9orf72 expansion mutation, no allergies or contraindications 
to barium, Metformin, or inactive ingredients* in the study 
medication, and no contraindications to study procedures. Female 
subjects must not be pregnant or breast-feeding. 
Phase 
2 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
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Page 7 of 61 
 
Inclusion Criteria 
1. Male or female subjects between 18-80 years of age, inclusive. 
2. Subjects have a diagnosis of probable or definite ALS in 
accordance with the Revisited El-Escorial Criteria (Cedarbaum, 
1999). 
3. Subjects have a diagnosis of C9orf72 positive ALS. 
4. Subjects must be currently on an oral diet and able to take foods, 
pills and liquids by mouth equivalent to a score of 4 or above on 
the Functional Oral Intake Scale (Crary, 2005).  
5. Subjects must have no known allergy to Metformin. 
6. Subjects or subject’s legally authorized representative must be 
willing and able to complete informed consent/assent and HIPAA 
authorization.  
7. Ability of the study subject or representative to comprehend and 
be informed of the nature of the study, as assessed by the PI or 
Co-Investigators. 
8. Availability to participate for the entire study duration. 
9. Female subjects of childbearing potential must have a negative 
urine pregnancy test prior to a videofluoroscopic swallowing 
study (VFSS) exam during Visit 1, 3, and 4.  
 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
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Page 8 of 61 
 
Exclusion Criteria:  
1. Subjects who score 3 or below on the Functional Oral Intake 
Scale (FOIS) (Crary, 2005). 
2. Subjects who do not carry the C9orf72 hexanucleotide repeat 
expansion as determined by previous laboratory analyses. 
3. Allergy to barium sulfate or Metformin 
4. Subjects with a history of clinically significant liver disease, 
renal disease, or any other medical condition judged to be 
exclusionary by the investigator. 
5. Subjects who are unwilling to sign informed consent or 
subjects who for any other reason in the judgment of 
investigator are unable to complete the study. 
6. Female subjects who have a positive urine pregnancy test 
ȕK&*DWVFUHHQLQJRUYLVLWDUHWU\LQJWREHFRPHSUHJQDQW
or are breastfeeding. 
7. Subjects with active cancer within the previous 2 years, except 
treated basal cell carcinoma of the skin. 
8. Subjects who have taken any experimental drug within 30 
days prior to enrollment or within 5 half-lives of the 
investigational drug –whichever is the longer period.   
9. Subjects with known history or presence of moderate or severe 
renal impairment as defined by an eGFR value below 30 
mL/min/1.73 m2. 
10. Subjects with hepatic impairment as defined by baseline 
elevations of serum aminotransferases greater than 5 times 
upper limit of normal or evidence of liver dysfunction (e.g., 
elevated bilirubin). 
11. Use of potentially hepatotoxic drugs: (e.g., allopurinol, 
methyldopa, sulfasalazine). 
12. Subjects with clinically significant abnormal laboratory values 
in the judgment of the investigator.  
13. Subject with implanted electrical device (i.e. cardiac 
pacemaker or a neurostimulator), metal or metallic clip(s) in 
their body (i.e. an aneurysm clip in the brain) that will be 
damaged by participation in the MRI portion of the study. 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
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Page 9 of 61 
 
14. Anything else that, in the opinion of the investigator, would 
place the subject at increased risk or preclude the subject’s full 
compliance with or completion of the study. 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
CONFIDENTIAL INFORMATION 
The information in this study protocol is confidential. Any disclosure, copying or distribution of the information contained within is strictly prohibited 
without written consent from University of Florida. 
Page 10 of 61 
 
 Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dr. James Wymer:  
x Disease Documentation 
x Comprehensive/brief physical exam 
x Comprehensive/brief neurologic exam 
x ALS Functional Rating Scale – Revised (ALSFRS-R) 
x BDI-Fast Screen 
x Eating Assessment Tool-10 (EAT-10) 
x Functional Oral Intake Scale (FOIS) 
x Forced Vital Capacity (FVC) 
x Peak Cough Flow 
x Body Mass Index 
x Adverse events 
x Clinical laboratory assessment, blood and urine 
x Lumbar puncture for CSF collection (20 ml volume on each 
study visit) 
x Vital signs/height and weight 
x Medical history 
x Concomitant medication 
x Inclusion/exclusion criteria 
Dr. David Vaillancourt:  
x MRI and DTI imaging of corticolumbar and corticobulbar 
tracts 
 
Nicole Herndon, M.S., CCC-SLP, BCS-S: 
x Videofluoroscopic Swallow Study (VFSS) 
 
 
 
 
 
 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
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Page 11 of 61 
 
Endpoints 
 
 
The primary endpoints of this study are: 
x To evaluate the tolerability and safety of Metformin 
administered over 24 weeks in subjects with ALS 
x To test the hypothesis that metformin can reduce RAN protein 
levels in patients with C9orf72 ALS 
 
Statistical Analysis 
 
The following safety and tolerability information will be included as 
summary statistics: 
x Type, incidence, and severity of adverse events 
x Physical and neurological examinations 
x Vital signs (heart rate, blood pressure, respiration rate, 
temperature), Height/Weight 
x Clinical laboratory tests (hematology, serum chemistry and 
urinalysis) 
x ALS Functional Rating Scale – Revised (ALSFRS-R) 
x Eating Assessment Tool -10 
x Functional Oral Intake Scale (FOIS) 
x BDI-Fast Screen  
 
Depending on the study enrollment rate, the Sponsor may elect to 
perform an interim analysis using available data from the subset of 
subjects who have completed the study at that time.  The results of 
this interim analysis will be used only for internal planning and 
possibly for sharing with regulatory authorities. The results of the 
interim analysis will have absolutely no influence on the subsequent 
conduct of the study. 
 
 
 
 

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
CONFIDENTIAL INFORMATION
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without written consent from University of Florida. 
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LIST OF ABBREVIATIONS 
Abbreviation
Definition
AE
adverse event
ALS
amyotrophic lateral sclerosis
ALSFRS-R
ALT
AST
BDI-FS
ALS Functional Rating Scale-Revised
Alanine Aminotransferase Test
Aspartate Aminotransferase Test
BDI-Fast Screen
BUN
Blood Urea Nitrogen
CBC
Complete Blood Count
CDE
Common Data Elements
CFR
Code of Federal Regulations
CMSU 
Clinical Materials Services Unit
CRF
case report form
CS
clinically significant
DM
data management
DIGEST
Dynamic Imaging Grade of Swallowing Toxicity Scoring System
EAT-10
Eating Assessment Tool -10
eCRF
electronic case report form
EDC
electronic data capture
FOIS
Functional Oral Intake Scale
FDA
Food and Drug Administration
FVC  
Forced Vital Capacity
GCP
Good Clinical Practice
HIPAA
Health Insurance Portability and Accountability Act
ICF
Informed Consent Form
ICH
International Council on Harmonization
MedDRA
Medical Dictionary for Regulatory Activities
NINDS
National Institute of Neurological Disorders and Stroke

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
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RAN proteins 
REDCap 
Repeat Associated Non-ATG (RAN) proteins 
Research Electronic Data Capture 
SAE 
SIT 
Serious Adverse Event 
Speech Intelligibility Test 
SOA  
Schedule of Activities 
VFSS 
Videofluoroscopic Swallow Study 
 
 

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
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without written consent from University of Florida.
Page 14 of 61
TABLE OF CONTENTS
PROTOCOL APPROVAL SIGNATORY PAGE........................................................2
INVESTIGATORS AND FACILITIES.......................................................................3
SYNOPSIS ...................................................................................................................4
LIST OF ABBREVIATIONS.....................................................................................12
TABLE OF CONTENTS ...........................................................................................14
BACKGROUND AND PHARMACOKINETICS.....................................................17
6.1.
Rationale.....................................................................................................................17
6.2.
Mechanism of Action .................................................................................................18
6.3.
Pharmacokinetics........................................................................................................18
6.4.
Safety ..........................................................................................................................19
STUDY OBJECTIVES ..............................................................................................20
7.1.
Primary Objectives .....................................................................................................20
STUDY DESIGN .......................................................................................................20
SELECTION AND ENROLLMENT OF SUBJECTS...............................................20
9.1.
Inclusion Criteria and Exclusion Criteria ...................................................................20
9.1.1.
Inclusion Criteria: .......................................................................................................20
9.1.2.
Exclusion Criteria:......................................................................................................21
9.2.
Study Enrollment Procedures .....................................................................................22
9.2.1.
Subject Recruitment and Retention ............................................................................23
9.2.2.
Screening Logs ...........................................................................................................23
9.2.3.
Randomization/Treatment Assignment ......................................................................24
DRUG PRODUCT .....................................................................................................25
10.1.
Drug Information........................................................................................................25
INVESTIGATIONAL PRODUCT ............................................................................26
11.1.
Handling of Study Drug, Packaging and Labeling.....................................................26

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
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11.2.
Concomitant Interventions..........................................................................................26
11.2.1.
Required Concomitant Medications/Interventions.....................................................26
11.2.2.
Prohibited Medications/Interventions.........................................................................27
11.3.
Subject Compliance....................................................................................................27
STUDY PROCEDURES............................................................................................28
12.1.
Schedule of Activities.................................................................................................28
12.2.
Timing of Study Activities .........................................................................................30
12.2.1.
Screening procedures..................................................................................................30
12.2.2.
On-Study/On-Intervention Evaluations/Procedures ...................................................32
12.3.
SPECIAL INSTRUCTIONS AND DEFINITIONS OF EVALUATIONS ...............39
12.3.1.
Protocol Deviations ....................................................................................................39
12.3.2.
Documentation of Amyotrophic Lateral Sclerosis .....................................................39
12.3.3.
Medical History ..........................................................................................................40
12.3.4.
Treatment History.......................................................................................................40
12.3.5.
Concomitant Medications/Treatments........................................................................40
12.3.6.
Clinical Assessments ..................................................................................................40
12.3.7.
Laboratory Evaluations...............................................................................................40
12.3.8.
Lumbar Puncture Analysis: ........................................................................................41
12.3.9.
Speech, Swallowing and Pulmonary Function:..........................................................41
12.3.10.
Imaging.......................................................................................................................43
12.3.11.
BDI-Fast Screen..........................................................................................................44
12.3.12.
Subject Adherence Assessments.................................................................................44
SAFETY AND ADVERSE EVENTS........................................................................44
13.1.
Adverse Experience Reporting and Follow Up..........................................................45
13.2.
Assessing Relationship to Study Drug .......................................................................46
13.3.
Procedures for Reporting Adverse Events..................................................................46

