Conditions: Amyotrophic Lateral Sclerosis
Keywords: ALS Placebo-Controlled Double-Blind Regimen Specific Appendix Lou Gehrig's Disease Trehalose Seelos Therapeutics
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 161
Healthy volunteers: no

Brief summary:
The HEALEY ALS Platform Trial is a perpetual multi-center, multi-regimen clinical trial evaluating the safety and efficacy of investigational products for the treatment of ALS.

Regimen E will evaluate the safety and efficacy of a single study drug, SLS-005 (Trehalose injection, 90.5 mg/mL for intravenous infusion) in participants with ALS.

Detailed description:
The HEALEY ALS Platform Trial is a perpetual multi-center, multi-regimen clinical trial evaluating the safety and efficacy of investigational products for the treatment of ALS. This trial is designed as a perpetual platform trial. This means that there is a single Master Protocol dictating the conduct of the trial. The HEALEY ALS Platform Trial Master Protocol is registered as NCT04297683.

Once a participant enrolls into the Master Protocol and meets all eligibility criteria, the participant will be eligible to be randomized into any currently enrolling regimen. All participants will have an equal chance of being randomized to any currently enrolling regimen.

If a participant is randomized to Regimen E SLS-005 - Trehalose, the participant will complete a screening visit to assess additional Regimen E eligibility criteria. Once Regimen E eligibility criteria are confirmed, participants will complete a baseline assessment and be randomized in a 3:1 ratio to either active SLS-005 or matching placebo.

Regimen E will enroll by invitation, as participants may not choose to enroll in Regimen E. Participants must first enroll into the Master Protocol and be eligible to participate in the Master Protocol before being able to be randomly assigned to Regimen E.

For a list of enrolling sites, please see the HEALEY ALS Platform Trial Master Protocol under NCT04297683.

Arms:
arm 1: SLS-005 is administered via infusion once weekly for 24 weeks. arm 2: Matching placebo is administered via infusion once weekly for 24 weeks.

Interventions: intervention 1: SLS-005 intervention 2: Matching Placebo
Intervention descriptions: intervention 1: Administration: Infusion Dose: 0.75 g/kg weekly intervention 2: Administration: Infusion Dose: equivalent weight-based volume as described for trehalose

Protocol document:
Prot_000.pdf: 
HEALEY ALS Platform Trial - Regimen E Trehalose 
NCT05136885 
Document Date: 03 Dec 2021 
 


REGIMEN-SPECIFIC APPENDIX E 
 
FOR SLS-005 – TREHALOSE 
 
 
Regimen-Specific Appendix Date: 
12/3/2021 
 Version Number: 
2.0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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TABLE OF CONTENTS 
TABLE OF CONTENTS ---------------------------------------------------------------------------------------------------------------------- 2 
SIGNATURE PAGE --------------------------------------------------------------------------------------------- 5 
LIST OF ABBREVIATIONS ------------------------------------------------------------------------------------ 6 
REGIMEN-SPECIFIC APPENDIX SUMMARY --------------------------------------------------------------- 7 
SCHEDULE OF ACTIVITIES – Placebo-controlled period --------------------------------------------------- 9 
SCHEDULE OF ACTIVITIES – Open Label Extension (Optional) ----------------------------------------------------- 12 
1. 
INTRODUCTION ---------------------------------------------------------------------------------------- 14 
1.1 Trehalose Background Information ------------------------------------------------------------------------------------- 14 
1.2 Nonclinical and Clinical Data ------------------------------------------------------------------------------------------- 14 
1.2.1 Nonclinical Experience -------------------------------------------------------------------------------------------- 14 
1.2.2 Nonclinical Literature ---------------------------------------------------------------------------------------------- 15 
1.2.3 Clinical Experience ------------------------------------------------------------------------------------------------- 15 
1.3 Trehalose Therapeutic Rationale ---------------------------------------------------------------------------------------- 16 
1.3.1 Rationale for the study --------------------------------------------------------------------------------------------- 16 
1.3.2 Rationale for the Dose, Dosing Regimen, and Route of Administration ------------------------------------- 16 
1.3.3 Rationale for the Patient Population ----------------------------------------------------------------------------- 17 
2. 
OBJECTIVES -------------------------------------------------------------------------------------------- 18 
2.1 Study Objectives and Endpoints ----------------------------------------------------------------------------------------- 18 
3. 
RSA DESIGN --------------------------------------------------------------------------------------------- 19 
3.1 Scientific Rationale for RSA Design ------------------------------------------------------------------------------------ 19 
3.2 End of Participation Definition ------------------------------------------------------------------------------------------ 19 
3.3 End of Regimen Definition ----------------------------------------------------------------------------------------------- 19 
4. 
RSA Enrollment ------------------------------------------------------------------------------------------ 20 
4.1 Number of Study Participants -------------------------------------------------------------------------------------------- 20 
4.2 Inclusion and Exclusion Criteria ---------------------------------------------------------------------------------------- 20 
4.2.1 RSA Inclusion Criteria ------------------------------------------------------------------------------- 20 
4.2.2 RSA Exclusion Criteria ------------------------------------------------------------------------------- 20 
4.3 Treatment Assignment Procedures -------------------------------------------------------------------------------------- 20 
4.4 Open Label Extension Eligibility ---------------------------------------------------------------------------------------- 20 


 
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5. 
INVESTIGATIONAL PRODUCT ----------------------------------------------------------------------- 21 
5.1 Investigational Product Manufacturer ---------------------------------------------------------------------------------- 21 
5.2 Labeling, Packaging, and Resupply ------------------------------------------------------------------------------------- 21 
5.3 Acquisition, Storage, and Preparation---------------------------------------------------------------------------------- 21 
5.4 Study Medication/Intervention, Administration, Escalation, and Duration ---------------------------------------- 22 
5.5 Justification for Dosage -------------------------------------------------------------------------------------------------- 22 
5.6 Dosage Changes ----------------------------------------------------------------------------------------------------------- 23 
5.7 Participant Compliance -------------------------------------------------------------------------------------------------- 23 
5.8 Overdose -------------------------------------------------------------------------------------------------------------------- 23 
5.9 Prohibited Medications --------------------------------------------------------------------------------------------------- 23 
5.10 Trehalose Known Potential Risks and Benefits ---------------------------------------------------------------------- 24 
5.10.1 Known Potential Risks ------------------------------------------------------------------------------ 24 
5.10.2 Known Potential Benefits --------------------------------------------------------------------------- 24 
6 
REGIMEN SCHEDULE --------------------------------------------------------------------------------- 25 
6.1 Baseline Visit --------------------------------------------------------------------------------------------------------------- 25 
6.2 Weekly Study Drug Infusion ---------------------------------------------------------------------------------------------- 26 
6.3 Week 2 Telephone Visit --------------------------------------------------------------------------------------------------- 27 
6.4 Week 4 and 8 Visits -------------------------------------------------------------------------------------------------------- 27 
6.4 Week 12 Telephone Visit -------------------------------------------------------------------------------------------------- 28 
6.5 Week 16 Visit --------------------------------------------------------------------------------------------------------------- 28 
6.6 Week 20 Telephone Visit -------------------------------------------------------------------------------------------------- 29 
6.7 Week 24 Visit or Early Termination Visit ------------------------------------------------------------------------------ 29 
6.8 Follow-Up Safety Call ---------------------------------------------------------------------------------------------------- 29 
6.9 Process for Early Terminations ----------------------------------------------------------------------------------------- 30 
6.10 Open Label Extension --------------------------------------------------------------------------------------------------- 30 
6.10.1 Weekly Investigational product Administration ---------------------------------------------------------------- 30 
6.10.2 Week 2 OLE Telephone Visit -------------------------------------------------------------------------------------- 31 
6.10.2 Week 4 OLE Visit --------------------------------------------------------------------------------------------------- 31 
6.10.3 Week 8 OLE Visit --------------------------------------------------------------------------------------------------- 32 
6.10.4  Week 12 OLE Telephone Visit ------------------------------------------------------------------------------------ 32 
6.10.5  Week 16 OLE Visit ------------------------------------------------------------------------------------------------- 33 
6.10.6  Week 20 OLE Telephone Visit ------------------------------------------------------------------------------------ 33 
6.10.7  Week 24 OLE Telephone Visit ------------------------------------------------------------------------------------ 34 


 
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6.10.8  Week 28 OLE Visit ------------------------------------------------------------------------------------------------- 34 
6.10.9  Week 40 OLE Visit ------------------------------------------------------------------------------------------------- 35 
6.10.10  Week 52 OLE Visit ----------------------------------------------------------------------------------------------- 35 
6.10.11 Follow-Up Safety Call -------------------------------------------------------------------------------------------- 36 
6.10.12 Process for Early Terminations from the OLE ---------------------------------------------------------------- 36 
7 
OUTCOME MEASURES AND ASSESSMENTS ------------------------------------------------------- 37 
7.1  ALSAQ-40 ----------------------------------------------------------------------------------------------------------------- 37 
7.2  Center for Neurologic Study Bulbar Function Scale ----------------------------------------------------------------- 37 
8.0 
BIOFLUID COLLECTION ------------------------------------------------------------------------------ 38 
8.1 CSF Biomarker Collection ----------------------------------------------------------------------------------------------- 38 
8.2 Serum Pharmacokinetic Assessments ----------------------------------------------------------------------------------- 38 
9 
REGIMEN-SPECIFIC STATISTICAL CONSIDERATIONS ------------------------------------------ 39 
Appendix I: The ALSAQ-40 ------------------------------------------------------------------------------------ 40 
Appendix II: The Bulbar Function Scale (CNS-BFS) ---------------------------------------------------------- 49 
REFERENCES ------------------------------------------------------------------------------------------------- 51 
 


 
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SIGNATURE PAGE 
I have read the attached Regimen-Specific Appendix (RSA) entitled, “REGIMEN E: Trehalose” 
dated December 3, 2021 (Version 2.0) and agree to abide by all described RSA procedures.  I 
agree to comply with the International Conference on Harmonisation Tripartite Guideline on 
Good Clinical Practice, applicable FDA regulations and guidelines identified in 21 CFR Parts 11, 
50, 54, and 312, central Institutional Review Board (IRB) guidelines and policies, and the Health 
Insurance Portability and Accountability Act (HIPAA). 
 
By signing the RSA, I agree to keep all information provided in strict confidence and to request 
the same from my staff. Study documents will be stored appropriately to ensure their 
confidentiality. I will not disclose such information to others without authorization, except to the 
extent necessary to conduct the study.  
 
 
 
 
Site Name: 
 
 
 
 
Site Investigator: 
 
 
 
 
Signed: 
 
 
 
 
 
Date: 
 
 
  
 
 
 
 
 
 
 


 
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LIST OF ABBREVIATIONS 
Abbreviation 
Definition 
AE 
Adverse Event  
ALSAQ-40 
Amyotrophic Lateral Sclerosis Assessment Questionnaire-40  
ASLFRS-R 
ALS Functional Rating Scale-Revised  
AUCinf 
Area Under the Curve to Time Infinity  
CNS-BFS 
Center for Neurologic Study Bulbar Function Scale  
FVC 
Forced Vital Capacity  
g 
Grams 
GLP 
Good Laboratory Practice  
HHD 
Hand-held Dynamometry  
LC-3 II 
Microtubule-associated Protein 1A/1B-light chain 3  
LPLV 
Last Patient Last Visit 
MTD 
Maximum Tolerated Dose 
OLE 
Open Label Extension 
OPMD 
Oculopharyngeal Muscular Dystrophy 
PK 
Pharmacokinetics 
RSA 
Regimen Specific Appendix 
SAE  
Serious Adverse Event 
SLS-005 
Trehalose 
SOA  
Schedule of Activities 
SOI 
Start of Infusion 
SVC 
Slow Vital Capacity  
TFEB 
Transcription Factor EB  
 
 
 
 
 
 
 
 
 
 
 
 


 
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REGIMEN-SPECIFIC APPENDIX SUMMARY 
Regimen-Specific Appendix E 
For SLS-005, also known as Trehalose injection, 90.5 mg/mL for intravenous infusion. 
Rationale and RSA Design 
Trehalose is a disaccharide that is well known for its protein-stabilizing properties (Elbein 2003, 
Emanuele 2014) and its ability to activate autophagy. Because of its known ability to reduce 
abnormal protein aggregations, trehalose was studied in several cellular and animal models of 
hereditary neurologic and muscular disorders. Three in vivo studies (Castillo et al (2013), Zhang 
et al (2014) and Li Y et al (2015)) demonstrated that trehalose is a potential protective agent in 
ALS. These studies were performed using the SOD1 mouse model with the G93T mutation or 
the G86R mutation.  In mice, trehalose can be used orally because they lack intestinal trehalase, 
the enzyme that catalyzes conversion of trehalose to two glucose molecules. Treatment with 
trehalose both oral and intraperitoneal delayed onset of disease, prolonged survival, preserved 
motor function and motor neurons in the spinal cord.  When trehalose is taken orally in humans, 
trehalase enzymes in the brush border of the gut actively cleave trehalose into 2 glucose 
molecules. The most effective way to ensure that adequate amounts of trehalose reaches the 
primary therapeutic target organs, brain and muscle cells, is to circumvent the extensive gut 
metabolism in humans by administering trehalose via IV infusion. 
This is a randomized double-blind placebo-controlled trial. Participants who qualify will be 
randomized 3:1 to receive weekly infusions of Trehalose Injection, 90.5 mg/mL for intravenous 
infusion at a dose of 0.75g/kg or placebo (sodium chloride injection, 0.9%, USP) intravenous 
infusion at an equivalent weight-based volume.  
Allocation to Treatment Regimens 
Participants must first be screened and determined to be eligible under the Master Protocol 
before they are randomized to a treatment regimen.  
As soon as pre-defined criteria for futility for the regimen are met, or the target number of 
randomized participants has been reached, enrollment will stop in the regimen.   
Number of Planned Participants and Treatment Groups 
The number of planned participants for this regimen is approximately 160.   
There are 2 treatment groups for this regimen, active and placebo. Participants will be 
randomized in a 3:1 ratio to active treatment or placebo (i.e., 120 active: 40 placebo).   
Planned Number of Sites 
Research participants will be enrolled from approximately 80 centers in the US.   
Treatment Duration 
The maximum duration of the placebo-controlled treatment period is 24 weeks.  
Follow-up Duration 
At the conclusion of the 24-week placebo-controlled treatment period of the study, all 
participants will either schedule a 28-day follow up phone call and end their participation in the 


 
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regimen or have the option to receive intravenous infusions of trehalose in the Open Label 
Extension (OLE) period of the study. The duration of the OLE period is planned for 52 weeks. 
Total Planned Trial Duration 
For participants completing the placebo-controlled treatment period of the study, the planned 
amount of time for a participant in the trial is up to 34 weeks, or about 8 months.  This duration 
assumes a 6-week screening window, a 24-week placebo-controlled treatment period, and a 4-
week safety follow-up period for those participants who do not enter the OLE. Participants will 
complete approximately 10 study visits during the placebo-controlled treatment period of the 
study, in addition to weekly infusions outside of these visits.  
If the participant opts into the subsequent OLE, the total planned amount of time for a participant 
in the trial is approximately 86 weeks. This duration assumes a 6-week screening window, 24-
week placebo-controlled treatment period, a 52-week OLE period, and a 4-week safety follow-up 
period. Participants will complete 10 study visits across the planned 52-week OLE period of the 
study, in addition to weekly infusions outside of these visits. 
  


 
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SCHEDULE OF ACTIVITIES – PLACEBO-CONTROLLED PERIOD  
 
As per the Schedule of Activities (SOA) below, visits must occur every 4 weeks and will be clinic-, phone- or telemedicine based, as 
applicable. In this regimen, participants receive a weekly infusion of trehalose or matching placebo. The infusions are performed at 
each site for at least the first 4 weeks then participant may be permitted, based on safety and tolerability assessments, to have infusions 
administered at home. There is a maximum 24-week duration of placebo-controlled treatment for a Regimen.  
 
Activity (page 1 of 2) 
Master 
Protocol or 
Regimen-
Specific 
Master 
Protocol 
Screening1 
Regimen 
Specific 
Screening1 
Baseline20 
Week 
221 
Week 4 
Week 8 
Week 12 
21 
Week 
1613 
Week 20 
21 
Week 
24 or 
Early 
Term. 
Visit 
Follow-Up 
Safety Call10 
Clinic 
Clinic 
Clinic 
Phone 
Clinic 
Clinic 
Phone 
Clinic 
Phone 
Clinic 
Phone 
-42 to -1 
Days15 
-41 to 0 
Day15 
Day 0 
Day 14 
±3 
Day 28 
±7 
Day 56 
±7 
Day 84 
±3 
Day 112 
±7 
Day 140 
±3 
Day 168 
±7 
28 days after 
last dose 3 
days 
Written Informed Consent2 
Master 
X 
X 
 
 
 
 
 
 
 
 
 
Written Informed Consent - OLE 
Master 
 
 
 
 
 
 
 
X 
 
 
 
Inclusion/Exclusion Review 
Master 
X 
X3 
 
 
 
 
 
 
 
 
 
ALS & Medical History 
Master 
X 
 
 
 
 
 
 
 
 
 
 
Demographics 
Master 
X 
 
 
 
 
 
 
 
 
 
 
Physical Examination 
Master 
X 
 
 
 
 
 
 
 
 
 
 
Neurological Exam 
Master 
X 
 
 
 
 
 
 
 
 
 
 
Vital Signs4 
Master 
X 
 
X 
 
X 
X 
 
X 
 
X 
 
Vital signs associated with 
infusion 5 
Regimen 
 
 
 
Weekly in conjunction with study drug administration from week 1 
through week 24 
 
 
Slow Vital Capacity 
Master 
X14 
 
X 
 
 
X 
 
X 
 
X 
 
Muscle Strength Assessment  
Master 
 
 
X 
 
 
X 
 
X 
 
X 
 
ALSFRS-R 
Master 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
 
ALSAQ-40 
Regimen 
 
 
X 
 
 
 
 
 
 
X 
 
CNS Bulbar Function Scale 
Regimen 
 
 
X 
 
 
X 
 
X 
 
X 
 
12-Lead ECG 
Master 
X 
 
 
 
 
 
 
 
 
X 
 
Clinical Safety Labs6, 17 
Master 
X 
 
X 
 
X 
X 
 
X 
 
X 
 
Biomarker Blood Collection 
Master 
 
 
X 
 
 
X 
 
X 
 
X 
 


 
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Activity (page 1 of 2) 
Master 
Protocol or 
Regimen-
Specific 
Master 
Protocol 
Screening1 
Regimen 
Specific 
Screening1 
Baseline20 
Week 
221 
Week 4 
Week 8 
Week 12 
21 
Week 
1613 
Week 20 
21 
Week 
24 or 
Early 
Term. 
Visit 
Follow-Up 
Safety Call10 
Clinic 
Clinic 
Clinic 
Phone 
Clinic 
Clinic 
Phone 
Clinic 
Phone 
Clinic 
Phone 
-42 to -1 
Days15 
-41 to 0 
Day15 
Day 0 
Day 14 
±3 
Day 28 
±7 
Day 56 
±7 
Day 84 
±3 
Day 112 
±7 
Day 140 
±3 
Day 168 
±7 
28 days after 
last dose 3 
days 
Biomarker Urine Collection17 
Master 
 
 
X 
 
 
X 
 
X 
 
X 
 
PK Blood Collection16 
Regimen 
 
 
X 
 
 
X 
 
 
 
 
 
DNA Collection8 (optional) 
Master 
 
 
X 
 
 
 
 
 
 
 
 
CSF Collection (optional) 
Master 
 
 
X 
 
 
 
 
X12 
 
 
 
Concomitant Medication Review 
Master 
X 
X 
X 
X  
X 
X 
X 
X 
X 
X 
 
Adverse Event Review7 
Master 
X 
X 
X 
X  
X 
X 
X 
X 
X 
X 
X 
Document changes in health, 
medications, and infusion-related 
adverse events 
Regimen  
 
 
 
Weekly in conjunction with study drug administration from week 1 
through week 24 
 
 
Columbia-Suicide Severity Rating 
Scale 
Master 
 
 
X 
 
X 
X 
 
X 
 
X 
 
Assignment to the Regimen 
Master 
X 
 
 
 
 
 
 
 
 
 
 
Randomization within the 
Regimen 
Master 
 
X 
 
 
 
 
 
 
 
 
 
Adjust Dose as Needed20 
Regimen 
 
 
 
 
X 
X 
 
X 
 
X19 
 
Administer/Dispense Study Drug 
Master 
 
 
X9 
Weekly IV infusion from week 1 through week 24 
X10 
 
Drug Accountability/Compliance 
Master 
 
 
 
Weekly in conjunction with study drug administration from week 1 
through week 2422 
X 
 
Exit Questionnaire 
Master 
 
 
 
 
 
 
 
 
 
X 
 
Vital Status Determination11 
Master 
 
 
 
 
 
 
 
 
 
X 
 
 


 
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1 Master Protocol Screening procedures must be completed within 42 days to 1 day prior to the Baseline Visit. The Regimen-Specific Screening Visit and Baseline Visit should be 
combined, if possible.   
2 During the Master Protocol Screening Visit, participants will be consented via the Platform Trial informed consent form (ICF). After a participant is randomized to a regimen, 
participants will be consented a second time via the regimen-specific ICF.  
3 At the Regimen Specific Screening Visit, participants will have regimen-specific eligibility criteria assessed.   
4 Vital signs include weight, systolic and diastolic pressure, respiratory rate, heart rate and temperature.  Height measured at Master Protocol Screening Visit only. If an infusion 
will occur at the completion of the visit, vital signs should be collected within 30 minutes of the start of infusion to serve as the pre-infusion vital signs collection.  
5 During each infusion, vital signs associated with the infusion (systolic and diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-infusion, 30 minutes (± 5 
mins) after the start of infusion (SOI), 60 minutes after the start of infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If a participant requires a 90-minute 
infusion duration due to receiving more than 2 bags of study drug an additional vital signs measurement is to be completed approximately 30 minutes (± 10 mins) after the end of 
the 90-minute infusion. 
6 Clinical safety labs include hematology (CBC with differential), complete chemistry panel, thyroid function and urinalysis. Hb A1C will be included in labs at baseline, week 16 
and week 24. Serum pregnancy testing will occur in women of child-bearing potential at the Master Protocol Screening Visit and as necessary during the study. Pregnancy testing 
is only repeated as applicable if there is a concern for pregnancy. 
7 Adverse events that occur after signing the master protocol consent form will be recorded.  
8 The DNA sample can be collected after the Baseline Visit if a baseline sample is not obtained or the sample is not usable. 
9 Administer first dose of study drug only after Baseline Visit procedures are completed. 
10 Participants will only have a Follow-Up Safety Call at this time if they do not continue on in the OLE.  Participants who continue into OLE will have a Follow-Up Safety Call 
after their last dose of investigational product during the OLE period. 
11 Vital status, defined as a determination of date of death or death equivalent or date last known alive, will be determined for each randomized participant at the end of the 
placebo-controlled portion of their follow-up (generally the Week 24 visit, as indicated). If at that time the participant is alive, his or her vital status should be determined again at 
the time of the last patient last visit (LPLV) of the placebo-controlled portion of a given regimen. We may also ascertain vital status at later time points by using publicly available 
data sources as described in section 8.15 of the Master Protocol. 
12  If the CSF collection is unable to be performed for logistical reasons, such as scheduling, at the Week 16 Visit, it may be performed at the Week 24 Visit. 
13 Visit may be conducted via phone or telemedicine with remote services instead of in-person if this is needed to protect the safety of the participant due to a pandemic or other 
reason. 
14 If required due to pandemic-related restrictions, Forced Vital Capacity (FVC) performed by a Pulmonary Function Laboratory evaluator or with a study-approved home 
spirometer, or sustained phonation using a study approved method may be used for eligibility (Master Protocol Screening ONLY).    
15 Master Protocol Screening and Regimen Specific Screening visit windows are relative to Baseline (Day 0). 
16   Participants completing the PK collection will have blood collected for PK analysis at Baseline (Day 0) and Week 8 (Day 56) at the following time points: pre-dose and 1 hour ± 5 
minutes, 2 hours ± 5 minutes, and 4 hours ± 5 minutes post start of infusion (SOI). At each time point, 4mL of blood will be collected.  
17 All urine samples must be collected prior to IP administration.  
19 Dose is adjusted only for those participants continuing into the OLE.  
20 The weight collected during each in clinic visit will be used to calculate the participant’s dose. The participant will remain on a stable dose until the next in clinic visit (Weeks 4, 8, 16) 
when weight be collected, at which point the dose may be adjusted if the participant’s weight has increased or decreased by 2kg. Additional information on dose adjustments is included in 
section 5.6 Dosing Changes. 
21 If the participant receives an in-clinic infusion within the visit window, this visit may be conducted while the participant is in clinic for their weekly infusion.   
22 Drug accountability will not be done at phone visits. Compliance will be automatically calculated in the EDC.  


 
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SCHEDULE OF ACTIVITIES – Open Label Extension (Optional) 
Activity (page 1 of 1) 
Master 
Protocol or 
Regimen-
Specific 
 
Open Label Extension (Optional)5 
Week 28 
Week 4 
Week 87 
Week 
128 
Week 167 
Week 
208 
 
Week 
248 
Week 287 
Week 407 
Week 
52 or 
Early 
Term. 
Visit 6, 7 
Follow-
Up 
Safety 
Call4, 6 
Clinic 
during 
Infusion 
Clinic 
Clinic 
Phone 
Clinic 
Phone 
Phone 
Clinic 
Clinic 
Clinic 
Phone 
 
Day 28 
±7 
Day 56 
±7 
Day 84 
±3 
Day 112 
±7 
Day 140 
±3 
Day168 
±3 
Day 196 
± 14 
Day 280 
± 14 
Day 364 
± 14 
28 days 
after last 
dose 3 
days 
Vital Signs1 
Master 
 
X 
X 
 
X 
 
 
X 
X 
X 
 
Vital signs associated with 
infusion9 
Regimen  
Weekly in conjunction with investigational product administration from OLE week 1 through OLE week 52 
 
 
Slow Vital Capacity 
Master 
 
X 
X 
 
X 
 
 
X 
X 
X 
 
ALSFRS-R 
Master 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
ALSAQ-40 
Regimen 
 
 
 
 
 
 
 
X 
 
X 
 
CNS Bulbar Function Scale 
Regimen 
 
 
X 
 
X 
 
 
X 
X 
X 
 
Clinical Safety Labs2 
Master 
 
X 
X 
 
X 
 
 
X 
X 
X 
 
Biomarker Blood Collection 
Master 
 
 
 
 
X 
 
 
X 
 
X 
 
Biomarker Urine Collection 
Master 
 
 
 
 
X 
 
 
X 
 
X 
 
Concomitant Medication 
Review 
Master 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Adverse Event Review3 
Master 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Document changes in health, 
medications, and infusion-
related adverse events 
Regimen  
Weekly in conjunction with Investigational Product administration from OLE week 1 through OLE week 52 
 
Columbia-Suicide Severity 
Rating Scale 
Master 
 
X 
X 
 
X 
 
 
X 
X 
X 
 


 
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Adjust Dose as Needed10 
Regimen  
 
X 
X 
 
X 
 
 
X 
X 
 
 
Administer/Dispense 
Investigational product 
Master 
Weekly IV infusion from OLE week 1 through week 52 
 
Drug 
Accountability/Compliance 
Master 
Weekly throughout study in conjunction with drug administration11 
 
1 Vital signs include weight, systolic and diastolic pressure, respiratory rate, heart rate and temperature. Height in cm measured at Master Protocol Screening Visit only. 
2 Clinical safety labs include hematology (CBC with differential), complete chemistry panel, thyroid function and urinalysis. HbA1c will be added to the safety labs at weeks 16, 
28 and 52.  Serum pregnancy testing will occur in women of child-bearing potential at the Master Protocol Screening Visit and as necessary during the study. Pregnancy testing is 
only repeated as applicable if there is a concern for pregnancy. 
3 Adverse events that occur after signing the master protocol consent form will be recorded.  
4 Participants who continue into OLE will have a Follow-Up Safety Call (as described in the body of this RSA) after their last dose of investigational product during the OLE 
period. 
5 The duration of the OLE is 52 weeks. 
6 Participants who continue into the OLE and then withdraw consent or early terminate will be asked to complete an Early Termination Visit and Follow-Up Safety Call as 
described in the body of this RSA.  
7 Visit may be conducted via phone or telemedicine with remote services instead of in-person if this is needed to protect the safety of the participant due to a pandemic or other 
reason.  
8 If the participant receives an in-clinic infusion within the visit window, this visit may be conducted while the participant is in clinic for their weekly infusion.   
9 During each infusion, vital signs associated with the infusion (systolic and diastolic pressure, respiratory rate, and heart rate) are to be measured pre-infusion, 30 minutes (± 5 
mins) after the start of infusion (SOI), 60 minutes after the start of infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If a participant requires a 90-minute 
infusion duration due to receiving more than 2 bags of study drug an additional vital signs measurement is to be completed approximately 30 minutes (± 10 mins) after the end of 
the 90-minute infusion. 
10 The weight collected during each in clinic visit will be used to calculate the participant’s dose. The participant will remain on a stable dose until the next in clinic visit (Weeks 
4, 8, 16, 28, 40) when weight be collected, at which point the dose may be adjusted if the participant’s weight has increased or decreased by 2kg. Additional information on dose 
adjustments is included in section 5.6 Dosing Changes. 
11 Drug accountability will not be done at phone visits. Compliance will be automatically calculated in the EDC.  


 
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1. INTRODUCTION 
Regimen E:  SLS-005 Trehalose  
1.1 Trehalose Background Information 
Trehalose is a disaccharide that is well known for its protein-stabilizing properties (Elbein 2003, 
Emanuele 2014) and its ability to activate autophagy (Rusmini 2019)). Because of its known 
ability to reduce abnormal protein aggregations, trehalose was studied in several cellular and 
animal models of hereditary neurologic and muscular disorders, especially those associated with 
CAG repeat expansion and polyalanine/polyglutamine accumulation (Tanaka 2004, Davies 
2006). This was the basis for human trials of intravenous (IV) trehalose in oculopharyngeal 
muscular dystrophy (OPMD) and spinocerebellar ataxia type 3 (SCA3 or Machado Joseph 
disease). 
Trehalose has been shown to penetrate both muscle and brain. In recent years it became clear 
that trehalose exposure results in the activation of cellular pathways that are relevant to its use as 
a potential treatment of ALS. Trehalose activates lysosomal and autophagic activity by various 
pathways and increases expression of biomarkers of autophagy such as microtubule-associated 
protein 1A/1B-light chain 3 (LC-3 II). It was shown that the basis of activation of these pathways 
is cellular glucose starvation (Mardones 2016). However, the mechanism of lysosomal-
autophagic activation by trehalose cannot be only glucose starvation. Trehalose also activates 
autophagy through the activation of Transcription Factor EB (TFEB), and relocation to the 
nucleus, a key factor in lysosomal and autophagy gene expression. (Rusmini 2019) Activation of 
TFEB is an emerging therapeutic target for a number of diseases with pathologic accumulation 
of storage material.  
Trehalose is not absorbed well in the human gut because of trehalase enzymes in the brush 
border that cleave the molecule into two glucose molecules. Oral ingestion of trehalose results in 
<0.5% absorption of trehalose. Seelos Therapeutics has developed IV trehalose to circumvent the 
breakdown of trehalose in the gut. In Study BBCO-001, an open-label Phase 2a study of 25 
OPMD patients with dysphagia, weekly trehalose treatments at a dose of 27 grams (g) for 6 
months was well tolerated. A single ascending dose study identified that clearance of trehalose is 
weight dependent and that the maximum tolerated dose (MTD) as 0.75 g/kg. This is the dose to 
be employed in this study. 
1.2 Nonclinical and Clinical Data 
1.2.1 Nonclinical Experience 
The safety and tolerability of trehalose has been extensively investigated. A detailed review of 
safety in animals is presented in the trehalose Investigator's Brochure (IB).  
Three-month IV Good Laboratory Practice (GLP) toxicology studies in rats and dogs at a dose of 
3.6 g/kg administered twice weekly showed no organ toxicity. A 6-month GLP toxicology study 
in rats with doses of 2.7, 5.4, and 10.8 g/kg demonstrated no organ toxicity. An inflammatory 
response at the end of the indwelling catheter was noted in a dose-dependent manner. Although 
there were an increasing number of observations with the increasing dose it was not a linear 
increase and thus it was considered by the reporting pathologist to a procedural related change 
due to manipulation of the catheter and not drug related. There was no systemic inflammatory 


 
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response noted. A 9-month chronic toxicity study in dogs using the same doses of 2.7, 5.4, and 
10.8 g/kg did not demonstrate any organ toxicity. 
1.2.2 Nonclinical Literature 
Three in vivo studies demonstrated that trehalose is a potential protective agent in ALS. These 
studies were performed using the SOD1 ‘classical’ mouse model with the G93T mutation or the 
G86R mutation.  In mice trehalose can be used orally because they lack intestinal trehalase. 
In the work of Castillo et al (2013), trehalose was administered to animals with a G86R SOD1 
mutation from age 35 days onward. The dose used was 3 weekly IP injections of 2 g/kg plus 3% 
trehalose in free drinking water. It was found that in female mice trehalose increased survival 
from a mean of 139 days in untreated animals to 154 days in trehalose treated animals. Male 
mice survival increased from 145 to 177 days. The level of SOD1 accumulation in the brain was 
lower when tested postmortem. In addition, there was preservation of motor neurons noted in the 
ventral horn of the spinal cord in trehalose treated animals.   
In a similar study, Zhang et al (2014) administered trehalose to animals with the G93A SOD 1 
mutation in the drinking water only (2% concentration) starting on day 60. Trehalose delayed 
mean onset of disease from day 95 to 112 (based on clinical observation in the animals). Mean 
survival was prolonged from 124 to 145 days of age; however, disease duration was unchanged.   
The number of preserved motor neurons in the spinal cord was assessed on day 120. 
Trehalose-treated animals had increased numbers of preserved motor neurons compared with 
untreated control animals. Neuronal SOD1 aggregates and p62 accumulation were decreased 
with trehalose treatment. Furthermore, histologic studies of muscle showed an increase in mean 
fiber size and evidence of preserved neuromuscular junctions, indicating reduced denervation of 
muscle tissue. 
Li Y et al (2015) evaluated 2 protocols for the study of trehalose in the SOD1-G93A mouse 
model (given in 2% solution in the drinking water). A short-term therapy protocol evaluated the 
effects after 30 days of treatment. Long-term therapy monitored the effects from onset to death. 
In both protocols, trehalose therapy was started on day 60. Disease onset, defined as the time 
when the animal dragged 1 leg, was significantly delayed by trehalose. Similarly, the time to 
reach grade 2 (2 affected limbs) was prolonged. Survival (defined as animal sacrifice time when 
it reached grade 4 of the disease) was delayed by 7 days, but this change did not reach statistical 
significance. In the short-term protocol motor function was improved as assessed by rotarod and 
hanging functional tests. There was a clear change in autophagy activation (levels of p62) in the 
spinal cord during the short-term protocol. 
1.2.3 Clinical Experience 
Previous clinical trials evaluating IV trehalose in healthy subjects and patients with OMPD and 
SCA3 are summarized below. Overall, administration of IV trehalose to adults appears safe and 
well tolerated. 
A total of 59 subjects have been exposed to trehalose: 18 healthy subjects, 25 OPMD patients, 15 
SCA3 patients, 1 adult with Sanfilippo Type B. Out of the 59, 11 patients received SLS-005 in 
the under expanded access use programs (10 subjects with OPMD and 1 with Sanfilippo type B).  
The healthy subjects received a single dose of 27, 54, or 81 g (6 subjects each dose), the OPMD 
patients received repeated weekly doses of 27 g trehalose for a duration of 6 to 18 months, the 


 
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SCA3 patients received repeated weekly doses of 13.5 or 27 g for a duration of 6 to 12 months, 
and the Sanfilippo patient is receiving 0.75 g/kg weekly, currently at over 4 months of treatment. 
Overall, trehalose was well tolerated.  
Study BB-TRE-101 studied the safety, tolerability, and pharmacokinetics (PK) of single 
ascending doses of SLS-005 in healthy subjects. The study demonstrated proportional linear 
kinetics for increasing doses of trehalose from 27 g to 81 g. In addition, it demonstrated a need 
for weight-based dosing since clearance increased with increasing weight. One (1) treatment-
emergent adverse event was assessed as possibly related elevated liver enzymes in 1 subject in 
the 81 g treatment group and was reported as a suspected unexpected serious adverse reaction 
(SUSAR). Based on the data from this study, along with supportive data from animal studies, the 
MTD was established as 0.75 g/kg.  
Study BBCO-001, an open-label study in patients with OPMD, assessed the safety, tolerability, 
PK, and efficacy of SLS-005 in patients treated for 6 months with weekly infusions of 27 grams. 
SLS-005 was generally safe and well tolerated. The most common adverse event (AE) was 
elevated urine glucose that occurred in 52% of patients. The AE of glycosuria was only reported 
from 1 site and, although the patients were asymptomatic, the investigator assessed the 
laboratory result as clinically significant. Increased glucose in urine is an anticipated result 
because trehalose is metabolized to glucose, however serum glucose did not exceed normal range   
There were 3 serious adverse events (SAEs) occurring in 2 patients: urinary tract infection, 
aspiration pneumonia, and aspiration leading to death. None of the SAEs were considered related 
to study drug.  
The subsequent Study BB-OPMD-301 was an open-label extension protocol to BBCO 001 that 
continued treatment of patients for more than 1 year. The safety profile in this study was similar 
to the initial study.  
Study BB-MJD-201, a Phase 2 double-blind, dose-controlled study in SCA3 patients, was an 
open-label study of 15 patients with SCA3 that evaluated the safety and tolerability of 2 different 
IV doses of SLS-005, 13.5 and 27 g, administered weekly. All AEs were considered mild in 
severity. The most common AE was transient glucosuria occurring in 20% (n=3) of patients. 
SLS-005 treatment was generally safe and well tolerated in this patient population.  
1.3 Trehalose Therapeutic Rationale 
1.3.1 Rationale for the study 
The nonclinical data generated to date for trehalose support its potential to be used as an 
effective treatment for ALS.  Through its effects on reducing proteinaceous 
aggregates/inclusions promoting autophagy and lysosomal pathways and reducing cell death, 
trehalose improved muscle strength and overall motor skills in animal models of OPMD, SCA3, 
and ALS. The clinical safety profile of trehalose and the nonclinical toxicology data support the 
investigation of trehalose as a potential treatment of ALS.   
1.3.2 Rationale for the Dose, Dosing Regimen, and Route of Administration 
Error! Reference source not found.If administered orally < 0.5% of ingested trehalose is 
absorbed into the blood stream. Therefore, to achieve therapeutic blood levels of trehalose, it is 
necessary to circumvent the metabolism in the human gastrointestinal tract. As such, Seelos has 