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
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Page 16 of 61
CRITERIA FOR INTERVENTION DISCONTINUATION.....................................48
STATISTICAL CONSIDERATIONS .......................................................................48
15.1.
General Design Issues.................................................................................................48
15.2.
Outcomes ....................................................................................................................48
15.3.
Primary and Secondary Outcomes: ............................................................................48
15.4.
Sample Size and Accrual............................................................................................49
15.5.
Data Monitoring..........................................................................................................49
15.6.
Data Analyses .............................................................................................................49
15.7.
Statistical Analysis......................................................................................................49
ETHICAL CONSIDERATIONS................................................................................50
16.1.
Basic Principles ..........................................................................................................50
16.2.
Institutional Review Board Review and Informed Consent.......................................50
16.3.
Revisions and/or Amendments to the Protocol ..........................................................51
16.4.
Study Termination ......................................................................................................52
16.5.
Delegation of Investigator Tasks................................................................................52
16.6.
Subject Confidentiality...............................................................................................52
REFERENCES ...........................................................................................................52
APPENDIX A................................................................................................................................54
APPENDIX B................................................................................................................................55
APPENDIX C................................................................................................................................56
APPENDIX D................................................................................................................................57
APPENDIX E ................................................................................................................................58
APPENDIX F.................................................................................................................................59
APPENDIX G................................................................................................................................60
APPENDIX H................................................................................................................................61

Protocol Number: UF2019001 Tolerability & Safety in ALS
Version No 0.9, December 16, 2023
Metformin
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BACKGROUND AND PHARMACOKINETICS
Metformin is an inexpensive, well tolerated, orally active biguanide that is known to cross the blood 
brain barrier. Dr. Ranum’s laboratory has discovered that Metformin can reduce RAN protein levels 
in cell culture and a C9orf72 BAC transgenic mouse model, and improved disease phenotypes and 
motor neuron survival in the C9orf72 ALS mice. This study is designed to evaluate the safety and 
tolerability of Metformin in the treatment of C9ORF72-expansion positive patients with amyotrophic 
lateral sclerosis. The study will also test the hypothesis that metformin can reduce RAN protein levels 
in patients with C9orf7 ALS.
6.1.
Rationale
The discovery that a G4C2 repeat expansion in the C9orf72 gene is the most common cause of both 
familial and sporadic amyotrophic lateral sclerosis (C9-ALS) presents an opportunity to better 
understand the molecular mechanisms of ALS and more than 40 similar microsatellite expansion 
diseases. In addition to ALS, these repeat expansion diseases also include Huntington’s disease, 
myotonic dystrophy types 1 and 2, fragile X syndrome and more than ten different spinocerebellar 
ataxias. Currently there are no effective treatment strategies for ALS or any of these disorders. 
Research in the Ranum laboratory identified two fundamental mechanisms in spinocerebellar ataxia 
type 8 (SCA8) that are now thought to play major roles in C9-ALS and other microsatellite expansion 
diseases. These mechanisms include: 1) bidirectional expression of the expanded repeat mutation 
resulting in the expression of sense and antisense expansion transcripts; and 2) that expansion 
mutations express toxic proteins in all three reading frames without the canonical AUG start codon 
through a novel process called repeat associated non-AUG (RAN) translation. The accumulation of 
RAN proteins in C9-ALS and a growing number of diseases highlights the need to develop drugs that 
effectively block their production.
More recently, the Ranum lab has generated unpublished preclinical data (Zu et al., 2019, manuscript 
in revision, APPENDIX H) showing RAN translation can be regulated both in vitro and in vivo 
through the protein kinase R (PKR) pathway. In cells, steady state levels of several types of RAN 
proteins are increased by PKR activation and decreased by PKR inhibition. Additionally, the Ranum 
lab previously generated a C9orf72 BAC (C9-BAC) transgenic mouse model that mimics the 
neuromuscular, neurological and molecular features of both C9-ALS.  Using these C9-BAC mice, 
they show that inhibiting PKR by AAV expression of a dominant-negative form of the protein, PKR-
K296R decreases RAN protein pathology in vivo and improves behavioral phenotypes. Additionally, 
the Ranum group has data showing Metformin, a widely used FDA-approved diabetes drug, also 
decreases PKR activation. Treatment of the C9orf72 BAC transgenic ALS mice with Metformin also 
decreased RAN protein levels, reduced neuroinflammation and improved behavioral and increased 
motor neuron survival in their C9-BAC mice. Based on these promising preclinical data, we now 
propose a study to test the safety and potential efficacy of Metformin in the treatment of C9-ALS 
patients.
Metformin is a widely used, well-tolerated drug that has been used for decades as a first-line defense 
for treating type 2 diabetes. Recently, it has also been shown to have beneficial effects on aging and 
neurodegenerative disorders. In Fragile X syndrome (FXS), Metformin normalizes protein translation 

 
 
Protocol Number: UF2019001 Tolerability & Safety in ALS 
Version No 0.9, December 16, 2023 
Metformin 
 
 
 
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Page 18 of 61 
 
by inhibiting the MAPK/Erk pathway indicating a connection with protein translation. Metformin 
also improved survival and behavioral phenotypes in Huntington’s disease mice, a microsatellite 
expansion disorder that also involves RAN protein accumulation.  
6.2. 
Mechanism of Action 
While there are multiple ongoing therapeutic strategies aimed at targeting individual C9-RAN 
proteins, there are no effective strategies targeting the production of these proteins.  Structured RNAs 
are a common theme in RAN translation as multiple expanded repeat motifs, including the C9 G4C2 
and G2C4 motifs, are known to express RAN proteins and form hairpin or G-quadruplex structures. 
The increased propensity of longer repeat tracts to adopt RNA structures may explain why longer 
repeat tracts are typically associated with higher levels of RAN protein accumulation and expression 
of RAN proteins in multiple reading frames. In investigating the effects of cellular stress pathways on 
RAN translation, we have discovered that hairpin-forming repeat expansions induce the 
phosphorylation of PKR which leads to an upregulation of RAN translation.  Additionally, by 
studying compounds that can alter RAN translation, we have discovered that Metformin decreases 
RAN translation by inhibiting levels of phosphor-PKR in cell culture models. Additionally, we have 
evidence that Metformin reduces RAN translation and ameliorates phenotypes in our C9-ALS mouse 
model. Taken together these data suggest that Metformin may be effective in the treatment of C9-
ALS.  
6.3. 
Pharmacokinetics 
We will be using Metformin 500 mg ER from Amneal Pharmaceutical or an equivalent supplier.   
Absorption 
The absolute bioavailability of a metformin hydrochloride 500 mg tablet given under fasting 
conditions is approximately 50% to 60%. Studies using single oral doses of metformin hydrochloride 
500 mg to 1,500 mg and 850 mg to 2,550 mg, indicate that there is a lack of dose proportionality with 
increasing doses, which is due to decreased absorption rather than an alteration in elimination. At 
usual clinical doses and dosing schedules of metformin hydrochloride, steady state plasma 
concentrations of metformin are reached within 24 to 48 hours and are generally <1 mcg/mL. 
Following a single oral dose of metformin hydrochloride extended-release tablets, C is achieved with 
a median value of 7 hours and a range of 4 to 8 hours. Peak plasma levels are approximately 20% 
lower compared to the same dose of metformin hydrochloride tablets, however, the extent of 
absorption (as measured by AUC) is comparable to metformin hydrochloride tablets.  
 
At steady-state, the AUC and C are less than dose proportional for metformin hydrochloride 
extended-release tablets within the range of 500 mg to 2,000 mg administered once daily. Peak 
plasma levels are approximately 0.6, 1.1, 1.4 and 1.8 mcg/mL for 500, 1,000, 1,500, and 2,000 mg 
once-daily doses, respectively. The extent of metformin absorption (as measured by AUC) from 
metformin hydrochloride extended-release tablets at a 2,000 mg once-daily dose is similar to the same 
total daily dose administered as metformin hydrochloride tablets 1,000 mg twice daily. After repeated 
administration of metformin hydrochloride extended-release tablets, metformin did not accumulate in 
plasma. Effect of food: Food decreases the extent of absorption and slightly delays the absorption of 
metformin, as shown by approximately a 40% lower mean peak plasma concentration (C ), a 25% 

 
 
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lower area under the plasma concentration versus time curve (AUC), and a 35-minute prolongation of 
time to peak plasma concentration (T) following administration of a single 850 mg tablet of 
metformin hydrochloride with food, compared to the same tablet strength administered fasting. 
Although the extent of metformin absorption (as measured by AUC) from the metformin 
hydrochloride extended-release tablets increased by approximately 50% when given with food, there 
was no effect of food on C and T of metformin. Both high and low fat meals had the same effect on 
the pharmacokinetics of metformin hydrochloride extended-release tablets. 
 
Distribution 
The apparent volume of distribution (V/F) of metformin following single oral doses of metformin 
hydrochloride 850 mg averaged 654 ± 358 L. Metformin is negligibly bound to plasma proteins. 
Metformin partitions into erythrocytes, most likely as a function of time. 
 
Metabolism  
Intravenous single-dose studies in normal subjects demonstrate that metformin is excreted unchanged 
in the urine and does not undergo hepatic metabolism (no metabolites have been identified in 
humans) nor biliary excretion. 
 
Elimination 
Renal clearance is approximately 3.5 times greater than creatinine clearance, which indicates that 
tubular secretion is the major route of metformin elimination. Following oral administration, 
approximately 90% of the absorbed drug is eliminated via the renal route within the first 24 hours, 
with a plasma elimination half-life of approximately 6.2 hours. In blood, the elimination halflife is 
approximately 17.6 hours, suggesting that the erythrocyte mass may be a compartment of distribution. 
 
6.4. 
Safety 
Metformin is contraindicated in patients with severe renal impairment (eGFR below 30 mL/min/1.73 
m2), hypersensitivity to Metformin, and acute or chronic metabolic acidosis, including diabetic 
ketoacidosis, with or without coma.  
See attached product information sheet (Appendix H) regarding the warnings and precautions (lactic 
acidosis, vitamin B12 deficiency, and hypoglycemia. 
The most commonly observed AEs associated with the use of Metformin are listed in table 1 of the 
product information sheet and include diarrhea, nausea/vomiting, flatulence, asthenia, indigestion, 
abdominal discomfort, and headache.  Diarrhea lead to discontinuation in 6% of patients.  
Postmarketing experience has identified cholestatic, hepatocellular, and mixed hepatocellular liver 
injury (see package insert attached). 
 

Protocol Number: UF2019001 Tolerability & Safety in ALS
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STUDY OBJECTIVES
7.1.
Primary Objectives
Metformin is an inexpensive, well-tolerated, orally active biguanide that is known to cross the blood 
brain barrier. Preclinical data generated in the Ranum lab show that Metformin can reduce RAN 
protein levels in cell culture and in a C9orf72 BAC transgenic mouse model. Additionally, they show 
that Metformin treatment improves behavioral phenotypes including DigiGait performance, decreases 
neuroinflammation and increases motor neuron survival in these mice.  These data provide the 
rationale for testing the therapeutic potential of Metformin in C9orf72 ALS patients.  Our primary 
objective is to evaluate the safety and tolerability of Metformin in the treatment of C9orf72-expansion 
positive patients with amyotrophic lateral sclerosis. An additional primary outcome measure will be 
to test if metformin reduces RAN protein levels which are produced from the C9orf72 expansion 
mutation in blood and CSF from C9orf72 ALS patients being treated with metformin.
STUDY DESIGN
Approximately 16 subjects with C9orf72 positive ALS will be enrolled. The subjects will be 
instructed in the use of Metformin and receive the first dose of Metformin under supervision of Dr. 
Wymer during Visit 1.
Subjects will then continue on Metformin per the dosing schedule for 24 weeks; with interim safety 
assessments at days 42, 84, 126 and 168 (+/- ~7 days).
Subjects will be screened to determine eligibility according to the specified inclusion/exclusion 
criteria.
SELECTION AND ENROLLMENT OF SUBJECTS
9.1.
Inclusion Criteria and Exclusion Criteria
9.1.1.
Inclusion Criteria:
Subjects meeting all the following criteria may be included in the study:
1. Male or female subjects between 18-80 years of age, inclusive.
2. Subjects have a diagnosis of possible, probable or definite ALS in accordance with 
the Revisited El-Escorial Criteria 
3. Subjects must be currently on an oral diet and able to take foods and liquids by mouth 
equivalent to a score of 4 or above on the Functional Oral Intake Scale (Crary, 2005). 
4. Subjects must have no known allergy to barium sulfate or Metformin.

 
 
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5. Subjects or subject’s legally authorized representative must be willing and able to 
complete informed consent/assent and HIPAA authorization.  
6. Ability to comprehend and be informed of the nature of the study, as assessed by the 
PI or Sub-Investigator. 
7. Availability to participate for the entire study duration. 
8. Female subjects of childbearing potential must have a negative urine pregnancy test at 
Screening and Visit 1, 3, and 4.   
9.1.2. 
Exclusion Criteria: 
Potential subjects meeting any of the following criteria will be excluded: 
1. Subjects who score 3 or below on the Functional Oral Intake Scale (Crary, 2005). 
2. Subjects who do not carry the C9orf72 hexanucleotide repeat expansion as 
determined by previous laboratory analysis. 
3. Subjects with known allergy to barium sulfate or Metformin. 
4. Subjects with a history of clinically significant liver disease, renal disease, or any 
other medical condition judged to be exclusionary by the investigator. 
5. Subjects who are unwilling to sign informed consent or subjects who for any other 
reason in the judgment of investigator are unable to complete the study. 
6. )HPDOHVXEMHFWVZKRKDYHDSRVLWLYHXULQHSUHJQDQF\WHVWȕK&*DWVFUHHQLQJRU
visit 1, 2, 3, and 4, or are trying to become pregnant or are breastfeeding. 
7. Subjects with active cancer within the previous 2 years, except treated basal cell 
carcinoma of the skin. 
8. Subjects who have taken any experimental drug within 30 days prior to enrollment or 
within 5 half-lives of the investigational drug – whichever is the longer period.   
9. Subjects with known history or presence of moderate or severe renal impairment as 
defined by an eGFR value below 30 mL/min/1.73 m2. 
10. Subjects with hepatic impairment as defined by baseline elevations of serum 
aminotransferases greater than 5 times upper limit of normal or evidence of liver 
dysfunction (e.g., elevated bilirubin). 
11. Use of potentially hepatotoxic drugs: (e.g., allopurinol, methyldopa, sulfasalazine). 
12. Subjects with clinically significant abnormal laboratory values in the judgment of the 
investigator.  

 
 
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13. Subject with implanted electrical device (i.e. cardiac pacemaker or a 
neurostimulator), metal or metallic clip(s) in their body (i.e., an aneurysm clip in the 
brain) that will be damaged by participation in the MRI portion of the study. 
14. Anything else that, in the opinion of the investigator, would place the subject at 
increased risk or preclude the subject’s full compliance with or completion of the 
study. 
Inclusion in the study is entirely voluntary and subjects may withdraw at any time for any 
reason. Possible reasons for withdrawal could include serious adverse events, adverse events, 
and subject’s choice. Subjects wishing to stop medication will immediately discontinue 
medication. Subjects who elect to discontinue medication will be asked to comply with end 
of study assessments (visit 4 on Table 3 - Schedule of Activities table) at a follow-up visit in 
an outpatient setting (to be determined by Investigator or Staff) at 14 +/- days after the last 
treatment Visit.    Subjects who withdraw due to an adverse event will be followed whenever 
possible until resolution of the adverse event. It may be appropriate for the subject to return 
to the site 2 weeks after the subject is off study to evaluate for resolution of any adverse 
events and to receive further information about options for future clinical care.  The 
Investigator will decide the course of action. 
9.2. 
Study Enrollment Procedures 
The following screening procedures will be conducted on each potential subject. 
x Obtain informed consent as evidenced by potential subject or subject’s legally 
authorized representative signing an informed consent. Informed consent will be 
signed prior to any study-related procedures and prior to screening per protocol. 
Informed consent will be obtained by one of the investigators and/or his or her 
designee. 
x Record medical history 
x Documentation of disease and C9orf72 mutation 
x Obtain vital signs (temperature, respiratory rate (RR), heart rate (HR), blood 
pressure (BP)) and weight/height.  
x Collect blood and urine for clinical laboratory evaluation (hematology, chemistry, 
liver function tests, urinalysis) and for additional molecular studies including 
RAN protein assessment, and urine for pregnancy test (females of childbearing 
potential only). For a complete list of all tests to be performed (Section 12.3.7, 
Laboratory Evaluations.) 

 
 
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x COVID nasal collection and testing will be completed to determine if the 
participant is COVD-19 negative. The results from the testing will be provided to 
the participant the next day when results have been placed in EPIC. 
x Complete physical and neurological examinations 
x ALS Functional Rating Scale – Revised (ALSFRS-R) 
x Eating Assessment Tool – 10 (EAT-10) 
x Functional Oral Intake Scale (FOIS) 
x BDI-Fast Screen  
x Collection of pretreatment AEs, if the subject is receiving Metformin pre-study  
x Principal Investigator’s/Sub-Investigator’s review of Inclusion/Exclusion criteria 
and all screening results/data to assess eligibility of each potential subject 
x Record/Review concomitant medications 
9.2.1. 
Subject Recruitment and Retention 
Details for recruitment procedures are provided in the protection of human subjects section. 
Briefly, ALS patients will be primarily identified and recruited from the University of 
Florida-Gainesville. Due to the rarity of the C9orf72 gene mutation, if insufficient patients 
are recruited, physicians at the University of Florida we are in frequent contact with 
physicians at other ALS centers in Florida and other parts of the US (Mayo-JAX, UF 
Jacksonville, Baptist Health, USF, and Miami, Johns Hopkins and Washington University) or 
other institutions and organizations. Generally, patients with the C9orf72 gene are well-
known to their attending neurologists. The PI, co-investigators, and study coordinator will be 
able to work with treating neurologists at these sites to identify potential participants. C9 
ALS subjects currently taking Metformin or previously prescribed Metformin may be 
considered. If a patient known to have the C9orf72 mutation is interested, contact 
information will be offered to the patient so that they can communicate with local study staff 
at will. We have a target recruitment schedule of only 16 patients and it is expected that there 
will be minimal need to reach out to other sites.  All other sites, if referring patients, will 
ONLY BE USED FOR RECRUITMENT purposes. All recruited patients will be seen at UF 
Gainesville.  The study will be advertised through clinicaltrials.gov and IRB approved UF 
listings. 
9.2.2. 
Screening Logs 
Screening logs to document reasons for ineligibility and reasons for nonparticipation of 
eligible subjects will be maintained to document number of subjects referred, referral source, 

 
 
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number screened, number failed due to ineligibility or nonparticipation of eligible subjects, 
and reasons for ineligibility or nonparticipation of eligible subjects.  
9.2.3. 
Randomization/Treatment Assignment 
Randomization is not applicable. This is an open label safety and tolerability trial. 

Protocol Number: UF2019001 Tolerability & Safety in ALS
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DRUG PRODUCT
10.1.
Drug Information
Table 2 Drug Information
Treatment
Drug Name
Metformin SR
Strength
500mg
Dosage Form
Oral tablet
Manufacturer
Dose
1 x 500 mg/day during week 1
2 x 500 mg/day during week 2
3 x 500 mg/day during week 3
4 x 500 mg/day during weeks 4-24
4 x 500 mg/day during Weeks 24-Week 
36 (Optional dosing)

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INVESTIGATIONAL PRODUCT
11.1.
Handling of Study Drug, Packaging and Labeling
The Investigative team will obtain a sufficient quantity of the study drug from the UF 
Pharmacy to allow completion of this study including 10% extra for replacements if pills are 
lost or misplaced by study subjects.
The study drug will be sent to the Investigator.  Each individual bottle with drug will be 
labeled in English containing at minimum:  Protocol Number, Drug Name, Strength, Does 
and Route of Administration, and cautionary statements required by the FDA about the use 
of this FDA approved drug to test the safety and tolerability of the drug in C9orf72 ALS 
patients, “Keep Out of Reach of Children”, storage conditions and batch/lot number.
The site pharmacy will keep a log of study drug kits received and dispensed identified by 
subject ID, subject initials, and date dispensed. Study drug will be stored at room temperature 
as directed by institutional SOP. The site pharmacist or coordinator as indicated in the 
delegation log will dispense study drug at the study visit #1, visit #2, visit 3 and visit 4.  
Subjects will begin treatment with Metformin at a dosage of 500 mg / day with an escalation 
of dosage by 500 mg every week to a maximal dosage of 2000 mg. Dosing will be twice 
daily from week 2 to week 24. In accordance with local regulatory requirements, the 
investigator, designated site staff, or head of the medical institution (where applicable) must 
document the amount of investigational product dispensed to study subjects, the amount 
received, and the amount destroyed upon completion of the study. The investigator is 
responsible for ensuring product accountability records are maintained throughout the course 
of the study. The inventory record will include details of Metformin study drug received and 
dispensed to subjects, batch, and ID numbers will be maintained by the Investigator.   
At the completion of the study, all unused study drug, including spares, will be retained by 
the study site. An accounting must be made of any drug deliberately or accidentally 
destroyed.
11.2.
Concomitant Interventions
All concomitant medications received by a subject during participation in the study will be 
recorded on the appropriate source documents.
11.2.1.
Required Concomitant Medications/Interventions
No concomitant medications or interventions are required per protocol.