 
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developed SLS-005 an IV formulation of trehalose for injection, 90.5 mg/mL to be evaluated in 
this study.  
In the single ascending dose study of IV trehalose in healthy subjects, the mean weight in the 54 g 
dose cohort was 74.5 kg. At the dose of 54 g, subjects received 0.72 g/kg. The area under the curve 
to time infinity (AUCinf) at that dose was 8,595±2,575 hour × µg/mL. Since clearance is dependent 
on body weight (i.e., clearance increases with increasing body weight), in order to achieve a 
consistent exposure in, at, or near the exposure at the MTD, 54 g/1 hour, a weight-based dose of 
0.75 g/kg should achieve an exposure within the clinically acceptable safety range. 
One of the proposed mechanisms of action of trehalose is to activate autophagy, the blockade of 
glucose receptors results in a starvation scenario within the cell (DeBosch 2016). Therefore, the 
MTD will be used to ensure significant exposure. Weekly dosing of IV trehalose is the only 
regimen to be evaluated in clinical trials to date (patients with OPMD and SCA3) and will be used 
in this trial. 
1.3.3 Rationale for the Patient Population 
The pathologic hallmark of the disease is aggregation of proteinaceous inclusions in motor 
neurons. The main composite of these inclusions is misfolded TDP43 (especially the 
phosphorylated form), which accumulates in the cytoplasm and is depleted in the nucleus. Patients 
with either sporadic or familial ALS accumulate TDP43 and patients with the SOD mutation 
accumulate SOD aggregates and thus have the potential to benefit from treatment with trehalose 
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2. OBJECTIVES 
2.1 Study Objectives and Endpoints  
Primary Efficacy Objective: 
To evaluate the efficacy of trehalose injection, 90.5 mg/mL for intravenous infusion as compared 
to placebo on ALS disease progression.  
Secondary Efficacy Objective: 
• To evaluate the effect of trehalose injection, 90.5 mg/mL for intravenous infusion on 
selected secondary measures of disease progression, including survival. 
Safety Objective: 
• To evaluate the safety of trehalose injection, 90.5 mg/mL for intravenous infusion for 
patients with ALS. 
Exploratory Efficacy Objective: 
• To evaluate the effect of trehalose injection, 90.5 mg/mL for intravenous infusion on 
selected biomarkers and endpoints. 
Primary Efficacy Endpoint: 
Change in disease severity as measured by the ALS Functional Rating Scale-Revised (ALSFRS-
R) total score using a Bayesian repeated measures model that accounts for loss to follow-up due 
to mortality.  
Secondary Efficacy Endpoints: 
• Change in respiratory function as assessed by slow vital capacity (SVC). 
• Change in muscle strength as measured isometrically using hand-held dynamometry 
(HHD) and grip strength. 
• Survival. 
Safety Endpoints: 
• Treatment-emergent adverse and serious adverse events. 
• Changes in laboratory values and treatment-emergent and clinically significant laboratory 
abnormalities. 
• Changes in ECG parameters and treatment-emergent and clinically significant ECG 
abnormalities. 
• Treatment-emergent suicidal ideation and suicidal behavior. 
Exploratory Efficacy Endpoints: 
• Changes in the biofluid biomarkers of neurodegeneration specified in the SOA. 
• Changes in the patient reported outcomes specified in the SOA. 


 
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3. RSA DESIGN 
This study is a multi-center, randomized, placebo-controlled trial, testing active dose of trehalose 
injection, 90.5 mg/mL for intravenous infusion (0.75 g/kg), given weekly versus placebo.  
Participants will be randomized 3:1 active: placebo. 
3.1 Scientific Rationale for RSA Design 
This RSA is designed to correspond with the design of the Master Protocol and the goals of the 
HEALEY ALS Platform Trial. 
3.2 End of Participation Definition 
A participant is considered to have ended his or her participation in the placebo-controlled period 
of the Regimen if they:  
• Complete planned placebo-controlled period visits, as described in the SOA, including 
participants on or off study drug 
• Early terminate from the study and complete the Early Termination Visit and Follow-Up 
Phone call as described in Section 6.9 
• Withdraw consent to continue participation in the study, or are lost to follow-up 
If a participant initiates open-label study drug in the OLE period, he or she is considered to have 
completed his or her participation in the OLE period of the Regimen if they choose to 
discontinue participation or if all planned OLE period visits, including the last visit or the last 
scheduled procedure shown in the SOA, have been completed. 
3.3 End of Regimen Definition 
The end of the placebo-controlled treatment period in a Regimen occurs when all randomized 
participants have completed their participation in the placebo-controlled period as defined in 
section 3.2. 
The end of the OLE period in a Regimen occurs when all participants who initiated open-label 
investigational product in the OLE period have completed their participation in the OLE period 
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4. RSA ENROLLMENT 
4.1 Number of Study Participants 
Approximately one hundred-sixty (160) participants will be randomized to this Regimen.   
4.2 Inclusion and Exclusion Criteria 
In order to be assigned to a Regimen, participants must meet the Master Protocol eligibility 
criteria.  In addition, participants meeting all of the following inclusion and exclusion criteria 
will be allowed to enroll in this Regimen:  
4.2.1 RSA Inclusion Criteria 
There are no additional RSA Inclusion Criteria from those described in the Master Protocol.   
4.2.2 RSA Exclusion Criteria  
1. Current diagnosis or healthcare professional-recommended treatment (medication, 
exercise or diet) of diabetes mellitus   
2. Master Protocol screening glucose >140 mg/dl 
3. Prior treatment with IV trehalose or known hypersensitivity to trehalose 
4. Current use of oral trehalose (see prohibited medication Section 5.9) 
5. Inability for participant to return to site for weekly drug administration, until approved 
for home infusions 
6. Master Protocol screening body weight >144 kilograms  
4.3 Treatment Assignment Procedures 
Each participant who meets all eligibility criteria for the Regimen will be randomized to receive 
either 0.75 g/kg of trehalose or equivalent weight-based volume of placebo by weekly 
intravenous infusion in the study’s 24-week placebo-controlled treatment period.   
4.4 Open Label Extension Eligibility 
Participants who complete the 24-week placebo-controlled treatment period on active or placebo 
study drug will be eligible to participate in the OLE.  


 
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5. INVESTIGATIONAL PRODUCT  
5.1 Investigational Product Manufacturer 
The investigational product is SLS-005 (trehalose injection, 90.5 mg/mL for intravenous 
infusion). This solution for IV infusion is administered once a week at a planned dose of 0.75 
g/kg. 
Trehalose is a stable, non-reducing disaccharide with 2 glucose molecules linked in an alpha 1,1 
configuration. It contains no less than 97.0% and no more than 102.0% of C12H22O11, 
calculated on the anhydrous basis. 
Trehalose Injection 90.5 mg/mL (an aqueous, sterile solution for IV infusion) is provided as a 
clear liquid in a 300 mL infusion bag. Each 300 mL bag contains 27.15 g of trehalose.  See the 
IB for further details on trehalose. 
Placebo is a sodium chloride injection, 0.9%, USP for intravenous infusion, provided as a clear 
liquid in a 300 mL infusion bag. Placebo is administered once a week in an equivalent weight-
based volume as described for trehalose dosing. 
5.2 Labeling, Packaging, and Resupply 
Trehalose will be provided in a 1 bag per kit configuration. Further details are provided in the 
Pharmacy Manual. 
Placebo will be provided in a 1 bag per kit configuration. Placebo has been manufactured by the 
drug product manufacturer in the same container closure (300-mL freeflex IV bag). 
5.3 Acquisition, Storage, and Preparation 
Acquisition 
Prior to study treatment, study drug will be supplied to the study site's pharmacy by the Sponsor 
or its designee. 
Shipment of study drug supplies for the study will be accompanied by shipment records 
describing the contents of the shipment, drug information, and other appropriate documentation. 
The shipment form will assist in maintaining current and accurate inventory records. 
The investigator must ensure the acknowledgement of receipt of the clinical trial material at the 
site, including that the material was received in good condition.  
Study Drug Storage 
The investigational drug and matching placebo will be stored in the study site’s pharmacy, at 
room temperature (20 to 25 ºC) with excursions permitted 15 to 30 °C. The Sponsor or its 
designee should be notified for any deviation from the storage conditions. 
Study Drug Preparation 
Trehalose Injection 90.5 mg/mL (an aqueous, sterile solution for IV infusion) is provided as a 
clear liquid in a 300 mL infusion bag containing 27.15 grams of trehalose. No preparation is 
required for administration of the study drug.  
Placebo is also provided as a clear liquid in a 300 mL infusion bag. No preparation is required 
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The dose of IV trehalose or placebo will be calculated based on participant body weight to 
determine the volume administered to each participant. Each participant’s weight should be 
measured consistently at each in-clinic visit. Shoes and heavy winter clothes (e.g. coats, 
sweaters, boots) should be removed prior to weighing. If shoes cannot be removed, then weight 
should be assessed with the participant’s shoes on at each clinic visit.  Doses are adjusted based 
on changes in body weight (2 kg increase or decrease) noted during clinical trial assessments. 
More than 1 bag may be needed per infusion based on the participant’s body weight. The 
intended SLS-005 total infusion time, excluding interruptions, is 60 minutes for infusion 
volumes up to 2 bags (i.e. up to 600mL) and should not exceed 70 minutes. For patients who 
require more than 2 bags (i.e. >600mL) of study drug the intended SLS-005 total infusion time 
should be increased to 90 minutes and not exceed 100 minutes.  Management of 
deviations/excursions is described in the Study Drug MOP. 
5.4 Study Medication/Intervention, Administration, Escalation, and Duration 
The participant will receive weekly infusions of study drug (trehalose or placebo). Although for 
most participants the total infusion time of SLS-005 is set at 60 minutes (+ 10 minutes), 
participants who require more than 2 bags of study drug the infusion time should be increased to 
90 minutes (+10 minutes) throughout the study. All infusions will be administered using an 
infusion pump and will be followed with an IV flush. The start calendar date and 24-hour clock 
time as well as the end calendar date and 24-hour clock time of each infusion will be recorded in 
the study database.  If an infusion is interrupted due to infusion issues, i.e. loss of IV site, the 
infusion can be completed once access has been re-established unless > 30 minutes have elapsed. 
If 30 or more minutes have elapsed the infusion should not be restarted, and the volume infused 
prior to the interruption recorded in the CRF as the total dose administered. 
If needed the use of an implanted venous access device (e.g. Mediport, PICC) or percutaneous 
indwelling catheter is permitted however small caliber indwelling catheters in peripheral vessels 
should only be inserted for single use. Participants are to be monitored for administration site 
reactions during study drug administration. Infusion site reactions or infusion reactions are to be 
recorded as AEs using the appropriate coding terms on the eCRF. 
If needed, remote services such as infusions at home can be considered after the participant has 
completed at least 4 weeks of in-clinic infusions, as long as the participant has had no adverse 
reaction to the infusions and all safety assessments are within acceptable limits. Decision to 
permit home infusions is at the discretion of the site investigator. Participants approved by their 
site investigator for weekly infusions outside of the study site will still be required to attend on-
site study visits at Week 16 (unless completed remotely due to pandemic-related or other 
reasons) and 24, and their associated IP infusions at these timepoints can be done in-clinic 
following completion of the study visit. Telephone visits at Weeks 2, 12 and 20 may be 
conducted in clinic if the participant is receiving their weekly infusions at the study site and the 
infusion will occur during the visit window.   
5.5 Justification for Dosage  
Based on the preclinical and clinical safety and PK data trehalose injection 90.5 mg/mL should 
be dosed using a weight-based dosing up to a maximum of 0.75 g/kg. Participants should receive 
trehalose injection 90.5 mg/mL over 60 + 10 minutes, with the exception noted above for more 
than 2 bags, using an infusion pump.  


 
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5.6 Dosage Changes 
If for any reason a participant is unable to receive a full dose this should be documented as per 
participant compliance. Doses are adjusted (increased or decreased) based on a weight change of 
2kg or greater from the previous in-clinic volume. A dosing chart, with the volume of study drug 
to be administered based off body weight to achieve a dose of 0.75 g/kg, is included in the Study 
Drug MOP.  
5.7 Participant Compliance 
Participants should be encouraged not to miss infusions if at all possible, as it may affect the 
efficacy of the drug. The dosing window is 1 week ± 3 days. There should be no fewer than 4 
days between study drug infusions.  
Compliance will be automatically calculated in the EDC for each weekly infusion.  
5.8 Drug Returns and Destruction 
At each in person visit the steps outlined in the Manual of Procedures must be followed for study 
drug accountability and compliance, as well as study drug return and destruction. 
5.9 Overdose 
Certain safety events that occur in association with investigational product may require 
reporting. These safety events include, but are not limited to, the following: 
• Overdose of the investigational product, where ‘overdose’ is defined as > 125% of the 
intended dose for a single treatment day infusion.  
• Suspected abuse/misuse of the investigational product 
• Inadvertent or accidental exposure to the investigational product 
• Medication error involving study drug (with or without participant exposure to the 
investigational product, e.g., name confusion) 
• The half-life of Trehalose Injection 90.5 mg/mL is approximately 1.5 hours, and there is no 
antidote. Trehalose is metabolized to 2 glucose molecules, there are no other metabolites. 
Supportive care with monitoring of vital signs, blood glucose levels, and liver enzymes 
should be considered. In 1 subject who received 81 g of Trehalose Injection 90.5 mg/mL, 
there was an increase in liver enzyme levels, but liver function remained normal and liver 
enzyme values returned to normal without treatment. 
These safety events should be reported to the Coordination Center whether they result in an 
AE/SAE or not. Safety events associated with an AE/SAE should also be reported in the EDC. 
The SI should also contact the Medical Monitor within 24 hours of the SI’s awareness.  
5.10 Prohibited Medications  
To date, there are no known pharmacokinetic drug interactions with trehalose. Participants must 
not take the following medications while on study drug in Regimen E: 


 
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• Oral trehalose is not permitted during the study. 
5.11 Trehalose Known Potential Risks and Benefits 
5.11.1 Known Potential Risks 
The safety profile from previous clinical trials of IV trehalose in adults shows no drug-related 
AEs other than mild, transient glycosuria which was asymptomatic and only 1 SAE, elevated 
liver enzymes, was considered possibly related to study drug due to temporal association of the 
changes and lack of alternative causation, however the event resolved without any intervention. 
Risks of study participation also includes the risk of an infusion reaction or infusion site 
reactions. There have been no infusion reactions to date in patients administered IV trehalose; 
however, 1 patient had an infusion site reaction (erythema). Toxicology data suggests that small 
indwelling catheters may result in vessel changes after multiple infusions. The inflammatory 
reaction noted was not associated with any generalized inflammation and there was no organ 
toxicity, thus it was considered a procedural complication of the indwelling catheter. If needed 
the use of an implanted venous access device (i.e. Mediport, PICC) or percutaneous indwelling 
catheter is permitted however small caliber indwelling catheters in peripheral vessels should be 
single use. Additional risks include those risks related to venipuncture (e.g., hematoma, bleeding, 
pain, infection), which are low, and the risk of progression of disease for patients assigned to 
receive placebo.  
5.11.2 Known Potential Benefits 
Given the relatively benign safety profile of trehalose noted thus far, and the lack of a highly 
effective treatment for ALS, treatment with trehalose for its potential to arrest or slow 
progression of ALS disease symptoms is justified.  


 
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6 REGIMEN SCHEDULE  
In addition to procedures in the Master Protocol, the following regimen-specific procedures will 
be conducted during the study: 
• ALSAQ-40 
• CNS Bulbar Function Scale 
• Serum PK parameter estimates 
• CSF biomarker LC3-II and Neurofilament light chain 
• HbA1c as part of the clinical safety labs 
Participants may be required to reconsent to the regimen if new procedures or information is 
added in the future. Should a participant need to reconsent, this should occur during the 
participant’s next in-person visit. If the participant’s next in-clinic visit is conducted remotely, 
reconsent may also be completed remotely using the following procedures: 
 
1. The site staff sends copy of the informed consent form to the participant. 
2. The participant reads through the consent form but does not sign.  
3. The Site Investigator, or other study staff member approved and delegated to obtain informed 
consent, calls the participant and reviews the informed consent form with the participant over 
the phone or via telemedicine.  
4. The participant signs the informed consent form and mails the original signed consent form 
back to the site. 
5. Once received at the site, the individual who consented the participant via phone signs the 
informed consent form. 
 
Modifications to Regimen Schedule  
Designated visits in the Schedule of Activities (i.e. Week 16) may be conducted via telemedicine 
(or phone if telemedicine is not available) with remote services instead of in-person if needed to 
protect the safety of the participant due to a pandemic or other reason. If a planned in-clinic visit 
is conducted via telemedicine (or phone if telemedicine is not available) with remote services, 
only selected procedures will be performed. Instructions on how to document missed procedures 
are included in the MOP.  
In addition to the procedures in the Master Protocol that should be conducted during the phone or 
telemedicine and remote visits, the following regimen-specific procedures should be completed:  
• CNS Bulbar Function Scale  
Details on collection of the CNS Bulbar Function Scale, dispensing IP during remote visits, and 
documenting subjects’ willingness to participate in OLE are described in the MOP. 
6.1 Baseline Visit 
This visit will take place on Day 0.  The following procedures will be performed for the regimen 
schedule: 


 
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• ALSAQ-40 
• CNS Bulbar Function Scale 
• Collect safety labs including for HbA1c evaluation 
• Collect urine, prior to start of infusion.  If urine cannot be collected prior to 
infusion, no urine should be collected at this visit 
• Confirm dosing volume based off Master Protocol Screening Visit weight 
• Administer first infusion of study drug in clinic after all Baseline procedures have 
been completed 
o During each infusion, vital signs associated with the infusion (systolic and diastolic 
pressure, respiratory rate, and heart rate are to be measured pre-infusion, 30 minutes (± 
5 mins) after the start of infusion (SOI), at the end of infusion (± 10 mins), and 90 
minutes (± 10 mins) after the start of infusion. If a participant requires a 90-minute 
infusion duration due to receiving more than 2 bags of study drug an additional vital 
signs measurement is to be completed approximately 30 minutes (±10 mins) after the 
end of the 90-minute infusion.   
• For applicable participants, collect PK blood sample (Pre-dose, 1-hour ± 5 
minutes, 2 hours ± 5 minutes, and 4 hours ± 5 minutes post start of infusion) 
6.2 Weekly Study Drug Infusion 
During the placebo-controlled treatment period, all participants will receive a weekly infusion of 
study drug (trehalose or placebo for 60 + 10 minutes, participants who require more than 2 bags 
of SLS-005 the infusion time is to be increased to 90 + 10 minutes) for the first 4 weeks of the 
study in clinic. Thereafter home infusions can be considered if the participant has had no adverse 
reaction to the infusion and all safety assessments are within acceptable limits.  
During the Week 8 in-clinic visit, PK assessments will occur prior to and after the infusion per 
the SOA. Therefore, if a participant transitions to home infusions after the first four weeks the 
site should coordinate the Week 8 infusion to occur in-clinic while the participant is in for the 
Week 8 in-clinic visit. The home infusion nurse will not collect any PK samples; therefore, the 
Week 8 PK samples would be missed if the Week 8 infusion does not occur in-clinic at the time 
of the Week 8 in-clinic visit. This would not result in a protocol deviation.  
Concurrent assessments such as vital signs, reporting of any infusion related adverse events, and 
reporting of new concomitant medications is to be completed weekly in conjunction with the 
study drug infusion. 
Blood pressure, respiration rate, and heart rate, should be conducted at four timepoints during 
each infusion:  (1) Pre-dose, (2) midway through infusion = 30 ± 5 min post start of infusion, (3) 
end of infusion = 60 ± 10 min post start of infusion, and (4) 90 ± 10 min post start of infusion. If 
a participant requires a 90-minute infusion duration due to receiving more than 2 bags of study 
drug an additional vital signs measurement is to be completed approximately 30 minutes (± 10 
mins) after the end of infusion. 
 