 
 
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11.2.2. 
Prohibited Medications/Interventions 
Concomitant medications and interventions will be reviewed at screening. The investigator 
may choose to exclude a subject from participation if a concomitant medication or 
intervention is judged likely to interfere with the study objectives or to affect the subject’s 
safety. 
The following drugs are known to interact with Metformin tablets (US Package Insert; 
revision 05/2018) and should not be administered with Metformin: 
x Agents that may increase Metformin blood concentration;  
x Agents that may decrease Metformin plasma concentrations;  
x Agents that may increase the risk of lactic acidosis; Carbonic anhydrase inhibitors 
may increase risk of lactic acidosis.  
x Agents that may impact glucose control and cause hyper or hypo glycemia; Taking 
Metformin with other drugs that lower blood sugar can raise your risk of 
hypoglycemia (low blood sugar). 
x Potentially hepatotoxic drugs: (e.g., allopurinol, methyldopa, sulfasalazine) 
x Drugs that reduce metformin clearance (such as ranolazine, vandetanib, dolutegravir, 
and cimetidine) may increase the accumulation of metformin. 
x Medications that interact with Metformin include digoxin, cimetidine, furosemide, 
nifedipine, amiloride, ranitidine, triamterene, morphine, quinidine, vancomycin, 
trimethoprim and procainamide.  
x Alcohol can potentiate the effect of metformin on lactate metabolism and should not 
be used in excess while taking Metformin.  
11.3. 
Subject Compliance 
Subject compliance will be assessed at all visits with direct and open questioning about 
compliance and adverse events. All returned medication will be counted. Greater than 10% 
noncompliance will prompt re-education of subject by the investigator on the importance of 
medication compliance. 
 
 
 
 
 

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STUDY PROCEDURES
12.1.
Schedule of Activities
The schedule of study activities is presented in Table 3.
Timeline:

 
 
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Table 3. Schedule of Activities: 
 
 
 
 
 
 
 
 
 
 
 
Evaluation 
Recruit 
& 
Screen 
Visit 1   
Visit 1  
Visit 2  
Visit 3  
Phone Call 
Visit 4  
Visit 4 
Phone Call 
Phone 
Call 
Day 1 
Day 2 
6 weeks 
12 weeks 
18 Weeks 
Day 1 
24 weeks 
Day 2 
24 weeks 
36 Weeks 
52 
weeks 
PM 
AM 
(42 days) 
(84 days) 
(126 days) 
(168 
days) 
(168 days) 
(252 days) 
(365 
days) 
Informed Consent 
X 
  
  
  
  
  
  
 
  
  
Inclusion/Exclusion 
Review 
X 
  
  
  
  
  
  
 
  
  
Documentation of Disease 
X 
  
  
  
  
  
  
 
  
  
Record/Review 
Concomitant Medications 
X 
 
X  
X 
X 
X 
X 
 
X 
 
Medical 
History/Demographics 
X 
 
X  
  
  
X 
  
 
X 
 
Vital Signs/Height and 
Weight 
X 
 
X 
X 
X 
  
X 
 
  
  
Comprehensive Physical 
and Neurological Exams 
X 
  
  
  
X 
  
X 
 
  
  
Brief Physical and 
Neurological Exam 
  
 
X  
X 
  
  
  
 
  
  
C9+ and RAN protein 
blood draw 
X 
 
X 
X 
X 
  
X 
 
  
  
Clinical Labs for safety 
(hematology, chemistry, 
liver function and 
urinalysis) 
X 
 
X 
X 
X 
  
X 
 
  
  
Urine Pregnancy Test 
(females of childbearing 
potential) 
X 
 
X  
X 
X 
  
X 
 
  
  
Lumbar Puncture (for 
RAN proteins and 
biomarkers) 
  
 
X  
X 
X 
  
X 
 
  
  
MRI/DTI imaging 
  
 
X 
  
  
  
 
X 
  
  
VFSS Penetration-
Aspiration Scale 
  
 X 
 
  
X 
  
 
X 
  
  
Functional Oral Intake 
Scale (FOIS) 
  
X 
 
  
X 
X 
 
X 
X 
X 
ALS Functional Rating 
Scale – Revised (ALSFRS-
R) 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
Eating Assessment Tool-10 
(EAT-10) 
  
X 
 
  
X 
X 
 
X 
X 
X 
Forced Vital Capacity 
(FVC) 
  
X 
 
  
X 
  
 
X 
  
  
Peak Cough Flow 
  
X 
 
  
X 
  
 
X 
  
  
Body Mass Index 
  
X  
 
  
X 
  
 
X 
  
  
BDI-FastScreen  
X 
  
  
X 
X 
 
X 
 
 
  
Adverse Event 
  
  
X 
X 
X 
X 
X 
 
X 
X 
Dispense Study Drug 
  
  
X 
X 
X 
  
 
 
  
  

 
 
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Drug 
Accountability/Compliance 
  
  
  
X 
X 
  
X 
 
  
  
 
Outcome Measures: 
Primary: 1. ALSFRS-R score 
Secondary: 2. Soluble GP in blood; 3. Swallowing physiology (PAS, DIGEST); 4. PFT’s 
(FVC, PCF); 5. MRI/DTI outcomes. 
12.2. 
Timing of Study Activities 
No study procedures will be performed prior to the signing of the Informed Consent Form 
(ICF). All subjects will sign an ICF prior to undergoing any study tests or procedures.  
Visit windows are consecutive calendar days and the target visit dates are calculated from the 
Baseline Visit, which should occur within 14 to 28 days of the Screening Visit. All clinic 
visits and calls after the Baseline visit will have a +/- 7 work day window. 
Inclusion in the study is entirely voluntary and subjects may withdraw at any time for any 
reason. Possible reasons for withdrawal could include serious adverse events, adverse events, 
and subject’s choice.  Subjects wishing to stop medication will immediately discontinue test 
article. Subjects who elect to discontinue medication will be asked to comply with end of 
study assessments (visit 4 on Table 3 Schedule of Activities) at a follow up visit in an 
outpatient setting (to be determined by Investigator or Staff) at 14 +/- days after the last 
treatment dose.  Subjects who withdraw due to an adverse event will be followed whenever 
possible until resolution of the adverse event. It may be appropriate for the subject to return 
to the site 2 weeks after the subject is off study to evaluate for resolution of any adverse 
events and to receive further information about options for future clinical care.  The 
Investigator will decide the course of action. 
12.2.1. 
Screening procedures 
At the screening visit, potential subjects will be informed about study procedures and will 
then sign an informed consent form. Inclusion/exclusion criteria will be reviewed and a 
medical history and comprehensive physical and neurological examination will be 
completed. Vital signs, height, weight, and concomitant medications will be recorded. Safety 
laboratory tests will be performed including complete blood count (CBC) with differential, 
electrolytes, BUN, creatinine, ALT, AST, total bilirubin, albumin, and a urine pregnancy test 
for women of childbearing potential. and COVID-19 nasal collection and testing.  
The following procedures will be performed during the screening visit: 
x Informed consent 
x Inclusion/exclusion assessment 

 
 
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x Documentation of Disease 
x Medical history/Demographics 
x Record concomitant medications 
x Clinical labs for safety (hematology, chemistry, liver functions, and urinalysis)  
x C9+ and RAN protein blood draw 
x Urine Pregnancy Test (females of childbearing potential) 
x COVID-19 nasal collection and testing - Due to COVID-19 Pandemic nasal 
collection and testing of the participants has been implemented to determine if the 
participant is COVID negative. The collection is done at UF Springhill collection site 
and testing is completed at Rocky Point Labs overnight with results placed in EPIC. 
The results of the testing will be provided to the participant once results are posted in 
EPIC. This will collection and testing will continue throughout the participants 
participation in the study at this visit. 
x Vital signs 
x Height and weight 
x Comprehensive physical and neurological examinations  
x ALS Functional Rating Scale – Revised (ALSFRS-R) 
x BDI-Fast Screen 
 
As part of the screening visit, the investigator will determine the competence of caregiver to 
administer the study drug at home. Teaching will begin on the importance of compliance and 
proper handling of study drug. All inclusion and exclusion criteria and safety laboratory tests 
will be reviewed by the investigator prior to scheduling the baseline visit (visit 1 on schedule 
of activities table). A paper log will be kept at the site to record all subjects screened for 
entry into the study. This information will also be captured electronically in the electronic 
data capture system (EDC) using REDCap. REDCap (Research Electronic Data Capture) is a 
secure, Web-based application designed to support traditional case report form data capture. 
Demographic characteristics of all subjects who are screened will be recorded whether or not 
they qualify for entry into the study. The reason for non-qualification will be recorded for all 
subjects who are not eligible. The reason for nonparticipation will also be recorded for 
subjects who are eligible but choose not to participate in the trial. If a subject fails screening 
for the study, the subject can be re-screened if the investigator determines it is appropriate to 
do so. At any time during the study, repeat laboratory tests can be obtained if the investigator 
or central laboratory believes that a laboratory test result is in error. Additional laboratory 
tests may also be obtained at any time during the study, at the discretion of the investigator. 
ALS Functional Rating Scale – Revised (ALSFRS-R) Questionnaire  

 
 
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The ALSFRS-R is a quickly administered (5 minute) ordinal rating scale (ratings 0-4) used to 
determine subjects’ assessment of their capability and independence in 12 functional 
activities/questions (Cedarbaum, 1999). All 12 activities are relevant in ALS. Initial validity 
was established by documenting that in subjects with ALS, change in ALSFRS-R scores 
correlated with change in strength over time, was closely associated with quality of life 
measures, and predicted survival (Appendix A). The test-retest reliability is greater than 0.88 
for all test items. The advantages of the ALSFRS-R are that the categories are relevant to 
ALS; it is a sensitive and reliable tool for assessing activities of daily living function in 
subjects with ALS; and it is quickly administered. In a recent trial employing the ALSFRS as 
a secondary outcome measure, placebo-treated patients showed a decline of 0.92 units per 
month, with a standard error of 0.08. With appropriate training the ALSFRS-R can be 
administered with high inter-rater reliability and test retest reliability. 
Beck Depression Inventory-Fast Screen (BDI-FS) (Appendix D) is a brief self-report 
inventory designed to evaluate depression in patients with medical illness. It has been found 
to be an efficient and effective tool for depression screening in the ALS population. The 
symptoms and prognosis of ALS have an immense impact on a patient’s life as well as their 
family and caregivers. Depression, when present, can significantly affect the life of patients 
and is more commonly seen in situations involving the threat of loss. BDI-FS is a short 7 
question self-report that can assist the investigator and staff in evaluating if patients are 
dealing with depression. The BDI-FS is evaluated at the time of the subject’s study visit by 
study staff. Each answer is scored on a scale of 0-3. Measures of 4-6 correspond to mild 
depression, 7-9 indicate moderate depression, and 10-21 indicates moderate-severe 
depression. Any measurement indicating depression, or any verbal suggestion of suicidality, 
will be reported to the investigator immediately. The investigator will address the results with 
the subject prior to their leaving the facility.  Any interventions deemed necessary will be 
addressed by a qualified physician or investigator at that time. 
12.2.2. 
 On-Study/On-Intervention Evaluations/Procedures 
12.2.2.1. Baseline, Visit 1, Day 1 pm and Visit 1, Day 2 am:  
The baseline visit (Visit 1, Day 1 pm and Visit 1, Day 2 am), will cover 2 days and include 
the first dose of study drug, and study drug dispensing. It will be scheduled within 14 to 28 
days after the screening visit. At this visit inclusion/exclusion criteria and concomitant 
medications will be reviewed. Vital signs, brief physical and neurologic examination, as well 
as study questionnaires and testing will be performed as noted in the schedule of activities. 
To lessen travel for participants during the COVID-19 pandemic, and after the pandemic, the 
Visit 1, Day 1 and Visit 1, Day 2 may take place in the same week as the Screening visit. If 
these visits are scheduled to take place in the same week, the *clinical labs for safety and 
*blood draw for C9+ and RAN proteins, and *urine pregnancy test, will not be repeated. 
The following documentation and procedures will be performed at Baseline (Visit 1, Day 1): 

 
 
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x FOIS 
x BDI-Fast Screen (BDI-FS) 
x VFSS – Penetration Aspiration Scale 
x EAT – 10 
x Forced Vital capacity (FVC)  
x Peak Cough Flow 
x Body Mass Index 
 
The following documentation and procedures will be performed at Baseline (Visit 1, Day 2): 
x Adverse event review 
x Inclusion/exclusion assessment 
x Record/review Concomitant medications 
x Medical history 
x Vital signs; weight and height 
x Brief physical exam 
x Brief neurologic exam 
x Clinical labs for safety (hematology, chemistry, liver functions, and urinalysis)  
x Blood draw for C9+ and RAN proteins 
x Lumbar puncture for RAN proteins and biomarkers 
x Urine Pregnancy Test (females of childbearing potential) 
x MRI/DTI imaging 
x ALSFRS-R 
x Dispense study drug 
Eating Assessment Tool -10 (EAT-10) 
EAT-10: The Eating Assessment Tool-10, is a validated 10-item patient report scale of 
perceived swallowing impairment (Belafsky et al. 2008).  The EAT-10 uses a 5-point ordinal 
rating scale where a patient rates their perceived degree of impairment for each item from 0 
(no impairment) to 4 (severe impairment) for a total range of scores between 0 (no perceived 
swallowing impairment) to 40 (severe impairment) (Appendix B).  One of the former PIs of 
this study (Dr. Plowman) recently determined that the EAT-10 was sensitive to identify ALS 
patients with severe dysphagia including aspiration. A Receiver Operating Curve analysis 
revealed that a cut point of 8 on the EAT-10 yielded a sensitivity of 86%, specificity of 72%, 

 
 
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likelihood ratio of 3.1, and negative predictive value of 95.5% for detecting aspiration in 
ALS (Plowman, et al., 2016). 
Functional Oral Intake Scale (FOIS) 
The FOIS is a simple 7-point scale (Crary, 2005 – see Appendix C) that describes the 
subject’s functional status with respect to oral intake, ranging from 1 (No oral intake) to 7 
(total oral intake with no restrictions). 
 
12.2.2.2. Study Visit 2, Week 6:  
All subjects will return at week 6 for a brief evaluation. The visit window is to be scheduled 
at 6 weeks (42 days) plus or minus 3 days. Participants will be seen for assessment of any 
adverse events, and concomitant medications. Study drug accountability and compliance will 
be assessed. Vital signs, brief physical and neurologic examination, as well as study 
questionnaires and testing will be performed as noted in the schedule of activities. 
The following procedures will be performed at Visit 2, Week 6: 
x Adverse events review 
x Record/review Concomitant medications 
x Vital signs, weight and height 
x ALSFRS-R 
x Brief physical exam 
x Brief neurologic exam 
x Clinical labs for safety (hematology, chemistry, liver functions, and urinalysis)  
x Blood draw for C9+ and RAN proteins 
x Lumbar puncture for RAN proteins and biomarkers 
x Dispense study medication 
x Drug Accountability/Compliance 
x BDI-Fast Screen (BDI-FS)  
x Urine Pregnancy Test (females of childbearing potential) 
12.2.2.3. Study Visit 3, Week 12:  
All subjects will return at week 12 for a mid-study evaluation. The visit window is to be 
scheduled at 12 weeks (84 days) plus or minus 3 days. Participants will be seen for 
assessment of any adverse events, and concomitant medications. Study drug accountability 
and compliance will be assessed. Vital signs, physical and neurologic examination, as well as 
study questionnaires and testing will be performed as noted in the schedule of activities. 
The following procedures will be performed at Visit 3, Week 12: 

 
 
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x Adverse events review 
x Record/review concomitant medications 
x Vital signs, weight and height 
x ALSFRS-R 
x Comprehensive physical exam 
x Comprehensive neurologic exam 
x Clinical labs for safety (hematology, chemistry, liver functions, and urinalysis)  
x Blood draw for C9+ and RAN proteins 
x Urine Pregnancy Test (females of childbearing potential) 
x COVID-19 nasal collection and testing may take place at this visit if travel time 
allows for the participant - Due to COVID-19 Pandemic, nasal collection and testing 
of the participants has been implemented to determine if the participant is COVID 
negative. The collection is done at UF Springhill collection site and testing is 
completed at Rocky Point Labs overnight with results placed in EPIC. The results of 
the testing will be provided to the participant once results are posted in EPIC. This 
collection and testing may continue throughout participation in the study at this visit.  
x Lumbar puncture for RAN proteins and biomarkers 
x FOIS 
x VFSS – Penetration Aspiration Scale 
x EAT – 10 
x Forced Vital capacity (FVC) 
x Peak Cough Flow 
x Body Mass Index 
x BDI-Fast Screen (BDI-FS) 
x Dispense study medication 
x Drug Accountability/Compliance 
12.2.2.4. Phone Call, Week 18: 
Subjects will be contacted by phone at Week 18 and the following study related evaluations 
will be completed: 
x ALSFRS-R 
x EAT-10 
x FOIS 

 
 
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x Adverse event review 
x Record/review concomitant medications 
12.2.2.5. Study Visit 4, Week 24 Day 1 or Day 2:  
All subjects will return at week 24 for a two-day end of study evaluation. The visit window is 
to be scheduled at 24 weeks (168 days) plus or minus 3 days. Participants will be seen for 
assessment of any adverse events, and concomitant medications. Study drug accountability 
and compliance will be assessed. Vital signs, physical and neurologic examination, as well as 
study questionnaires and testing will be performed as noted in the schedule of activities. 
The following documentation and procedures will be performed at Visit 4, Week 24, on Day 
1 or Day 2: 
x Adverse events 
x Record/review concomitant medications 
x Vital signs, weight and height 
x ALSFRS-R 
x Comprehensive physical exam 
x Comprehensive neurologic exam 
x Clinical labs for safety (hematology, chemistry, liver functions, and urinalysis) – Due 
to the COVID-19 Pandemic, and only for the participants currently enrolled (3) in this 
study, the clinical labs for safety will be drawn at a local lab or physician’s office and 
will be shipped as needed. If there is any charge for the draw the participant may 
either pay the invoice and be reimbursed or have the lab contact the study team in the 
ICF to pay the invoice directly. This modification to the protocol is to prevent 
potential exposure to COVID-19 by minimizing participant travel to our site. 
x Blood draw for C9+ and RAN proteins – Due to the COVID-19 Pandemic, and only 
for the participants currently enrolled (3) in this study, additional blood samples will 
be collected and will be shipped to the Ranum Lab for analysis. The kits, drawing 
instructions, and shipping documents will be sent to the physician or the participant to 
complete this procedure. If there is a charge for the blood draw the participant may 
either pay the invoice and be reimbursed or have the lab contact the study team in the 
ICF to pay the invoice directly. This modification to the protocol is to prevent 
potential exposure to COVID-19 by minimizing participant travel to our site. 
x Urine Pregnancy Test (females of childbearing potential) 
x COVID-19 nasal collection and testing may take place at this visit if travel time 
allows for the participant - Due to COVID-19 Pandemic, nasal collection and testing 
of the participants has been implemented to determine if the participant is COVID 

 
 
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negative. The collection is done at UF Springhill collection site and testing is 
completed at Rocky Point Labs overnight with results placed in EPIC. The results of 
the testing will be provided to the participant once results are posted in EPIC. This 
collection and testing may continue throughout participation in the study at this visit.  
x Lumbar puncture for RAN proteins and biomarkers – Due to the COVID-19 
Pandemic, and only for the participants currently enrolled (3) in this study, this 
procedure may be completed in the participant’s home area and the samples shipped 
to the Ranum Lab for analysis. The procedure will be completed by a licensed 
physician who is qualified in the proper procedures and has agreed to complete the 
lumbar puncture per the protocol. The lumbar puncture kit and needle will be shipped 
to the physician for this procedure. This modification to the protocol is to prevent 
potential exposure to COVID-19 by minimizing participant travel to our site.  
x Drug Accountability/Compliance 
x FOIS 
x VFSS – Penetration Aspiration Scale 
x EAT – 10 
x Forced Vital capacity (FVC) 
x Peak Cough Flow 
x Body Mass Index 
x BDI-Fast Screen (BDI-FS) 
x MRI/DTI imaging 
12.2.2.6. Phone call, Week 36: 
Subjects will be contacted by phone at Week 36 and the following study related evaluations 
will be completed: 
x Adverse events review 
x Record/review concomitant medications 
x ALSFRS-R 
x EAT – 10 
x FOIS 
12.2.2.7. Phone call, Week 52 (Follow-up):  
Subjects will be contacted by phone at week 52 for an end of study evaluation. The phone 
call window is to be scheduled at 52 weeks plus or minus 3 days.   
x Adverse events review 

 
 
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x Record/review concomitant medications 
x ALSFRS-R 
x EAT – 10 
x FOIS 
 
Missed Visits: If a subject fails to appear for a scheduled visit, the site will contact the 
subject. The site will stress the importance of the evaluation and reschedule within a window 
of +/- 3 days if possible. 
12.2.2.8. Study Medication/Intervention Discontinuation Evaluations/Procedures 
The investigator must document within 24 hours any subject permanently discontinuing 
study drug, documenting the reasons for discontinuation in the CRF and on the appropriate 
source documents. Subjects will be asked to comply with end of study assessments (visit 4 on 
schedule of activities table) at a follow-up visit in an outpatient setting (to be determined by 
Investigator or Staff) at 14 +/- days after the last treatment dose.  The outcome measures will 
be documented at the study visit(s) following discontinuation of study drug. Subjects who 
have discontinued study drug will be removed from ongoing participation in the study. 
Inclusion in the study is entirely voluntary and subjects may withdraw at any time for any 
reason. Possible reason for withdrawal could include serious adverse events, adverse events, 
and subject’s choice.  Subjects wishing to stop medication will immediately discontinue test 
drug. Subjects who elect to discontinue medication will be asked to comply with end of study 
assessments (visit 4 on schedule of activities table) at a follow up visit in an outpatient setting 
(to be determined by Investigator or Staff) at 14 +/- days after the last treatment dose.   
Subjects who withdraw due to an adverse event will be followed whenever possible until 
resolution of the adverse event. It may be appropriate for the subject to return to the site 2 
weeks after the subject is off study to evaluate for resolution of any adverse events and to 
receive further information about options for future clinical care.  The Investigator will 
decide the course of action. 
All serious adverse events (SAEs), whether or not the event is deemed drug-related, will be 
reported on the SAE Report Form.   
12.2.2.9. Final On-Study Evaluations 
Subjects who elect to discontinue medication will be asked to comply with end of study 
assessments (see visit 4 on schedule of activities table) at a follow-up visit in an outpatient 
setting (to be determined by Investigator or Staff) at 7 +/- days after the last treatment visit.   
Subjects who withdraw due to an adverse event will be followed whenever possible until 
resolution of the adverse event. It may be appropriate for the subject to return to the site 2 
weeks after the subject is off study to evaluate for resolution of any adverse events and to 