  


 
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If the infusion is done at home, confirmation of infusion and information on infusion-related 
adverse events, and changes in health and medications will be provided to study site by the 
home infusion nurse via study provided source documents. Site staff will then enter the data 
into the EDC and follow up with the participant as needed to document any changes in AEs 
or concomitant medications in the EDC.  
 
6.3 Week 2 Telephone Visit  
This visit will take place 14 ± 3 days after the Baseline Visit. No regimen-specific procedures 
will be performed at this visit. 
 
If the participant will receive their in-clinic infusion within the Week 2 visit window, this visit 
may occur in-clinic at the time of the infusion visit.  
 
6.4 Week 4 and 8 Visits 
Study drug should not be administered until after all study visit procedures are complete. 
These visits will take place on Days 28 ± 7 and 56 ± 7 days, respectively.  The following 
procedures will be performed for the regimen schedule: 
• Collect urine, prior to start of infusion.  If urine cannot be collected prior to 
infusion, no urine should be collected at this visit 
• Collect safety labs  
• CNS Bulbar Function Scale [Week 8 only] 


 
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• Adjustment of dosing volume based on most recent weight measurement as 
necessary 
• Administer infusion of study drug in clinic after all study procedures have been 
completed, provided at least 4 days have passed since prior infusion. 
• During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-
infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If a 
participant requires a 90-minute infusion duration due to receiving more than 2 
bags of study drug an additional vital signs measurement is to be completed 
approximately 30 minutes (± 10 mins) after the end of infusion.   
• For applicable participants, collect PK blood sample (Pre-dose, 1-hour ± 5 minutes, 2 
hours ± 5 minutes, and 4 hours ± 5 minutes post start of infusion) [Week 8 only] 
6.4 Week 12 Telephone Visit 
This visit will take place 84 ± 3 days after the Baseline Visit. No regimen-specific procedures 
will be performed at this visit. 
If the participant will receive their infusion in clinic and within the Week 12 visit window, this 
visit may occur in-clinic at the time of the infusion visit.  
 
6.5 Week 16 Visit  
Study drug should not be administered until after study visit procedures are complete. 
This visit will take place on Day 112 ± 7 days.  The following procedures will be performed for 
the regimen schedule: 
• Document participant’s willingness to participate in the OLE 
o If OLE consent is not obtained at Week 16, it may be obtained at Week 24. 
 
• CNS Bulbar Function Scale 
• Collect safety labs including for HbA1c evaluation 
• Collect urine, prior to start of infusion.  If urine cannot be collected prior to 
infusion, no urine should be collected at this visit 
• Adjustment of dosing volume based on most recent weight measurement as necessary 
• 
Administer infusion of study drug in clinic after all study procedures have been completed, 
provided at least 4 days have passed since prior infusion. 
• During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-
infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If a 


 
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participant requires a 90-minute infusion duration due to receiving more than 2 
bags of study drug an additional vital signs measurement is to be completed 
approximately 30 minutes (± 10 mins) after the end of infusion.   
6.6 Week 20 Telephone Visit 
This visit will take place 140 ± 3 days after the Baseline Visit. No regimen-specific procedures 
will be performed at this visit. 
If the participant will receive their infusion in clinic and within the Week 20 visit window, this 
visit may occur in-clinic at the time of the infusion visit.  
 
6.7 Week 24 Visit or Early Termination Visit 
Study drug should not be administered until after study visit procedures are complete. 
This visit will take place on Day 168 ± 7 days.  The following procedures will be performed for 
the regimen schedule: 
• ALSAQ-40 
• CNS Bulbar Function Scale 
• Collect urine, prior to start of infusion.  If urine cannot be collected prior to 
infusion, no urine should be collected at this visit 
• Collect safety labs including for HbA1c evaluation  
• Adjustment of dosing volume based on most recent weight measurement as 
necessary (only if participant is continuing in the Open Label Extension) 
• Administer infusion (Drug is only administered at this visit if the participant is 
continuing in the Open Label Extension.) 
• During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-
infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If a 
participant requires a 90-minute infusion duration due to receiving more than 2 
bags of study drug an additional vital signs measurement is to be completed 
approximately 30 minutes (± 10 mins) after the end of infusion.   
o Note: The first OLE infusion should occur in-clinic following completion of 
the Week 24 visit. The participant’s final infusion of the placebo-controlled 
treatment period should be scheduled and completed at least 4 days prior to 
the Week 24 Visit.  
6.8 Follow-Up Safety Call 
Participants will have a Follow-Up Safety Call 28±3 days after their last dose of study drug. 
Only those participants NOT continuing on in the Open Label Extension will have the Follow-
Up Safety Call following the end of their participation in the placebo-controlled portion of the 
trial.    


 
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The following procedures will be performed: 
• Assess and document AEs 
6.9 Process for Early Terminations  
Participants who early terminate from the study and do not complete the protocol per ITT will be 
asked to be seen for an in-person Early Termination Visit and complete a Follow-Up Safety Call.  
The in-person Early Termination Visit should be scheduled as soon as possible after a participant 
early terminates. If the participant early terminates during the placebo-controlled portion of the 
Regimen, all assessments that are collected at the Week 24 in-clinic visit should be conducted. 
The Follow-Up Safety Call should be completed approximately 28 days after the last dose of 
study drug.   
If the Early Termination Visit occurs approximately 28±3 days after the last dose of study drug, 
the information for the Follow-Up Safety Call can be collected during the Early Termination 
Visit, and a separate Follow-Up Safety Call does not need to be completed. If the in-person Early 
Termination Visit does not occur within 28±3 days of the last dose of study drug, the Follow-Up 
Safety Call should occur approximately 28 days after the last dose of study drug and the Early 
Termination Visit will be completed after the Follow-Up Safety Call.  
If a participant decides to discontinue study drug, but will complete the protocol per ITT, an in-
person Early Termination Visit and Follow-Up Safety Call is not necessary. 
6.10 Open Label Extension  
Participants who have completed the placebo-controlled portion of the trial on study drug, active 
drug or placebo, will be eligible to continue in the Open Label Extension (OLE) as outlined in 
the SOA. Participants will first be asked about their desire to continue in the OLE at the 
Regimen-Specific Screening Visit.  Participants will consent to participate in the OLE at the 
week 16 visit, after the site investigator reviews the OLE procedures in detail. If OLE consent is 
not obtained during the week 16 visit, OLE consent may be obtained at the week 24 visit.  
Modifications to Regimen Schedule  
Designated visits in the Schedule of Activities for the OLE (i.e. Week 8, Week 16, Week 28, and 
Week 40) may be conducted via telemedicine (or phone if telemedicine is not available) with 
remote services instead of in-person if needed to protect the safety of the participant due to a 
pandemic or other reason. If a planned in-clinic visit is conducted via telemedicine (or phone if 
telemedicine is not available) with remote services, only selected procedures will be performed. 
Instructions on how to document missed procedures are included in the MOP.  
In addition to the procedures in the Master Protocol that should be conducted during the phone or 
telemedicine and remote visits, the following regimen-specific procedures should be completed:  
• CNS Bulbar Function Scale  
• ALSAQ-40 
6.10.1 Weekly Investigational product Administration 
During the OLE all participants will receive a weekly infusion of investigational product 
(trehalose for 60 + 10 minutes, for participants who require more than 2 bags of SLS-005 the 


 
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infusion time is increased to 90 + 10 minutes) for the first 4 weeks of the study in clinic. 
Thereafter home infusions can be considered if the participant has had no adverse reaction to the 
infusion and all safety assessments are within acceptable limits. Concurrent assessments such as 
vital signs, reporting of any infusion related adverse events including Key Study Events (see 
section 10.3 of Master Protocol) and reporting of new concomitant medications should also be 
done weekly in conjunction with the investigational product infusion. 
6.10.2 Week 2 OLE Telephone Visit  
This visit will take place 14 ± 3 days the Week 24 Visit of the placebo-controlled portion of the 
trial. If the participant will receive their in-clinic infusion within the OLE Week 2 visit window, 
this visit may occur in-clinic at the time of the infusion visit.  
The following procedures should be performed: 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
 
6.10.2 Week 4 OLE Visit 
This visit will take place in-person 28 ± 7 days after the Week 24 Visit of the placebo-controlled 
portion of the trial. The following procedures will be performed: 
• 
Collect weight 
• 
Perform SVC 
• 
Administer ALSFRS-R questionnaire 
• 
Collect blood samples for Clinical Safety Labs and, for WOCBP, for pregnancy test if 
applicable 
• 
Collect urine, prior to start of infusion. If urine cannot be collected prior to infusion, no urine 
should be collected at this visit. 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
• 
Administer the C-SSRS Since Last Visit questionnaire  
• 
Adjustment of dosing volume based on most recent weight measurement as necessary 
• Administer infusion of investigational product in clinic after all study procedures have been 
completed, provided at least 4 days have passed since prior infusion. 
o During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-
infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If 
a participant requires a 90-minute infusion duration due to receiving more 
than 2 bags of study drug an additional vital signs measurement is to be 
completed approximately 30 minutes (± 10 mins) after the end of infusion.   


 
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6.10.3 Week 8 OLE Visit 
This visit will take place in-person at 56 ± 7 days after the Week 24 Visit of the placebo-
controlled portion of the trial. The following procedures will be performed: 
• 
Collect weight 
• 
Perform SVC 
• 
Administer ALSFRS-R questionnaire 
• 
CNS Bulbar Function Scale 
• 
Collect blood samples for Clinical Safety Labs and, for WOCBP, for pregnancy test if 
applicable 
• 
Collect urine, prior to start of infusion. If urine cannot be collected prior to infusion, no urine 
should be collected at this visit. 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
• 
Administer the C-SSRS Since Last Visit questionnaire  
• 
Adjustment of dosing volume based on most recent weight measurement as necessary 
• Administer infusion of investigational product in clinic after all study procedures have been 
completed, provided at least 4 days have passed since prior infusion. 
o During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-
infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If 
a participant requires a 90-minute infusion duration due to receiving more 
than 2 bags of study drug an additional vital signs measurement is to be 
completed approximately 30 minutes (± 10 mins) after the end of infusion.   
 
6.10.4  Week 12 OLE Telephone Visit 
This visit will take place in clinic or via phone at 84 ± 3 days after the Week 24 Visit of the 
placebo-controlled portion of the trial. If the participant will receive their in-clinic infusion 
within the OLE Week 12 visit window, this visit may occur in-clinic at the time of the infusion 
visit. 
The following procedures will be performed: 
• 
Administer ALSFRS-R questionnaire 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
 


 
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6.10.5  Week 16 OLE Visit 
Investigational product should not be administered until after study visit procedures are 
complete. 
This visit will take place in-person at 112 ± 7 days after the Week 24 Visit of the placebo-
controlled portion of the trial. The following procedures will be performed: 
• 
Collect weight 
• 
Perform SVC 
• 
Administer ALSFRS-R questionnaire 
• 
CNS Bulbar Function Scale 
• 
Collect blood samples for Clinical Safety Labs including Hb1Ac and, for WOCBP, for 
pregnancy test if applicable 
• 
Collect urine, prior to start of infusion. If urine cannot be collected prior to infusion, no urine 
should be collected at this visit. 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
• 
Administer the C-SSRS Since Last Visit questionnaire  
• 
Collect blood sample for biomarker analyses 
• 
Adjustment of dosing volume based on weight measurement as necessary 
• Administer infusion of investigational product in clinic after all study procedures have been 
completed, provided at least 4 days have passed since prior infusion. 
o During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate) are to be measured pre-
infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If 
a participant requires a 90-minute infusion duration due to receiving more 
than 2 bags of study drug an additional vital signs measurement is to be 
completed approximately 30 minutes (± 10 mins) after the end of infusion.   
 
6.10.6  Week 20 OLE Telephone Visit 
This visit will take place in clinic or via phone at 140 ± 3 days after the Week 24 Visit of the 
placebo-controlled portion of the trial. If the participant will receive their in-clinic infusion 
within the OLE Week 20 visit window, this visit may occur in-clinic at the time of the infusion 
visit. 
The following procedures will be performed: 
• 
Administer ALSFRS-R questionnaire 
• 
Review concomitant medications 


 
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• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
 
 
6.10.7  Week 24 OLE Telephone Visit 
This visit will take place in clinic or via phone at 168 ± 3 days after the Week 24 Visit of the 
placebo-controlled portion of the trial. If the participant will receive an in-clinic infusion within 
the OLE Week 24 visit window, this visit may occur in-clinic at the time of the infusion visit. 
The following procedures will be performed: 
• 
Administer ALSFRS-R questionnaire 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
 
6.10.8  Week 28 OLE Visit 
Investigational product should not be administered until after study visit procedures are 
complete. 
The Week 28 OLE Visit will take place in-person 196 ± 14 days after the Week 24 Visit of the 
placebo-controlled portion of the trial. The following procedures will be performed: 
• 
Collect weight 
• 
Perform SVC 
• 
Administer ALSFRS-R questionnaire 
• 
ALSAQ-40 
• 
CNS Bulbar Function Scale 
• 
Collect blood samples for Clinical Safety Labs including HbA1c and, for WOCBP, for 
pregnancy test if applicable 
• 
Collect urine, prior to start of infusion. If urine cannot be collected prior to infusion, no urine 
should be collected at this visit. 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
• 
Administer the C-SSRS Since Last Visit questionnaire  
• 
Collect blood sample for biomarker analyses 
• 
Adjustment of dosing volume based on most recent weight measurement as necessary 
• Administer infusion of investigational product in clinic after all study procedures have been 
completed, provided at least 4 days have passed since prior infusion. 
o During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-


 
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infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If 
a participant requires a 90-minute infusion duration due to receiving more 
than 2 bags of study drug an additional vital signs measurement is to be 
completed approximately 30 minutes (± 10 mins) after the end of infusion.   
 
6.10.9  Week 40 OLE Visit 
The Week 40 OLE Visit will take place in-person 280 ± 14 days after the Week 24 Visit of the 
placebo-controlled portion of the trial. The following procedures will be performed: 
• 
Collect weight 
• 
Perform SVC 
• 
Administer ALSFRS-R questionnaire 
• 
CNS Bulbar Function Scale 
• 
Collect blood samples for Clinical Safety Labs and, for WOCBP, for pregnancy test if 
applicable 
• 
Collect urine, prior to start of infusion. If urine cannot be collected prior to infusion, no urine 
should be collected at this visit. 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
• 
Administer the C-SSRS Since Last Visit questionnaire 
• 
Adjustment of dosing volume based on weight measurement as necessary 
• Administer infusion of investigational product in clinic after all study procedures have been 
completed, provided at least 4 days have passed since prior infusion. 
o During each infusion, vital signs associated with the infusion (systolic and 
diastolic pressure, respiratory rate, and heart rate)  are to be measured pre-
infusion, 30 minutes (± 5 mins) after the start of infusion (SOI), at the end of 
infusion (± 10 mins), and 90 minutes (± 10 mins) after the start of infusion. If 
a participant requires a 90-minute infusion duration due to receiving more 
than 2 bags of study drug an additional vital signs measurement is to be 
completed approximately 30 (± 10 mins) minutes after the end of infusion.   
 
6.10.10  Week 52 OLE Visit 
The Week 52 OLE Visit will take place in-person 364 ± 14 days after the Week 24 Visit of the 
placebo-controlled portion of the trial. The following procedures will be performed: 
• 
Collect vital signs including weight 
• 
Perform SVC 
• 
Administer ALSFRS-R questionnaire 


 
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• 
ALSAQ-40 
• 
CNS Bulbar Function Scale 
• 
Collect blood and urine samples for Clinical Safety Labs including HbA1c and, for WOCBP, 
for pregnancy test if applicable 
• 
Review concomitant medications 
• 
Assess and document AEs, including Key Study Events (see section 10.3 of Master Protocol) 
Administer the C-SSRS Since Last Visit questionnaire  
• 
Collect blood sample for biomarker analyses 
 
6.10.11 Follow-Up Safety Call 
Participants will have a Follow-Up Safety Call 28±3 days after their last dose of investigational 
product.  
The following procedures will be performed: 
• Assess and document AEs 
 
6.10.12 Process for Early Terminations from the OLE  
Participants who early terminate from the study be asked to be seen for an in-person Early 
Termination Visit and complete a Follow-Up Safety Call.  
The in-person Early Termination Visit should be scheduled as soon as possible after a participant 
early terminates. If the participant early terminates during the OLE period, all assessments that 
are collected at the OLE Week 52 in-clinic visit should be conducted. The Follow-Up Safety Call 
should be completed approximately 28 days after the last dose of investigational product.   
If the Early Termination Visit occurs approximately 28±3 days after the last dose of 
investigational product, the information for the Follow-Up Safety Call can be collected during 
the Early Termination Visit, and a separate Follow-Up Safety Call does not need to be 
completed. If the in-person Early Termination Visit does not occur within 28±3 days of the last 
dose of investigational product, the Follow-Up Safety Call should occur approximately 28 days 
after the last dose of investigational product and the Early Termination Visit will be completed 
after the Follow-Up Safety Call.  
 


 
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7 OUTCOME MEASURES AND ASSESSMENTS 
7.1  ALSAQ-40 
The Amyotrophic Lateral Sclerosis Assessment Questionnaire-40 (ALSAQ-40) is a participant 
self-report health status patient-reported outcome. The ALSAQ-40 consists of forty questions 
that are specifically used to measure the subjective well-being of participants with ALS and 
motor neuron disease. 
Participants will be handed the questionnaire and asked to write their answers themselves.  
Caregivers can also help, if needed. 
7.2  Center for Neurologic Study Bulbar Function Scale 
The Center for Neurologic Study Bulbar Function Scale (CNS-BFS) is a participant self-report 
scale that has been developed for use as an endpoint in clinical trials and as a clinical measure for 
evaluating and following ALS participants. The CNS-BFS consists of three domains 
(swallowing, speech, and salivation), which are assessed with a 21-question, self-report 
questionnaire. 
Participants will be handed the questionnaire and asked to write their answers themselves.  
Caregivers can also help, if needed. 