 
 
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receive further information about options for future clinical care.  The Investigator will 
decide the course of action. 
12.2.2.10. Off-Study Requirements 
Study medication will be distributed at Week 24 (due to COVID-19 pandemic and travel 
difficulties). Drug will be discontinued and collected at Week 36. Study drug will be ordered 
by the Co-Investigator, dispensed by Investigational Drug Service and a study team member 
will ship it to the participants (3).  There are no additional risks of extending the medication 
by 12 weeks. The subject will be contacted by phone at weeks 18 and 36 to evaluate for 
resolution of any adverse events and to receive further information about options for future 
clinical care. In addition to this contact there will be another phone contact at weeks 36 and 
52 to monitor long term health and follow-up on clinical outcome. This phone call will be at 
the discretion of the Investigator. Subjects may be contacted for follow-up safety evaluations, 
including possible blood draw for further study. 
12.2.2.11. Pregnancy 
Women who are pregnant will not be enrolled in the study.  Metformin has not been well 
studied in pregnancy and the Metformin US Package Insert notes that based on animal 
studies Metformin was not teratogenic. A recent review of RCT data reveal no evidence of an 
increase in congenital malformations or miscarriages when Metformin is started before 
pregnancy and continued to term (Hyer et al 2018). 
Following administration of study drug, any known cases of pregnancy in female subjects or 
female partners of male subjects, will be reported until 30 days after the subject completes or 
withdraws from the study.  (Section 13.3; Procedures for Reporting Adverse Events) 
 
12.3. 
SPECIAL INSTRUCTIONS AND DEFINITIONS OF 
EVALUATIONS 
12.3.1. 
Protocol Deviations  
Missed visits and any procedures not performed (not attempted) for any reason will be 
reported as protocol deviations. Details regarding any deviations will be documented. 
12.3.2. 
Documentation of Amyotrophic Lateral Sclerosis 
To be eligible for participation in this trial, participants must have ALS defined as probable 
or definite ALS in accordance with the Revisited El-Escorial Criteria (Cedarbaum,1999). 
These criteria will be clearly designated on source documentation and in the electronic case 
report forms (eCRFs). 

 
 
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12.3.3. 
Medical History 
A comprehensive medical history will be obtained at the screening visit and reviewed for any 
exclusionary criteria. 
12.3.4. 
Treatment History 
Medications will be reviewed for concomitant therapies that would be exclusionary. 
12.3.5. 
Concomitant Medications/Treatments 
Current medications will be reviewed for contraindicated therapies. 
12.3.6. 
Clinical Assessments 
Patient Reported Outcomes: The Eating Assessment Tool 1091 (Appendix B) and the 
ALSFRS-R (Appendix A) will be administered at each time point to track self-reported 
bulbar impairment profiles. To facilitate efficiency, questionnaires will be sent to patient’s 
homes one-week prior to appointments for completion. 
12.3.7. 
Laboratory Evaluations 
Blood specimens will be collected by qualified study center personnel at Screening, Visit 1, 
2, 3, and Visit 4 as described under study procedures and sent to Shands for analysis for the 
following safety laboratory evaluations: hematology (complete blood count with differential), 
chemistry (electrolytes, BUN, creatinine, ALT, AST, total bilirubin, albumin). Urine will be 
collected for urinalysis.  
In females, of childbearing potential, urine will be collected for pregnancy test during 
recruitment/screening, visit 1, visit 3 and visit 4. Prior to each VFSS, women of childbearing 
potential will be asked to perform the urine pregnancy test in a private bathroom located near 
the exam room.   
C9orf72 testing will be performed using the repeat-primed PCR assay and other molecular 
assays as needed in the Ranum lab. 
RAN protein levels:  Isolation of peripheral blood lymphocytes (PBLs) for protein and other 
molecular studies will be performed in the Ranum and the Garrett laboratories. C9 RAN 
proteins present in the blood will be analyzed using highly sensitive MesoScale Discovery 
(MSD) electrochemiluminesence assays using custom antibodies generated and characterized 
by the Dr. Ranum’s group.  The MSD assays has been optimized for the detection of C9 
RAN proteins. The remainder of the samples will be stored in the Center for NeuroGenetics 
bank for additional molecular or biochemical studies for an indefinite period of time.   
 

 
 
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Abnormal laboratory test results and positive pregnancy test results will be flagged by the 
laboratory.  All clinically important abnormal laboratory tests occurring during the study will 
be followed until they resolve (return to normal or baseline values) or stabilize, or until they 
are considered by the Investigator to be no longer clinically significant. 
COVID-19 nasal collection and testing may take place at this visit if travel time allows for 
the participant - Due to COVID-19 Pandemic, nasal collection and testing of the participants 
has been implemented to determine if the participant is COVID negative. The collection is 
done at UF Springhill collection site and testing is completed at Rocky Point Labs overnight 
with results placed in EPIC. The results of the testing will be provided to the participant once 
results are posted in EPIC. This collection and testing will continue throughout participation 
in the study.  
 
12.3.8. 
Lumbar Puncture Analysis: 
CSF fluid will be drawn from the patient for the following disease evaluations: 
 
Inflammation and RAN protein levels: Neuroinflammation markers and/or C9 RAN proteins 
present in CSF will be analyzed using a highly sensitive MesoScale Discovery (MSD) 
electrochemiluminesence assays and other assays. For some of these studies custom 
antibodies generated and characterized by the PI will be used.  The RAN MSD assays have 
been optimized for the detection of the C9 GP RAN proteins. These and other molecular 
studies will be done in the Ranum. Any remaining CSF will be stored indefinitely in the 
Center for NeuroGenetics bank for future studies.   
 
Due to the COVID-19 Pandemic, and only for the participants currently enrolled (3) in this 
study, this procedure may be completed in the participant’s home area and the samples 
shipped to the Ranum Lab for analysis. The lumbar puncture will be completed by a licensed 
physician who is qualified to perform this procedure and agrees to complete the procedure 
per the protocol. 
 
Metabolomics: The level of Metformin in the CSF and levels of various metabolomics 
components will be analyzed by traditional liquid chromatography tandem mass 
spectrometry of (UPLC/MS/MS) in the Garrett lab.  
12.3.9. 
Speech and Swallowing Function: 
The following will be performed at the Fixel Institute for Neurological Diseases, in room 
1010.6 as directed by Nicole Herndon, a Certified Speech Language Pathologist. The 
following procedures and outcomes will be performed and derived: 
 

 
 
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x Videofluoroscopic Swallow Study (VFSS): VFSS will be performed using a 
Phillips BV Endura fluoroscopic C-arm unit (GE OEC 8800 Digital Mobile 
C-Arm system type 718074) that is housed at Fixel and used for clinical and 
research purposes. Fluoroscopic data will be captured in high resolution at 30 
frames per second and in the lateral field of view using a TIMS Dicom 
(Version 3.2, TIMS Medical, TM, Chelmsford, MA). Patients will be seated 
and will complete a standardized bolus protocol consisting of: 1) three 5ml 
thin liquid barium; 2) a regular sip of thin liquid taken from a cup containing 
90cc of thin liquid; 3) consecutive cup sip of remaining barium; 4) three 
teaspoons of thin honey consistency barium; 5) two teaspoons of pudding-
thick barium; 6) graham cracker with pudding-thick barium; and a 7) 13mm 
barium tablet (EZ-Disk). To ensure patient safety, a bail out criterion will be 
utilized and the VFSS will cease on the third episode of frank aspiration or 
residue greater than 75% of either vallecular or pyriform housing that is 
unable to be cleared with strategies. Once the study is completed, recordings 
will be saved and digitally backed up for subsequent analyses. 
x VFSS Outcomes: Two independent, blinded and trained raters will perform 
analyses on the data for every bolus using validated procedures. Discrepancies 
between raters will be flagged and resolved at a consensus meeting and three 
swallows from each evaluation will be repeat rated at random for evaluation 
of intra-rater reliability. VFSS measures are summarized below and include 
indices of Global Swallowing Function (Appendix E) and Swallowing Safety 
(Appendix F).  
x Global Swallowing Function: The validated Dynamic Imaging Grade of 
Swallowing Toxicity (DIGEST) will be performed on all collected 
videofluoroscopic swallowing studies to assess global swallowing function 
(Hutcheson and Fuller, 2015). The DIGEST total score is determined using 
the composite of individual airway safety and bolus efficiency subscores 
(range: 0-4). The DIGEST total is rated on a 5-point ordinal score ranging 
from 0 (no dysphagia) to 4 (life-threatening dysphagia).  For statistical 
analysis, DIGEST total scores will be compared within individuals between 
the Visit 1 (baseline), Visit 3 (12 weeks) and Visit 4 (24 weeks). DIGEST 
WRWDOVFRUHVRILQGLFDWHIXQFWLRQDOVZDOORZLQJDQGVFRUHVLQGLFDWH
dysphagia. The DIGEST scoring schema is provided in Appendix E.  
x Swallowing Safety: The Penetration-Aspiration scale [(PAS), (Rosenbek et 
al., 1996)] is a validated eight-point ordinal scale indexing the degree of 
airway invasion during swallowing, the participant’s response, and whether 
the invasive material is successfully ejected from the airway. PAS scores of 1 
and 2 are considered safe, while scores between 3 and 5 indicate penetration 
of material in the upper airway at or above the level of the true vocal folds. 

 
 
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PAS scores greater than 6 indicate aspiration of material below the level of the 
true vocal folds into the airway. For statistical comparisons across airway 
safety status levels the convention is as follow: safe swallowing = PAS <2 and 
unsafe swallowing = PAS>3.  
 