 
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8.0 BIOFLUID COLLECTION 
CSF Biomarker and serum PK samples will be collected as per the schedule of activities  
8.1 CSF Biomarker Collection 
The CSF for neurofilament light chain concentration and microtubule-associated protein 1A/-
light chain 3 (LC3-II) will be obtained from participants who consent to lumbar puncture.  Four 
cryovials containing 0.25 mL of CSF (for a total of 1.0 mL) will be provided from the Baseline 
and Week 16 Visits for analysis. Details regarding sample collection preparation and shipment 
will be provided in laboratory manual.    
8.2 Serum Pharmacokinetic Assessments 
Not all sites may have the capacity to collect PK for this regimen, therefore PK will only be 
collected on participants who are enrolled at a site capable of performing the PK collection 
procedures.  
The first thirty-six (36) participants randomized within the regimen to reach the Week 8 visit at 
sites participating in the PK collection will complete the PK collection procedure. Any participant 
not able to complete Week 8 PK will be replaced. 
Participants completing the PK collection will have blood collected for PK analysis at Baseline 
(Day 0) and Week 8 (Day 56) at the following time points: pre-dose and 1 hour ± 5 minutes, 2 
hours ± 5 minutes, and 4 hours ± 5 minutes post start of infusion (SOI). At each time point, 4mL 
of blood will be collected.  


 
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9 
REGIMEN-SPECIFIC STATISTICAL CONSIDERATIONS 
9.1  Deviations from the Default Master Protocol Trial Design 
The statistical design for this regimen will be in accordance with the default statistical design 
described in Appendix I of the Master Protocol with only one deviation. This regimen will not 
include interim analyses for early success.  
9.2  Regimen Specific Operating Characteristics  
Clinical trial simulation is used to quantify operating characteristics for this regimen (refer to the 
regimen SAP for further details).   
9.3  Sharing of Controls from other Regimens 
The primary analysis of this regimen will include sharing of all controls from the other 
regimens.  This is justified by the minor differences in inclusion/exclusion criteria of the RSA, 
such that there are no expected systematic differences in the primary endpoint between the 
controls across regimens.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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APPENDIX I: THE ALSAQ-40 
 
 
 
 
 
 
 
 


 
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APPENDIX II: THE BULBAR FUNCTION SCALE (CNS-BFS) 
BULBAR FUNCTION SCALE (CNS-BFS) 
SIALORRHEA 
Does 
Not 
Apply 
(1) 
Applies 
Rarely 
(2) 
   Applies 
Occasionally 
(3) 
Applies 
Frequently  
(4) 
Applies 
Most of 
the Time 
(5) 
 
1. Excessive saliva is a concern to 
me. 
 
 
 
 
 
2. I take medication to control 
drooling. 
 
 
 
 
 
3. Saliva causes me to gag or 
choke. 
 
 
 
 
 
4. Drooling causes me to be 
frustrated or embarrassed. 
 
 
 
 
 
5. In the morning I notice saliva on 
my pillow. 
 
 
 
 
 
6. My mouth needs to be dabbed to 
prevent drooling. 
 
 
 
 
 
7. My secretions are not 
manageable. 
 
 
 
 
 
  
TOTAL Sialorrhea Score: _____ 
SPEECH 
Does 
Not 
Apply 
(1) 
Applies 
Rarely 
(2) 
   Applies 
Occasionally 
(3) 
Applies 
Frequently  
(4) 
Applies 
Most of 
the Time 
(5) 
Unable to 
Communicate 
by Speaking 
(6) 
1. My speech is difficult to 
understand. 
 
 
 
 
 
 
2. To be understood I repeat myself. 
 
 
 
 
 
 
3. People who understand me tell 
other people what I said.  
 
 
 
 
 
 


 
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4. To communicate I write things 
down or use devices such as a 
computer. 
 
 
 
 
 
 
5. I am talking less because it takes 
so much effort to speak. 
 
 
 
 
 
 
6. My speech is slower than usual. 
 
 
 
 
 
 
7. It is hard for people to hear me. 
 
 
 
 
 
 
  
TOTAL Speech Score: _____ 
SWALLOWING 
Does 
Not 
Apply 
(1) 
Applies 
Rarely 
(2) 
   Applies 
Occasionally 
(3) 
Applies 
Frequently  
(4) 
Applies 
Most of 
the Time 
(5) 
  
    Feeding tube is in place 
1. Swallowing is a problem. 
 
 
 
 
 
2. Cutting my food makes it easier 
to chew and swallow. 
 
 
 
 
 
3. To get food down I have 
switched to a soft diet. 
 
 
 
 
 
4. After swallowing I gag or choke. 
 
 
 
 
 
5. It takes longer to eat. 
 
 
 
 
 
6. My weight is dropping because I 
can't eat normally. 
 
 
 
 
 
7. Food gets stuck in my throat. 
 
 
 
 
 
  
TOTAL Swallowing Score: _____ 
OVERALL SCORE: _____ 
 


 
HEALEY ALS Platform Trial  
Regimen-Specific Appendix E, Trehalose 
 
Version 2.0 12/03/2021  
CONFIDENTIAL 
Page 51 of 51 
REFERENCES 
1. Elbein AD, Pan YT, Pastuszak I, et al. New insights on trehalose: a multifunctional 
molecule. Glycobiology 2003;13(4):17R-27R. 
2. Emanuele E. Can Trehalose Prevent Neurodegeneration? Insights from Experimental 
Studies. Current Drug Targets 2014;15(5):1-7. 
3. Rusmini P, Cortese K, Crippa V, et al. Trehalose induces autophagy via lysosomal-mediated 
TFEB activation in models of motoneuron degeneration. Autophagy 2019;15(4):631-651. 
4. Tanaka M, Machida Y, Niu S, et al. Trehalose alleviates polyglutamine-mediated pathology 
in a mouse model of Huntington disease. Nature Medicine 2004;10(2):148-154. 
5. Davies JE, Sarkar S, Rubinsztein DC. Trehalose reduces aggregate formation and delays 
pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy. Human 
Molecular Genetics 2006;15(1):23-31. 
6. Mardones P, Rubinsztein DC, Hetz C. Mystery solved: Trehalose kickstarts autophagy by 
blocking glucose transport. Science Signaling 2016;9(416):fs2. 
7. Castillo K, Nassif M, Valenzuela V, et al. Trehalose delays the progression of amyotrophic 
lateral sclerosis by enhancing autophagy in motoneurons. Autophagy 2013;9(9):1308-1320.  
8. Zhang X, Chen S, Song L, et al. MTOR-independent, autophagic enhancer trehalose 
prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model 
of amyotrophic lateral sclerosis. Autophagy 2014;10(4):588-602. 
9. Li Y, Guo Y, Wang X, et al. Trehalose decreases mutant SOD1 expression and alleviates 
motor deficiency in early but not end-stage amyotrophic lateral sclerosis in a SOD1-G93A 
mouse model. Neuroscience 2015;298:12-25.  
10. DeBosch BJ, Heitmeier MR, Mayer AL, et al. Trehalose inhibits solute carrier 2A (SLC2A) 
proteins to induce autophagy and prevent hepatic steatosis. Science Signaling 
2016;9(416):ra21. 
 
 

SAP_001.pdf: 
HEALEY ALS Platform Trial - Regimen E Trehalose 
NCT05136885 
Document Date: 10 Oct 2023 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 1 of 37 
 
 
RGE REGIMEN-SPECIFIC STATISTICAL ANALYSIS PLAN (R-SAP) 
Master Protocol 
Platform Trial for the Treatment of Amyotrophic Lateral 
Sclerosis (ALS):  A perpetual multi-center, multi-regimen, 
clinical trial evaluating the safety and efficacy of 
investigational products for the treatment of ALS 
Regimen 
RGE: Trehalose 
Regimen Partner 
Seelos Therapeutics, Inc. 
Regulatory Sponsor 
Merit E. Cudkowicz, MD 
Master Protocol Version 
4.0, 31 Aug 2020 
RSA Version 
2.0, 03 Dec 2021 
Master SAP Version 
2.0, 01 Aug 2020 
R-SAP Version 
2.0, 10 Oct 2023 
 
SAP APPROVAL SIGNATURES 
 
 
Merit E. Cudkowicz, MD, MSc 
Principal Investigator and Regulatory Sponsor 
Date 
 
 
Sabrina Paganoni, MD, PhD 
Co-Principal Investigator 
Date 
 
 
Warren Wasiewski, MD 
Senior Neurological Consultant, Seelos Therapeutics, Inc. 
Date 
10-Oct-2023
11-Oct-2023
11-Oct-2023


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 2 of 37 
 
 
 
Ben Saville, PhD 
Senior Statistical Scientist, Berry Consultants 
Date 
 
 
Eric A. Macklin, PhD 
Study Biostatistician 
Date 
 
 
Suzanne Hendrix, PhD 
President & CEO, Pentara Corporation 
Date 
12-Oct-2023
10-Oct-2023
13-Oct-2023


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 3 of 37 
 
SAP REVISION HISTORY 
Version 
Date 
Description of Changes 
1.0 
13 Apr 2023 Initial version 
2.0 
10 Oct 2023 
Revision of Section 1.0 Governing Documents to add a reference 
to analyses specific to the open-label extension period. 
Revision of Section 2.4 Participant Flow to add a description of 
participation during the open-label extension period. 
Revision of Section 2.6 Treatment Allocation to add a description 
of treatment during the open-label extension period. 
Addition of Section 2.10 RGE Schedule of Activities (SoA) 
during the Open-label Extension Period with footnotes. 
Revision of Section 4.1.3 Exploratory Efficacy Endpoints to 
switch from ALSAQ-40 symptom index to ALSAQ-40 total 
score, to specify that time to clinical events is restricted to the 
placebo-controlled period, to add analyses of survival defined as 
freedom from death and PAV and freedom from death alone from 
baseline to the last participant's last visit in the placebo-controlled 
period, and to add long-term CAFS analyses of ALSFRS-R and 
SVC as functional measures and freedom from death and PAV or 
from death alone as survival components. 
Revision of Section 4.3 Safety Endpoints to add tabulations of 
adverse events and clinically significant ECG and lab 
abnormalities that occurred after crossover into the OLE period. 
Revision of Section 5.4 Biofluid Biomarkers of 
Neurodegeneration to specify a secondary analysis of serum NfL 
as an endpoint that excludes measured concentrations less than 
4 ng/mL if they are less than 2% of the maximum measured 
concentration for a given participant. 
Revision of Section 5.5 ALSAQ-40 to define calculation of the 
ALSAQ-40 total score and remove reference to an ALSAQ-40 
symptom index. 
Revision of Section 6.1 Analysis Sets to define the Efficacy 
Relyvrio Free (ERF) analysis set that excludes participants who 
used sodium phenylbutyrate/taurursodiol at baseline or during 
their follow-up in the placebo-controlled period. 
Revision of Section 6.2 Baseline Characteristics to switch from 
ALSAQ-40 symptom index to ALSAQ-40 total score. 


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Version 2.0, 10 Oct 2023 
 
Page 4 of 37 
 
Version 
Date 
Description of Changes 
2.0 
(continued) 
10 Oct 2023 
Revision of Section 6.3 Primary Efficacy Analysis and 
Supportive Analyses to specify that baseline log-transformed 
serum NfL will be handled the same as previously specified 
covariates as described in Section 2.2.5 of the MPRDR and to 
add the EPP and ERF analysis sets for additional sensitivity 
analysis. 
Revision of Section 6.5.2 Combined Assessment of Function and 
Survival to add the ERF analysis set as an additional sensitivity 
analysis and to add long-term CAFS analyses of ALSFRS-R and SVC 
as functional measures and freedom from death and PAV or from death 
alone as survival components. 
Revision of Section 6.5.3 Repeated-measures Model to switch 
from ALSAQ-40 symptom index to ALSAQ-40 total score and to 
add the ERF analysis set as an additional sensitivity analysis of 
ALSFRS-R total score and SVC. 
Revision of Section 6.5.4 Random-slopes Model to switch from 
ALSAQ-40 symptom index to ALSAQ-40 total score and to fix 
omission of baseline NfL in the list of covariates in the model 
specification and template SAS code. 
Revision of Section 6.7.3 Concomitant Medication Use to add 
time to event summaries of post-baseline initiation of edaravone, 
riluzole, or sodium phenylbutyrate/taurursodiol individually and 
in combination among the subset of participants who were not on 
a given drug or combination of drugs at baseline. 
Deletion of Section 6.5.10 Comparison to External Controls. 
 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 5 of 37 
 
ABBREVIATIONS 
ALD 
After Last Dose 
ALP 
Alkaline Phosphatase 
ALS 
Amyotrophic Lateral Sclerosis 
ALSAQ-40 
Amyotrophic Lateral Sclerosis Assessment Questionnaire, 40-item version 
ALSFRS-R 
Amyotrophic Lateral Sclerosis Functional Rating Scale, Revised 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
ATC 
WHODrug Anatomical, Therapeutic, and Chemical class 
BLQ 
Below the Limit of Quantitation 
BMI 
Body Mass Index 
C-SSRS 
Columbia Suicide Severity Rating Scale 
CAFS 
Combined Assessment of Function and Survival 
CBC 
Complete Blood Count 
CKD 
Chronic Kidney Disease 
COVID-19 
Coronavirus Disease 2019 
CNS-BFS 
Center for Neurologic Study Bulbar Function Scale 
CSF 
Cerebrospinal Fluid 
CTCAE 
Common Terminology Criteria for Adverse Events  
delta-FRS 
Pre-baseline Slope in ALSFRS-R  
DAP 
Data Analysis Plan 
DNA 
Deoxyribonucleic Acid 
DRR 
Disease Rate Ratio 
ECC 
Efficacy Concurrent Control 
ECM 
Efficacy Common Mode of Administration 
ECG 
Electrocardiography or Electrocardiogram 
eGFR 
Estimated Glomerular Filtration Rate 
ENCALS 
European Network for the Cure of ALS  
EPP 
Efficacy Per-protocol 
ERO 
Efficacy Regimen-only 
FAS 
Full Analysis Set 
FVC 
Forced Vital Capacity 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 6 of 37 
 
ABBREVIATIONS (continued) 
GLI 
Global Lung Initiative 
HbA1c 
Hemoglobin A1c 
hCG 
Human Chorionic Gonadotropin 
HHD 
Hand-held Dynamometry 
ICF 
Informed Consent Form 
ITT 
Intention-to-treat Principle 
IV 
Intravenous 
LC3-II 
Lipid Modified Form of Microtubule-associated Proteins 1A/1B Light Chain 3B  
M-SAP 
Master Statistical Analysis Plan 
MDRD 
Modification of Diet in Renal Disease 
MedDRA 
Medical Dictionary for Regulatory Activities 
MP 
Master Protocol 
MPRDR 
ALS Master Protocol Recommended Statistical Analysis, Design and 
Simulation Report 
NCI 
National Cancer Institute 
NEALS 
Northeast ALS 
NfL 
Neurofilament Light Chain 
OLE 
Open-label Extension 
PAV 
Permanent Assisted Ventilation 
PK 
Pharmacokinetics 
PRO-ACT 
Pooled Resource Open-Access ALS Clinical Trials 
RBC 
Red Blood Cell 
RDW 
RBC Distribution Width 
RGE 
Regimen E (trehalose) 
RSA 
Regimen-specific Appendix 
R-SAP 
Regimen-specific Statistical Analysis Plan 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SoA 
Schedule of Activities 
SOC 
MedDRA System Organ Class 
SOI 
Start of Infusion 
 


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Version 2.0, 10 Oct 2023 
 
Page 7 of 37 
 
ABBREVIATIONS (continued) 
SRO 
Safety Regimen-only 
STF 
Safety and Tolerability Full 
STN 
Safety and Tolerability Narrow 
SVC 
Slow Vital Capacity 
TBL 
Total Bilirubin 
TEAE 
Treatment-emergent Adverse Event 
TRICALS 
Treatment Research Initiative to Cure ALS 
TSH 
Thyroid Stimulating Hormone 
ULN 
Upper Limit of Normal 
WBC 
White Blood Cell 
WHODrug 
World Health Organization Drug Dictionary Enhanced 
 


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Version 2.0, 10 Oct 2023 
 
Page 8 of 37 
 
Table of Contents 
1. 
Governing Documents ...................................................................................................................... 10 
2. 
Study Design ..................................................................................................................................... 10 
2.1 
Overview ....................................................................................................................................................... 10 
2.2 
Study Objectives ........................................................................................................................................... 11 
2.3 
Study Population ........................................................................................................................................... 11 
2.4 
Participant Flow ............................................................................................................................................ 11 
2.5 
Regimen Allocation ....................................................................................................................................... 12 
2.6 
Treatment Allocation ..................................................................................................................................... 12 
2.7 
Treatment Administration ............................................................................................................................. 12 
2.8 
Allocation Concealment ................................................................................................................................ 12 
2.9 
RGE Schedule of Activities (SoA) during the Placebo-controlled Period .................................................... 12 
2.10 
RGE SoA during the Open-label Extension Period .................................................................................. 15 
3. 
General Considerations for Data Analysis ..................................................................................... 16 
3.1 
Statistical Software ........................................................................................................................................ 16 
3.2 
Summary Statistics ........................................................................................................................................ 16 
3.3 
Precision ........................................................................................................................................................ 16 
3.4 
Transformations ............................................................................................................................................ 17 
3.5 
Multiplicity Adjustments ............................................................................................................................... 17 
3.6 
Missing Data ................................................................................................................................................. 17 
4. 
Study Endpoints ............................................................................................................................... 17 
4.1 
Efficacy Endpoints ........................................................................................................................................ 17 
4.1.1 
Primary Efficacy Endpoint .................................................................................................................. 17 
4.1.2 
Key Secondary Efficacy Endpoints ..................................................................................................... 17 
4.1.3 
Exploratory Efficacy Endpoints ........................................................................................................... 18 
4.2 
Pharmacokinetic Endpoints ........................................................................................................................... 19 
4.3 
Safety Endpoints ........................................................................................................................................... 19 
5. 
Measurement Definitions ................................................................................................................. 19 
5.1 
ALSFRS-R .................................................................................................................................................... 19 
5.2 
SVC ............................................................................................................................................................... 19 
5.3 
HHD and Grip Strength ................................................................................................................................. 19 
5.4 
Biofluid Biomarkers of Neurodegeneration .................................................................................................. 20 
5.5 
ALSAQ-40 .................................................................................................................................................... 20 
5.6 
CNS-BFS ....................................................................................................................................................... 20 
5.7 
Survival ......................................................................................................................................................... 20 
5.8 
King's ALS Clinical Staging System ............................................................................................................. 21 
5.9 
Hospitalization and Other Clinical Events .................................................................................................... 21 
5.10 
TRICALS Risk Profile ............................................................................................................................. 22 
5.11 
Pharmacodynamic Biomarkers ................................................................................................................. 22 
5.12 
Clinical Safety Laboratory Tests .............................................................................................................. 22 
6. 
Statistical Methodology .................................................................................................................... 23 
6.1 
Analysis Sets ................................................................................................................................................. 23 
6.2 
Baseline Characteristics ................................................................................................................................ 24 
6.3 
Primary Efficacy Analysis and Supportive Analyses .................................................................................... 24 
6.4 
Interim Analysis ............................................................................................................................................ 25 
6.5 
Secondary Efficacy Analyses ........................................................................................................................ 25 
6.5.1 
Hierarchical Testing ............................................................................................................................. 25 
6.5.2 
Combined Assessment of Function and Survival ................................................................................ 26 
6.5.3 
Repeated-measures Model ................................................................................................................... 27 
6.5.4 
Random-slopes Model ......................................................................................................................... 29 
6.5.5 
Survival and Time to Clinical Events .................................................................................................. 30 
6.5.6 
HHD0 and HHD02 ............................................................................................................................... 31 
6.5.7 
Placebo Multiple Imputation................................................................................................................ 31 
6.5.8 
Inverse Probability Weights ................................................................................................................. 31 
6.5.9 
Principal Stratification ......................................................................................................................... 31 


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6.5.10 
Additional Sensitivity Analyses of Primary and Key Secondary Outcomes ....................................... 32 
6.5.11 
Subgroup Analyses .............................................................................................................................. 32 
6.5.12 
Comparison of Controls across Regimens ........................................................................................... 33 
6.5.13 
Pharmacokinetic Analyses ................................................................................................................... 33 
6.5.14 
Pharmacodynamic Biomarker Analyses .............................................................................................. 33 
6.6 
Safety Analyses ............................................................................................................................................. 33 
6.6.1 
Treatment-emergent Adverse Events ................................................................................................... 33 
6.6.2 
Safety Labs .......................................................................................................................................... 34 
6.6.3 
ECG Results......................................................................................................................................... 34 
6.6.4 
Vital Signs and Weight ........................................................................................................................ 34 
6.6.5 
Suicidality ............................................................................................................................................ 34 
6.7 
Other Analyses .............................................................................................................................................. 34 
6.7.1 
Participant Disposition ......................................................................................................................... 34 
6.7.2 
Study Drug Compliance and Tolerance ............................................................................................... 35 
6.7.3 
Concomitant Medication Use .............................................................................................................. 35 
6.7.4 
Medical History ................................................................................................................................... 35 
6.7.5 
Blindedness .......................................................................................................................................... 35 
6.7.6 
Impact of COVID-19 Pandemic .......................................................................................................... 35 
7. 
Validation .......................................................................................................................................... 36 
7.1 
Primary Efficacy Analysis ............................................................................................................................. 36 
7.2 
Secondary, Exploratory, and Safety Analyses ............................................................................................... 36 
8. 
References ......................................................................................................................................... 36 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 10 of 37 
 
1. Governing Documents 
This Regimen-specific Statistical Analysis Plan (R-SAP) for the trehalose regimen (RGE) 
specifies any modification from the default outcome measures, analysis samples, and planned 
analyses for the placebo-controlled period of the HEALEY ALS Platform Trial as specified in 
the Master SAP (M-SAP) and adds analyses specific to the open-label extension (OLE) period. 
The M-SAP and this R-SAP supplement the Master Protocol, the "ALS Master Protocol 
Recommended Statistical Analysis, Design and Simulation Report" (Appendix 1 to the Master 
Protocol), and the RGE Regimen-specific Appendix (RSA). Please refer to the Master Protocol 
and the RGE RSA for details on the rationale for the study design, eligibility criteria, conduct of 
the trial, clinical assessments and schedule of assessments, definitions and reporting of adverse 
events, data management conventions, and regulatory oversight and compliance procedures. The 
"ALS Master Protocol Recommended Statistical Analysis, Design and Simulation Report" 
(MPRDR) and any regimen-specific deviations described in the RGE RSA and this R-SAP are 
authoritative in defining the primary and interim analyses. In case of discrepancies between the 
RGE RSA and this R-SAP concerning use of shared placebos, this R-SAP is authoritative. In 
case of discrepancies between either SAP and the Master Protocol and the RGE RSA concerning 
matters of analysis other than the primary and interim analyses and use of shared placebos, the 
M-SAP and this R-SAP are authoritative. In case of discrepancies between the M-SAP and this 
R-SAP, this R-SAP is authoritative. On all matters not related to analysis, the Master Protocol 
and the RGE RSA are authoritative. The following table describes relationships among the 
relevant documents in adjudicating possible discrepancies with higher numbers indicating greater 
authority. 
Issues potentially requiring adjudication 
Master 
Protocol 
RGE  
RSA 
MPRDR 
M-SAP 
RGE  
R-SAP 
Use of shared placebos 
1 
4 
2 
3 
5 
Primary and interim analysis specifications not 
related to use of shared placebo 
1 
5 
4 
2 
3 
Statistical analysis specifications not related to use 
of shared placebo or primary and interim analyses 
1 
3 
2 
4 
5 
All matters not related to statistical analysis 
4 
5 
1 
2 
3 
2. Study Design 
2.1 
Overview 
The HEALEY ALS Platform Trial is a perpetual multi-center, multi-regimen clinical trial 
evaluating the safety and efficacy of investigational products for the treatment of ALS. RGE 
evaluates the safety and efficacy of trehalose administered weekly via intravenous (IV) infusion 
at a dosage of 0.75 g/kg vs. placebo. The RGE RSA describes the nature of the intervention and 
its mechanism of action, the mode and frequency of administration, additional eligibility criteria 
beyond those specified in the Master Protocol, additional enrollment procedures, and additions 
and modifications of safety and efficacy assessments relative to those outlined in the Master 
Protocol. 


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2.2 
Study Objectives 
Primary Efficacy Objective: 
• To evaluate the efficacy of trehalose injection, 90.5 mg/mL for IV infusion as compared 
to placebo on ALS disease progression. 
Secondary Efficacy Objectives: 
• To evaluate the effect of trehalose injection, 90.5 mg/mL for IV infusion on selected 
secondary measures of disease progression, including survival 
Safety Objectives: 
• To evaluate the safety of trehalose injection, 90.5 mg/mL for IV infusion for patients with 
ALS 
Exploratory Efficacy Objectives: 
• To evaluate the effect of trehalose injection, 90.5 mg/mL for IV infusion on selected 
biomarkers and endpoints. 
2.3 
Study Population 
In addition to eligibility criteria specified in the Master Protocol, participants in RGE must not 
have a current diagnosis of or be treated for diabetes mellitus, have a screening serum glucose 
level greater than or equal to 140 mg/dL, have previously been treated with IV trehalose or have 
a known hypersensitivity to trehalose, currently be taking oral trehalose, be unable to return to 
the clinic for weekly infusions prior to approval for home infusions, or have a screening body 
weight greater than 144 kg. Detailed criteria are specified in the Master Protocol and the RGE 
RSA. 
Participants will be recruited from approximately 60 centers located throughout the US that are 
part of the Northeast ALS (NEALS) Consortium. 
2.4 
Participant Flow 
Participants in RGE follow the consenting, Master screening, regimen assignment, regimen-
specific screening, randomization to active or placebo treatment, and follow-up procedures and 
timing described in the M-SAP for the placebo-controlled period. 
Participants in RGE who complete the placebo-controlled period may extend their participation 
into the OLE period. Consent to the OLE will be obtained prior to RGE screening and will 
typically be confirmed at the Week 16 visit. Participants determined eligible to enter the OLE 
will be dispensed OLE study drug at the Week 24 visit. This visit will serve as Day 0 for the 
OLE period. Note that references to "baseline" refer to the Baseline visit of the placebo-
controlled period. References to the start of participation in the OLE will refer to OLE Day 0. 
The duration and visit schedule of the OLE is not specified in the Master Platform protocol, but 
the guidance is to replicate the visit schedule of the RCT period over the first 24 weeks of the 
OLE, and the OLE period is typically extended to 52 weeks. Under that schedule, participants 
who are dispensed OLE study drug would be seen in clinic 4, 8, 16, 28, 40, and 52 weeks after 
OLE Day 0 and would complete telephone visits at 2, 12, 20, and 24 weeks after OLE Day 0 and 


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Page 12 of 37 
 
28 days after last dose of study drug or as otherwise specified in the applicable RSA. The OLE 
visit schedule and duration for a given regimen are specified in the applicable RSA. 
Detailed descriptions of study procedures and timing are specified in the Master Protocol and the 
RGE RSA. 
2.5 
Regimen Allocation 
Participants in RGE are those determined eligible for Master Protocol-level inclusion and 
exclusion criteria and randomly assigned to RGE, stratified by use of riluzole, edaravone, both, 
or neither at the time of screening for the Master Protocol. Details of regimen assignment are 
described in the Platform Trial Regimen Assignment Plan. 
2.6 
Treatment Allocation 
Participants in RGE are randomly allocated in a 3:1 ratio to weekly IV trehalose at a dosage of 
0.75 g/kg or placebo treatment based on a pre-specified permuted-block randomization schedule, 
stratified by use of riluzole, edaravone, both, or neither at the time of screening for the Master 
Protocol. All participants who enter the OLE period receive weekly IV trehalose at a dosage of 
0.75 g/kg starting at OLE Day 0. 
2.7 
Treatment Administration 
Trehalose and placebo are supplied as sterile fluids for IV administration. Each infusion bag of 
active study drug contains 300 mL of 90.5 mg/mL trehalose in aqueous solution. Each infusion 
bag of placebo contains 300 mL of US Pharmacopeia grade 0.9% sodium chloride in aqueous 
solution. 
The first infusion of study drug should be administered while in the office/clinic on the day of 
the Baseline Visit after all visit assessments are complete. 
Additional details of treatment administration are described in the RGE RSA. 
2.8 
Allocation Concealment 
Allocation concealment is the same as described in the M-SAP. Both active and placebo infusion 
bags are identical in appearance. 
2.9 
RGE Schedule of Activities (SoA) during the Placebo-controlled Period 
Activity 
MP 
Scrn1 
RGE 
Scrn1 
Base-
line20 
Week 
221 
Week 
4 
Week 
8  
Week 
1221 
Week 
1613 
Week 
2021 
Week 
24/ET 
Final 
Call10 
Clinic Clinic Clinic Phone Clinic Clinic Phone Clinic Phone Clinic Phone 
-42d to 
-1d15 
-41d to 
0d15 
Day  
0 
Day 
14 ±3 
Day 
28 ±7 
Day 
56 ±7 
Day 
84 ±3 
Day 
112 ±7 
Day 
140 ±3 
Day 
168 ±7 
28d ±3 
ALD 
Written Informed Consent2 
X 
X 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion Review 
X 
X3 
 
 
 
 
 
 
 
 
 
ALS & Medical History 
X 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
Physical Examination 
X 
 
 
 
 
 
 
 
 
 
 
Neurological Exam 
X 
 
 
 
 
 
 
 
 
 
 
Vital Signs4 
X 
 
X 
 
X 
X 
 
X 
 
X 
 
Vital Signs at Infusions5 
 
 
 
At weekly IV infusions Weeks 1 to 24 
 
 


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Page 13 of 37 
 
Activity 
MP 
Scrn1 
RGE 
Scrn1 
Base-
line20 
Week 
221 
Week 
4 
Week 
8  
Week 
1221 
Week 
1613 
Week 
2021 
Week 
24/ET 
Final 
Call10 
Clinic Clinic Clinic Phone Clinic Clinic Phone Clinic Phone Clinic Phone 
-42d to 
-1d15 
-41d to 
0d15 
Day  
0 
Day 
14 ±3 
Day 
28 ±7 
Day 
56 ±7 
Day 
84 ±3 
Day 
112 ±7 
Day 
140 ±3 
Day 
168 ±7 
28d ±3 
ALD 
Slow Vital Capacity 
X14 
 
X 
 
 
X 
 
X 
 
X 
 
Muscle Strength Assessment  
 
 
X 
 
 
X 
 
X 
 
X 
 
ALSFRS-R 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
 
ALSAQ-40 
 
 
X 
 
 
 
 
 
 
X 
 
CNS Bulbar Function Scale 
 
 
X 
 
 
X 
 
X 
 
X 
 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
X 
 
Clinical Safety Labs6, 17 
X 
 
X 
 
X 
X 
 
X 
 
X 
 
Biomarker Blood Collection 
 
 
X 
 
 
X 
 
X 
 
X 
 
Biomarker Urine Collection17 
 
 
X 
 
 
X 
 
X 
 
X 
 
PK Blood Collection14 
 
 
X 
 
 
X 
 
 
 
 
 
DNA Collection8 (optional) 
 
 
X 
 
 
 
 
 
 
 
 
CSF Collection (optional) 
 
 
X 
 
 
 
 
X12 
 
 
 
Concomitant Medication Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Adverse Event Review7 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical Review at Infusions 
 
 
 
At weekly IV infusions Weeks 1 to 24 
 
 
Suicidality C-SSRS 
 
 
X 
 
X 
X 
 
X 
 
X 
 
Assignment to Regimen 
X 
 
 
 
 
 
 
 
 
 
 
Randomization within Regimen 
 
 
X 
 
 
 
 
 
 
 
 
Adjust Dose as Needed20 
 
 
 
 
X 
X 
 
X 
 
X19 
 
Administer/Dispense Study Drug 
 
 
X9 
At weekly IV infusions Weeks 1 to 24 
X10 
 
Drug Accountability/Compliance 
 
 
 
At weekly IV infusions Weeks 1 to 2422 
X 
 
Exit Questionnaire 
 
 
 
 
 
 
 
 
 
X 
 
Vital Status Determination11 
 
 
 
 
 
 
 
 
 
X 
 
Abbreviations: ALD = after last dose, ALS = amyotrophic lateral sclerosis, ALSAQ-40 = ALS 
Assessment Questionnaire, ALSFRS-R = ALS Functional Rating Scale Revised, CSF = 
cerebrospinal fluid, C-SSRS = Columbia-Suicide Severity Rating Scale, d = day, DNA = 
deoxyribonucleic acid, ECG = electrocardiogram, ET = early termination, IV = intravenous, MP 
= Master Protocol, PK = pharmacokinetic, RGE = the trehalose regimen, Scrn = Screening Visit. 
1 Master Protocol Screening procedures must be completed within 42 days to 1 day prior to the 
Baseline Visit. The Regimen-Specific Screening Visit and Baseline Visit should be combined, if 
possible.   
2 During the Master Protocol Screening Visit, participants will be consented via the Platform 
Trial informed consent form (ICF). After a participant is randomized to a regimen, participants 
will be consented a second time via the regimen-specific ICF.  
3 At the Regimen Specific Screening Visit, participants will have regimen-specific eligibility 
criteria assessed. 
4 Vital signs include weight, systolic and diastolic blood pressure, respiratory rate, heart rate, and 
temperature. Height measured at Master Protocol Screening Visit only. If an infusion will occur 
at the completion of the visit, vital signs should be collected within 30 minutes of the start of 
infusion to serve as the pre-infusion vital signs collection. 


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5 During each infusion, vital signs associated with the infusion (systolic and diastolic blood 
pressure, respiratory rate, and heart rate) are to be measured pre-infusion, 30 minutes (± 5 mins) 
after the start of infusion, 60 minutes after the start of infusion (± 10 mins), and 90 minutes (± 10 
mins) after the start of infusion. If a participant requires a 90-minute infusion duration due to 
receiving more than 2 bags of study drug, an additional vital signs measurement is to be 
completed approximately 30 minutes (± 10 mins) after the end of the 90-minute infusion. 
6 Clinical safety labs include hematology (CBC with differential), complete chemistry panel, 
thyroid function, and urinalysis. Hemoglobin A1C (HbA1c) will be included in labs at baseline, 
week 16, and week 24. Serum pregnancy testing will occur in women of child-bearing potential 
at the Master Protocol Screening Visit and as necessary during the study. Pregnancy testing is 
only repeated as applicable if there is a concern for pregnancy. 
7 Adverse events that occur after signing the Master Protocol consent form will be recorded. 
8 The DNA sample can be collected after the Baseline Visit if a baseline sample is not obtained 
or the sample is not usable. 
9 Administer first dose of study drug only after Baseline Visit procedures are completed. 
10 Participants will only have a Follow-Up Safety Call at this time if they do not continue into the 
OLE. Participants who continue into OLE will have a Follow-Up Safety Call after their last dose 
of investigational product during the OLE period. 
11 Vital status, defined as a determination of date of death or death equivalent or date last known 
alive, will be determined for each randomized participant at the end of the placebo-controlled 
portion of their follow-up (generally the Week 24 visit, as indicated). If at that time the 
participant is alive, his or her vital status should be determined again at the time of the last 
patient last visit (LPLV) of the placebo-controlled portion of a given regimen. We may also 
ascertain vital status at later time points by using publicly available data sources as described in 
section 8.15 of the Master Protocol. 
12 If the CSF collection is unable to be performed for logistical reasons, such as scheduling, at 
the Week 16 Visit, it may be performed at the Week 24 Visit. 
13 Visit may be conducted via phone or telemedicine with remote services instead of in-person if 
this is needed to protect the safety of the participant due to a pandemic or other reason. 
14 If required due to pandemic-related restrictions, Forced Vital Capacity (FVC) performed by a 
Pulmonary Function Laboratory evaluator or with a study-approved home spirometer, or 
sustained phonation using a study approved method may be used for eligibility (Master Protocol 
Screening ONLY). 
15 Master Protocol Screening and Regimen Specific Screening visit windows are relative to 
Baseline (Day 0). 
16 Participants completing the PK collection will have blood collected for PK analysis at Baseline 
(Day 0) and Week 8 (Day 56) at the following time points: pre-dose and 1 hour ± 5 minutes, 2 
hours ± 5 minutes, and 4 hours ± 5 minutes post start of infusion. At each time point, 4 mL of 
blood will be collected.  
17 All urine samples must be collected prior to IP administration. 
19 Dose is adjusted only for those participants continuing into the OLE.  


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20 The weight collected during each in-clinic visit will be used to calculate the participant’s dose. 
The participant will remain on a stable dose until the next in-clinic visit (Weeks 4, 8, 16) when 
weight is collected, at which point the dose may be adjusted if the participant’s weight has 
increased or decreased by 2 kg. Additional information on dose adjustments is included in 
Section 5.6 Dosing Changes of the RGE RSA. 
21 If the participant receives an in-clinic infusion within the visit window, this visit may be 
conducted while the participant is in-clinic for their weekly infusion. 
22 Drug accountability will not be done at phone visits. Compliance will be automatically 
calculated in the EDC.  
2.10 RGE SoA during the Open-label Extension Period 
Activity 
Week 
28 
Week 
4 
Week 
87 
Week 
128 
Week 
167 
Week 
208 
Week 
248 
Week 
287 
Week 
407 
Week 
52/ET6
, 7 
Follow
-Up 
Safety 
Call4, 6 
Clinic 
during 
Infusio
n 
Clinic 
Clinic 
Phone 
Clinic 
Phone 
Phone 
Clinic 
Clinic 
Clinic 
Phone 
 
Day 28 
±7 
Day 56 
±7 
Day 84 
±3 
Day 
112 ±7 
Day 
140 ±3 
Day16
8 ±3 
Day 
196 ± 
14 
Day 
280 ± 
14 
Day 
364 ± 
14 
28d ±3 
ALD 
Vital Signs1 
 
X 
X 
 
X 
 
 
X 
X 
X 
 
Vital signs associated with infusion9 
Weekly in conjunction with investigational product administration from OLE week 
1 through OLE week 52 
 
 
Slow Vital Capacity 
 
X 
X 
 
X 
 
 
X 
X 
X 
 
ALSFRS-R 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
ALSAQ-40 
 
 
 
 
 
 
 
X 
 
X 
 
CNS Bulbar Function Scale 
 
 
X 
 
X 
 
 
X 
X 
X 
 
Clinical Safety Labs2 
 
X 
X 
 
X 
 
 
X 
X 
X 
 
Biomarker Blood Collection 
 
 
 
 
X 
 
 
X 
 
X 
 
Biomarker Urine Collection 
 
 
 
 
X 
 
 
X 
 
X 
 
Concomitant Medication Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Adverse Event Review3 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Document changes in health, 
medications, and infusion-related AEs 
Weekly in conjunction with Investigational Product administration from OLE week 1 
through OLE week 52 
 
Suicidality C-SSRS 
 
X 
X 
 
X 
 
 
X 
X 
X 
 
Adjust Dose as Needed10 
 
X 
X 
 
X 
 
 
X 
X 
 
 
Administer/Dispense Study Drug 
Weekly IV infusion from OLE week 1 through week 52 
 
Drug Accountability/Compliance 
Weekly throughout study in conjunction with drug administration11 
 
Abbreviations: AE = adverse event, ALD = after last dose, ALSAQ-40 = ALS Assessment 
Questionnaire, ALSFRS-R = ALS Functional Rating Scale Revised, C-SSRS = Columbia-
Suicide Severity Rating Scale, d = day, ET = early termination. 
1 Vital signs include weight, systolic and diastolic pressure, respiratory rate, heart rate and 
temperature. Height in cm measured at Master Protocol Screening Visit only. 
2 Clinical safety labs include hematology (CBC with differential), complete chemistry panel, 
thyroid function and urinalysis. HbA1c will be added to the safety labs at weeks 16, 28 and 52.  