12.3.9.1 Pulmonary Function 
 
The following will be performed by trained clinical research coordinators at the Fixel 
Institute for Neurological Diseases:  
  
x Voluntary Peak Cough Flow Testing: Participants will be seated with a nose 
clip in place and instructed to take a deep breath in and “cough hard like you 
have something stuck in your throat”. To reduce the potential for labial air 
leakage due to reduced orofacial muscle tone, a rubber flanged mouthpiece 
(MTH6400), will be attached to the end of a Mini-Wright CE0120 analogue 
peak flow meter device. The patient will perform three trials inter-spaced with 
a one-minute rest break. Outcomes will include peak expiratory flow (L/min) 
(PEF). The highest obtained values will be used.  
x Pulmonary Function Testing: Forced vital capacity (% predicted) will be 
assessed using a hand-held digital spirometer (MD Spiro Micro 1 Spirometer).  
12.3.10. 
Imaging 
The following will be performed on the Siemens or Phillips 3T system at the University of 
Florida under the direction of Dr. Vaillancourt: 
 
x Measurement of brain structure and brain function: This experiment will 
require the participant to lie down on a platform that will move into an 
enclosed magnetic resonance imaging system for both functional magnetic 
resonance imaging (fMRI) and structural imaging (diffusion, T1 and T2 
weighted). fMRI is a technique used to non-invasively measure changes in 
brain activity in relation to performance of a specific task. T1, T2, and 
diffusion imaging examine the macrostructure and microstructure of the brain 
tissue non-invasively.  In brain structure measures, the participant will lie still 
in the MRI without performing a task. In brain function measures, the 
participant will perform a motor task. The participant will be asked to practice 
the experimental tasks described below until they feel comfortable performing 
them. During this task the participant will use a small pinch grip device that 
they will hold between the thumb and index finger. Once inside the scanner, 
the participant will be asked to lie still while the study staff perform a scan to 
provide an anatomical picture of the brain. The participant will be asked to 

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complete a series of tasks during which study staff will obtain pictures of the 
functional changes in brain activity. During this task the participant will move 
their hand under different conditions. In one condition the participant will be 
asked to perform the task with no sensory information about the task. Study 
staff will refer to this task as a “self-initiated” task. No stimulus will be 
presented in this condition. In another condition the participant will be asked 
to respond to a stimulus, which will give them information regarding the 
timing of their movement or the extent to which they should contract their 
muscles. Study staff will refer to this task as an “auditory cued task” or a 
“visually cued task”. The participants’ goal during all movement conditions 
will be to move as quickly as possible. 
12.3.11.
BDI-Fast Screen
The BDI-Fast Screen, which assesses depression (Appendix D), will be administered at Visit 
2, Visit 3, Phone call at 18 weeks, Visit 4, and Phone call at week 36.  Subject indicating 
depression will be counseled by a physician prior to leaving the clinic if they are at UF and 
may be referred to a mental health professional.
12.3.12.
Subject Adherence Assessments
Adherence to study requirements and compliance with medication will be assessed at the 
study visit through direct questioning by staff personnel.
SAFETY AND ADVERSE EVENTS
The AE definitions and reporting procedures provided in this protocol comply with all 
applicable International Conference on Harmonization (ICH) guidelines. Study staff will 
carefully monitor each subject throughout the study for possible AEs. All AEs will be 
documented on CRFs designed specifically for this purpose. It is also important to report all 
AEs, especially those that result in permanent discontinuation of the investigational product 
being studied, whether serious or non-serious.
Safety Review Plan and Monitoring- Oversight of participant safety includes review of 
adverse events as well as study progress, data integrity and study outcomes. The study will 
engage a physician safety monitor to discuss issues regarding side effects and adverse events. 
The safety monitor is external to the clinical study and is free of any conflicts of interest to 
maintain independence and objectivity.
Safety and study progress reviews

 
 
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1. Adverse events will be reviewed by the Principal Investigator, the Co-
Principal Investigator(s) and the Safety Officer  
 
 
 
2. Study progress will be reviewed by the study investigators on a quarterly 
 
 
basis (e.g. Recruitment, retention, protocol adherence). 
 
 
 
3. For each Continuing Review period, a report will include  
       
a. list and summary of adverse events 
 
 
b. whether adverse events rates are consistent with pre-study  
 
 
assumptions 
 
 
c. Summary of recruitment and retention and reason for withdrawals 
 
 
d. whether the study is on track to be completed and accomplish the 
 
 
stated aims.  
 
13.1. 
Adverse Event Reporting and Follow Up 
Subjects will be instructed to inform clinic personnel of any untoward medical symptoms 
and/or events that may arise during the course of the study.  If an adverse event is 
experienced by a subject, then the subject will be questioned concerning symptoms that may 
have occurred after the administration of the study drug.  The incidence, severity and 
duration of all AEs will be recorded according to the following scale: 
Mild 
Adverse event resulting in discomfort, but not sufficient to cause 
interference in normal daily activities 
Moderate 
Adverse event resulting in discomfort that is sufficient to cause 
interference in daily activities. 
Severe 
Adverse event resulting in discomfort causing an inability to carry out 
normal daily activities 
Subjects who withdraw due to an adverse event will be followed whenever possible until 
resolution of the adverse event. It may be appropriate for the subject to return to the site 2 
weeks after the subject is off study to evaluate for resolution of any adverse events and to 
receive further information about options for future clinical care.  The Investigator will 
decide the course of action. 

 
 
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13.2. 
Assessing Relationship to Study Drug 
The Principal Investigator/Sub Investigators will assess the relationship of all adverse 
reactions to the drug, using the following scale: 
Probable 
A clinical event, including laboratory test abnormality, with a 
reasonable time sequence to drug administration, unlikely to be 
attributed to concurrent disease or other drugs or chemicals, and 
which follows a clinically reasonable response on withdrawal. 
Possible 
A clinical event, including laboratory test abnormality, with a 
reasonable time sequence to drug administration, but which could 
also be explained by concurrent disease or other drugs or chemicals.  
Information on drug withdrawal may be lacking or unclear. 
Unlikely 
A clinical event, including laboratory test abnormality, with a 
temporal relationship to drug administration which makes a causal 
relationship improbable, and which other drugs, chemicals or 
underlying disease provide plausible explanation. 
Unrelated 
This category is applicable to AEs which are judged to be clearly 
and incontrovertibly due to extraneous causes (diseases, 
environment, etc.) and do not meet the criteria for drug relationship 
listed for the above-mentioned conditions. 
 
Subjects who withdraw due to an adverse event will be followed whenever possible until 
resolution of the adverse event. It may be appropriate for the subject to return to the site 2 
weeks after the subject is off study to evaluate for resolution of any adverse events and to 
receive further information about options for future clinical care.  The Investigator will 
decide the course of action. 
13.3. 
Procedures for Reporting Adverse Events 
Subjects will be instructed to inform clinic personnel of the AEs that may arise during the 
course of the study.  Treatment of any AEs will be administered under the direction of the 
Investigator. 
All symptoms will be recorded by clinic staff and will be reviewed by the Investigator or 
Sub-Investigators prior to any subsequent dosing. 
When appropriate, medical tests and examinations will be performed to document resolution 
of the event(s). 

 
 
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Adverse events will be coded into the Preferred Term (PT), classified according to the 
current version of Medical Dictionary for Regulatory Activities (MedDRA) with System 
Organ Classification (SOC) and reported with severity, duration, onset time and relationship 
to study drug and action taken. 
Female subjects of child-bearing potential must have a negative pregnancy test at all visits 
where VFSS will be performed.  Following administration of study drug, any known cases of 
pregnancy in female subjects will be reported until 30 days after the subject completes or 
withdraws from the study.  The pregnancy will be reported immediately by phone and by 
faxing/emailing a completed Pregnancy Report to the Principal Investigator or study team 
within 24 hours of knowledge of the event.  The pregnancy will not be processed as a serious 
adverse event (SAE); however the Principal Investigator will follow the subject until 
completion of the pregnancy and must assess the outcome in the shortest possible time but 
not more than 30 days after completion of pregnancy.  If the outcome of the pregnancy meets 
the criteria for immediate classification of an SAE (e.g., spontaneous or therapeutic abortion 
[any congenital anomaly detected in an aborted fetus is to be documented] stillbirth, neonatal 
death, or congenital anomaly), the Principal Investigator will report the event by phone and 
by faxing a completed SAE form to the IRB and the FDA within 24 hours of knowledge of 
the event. 
All serious adverse events (SAEs), whether or not the event is deemed drug-related, will be 
reported on the SAE Report Form   
 
The Investigator will be responsible for notifying the IRB and regulatory agencies.    
If a subject experiences a non-serious Adverse Event: 
All clinical adverse events are recorded in the CRFs designed specifically for this purpose. It 
is important to report all AEs, especially those that result in permanent discontinuation of the 
investigational product being studied, whether serious or non-serious. 
The site should fill out the CRF and enter the non-serious AE information into the online 
Adverse Event Reporting System within 5 working days/7 calendar days of the site learning 
of a new AE or receiving an update on an existing AE.  
Expected adverse events for Metformin are provided in Section 6.4; Safety.  

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CRITERIA FOR INTERVENTION DISCONTINUATION
Subjects will be discontinued from the study under the following circumstances:
x
If the subject is hemodynamically unstable
x
If discontinuation suspension is recommended by treating physician
x
At the subject’s request
STATISTICAL CONSIDERATIONS
15.1.
General Design Issues
This open label study has been designed to assess the safety and tolerability of Metformin in
individuals with C9orf72 positive ALS.  The study will be performed at the University of 
Florida. The anticipated time from study enrollment until completion of data analyses is 
approximately 10-18 months. The inclusion criteria are intended to provide a cohort of 
subjects with ALS who are appropriate candidates for treatment with Metformin.  Exclusion
criteria prohibit enrollment of subjects with certain medical conditions, allergies to study 
drug or females that are pregnant or breastfeeding.  
15.2.
Outcomes
15.3.
Primary and Secondary Outcomes: 
The primary objective of this study is to assess the safety and tolerability of Metformin
administered twice daily.
Additional outcomes:
x
Type, incidence, and severity of adverse events
x
Physical and neurological examinations
x
Vital signs (heart rate, blood pressure, respiration rate, temperature), Height/Weight
x
Clinical laboratory tests (hematology, serum chemistry and urinalysis)
x
BDI-Fast Screen 
x
Impact on swallowing
x
Impact on respiratory testing
x
Impact on RAN protein levels
x
Impact on neuroimaging

 
 
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15.4. 
Sample Size and Accrual 
Using a conservative value, screening 50 individuals affected by ALS should yield the 16 
study subjects for this protocol. Participants will be recruited from UF and multiple clinical 
sites in the state of Florida and throughout the US. Interested participants will be contacted 
by the investigators or their staff and invited to participate. These recruitment strategies will 
include a mechanism by which the subjects can provide their contact information. 
15.5. 
Data Monitoring 
Data Monitoring will be conducted according to Good Clinical Practice and applicable 
government regulations. The investigator agrees to allow monitors access to the clinical 
supplies, dispensing and storage areas, and the clinical files of the study subjects and, if 
requested, agrees to assist the monitors. 
Safety monitoring will include careful assessment and appropriate reporting of adverse 
events. Medical monitoring will include contemporaneous assessment of serious adverse 
events. 
15.6. 
Data Analyses 
15.7. 
Data analyses will be performed by the investigative team 
including individuals working in the Ranum laboratory with the 
exception that Dr. Ranum will not perform statistical analyses. 
General data analysis including impact on swallowing and 
respiration, and manuscript writing will also be performed by Co-
Investigator Emily K. Plowman at Ohio State University. 
Statistical Analysis 
Statistical analysis will be performed by investigators in the laboratories of Drs. Wymer, 
Ranum, Vaillancourt or Garrett.  Because Dr. Ranum has a potential conflict of interest as an 
inventor of a pending patent and as a founder of RanTran that may license this technology 
from UF in the future, she will not perform any of the specific statistical tests of the data 
generated during the course of this study.  Summary statistics will be reported for safety and 
tolerability endpoints as listed above (Section 15.2.1) as well as subject characteristics 
(demographics, ALS Functional Rating Scale – Revised (ALSFRS-R), RAN protein levels, 
respiratory, and bulbar dysfunction).   
Depending on the study enrollment rate, the PI may elect to perform an interim analysis 
using available data from the subset of subjects who have completed the study at that 
time.  The results of this interim analysis will be used only for internal planning and possibly 
for sharing with regulatory authorities. The results of the interim analysis will have 
absolutely no influence on the subsequent conduct of the study. 