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Serum pregnancy testing will occur in women of child-bearing potential at the Master Protocol 
Screening Visit and as necessary during the study. Pregnancy testing is only repeated as 
applicable if there is a concern for pregnancy. 
3 Adverse events that occur after signing the master protocol consent form will be recorded.  
4 Participants who continue into OLE will have a Follow-Up Safety Call (as described in the 
body of the RGE RSA) after their last dose of investigational product during the OLE period. 
5 The duration of the OLE is 52 weeks. 
6 Participants who continue into the OLE and then withdraw consent or early terminate will be 
asked to complete an Early Termination Visit and Follow-Up Safety Call as described in the 
RGE RSA.  
7 Visit may be conducted via phone or telemedicine with remote services instead of in-person if 
this is needed to protect the safety of the participant due to a pandemic or other reason.  
8 If the participant receives an in-clinic infusion within the visit window, this visit may be 
conducted while the participant is in clinic for their weekly infusion.   
9 During each infusion, vital signs associated with the infusion (systolic and diastolic pressure, 
respiratory rate, and heart rate) are to be measured pre-infusion, 30 minutes (± 5 mins) after the 
start of infusion (SOI), 60 minutes after the start of infusion (± 10 mins), and 90 minutes (± 10 
mins) after the start of infusion. If a participant requires a 90-minute infusion duration due to 
receiving more than 2 bags of study drug an additional vital signs measurement is to be 
completed approximately 30 minutes (± 10 mins) after the end of the 90-minute infusion. 
10 The weight collected during each in clinic visit will be used to calculate the participant’s dose. 
The participant will remain on a stable dose until the next in clinic visit (Weeks 4, 8, 16, 28, 40) 
when weight be collected, at which point the dose may be adjusted if the participant’s weight has 
increased or decreased by 2 kg. Additional information on dose adjustments is included in 
Section 5.6 Dosing Changes of the RGE RSA. 
11 Drug accountability will not be done at phone visits. Compliance will be automatically 
calculated in the EDC. 
3. General Considerations for Data Analysis 
3.1 
Statistical Software 
Statistical software use for analyses is the same as described in the M-SAP. 
3.2 
Summary Statistics 
Data summaries are the same as described in the M-SAP. 
Summary statistics for time-to-event endpoints will include the percentage of subjects having the 
event and relevant percentiles of the survival curve depending on frequency of a given event. 
3.3 
Precision 
Precision of reported results is the same as described in the M-SAP. 


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3.4 
Transformations 
Data transformations are the same as described in the M-SAP. 
3.5 
Multiplicity Adjustments 
The primary endpoint will be tested first, and key secondary endpoints will be tested next in a 
fixed sequence with overall family-wise error rate controlled by a closed-testing procedure as 
indicated in Section 6.5.1 Hierarchical Testing below. Exploratory endpoints and safety 
evaluations will report unadjusted p-values. 
3.6 
Missing Data 
Handling of missing data is the same as described in the M-SAP except as indicated below. 
Missing baseline covariates will be mean imputed. Any transformed covariates will be 
transformed prior to mean imputation. Clinic-based assessments that are missing due to COVID-
19 restrictions or disruptions are considered missing completely at random. 
Handling of missing data depends upon the type and pattern of missing data (monotone, non-
monotone, deaths), the endpoint analyzed (ALSFRS-R, SVC, HHD, etc.), the method of analysis 
(continuous, time to event), and the reason for missing data (related to lack of efficacy, related to 
tolerability, administrative reasons unrelated to lack of efficacy or tolerability, and unknown 
reasons). Deaths are considered a special type of missing data. 
For analysis of continuous endpoints, the method described in the M-SAP for handling missing 
data will be followed. 
For time-to-event analyses that depend on scheduled assessments, participants lacking any post-
baseline assessment not due to death or disease progression will be censored at baseline and 
functionally omitted from the analysis. If data are missing not due to death, no imputation will be 
made for the primary analysis. As a sensitivity analysis, multiple imputations as described in 
Section 6.5.10 Placebo Multiple Imputation below will be used. All subjects who die or reach a 
death equivalent before reaching the pre-specified time-to-event will be considered as having the 
unfavorable event being analyzed at the time of death or death equivalent. 
4. Study Endpoints 
4.1 
Efficacy Endpoints 
4.1.1 Primary Efficacy Endpoint 
The primary efficacy endpoint is the same as described in the M-SAP. 
4.1.2 Key Secondary Efficacy Endpoints 
The following key secondary efficacy endpoints will be evaluated hierarchically (see Section 
6.5.1): 
• Combined analysis of function and survival (CAFS) using ALSFRS-R total score as the 
functional measure, freedom from death and permanent assisted ventilation (PAV) as the 
survival component, evaluated to the Week 24 time point, and adjusting the rank-sums for 
baseline covariates of pre-baseline slope of ALSFRS-R, time since symptom onset, use of 
edaravone, use of riluzole, and log-transformed serum NfL level,  


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• Change from baseline to Week 24 in ALS functioning as assessed using ALSFRS-R total 
score change from baseline,  
• Change from baseline to Week 24 in respiratory function as assessed by slow vital capacity 
(SVC) and measured as percent predicted using Global Lung Initiative (GLI) normal values,  
• Change from baseline to Week 24 in upper limb muscle strength as assessed isometrically 
using hand-held dynamometry (HHD) and grip strength and measured as average percent 
change from baseline among maneuvers with non-zero strength at baseline,  
• Change from baseline to Week 24 in lower limb muscle strength as assessed isometrically 
using HHD and measured as average percent change from baseline among maneuvers with 
non-zero strength at baseline, and  
• Survival defined as freedom from death and PAV from baseline to Week 24. 
4.1.3 Exploratory Efficacy Endpoints 
The following exploratory efficacy endpoints will be evaluated: 
• Change from baseline to Week 24 in ALSFRS-R domain scores: bulbar, fine motor, gross 
motor, respiratory, and fine and gross motor combined,  
• Change from baseline to Week 24 in combined upper and lower limb muscle strength as 
assessed isometrically using HHD and grip strength and measured as average percent change 
from baseline among muscles or muscle groups with non-zero strength at baseline,  
• Time to loss of measurable strength of the first (HHD0) and second (HHD02) muscle or 
muscle group with non-zero strength at baseline,  
• Change from baseline to Week 24 in biofluid biomarkers of neurodegeneration, 
neuromuscular degeneration, and autophagy: serum creatinine, serum and cerebrospinal fluid 
(CSF) neurofilament light chain (NfL), and the lipid modified form of microtubule-
associated proteins 1A/1B light chain 3B (LC3-II) in CSF,  
• Change in patient-reported outcomes: ALSAQ-40 physical mobility, independence in 
activities of daily living, eating and drinking, communications, and emotional reactions 
domain scores and ALSAQ-40 total score, and CNS-BFS total score,  
• Time to clinical events during the placebo-controlled period: first hospitalization due to a 
serious adverse event (SAE), first hospitalization due to an ALS-related SAE, first use of 
assisted ventilation, first placement of a feeding tube, first time reaching King's stage 4a or 
4b, and first instance of any of the following events: hospitalization for an SAE, feeding tube 
placement, tracheostomy, initiation of PAV, or death, 
• Survival defined as freedom from death and PAV and freedom from death alone from 
baseline to the last participant's last visit in the placebo-controlled period (RCT LPLV) 
analyzed in the ERO analysis set, 
• CAFS analyses of ALSFRS-R total score and SVC as functional measures, freedom from 
death and PAV or from death alone as survival components, evaluated to the RCT LPLV 
time point, and adjusting the rank-sums for baseline covariates of pre-baseline slope of 
ALSFRS-R, time since symptom onset, use of edaravone, use of riluzole, and log-
transformed serum NfL level analyzed in the ERO analysis set. 


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4.2 
Pharmacokinetic Endpoints 
The following pharmacokinetic endpoints will be evaluated: 
• Serum trehalose concentration at the following time points: pre-dose, 60 (±5) min after start 
of infusion (SOI), 120 (±5) min after SOI, and 240 (±5) min after SOI. 
4.3 
Safety Endpoints 
The safety endpoints are the same as those described in the M-SAP with the addition of 
tabulations of adverse events and clinically significant ECG and lab abnormalities that occurred 
after crossover into the OLE period. 
5. Measurement Definitions 
5.1 
ALSFRS-R 
The definitions of ALSFRS-R scores are the same as described in the M-SAP with the 
clarification that scores will be considered missing in cases of item nonresponse. Pre-baseline 
slope in ALSFRS-R (delta-FRS) is defined as 48 minus the baseline ALSFRS-R total score then 
divided by the number of months from onset of symptomatic weakness to the Baseline Visit. The 
number of months will be calculated as the difference in days from onset of symptomatic 
weakness to the Baseline Visit multiplied by 12 / 365.25. The date of onset of symptomatic 
weakness will be imputed as the fifteenth day of a month if not specified more precisely. 
5.2 
SVC 
The derivation of SVC percent-predicted of normal is the same as described in the M-SAP with 
age calculated as number of days from date of birth to the date of a given SVC assessment 
divided by 365.25 and with the following correspondence between self-identified race and race 
defined by the GLI classification: 
Self-identified Race 
GLI-defined Race 
American Indian or Alaska Native 
Mixed/Other 
Asian 
South East Asian 
Black or African American 
African American 
Native Hawaiian or Other Pacific Islander 
Mixed/Other 
White 
Caucasian 
Unknown 
Caucasian 
Not reported 
Caucasian 
More than one race indicated 
Mixed/Other 
5.3 
HHD and Grip Strength 
The derivation of HHD upper and lower extremity scores and HHD0 are the same as described in 
the M-SAP with the revision that HHD0 is a composite endpoint with death or death equivalent, 
whichever occurs first. 
A second HHD time-to-event endpoint is defined as the time from the Baseline Visit to the 
second post-baseline occurrence of a muscle with a strength recording of 0 among those muscles 


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that were non-zero at baseline or time to death or death equivalent, whichever occurs first 
(HHD02).  
Time at risk for HHD0 and HHD02 will be censored at the last date that an HHD assessment was 
performed up to the end of the Week 24 Visit window. 
5.4 
Biofluid Biomarkers of Neurodegeneration 
Blood biomarkers of neurodegeneration and neuromuscular degeneration will be assayed. These 
will include serum creatinine and serum and CSF neurofilament light chain (NfL). Serum 
creatinine will be assayed by the kinetic Jaffe method (test 001370, Labcorp, Burlington, NC). 
NfL will be assayed by single-molecule array (Simoa; Quanterix, Billerica, MA). Levels of 
serum and CSF NfL that are reported to be below the limit of quantitation will be imputed at the 
limit of quantitation. All serum NfL results that pass Quanterix quality control criteria will be 
analyzed for primary analyses using serum NfL, both as a baseline covariate and as an endpoint. 
A secondary analysis of serum NfL as an endpoint will exclude measured concentrations less 
than 4 ng/mL if they are less than 2% of the maximum measured concentration for a given 
participant. Levels of serum and CSF NfL will be log-transformed in all analyses. 
5.5 
ALSAQ-40 
The description of the ALSAQ-40 instrument and item-level scores are the same as described in 
the M-SAP. Each of the five domains will be scored as the mean of all domain-specific items 
multiplied by 25 (range 0 to 100). An overall total score will be calculated as the mean of all 40 
items multiplied by 25 (range 0 to 100; Jenkinson et al. 2003). Domain scores and the total score 
will be missing if more than 20% of the applicable items are missing; otherwise, item non-
response will be mean-imputed from other completed items from the same assessment. Higher 
scores indicate worse quality of life. 
5.6 
CNS-BFS 
The definition of CNS-BFS total score is the same as described in the M-SAP. 
5.7 
Survival 
The primary definition of survival time is the same as described in the M-SAP with the 
clarifications that PAV is defined as more than 22 hours per day of noninvasive or invasive 
mechanical ventilation for more than seven consecutive days, that time at risk should not be 
censored at date of consent withdrawal, and that time at risk should be censored at date last 
known alive. The date of PAV initiation, where applicable, will be imputed as the fifteenth day 
of a month if not specified more precisely. Any participant on PAV at baseline will be censored 
at baseline. A secondary survival endpoint of death alone, independent of any death equivalent 
(including a death equivalent that occurs prior to baseline), is also defined and will be censored 
at the Week 24 Visit if completed or at the earlier of the end of the Week 24 Visit window or 
date last known alive if a Week 24 visit is not completed. An exploratory survival endpoint of 
death or death-equivalent will censor at the earlier of last known PAV-free or last known alive. 
Time at risk for the composite endpoint of death or death equivalent and time at risk for the 
endpoint of death alone will be measured from each participant's Baseline Visit. Time at risk will 
be censored at two time points: (1) at the Week 24 Visit as defined in the M-SAP, and (2) at a 


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subsequent assessment of death or death equivalent scheduled approximately at the end of 
placebo-controlled follow-up of the last RGE participant. The primary analysis of survival will 
evaluate PAV-free survival to the Week 24 Visit time point. 
5.8 
King's ALS Clinical Staging System 
The King's ALS Clinical Staging System (Roche et al. 2012) is a 4-level ordinal scale with the 
first three levels indicating the number (1, 2, or 3) of distinct central nervous system regions 
(bulbar, upper limb, and lower limb) with neuromuscular dysfunction and levels 4a and 4b 
indicating nutritional or respiratory failure secondary to ALS, respectively. 
Participants will be classified to King's stage 1, 2, 3, 4a, or 4b based on scores from ALSFRS-R 
assessments according to a published derivation (Balendra et al. 2014). Bulbar involvement is 
defined as a score less than 4 on any of the ALSFRS-R questions in the bulbar domain (questions 
1, 2, and 3). Upper limb involvement is defined as a score less than 4 on either of the ALSFRS-R 
questions related to hand function (questions 4 and 5A). Lower limb involvement is defined as a 
score less than 4 on the ALSFRS-R question about walking (question 8). Nutritional failure is 
defined as responding that the participant uses gastrostomy for greater than 50% of their 
nutrition. Respiratory failure is defined as a score of 0 on the ALSFRS-R question addressing 
dyspnea (question 10 or R-1) or a score less than 4 on the ALSFRS-R question about use of 
mechanical ventilation (question 12 or R-3). Participants without evidence by ALSFRS-R scores 
of involvement of any of the three central nervous system regions will be scored as King's stage 
1 due to their confirmed diagnosis with ALS. Participant may meet criteria for both King's stage 
4a and 4b. 
5.9 
Hospitalization and Other Clinical Events 
Times to the following clinically relevant events are defined: 
• Time to first hospitalization due to a serious adverse event (SAE),  
• Time to first hospitalization due to an ALS-related SAE,  
• Time to first use of assisted ventilation,  
• Time to first placement of a feeding tube,  
• Time to King's stage 4a or 4b, and  
• Time to first instance of hospitalization for an SAE, feeding tube placement, tracheostomy, 
initiation of PAV, or death. 
Time at risk for each event will be measured from each participant's Baseline Visit. Time to first 
hospitalization excludes hospitalizations for elective procedures. ALS-related SAEs are those 
indicated as related to ALS disease progression by the site investigator. Participants who are 
already using assisted ventilation or have a feeding tube at the time of the Baseline Visit will be 
excluded from analysis of those endpoints. Death or death equivalent will be considered an 
outcome for each of the events listed, forming a composite endpoint. 
Time at risk for these events will be censored at the Week 24 Visit, if completed, the date of 
consent withdrawal, if withdrawn, or the last date at which the status of each endpoint is known 
prior to the end of the Week 24 Visit window for participants lost to follow-up. Time to King's 
stage 4a or 4b is interval censored between ALSFRS-R assessments. 