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The Suicidal Ideation Score is a numerical score derived from the categories 1-7 on BDI-
FastScreen (BDI-FS) present at the assessment.  It will be reported as a numerical score and 
will be provided in the listing as well.  If no ideation; then score is zero.
No formal statistical tests will be performed to compare Screening vs. Visit 1 vs Visit 2 vs 
Visit 3 on tolerability and safety endpoints.
Summary statistics will also include concomitant medications.  No efficacy analysis will be 
performed.
Additional statistical and alternate tests may be performed, if necessary.  
ETHICAL CONSIDERATIONS
16.1.
Basic Principles
This research will be carried out in accordance with Good Clinical Practice (GCP) as set out 
by the International Council for Harmonization (ICH), the basic principles defined in the 
U.S. Code of Federal Regulations (21 CFR Part 312), the Belmont Report, Directive 
2001/20/EC (Europe), The Tri-Council Policy Statement and the principles enunciated in the 
most recent version of the World Medical Association Declaration of Helsinki.
16.2.
Institutional Review Board Review and Informed Consent
The protocol and the informed consent document and any subsequent modifications will be 
reviewed and approved by the IRB responsible for oversight of the study. A signed consent 
form, approved by the IRB, will be obtained from the subject by Study Investigators and/or 
their IRB-approved designee. 
For subjects who cannot provide consent for themselves, such as those below the legal age, a 
parent, legal guardian, or person with power of attorney, must sign the consent form; 
additionally, the subject's assent must also be obtained if he or she is able to understand the 
nature, significance, and risks associated with the study. 
Written informed consent will be obtained from each participant before any study-specific 
procedures or assessments are performed and after the aims, methods, anticipated benefits 
and potential hazards are explained.   The Investigator will keep the original consent forms 
and copies will be given to the participants.  It will also be explained to the participants that 
they are free to refuse entry into the study and free to withdraw from the study at any time 
without prejudice to future treatment.  Written and/or oral information about the study in a 
language understandable to the participant will be given to all participants.  HIPAA 
guidelines for confidentiality and the principles of medical ethics will be adhered to during 
the study. 
16.2(a) Informed consent procedure: 

 
 
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The investigator will inquire if the patient is interested in hearing about the study.  If the 
patient wants to hear information, the staff member will introduce himself/herself to the 
patient and/or legally authorized representative, if applicable. The informed consent session 
will take place in a private setting. The consent form may be read aloud to the potential 
subject(s) or the potential subject(s) may be asked to read the consent in the presence of the 
staff member. Adequate time must be given to the potential subject or legal representative to 
read the consent form and to ask any questions. 
All questions must be answered regarding the research before the document is signed. 
The person obtaining consent will acknowledge that the potential subject/legal 
representative verbalizes understanding of the research and the research related 
procedures. 
A copy of the fully signed consent form will be given to the potential subject/legal 
representative, the original signed consent form will be filed in the potential subject’s 
research file, and the consent form will be scanned into the patient’s electronic 
medical record. 
 
16.3. 
Revisions and/or Amendments to the Protocol 
All revisions and/or amendments to this protocol must be documented and approved in 
writing by the Investigator. If the revision/amendment will affect subject safety and/or study 
design, then the amendment will be re-submitted to the IRB for approval.  Administrative 
changes (i.e., typographical errors, discrepancies, clarifications) will also be submitted to the 
IRB, but may not require approval.  A copy of the IRB’s approval documents will be 
included in the final report.  
For revisions or amendments to the protocol that substantially alter the study design after 
initiation of the study, the Investigator will decide whether a revised and approved ICF will 
be needed for continued participation. 
16.3(a) Signing of revised informed consent forms 
 
If a consent form is revised while patients are enrolled in the study, and the IRB states 
that patients need to be re-consented, all study subjects currently enrolled and 
participating in the study must be advised of the changes, have their questions 
answered, and sign the revised version of the consent form if they wish to continue their 
participation in the study. The staff member who advised the subject of the changes and 
re-consented the subject must also sign the consent form. 
A copy of the signed revised consent form will be given to the subject/legal 

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representative, the original revised consent form will be filed in the subject’s binder, and 
the revised consent form will be scanned into the subjects electronic medical record 
unless the study is of a sensitive nature and the IRB has approved not scanning the 
consent into the subject’s medical record.
16.4.
Study Termination
The Investigators reserve the right to discontinue the study at the clinical study site for safety 
or administrative reasons at any time. Should the study be terminated and/or the clinical 
study site closed for any reason, all documentation and study medication pertaining to the 
study must be retained at the site.
16.5.
Delegation of Investigator Tasks
The Investigator may delegate tasks as appropriate to individuals who are qualified by 
education, training and experience (and state licensure where relevant) to perform the 
delegated task, as described in the FDA Guidance for Industry on Investigator 
Responsibilities – Protecting the Rights, Safety, and Welfare of Study Subjects, October 
2009.
16.6.
Subject Confidentiality
All laboratory specimens, evaluation forms, reports, video recordings, and other records that 
leave the clinical study site will be identified only by the study specific Subject Identification 
Number (SID) to maintain subject confidentiality. All records will be kept in a locked file 
cabinet. All computer entry and networking programs will be done using study specific SIDs 
only. Clinical information will not be released without written permission of the subject, 
except as necessary for monitoring by IRB and the FDA.
REFERENCES
Cedarbaum JM, Stambler N, Malta E, Fuller C, Hilt D, Thurmond B, et al. The ALSFRS-R: 
a revised ALS functional rating scale that incorporates assessments of respiratory function. 
BDNF ALS Study Group (Phase III). J Neurol Sci 1999;169(1-2):13-21.
Crary MA, Carnaby-Mann GD, Groher ME. Initial psychometric assessment of a functional 
oral intake scale for dysphagia in stroke patients. Arch Phys Med Rehabil 2005;86:1516-
1520.  
Rosenbek JC, Robbins JA, Roecker EB, Coyle JL, Wood JL. A penetration-aspiration scale. 
Dysphagia 1996;11(2):93-8.
Watanabe H, Atsuta N, Nakamura R, Hirakawa A, Watanabe H, Ito M, Senda J, Katsuno M, 
Izumi Y, Morita M and others. Factors affecting longitudinal functional decline and survival 

 
 
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Page 53 of 61 
 
in amyotrophic lateral sclerosis patients. Amyotroph Lateral Scler Frontotemporal Degener. 
2015;16(3-4):230-236. 
 
 
 

 
 
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APPENDIX A 
ALS Functional Rating Scale – Revised 
 

 
 
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APPENDIX B 
Eating Assessment Tool (EAT-10) 
 
Please answer each of the ten questions listed below by circling the appropriate 
number that you feel best describes how you feel, with:  0= no problem at all; 4= 
severe problems. 
 
 
Circle the appropriate response: 
To what extent are the following scenarios problematic for you? 
0= No Problem  
4=Severe Problem  
1. My swallowing problem has caused me to lose weight. 
0 
1 
2 
3 
4 
2. My swallowing problem interferes with my ability to go out for meals. 
0 
1 
2 
3 
4 
3. Swallowing liquid takes extra effort. 
0 
1 
2 
3 
4 
4. Swallowing solids takes extra effort. 
0 
1 
2 
3 
4 
5. Swallowing pills takes extra effort. 
0 
1 
2 
3 
4 
6. Swallowing in painful. 
0 
1 
2 
3 
4 
7. The pleasure of eating is affected by my swallowing. 
0 
1 
2 
3 
4 
8. When I swallow food sticks in my throat. 
0 
1 
2 
3 
4 
9. I cough when I eat.  
0 
1 
2 
3 
4 
10. Swallowing is stressful. 
0 
1 
2 
3 
4 
                                                                                                                                        Total EAT-10 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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APPENDIX C 
FUNCTIONAL ORAL INTAKE SCALE (FOIS) 
 
 
 
 
 
 

 
 
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APPENDIX D 
 

 
 
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APPENDIX E 
DIGEST Scoring System 
 
 
 

 
 
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APPENDIX F 
Penetration-Aspiration Scale (PAS) 
Score    Description of Events                                                                                  
1           Material does not enter airway.    
2           Material enters the airway, remains above the vocal folds.    
3           Material enters the airway, remains above the vocal folds, and is not ejected from the 
airway.    
4           Material enters the airway, contacts the vocal folds, and is ejected from the airway.    
5           Material enters the airway, contacts the vocal folds, and is not ejected from the 
airway.    
6           Material enters the airway, passes below the vocal folds, and is ejected into the 
larynx or out of the airway.    
7           Material enters the airway, passes below the vocal folds, and is not 
ejected from the trachea despite effort.    
8           Material enters the airway, passes below the vocal folds, and no effort is made to 
eject.    
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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APPENDIX G 
 
Metformin inhibits RAN translation through PKR pathway and corrects 
ALS/FTD phenotypes in C9orf72 mouse model 
 
Tao Zu1,2,5, Olgert Bardhi1,2,5, Shu Guo1,2,5, Daniel A. Ryskamp1,2,5, Solaleh Khoramian 
Tusi1,2,5, Kelena Klippel1,2,5, Paramita Chakrabarty4, Lien Nguyen1,2,5, Todd 5 E. Golde4,6, 
Nahum Sonenberg7, Laura P.W. Ranum1-5* 
 
1Center for NeuroGenetics, College of Medicine; 2,3Departments of Molecular Genetics & 
Microbiology and Neurology, College of Medicine; 4Center for Translation Research in 
Neurodegenerative Disease and Department of Neuroscience, College of Medicine; 5Genetics 
Institute, 6McKnight Brain Institute, University of Florida, Gainesville, FL, USA; 
7Department of Biochemistry, McGill University, Montreal, Canada 
 
MANUSCRIPT ATTACHED  
 
 

 
 
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APPENDIX H 
 
Drug Pamphlet for Metformin 500 mg ER from Asend Laboratories, LLC and Amneal 
Pharmaceuticals, LLC attached