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5.10 TRICALS Risk Profile 
The Treatment Research Initiative to Cure ALS (TRICALS) Risk Profile is based on the the 
European Network for the Cure of ALS (ENCALS) survival prediction model (Westeneng et al. 
2018). The TRICALS score will be calculated at baseline as follows: 
Profile = 0.474ൣ(𝑉𝑉𝑉𝑉100
⁄
)−1 + (𝑉𝑉𝑉𝑉100
⁄
)−1/2൧−2.376ൣ(𝐷𝐷𝐷𝐷10
⁄
)−1/2 + 𝑙𝑙𝑙𝑙(𝐷𝐷𝐷𝐷10
⁄
)൧
−1.839(𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑+ 0.1)−1/2 −0.264(𝐴𝐴𝐴𝐴𝐴𝐴100
⁄
) + 0.271 Bulbar
+ 0.238 Definite + 0.415 FTD 
where VC is vital capacity in units of percent-predicted using GLI norms as defined in Section 
5.2, DD is diagnostic delay calculated as (date of diagnosis – date of symptom onset) / 365.25 x 
12, dFRS is pre-baseline slope of ALSFRS-R total score as defined in Section 5.1, AAO is age at 
symptom onset calculated as (date of symptom onset – date of birth) / 365.25, Bulbar is an 
indicator of initial site of onset in the bulbar region, Definite is an indicator for classification as 
definite ALS by revised El Escorial criteria, and FTD is an indicator for frontotemporal 
dementia. While participants are not evaluated for frontotemporal dementia at baseline, given 
that presence of dementia defined broadly is an exclusion criterion at screening, TRICALS 
scores will be calculated assuming that participants do not have frontotemporal dementia. 
5.11 Pharmacodynamic Biomarkers 
CSF LC3-II levels in samples collected at Baseline and Weeks 16 or 24 will be assayed. The 
method of analysis of CSF LC3-II will be specified once known. Levels of CSF LC3-II that are 
reported to be below the limit of quantitation will be imputed at the limit of quantitation. Levels 
of CSF LC3-II will be log-transformed in all analyses. 
5.12 Clinical Safety Laboratory Tests 
Clinical safety labs include hematology, blood chemistry panel, liver function tests, thyroid 
function, urinalysis, and pregnancy testing in women of childbearing potential as specified in 
Section 9.1.2 Clinical Safety Laboratory Tests of the Master Protocol: 
• Hematology: hematocrit, hemoglobin, platelet count, red blood cell (RBC) count, mean 
corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin 
concentration, RBC distribution width (RDW), RBC morphology, white blood cell (WBC) 
count, and counts and percentages of basophils, eosinophils, lymphocytes, monocytes, and 
neutrophils; 
• Blood chemistry panel: bicarbonate, chloride, potassium, sodium, calcium, magnesium, 
phosphate, blood urea nitrogen, creatinine, estimated glomerular filtration rate (eGFR) 
calculated using the Modification of Diet in Renal Disease (MDRD) four-variable equation, 
creatinine clearance calculated using the Cockcroft-Gault equation, glucose, and hemoglobin 
A1c; 
• Liver function tests: alanine aminotransferase (ALT [SGPT]), aspartate aminotransferase 
(AST [SGOT]), alkaline phosphatase (ALP), albumin, total protein, total bilirubin (TBL); 
• Thyroid function tests: thyroid-stimulating hormone (TSH); 
• Urinalysis: clarity, color, specific gravity, pH, microalbumin, protein, glucose, ketones, 
bilirubin, urobilinogen, nitrite, leukocyte esterase, and blood; and  


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• Pregnancy: qualitative and quantitative serum human chorionic gonadotropin (hCG). 
Clinical safety labs will also include derived measures of potential drug-induced liver injury 
(DILI), including those that potentially meet the Hy’s law criteria, as distinct safety lab 
outcomes. 
Three potential DILI criteria will be defined: 
• ALT or AST >3x ULN with TBL >1.5x ULN 
• AST or ALT >3x ULN with TBL >2x ULN 
• AST or ALT >3x ULN with TBL >2x ULN and ALP <2x ULN (potential Hy’s Law cases) 
where ULN is upper level of normal and all levels are measured on the same day. 
6. Statistical Methodology 
6.1 
Analysis Sets 
The ITT analysis set is henceforth referred to as the Full Analysis Set (FAS) and defined as 
follows: 
• 
Full Analysis Set (FAS): Participants who were randomized within RGE plus placebo 
participants from regimens A-D who had an opportunity to complete placebo-controlled 
follow-up, classified according to their randomized treatment assignment. Participants who 
discontinue study drug but remain in the study will be included in the FAS. Observations 
completed after regimen data lock are excluded. Participants determined to not meet ALS 
diagnostic criteria are excluded. 
The definition of the STF analysis set is revised as follows: 
• 
Safety Full (STF) Set: Participants who initiated treatment within RGE plus placebo 
participants from regimens A-D who are not known to be ineligible for RGE and who 
initiated treatment in their respective regimen, classified according to the treatment they 
actually received. Observations made after premature permanent discontinuation of study 
drug are included in this sample, should such participants remain on study. Observations 
completed after regimen data lock are excluded. 
An analysis set restricting shared placebo participants to those regimens in which study drug is 
administered by the same route as RGE is defined as follows: 
• Efficacy Common Mode of Administration (ECM) Set: The subset of participants in the FAS 
who are in regimens in which study drug is administered by the same route as RGE. 
The definitions of the ECC, ERO, STN, and SRO analysis sets are the same as described in the 
M-SAP with reference to the ITT analysis set now referencing the FAS analysis set. 
• 
Efficacy Per-protocol (EPP) Set: The subset of participants in the FAS analysis set who 
initiated study treatment and who were not involved in protocol deviations related to 
eligibility, treatment, follow-up, assessment, or use of other disease-modifying treatments for 
ALS, classified according to the treatment they actually received. Final specification of 
participants included vs. excluded from the EPP analysis set will be defined prior to data 
lock. Inclusion or exclusion from the EPP analysis set of any participant for whom treatment 


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assignment was unblinded prior to data lock will be governed by the prespecified criteria 
above. If a participant's data are truncated for inclusion in the EPP analysis set due to a time-
dependent event (e.g., non-adherence to protocol-specified dosing, initiation of a prohibited 
medication, initiation of edaravone, riluzole, or sodium phenylbutyrate/taurursodiol), clinical 
events observed up to 28 days after the censoring event will be included in the EPP analysis 
set. Data from placebo participants shared from other regimens will not be truncated due to 
non-adherence to protocol-specified dosing. 
An analysis set that excludes participants who used sodium phenylbutyrate/taurursodiol during 
their follow-up for the placebo-controlled period is defined as follows: 
• Efficacy Relyvrio Free (ERF) Set: The subset of participants in the ERO who were not using 
sodium phenylbutyrate/taurursodiol at baseline and did not start using sodium 
phenylbutyrate/taurursodiol after baseline and prior to completion of their Week 24 visit or 
death, earlier termination, withdrawal, or loss to follow-up prior to the end of the Week 24 
visit window if they did not complete a Week 24 visit. 
Applicable analysis sets (FAS, ECC, EPP, and STF) will include shared placebo participants 
from regimens A, B, C, and D. As RGE is the only regimen among the contributing regimens 
that is administered as an IV infusion, the ERO and ECM analysis sets are synonymous and only 
the ERO analysis set will be referenced. Similarly, the SRO and STN analysis sets are 
synonymous and only the SRO analysis set will be referenced. 
6.2 
Baseline Characteristics 
The baseline characteristics summarized for participants randomized within RGE are the same as 
those specified in the M-SAP with the addition of ALSAQ-40 total and domain scores, CNS-
BFS total score, King's stage, TRICALS score, weight, body mass index (BMI), serum creatinine 
concentration, serum NfL concentration, and baseline use of sodium phenylbutyrate/taurursodiol. 
6.3 
Primary Efficacy Analysis and Supportive Analyses 
The primary analysis for RGE is a Bayesian shared-parameter, repeated-measures model of 
ALSFRS-R that accounts for loss of follow-up due to mortality. Details of the model, including 
documentation of operating characteristics under a range of scenarios, are provided in the 
MPRDR with the addition of baseline log-transformed serum NfL as a covariate. Baseline log-
transformed serum NfL will be handled the same as previously specified covariates with respect 
to standardization, imputation of missing values, model for covariate effect, and prior 
distributions as described in Section 2.2.5 of the MPRDR. The Bayesian shared-parameter, 
repeated-measures model will be applied to the FAS analysis set as the primary analysis and to 
the ECC, ERO, EPP, and ERF analysis sets as sensitivity analyses. 
The estimand of the primary analysis is the relative rate of disease progression (the "disease rate 
ratio" or DRR) of active treatment relative to placebo in the FAS population under an assumption 
that active treatment slows mean time to death or death equivalent by the same proportion as 
treatment slows the mean rate of functional progression as measured by change in ALSFRS-R 
total score over time. The estimand is defined by the following attributes: 
• Treatment: weekly IV infusion of trehalose at a dosage of 0.75 g/kg vs. placebo. 
• Population: FAS population as defined in Section 6.1. 


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• Variables: time to death or death equivalent and rate of change in ALSFRS-R total score 
from baseline to the Week 24 Visit. 
• Intercurrent event 1: treatment discontinuation due to death or death equivalent: no ALSFRS-
R data from participants who reach the death or death equivalent endpoint are included in the 
analysis, handled via mortality component in model, composite variable strategy approach. 
• Intercurrent event 2: treatment discontinuation not due to death or death equivalent: handled 
via treatment policy approach, all data will be used including data collected during the 
placebo-controlled period after treatment discontinuation regardless of concomitant 
medication, for those participants who have not been censored due to mortality. Missing data 
post-treatment will not be imputed, handled via missing at random assumption. 
• Population-level summary: mean ratio of hazard or progression rate of active treatment 
relative to placebo. 
6.4 
Interim Analysis 
RGE will be considered for early stopping for futility according to the interim analysis schedule 
and definition specified in the MPRDR. RGE will not be stopped early for success. 
6.5 
Secondary Efficacy Analyses 
6.5.1 Hierarchical Testing 
Primary inference for secondary efficacy endpoints will be based on analysis of the FAS. The 
primary CAFS analysis is described in Section 6.5.2. Primary estimates and inference for 24-
week change of continuous endpoints will be by a mixed model repeated-measures analysis (see 
Section 6.5.3 below). Primary estimates and inference for time-to-event endpoints will be by 
Kaplan-Meier product-limit estimates and log-rank test (see Section 6.5.5 below). The sequence 
for testing secondary efficacy endpoints is the following: 
1. CAFS using ALSFRS-R total score as the functional measure, freedom from death and 
PAV as the survival component, evaluated to the Week 24 time point, and adjusting the 
rank-sums for baseline covariates of pre-baseline slope of ALSFRS-R, time since 
symptom onset, use of edaravone, use of riluzole, and log-transformed serum NfL level,  
2. Change from baseline to Week 24 in ALS functioning as assessed using ALSFRS-R total 
score change from baseline in the FAS,  
3. Change from baseline to Week 24 in respiratory function as assessed by slow vital 
capacity (SVC) and measured as percent predicted using Global Lung Initiative normal 
values in the FAS,  
4. Change from baseline to Week 24 in upper limb muscle strength as assessed 
isometrically using HHD and grip strength and measured as average percent change from 
baseline among maneuvers with non-zero strength at baseline in the FAS,  
5. Change from baseline to Week 24 in lower limb muscle strength as assessed 
isometrically using HHD and measured as average percent change from baseline among 
maneuvers with non-zero strength at baseline in the FAS, and  


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6. Survival defined as freedom from death and permanent assisted ventilation (PAV) from 
baseline to Week 24 in the FAS. 
If the primary analysis indicates a significant slowing in disease progression, then each 
secondary efficacy endpoint in succession would be declared significant in the specified 
sequence using a comparison-wise criterion of two-tailed p < 0.05. After the first failure to 
declare significance, no endpoints lower in the hierarchy can be significant. This sequential 
closed-testing procedure controls the overall type 1 error rate at 5%. Nominal comparison-wise 
p-values for secondary efficacy endpoints will also be reported. 
6.5.2 Combined Assessment of Function and Survival 
The first key secondary analysis for RGE is a Combined Assessment of Function and Survival 
(CAFS). CAFS is a joint-rank test of a composite endpoint combining function as measured by 
change in ALSFRS-R total scores and survival as measured by time to death or death equivalent. 
CAFS will be applied to the FAS as the principal CAFS analysis with the addition of baseline 
log-transformed serum NfL level as a covariate (see MPRDR for more details) and to the ECC, 
ERO, and ERF analysis sets as sensitivity analyses. 
The estimand is the stochastic probability that a randomly selected active participant will rank 
higher than a randomly selected placebo participant based on time to death or death equivalent or 
change from baseline in ALSFRS-R in the FAS. The estimand is defined by the following 
attributes: 
• Treatment: weekly IV infusion of trehalose at a dosage of 0.75 g/kg vs. placebo. 
• Population: FAS as defined in Section 6.1. 
• Variables: the rank-sum score for each participant derived from comparing each study 
participant’s outcome to all others in the analysis set in a series of pairwise comparisons, 
based on first on survival up to Week 24 and second on absolute change from baseline in 
ALSFRS-R total score. 
• Intercurrent event 1: treatment discontinuation due to death or death equivalent: no ALSFRS-
R data from participants who reach the death or death equivalent endpoint are included in the 
analysis, handled via mortality component in model, composite variable strategy approach. 
• Intercurrent event 2: treatment discontinuation not due to death or death equivalent: handled 
via a treatment policy approach, all data will be used including data collected during the 
placebo-controlled period after treatment discontinuation regardless of concomitant 
medication, for those participants who have not been censored due to mortality. Pairs of 
participants will be ranked at the maximum follow-up time at which both participants have 
an observation during the placebo-controlled period. Missing data post-treatment will not be 
imputed and will be handled via a missing at random assumption. 
• Population-level summary: the ANCOVA derived least-square mean rank-sum score for each 
treatment group, the LS mean estimated difference in rank-sum scores (adjusted for 
covariates: time since symptom onset, delta-FRS, baseline riluzole use, baseline edaravone 
use, and baseline log-transformed serum NfL) as well as the corresponding 2-sided p-value 
will be presented. A Gehan-Wilcoxon rank sum test will be performed for the FAS as a 
sensitivity analysis to the primary analysis. 


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Inference from this analysis is supportive of inference from the Bayesian shared-parameter, 
repeated-measures model described in MPRDR. 
Including the analysis above, all combinations of the following elements will be tested in 
separate joint-rank analyses: 
1. Survival endpoint: (a) death or death-equivalent, and (b) death alone. 
2. Function endpoint: (a) ALSFRS-R total score, and (b) SVC percent-predicted. 
3. Analysis: (a) Wilcoxon rank-sum test by t-distribution approximation, and (b) adjusted mixed 
model or linear regression controlling for time since ALS symptom onset, delta-FRS, baseline 
log-transformed serum NfL level, use of riluzole at baseline, and use of edaravone at baseline, 
and including a random effect of regimen in all analyses that include at least three regimens or a 
fixed effect of regimen otherwise. 
Additional CAFS analyses of long-term change in function and survival will be performed using 
ALSFRS-R total score and SVC percent-predicted as functional measures, freedom from death 
and PAV and freedom from death alone as survival components, both evaluated to the RCT 
LPLV time point and adjusting the rank-sums for baseline covariates of pre-baseline slope of 
ALSFRS-R, time since symptom onset, use of edaravone, use of riluzole, and log-transformed 
serum NfL level analyzed in the ERO analysis set. 
6.5.3 Repeated-measures Model 
The specification of the repeated-measures linear mixed model and the primary linear contrast 
for estimating differences in 24-week change from baseline in a given continuous efficacy 
endpoint (ALSFRS-R total and domain scores, SVC, HHD upper extremity, lower extremity, and 
global average percentages, serum creatinine, serum NfL, ALSAQ-40 total and domain scores, 
and CNS-BFS total score) are revised from those specified in the M-SAP to include a main effect 
of treatment and its interaction with visit as additional covariates and to add the ERF analysis set 
for analysis of ALSFRS-R total score and SVC. 
The model will include fixed terms for discrete visit, treatment group, treatment group × visit 
interaction, centered time since symptom onset and centered time since symptom onset × visit 
interaction, centered delta-FRS and centered delta-FRS × visit interaction, centered baseline 
riluzole use and centered baseline riluzole × visit interaction, centered baseline edaravone use 
and centered baseline edaravone × visit interaction, and centered baseline log-transformed serum 
NfL and centered baseline log-transformed serum NfL × visit interaction. The following 
equations describe the model with regimen random effects: 
𝑌𝑌𝑖𝑖𝑖𝑖= 𝑎𝑎𝑘𝑘(𝑖𝑖) + 𝛾𝛾1𝑡𝑡𝑖𝑖+ 𝛾𝛾2,𝑗𝑗𝑣𝑣𝑗𝑗+ 𝜸𝜸𝟑𝟑
′ 𝒛𝒛𝒊𝒊+ 𝛾𝛾4,𝑗𝑗 𝑡𝑡𝑖𝑖𝑣𝑣𝑗𝑗+ 𝜸𝜸𝟓𝟓,𝒋𝒋
′ 𝒛𝒛𝑖𝑖 𝑣𝑣𝑗𝑗+ 𝜖𝜖𝑖𝑖𝑖𝑖 
𝑎𝑎𝑘𝑘 ~ 𝑁𝑁(0, 𝜎𝜎𝑟𝑟
2), 𝝐𝝐𝒊𝒊∙ ~ 𝑁𝑁(𝟎𝟎, 𝑹𝑹), Cov൫𝑏𝑏𝑘𝑘(𝑖𝑖), 𝜖𝜖𝑖𝑖𝑖𝑖൯= 0 
(eqn. 1)
where Yij is a given efficacy endpoint measured in participant i at visit j, ak(i) is a random 
intercept for regimen k to which participant i was assigned, vj is an indicator variable for visit j, zi 
is the vector of covariates (centered time since onset, centered delta-FRS, centered baseline 
riluzole use, centered baseline edaravone use, and centered baseline log-transformed serum NfL) 
for participant i, ti is an indicator variable for treatment t to which participant i was assigned, γ1, 
γ2,j, γ3, γ4,j, and γ5,j are estimated parameters and vectors of parameters for the fixed effects, and 
ϵij is the residual for participant i at visit j. The regimen-specific random effects are normally 


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distributed with mean 0 and variance σ2r. The vector of residuals for a given participant are 
normally distributed with mean 0 and an unstructured covariance matrix R. The regimen-specific 
random effect for a given participant and residuals for that participant are uncorrelated. 
The following SAS code specifies the model: 
proc mixed data=xxx method=reml; 
  class regimen id trtrnd visit; 
  model Value = trtrnd|visit  
                sx2bl|visit dFRS|visit rlz|visit edv|visit NfL|visit  
                / solution cl; 
  random intercept / subject=regimen type=un; 
  repeated visit / subject=id type=un; 
where id is a participant study identifier, trtrnd is the randomly assigned treatment group, 
visit is the visit identifier, Value is value of the efficacy endpoint being tested for a given 
participant at a given visit, sx2bl is years since ALS symptom onset, dFRS is pre-baseline slope , 
rlz is an indicator of riluzole use at baseline, edv is an indicator of edaravone use at baseline, 
and NfL is baseline log-transformed serum NfL level. Covariates will be centered at their sample 
means. The primary estimate will be the treatment-dependent difference in change from baseline 
to the Week 24 Visit. The estimate and its 95% Wald confidence bounds will be obtained by a 
linear contrast of adjusted means. The following SAS code specifies the linear contrast for an 
endpoint measured every 8 weeks and assuming that the sort order for treatment group has the 
active group last and visits are sorted chronologically: 
  estimate "3|Act vs Plb|dWk 24" trtrnd*visit 1 0 0 -1  -1 0 0 1 / cl; 
A significant difference in 24-week change from baseline in the direction of greater 
improvement or less worsening among participants randomized to active treatment would 
support inference of benefit from active treatment for the efficacy endpoint being tested. 
The estimand estimated by the primary linear contrast of the repeated-measures linear mixed 
model is the mean difference in 24-week change from baseline of a given continuous efficacy 
endpoint in the active treatment group relative to the placebo group in the FAS. The estimand is 
defined by the following attributes: 
• Treatment: weekly IV infusion of trehalose at a dosage of 0.75 g/kg vs. placebo. 
• Population: FAS as defined in Section 6.1. 
• Variables: absolute change in endpoint from baseline to the Week 24 Visit. 
• Intercurrent event: treatment discontinuation: handled via a treatment policy approach, all 
data will be used including data collected during the placebo-controlled period after 
treatment discontinuation. Missing data post-treatment, including data missing due to death, 
will not be imputed, and will be handled via a missing at random assumption. 
• Population-level summary: difference in least-square means of active treatment relative to 
placebo. 
Inference from these analyses is supportive of inference from the Bayesian shared-parameter, 
repeated-measures model. 


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6.5.4 Random-slopes Model 
The specification of the random-slopes linear mixed model and the primary linear contrast for 
estimating differences in mean rate of progression in a given continuous efficacy endpoint 
(ALSFRS-R total and domain scores, SVC, HHD upper extremity, lower extremity, and global 
average percentages, serum creatinine, serum NfL, ALSAQ-40 total and domain scores, and 
CNS-BFS total score) are revised from those specified in the M-SAP to include a main effect of 
treatment and its interactions with study month as additional covariates and to specify that study 
months are calculated as the difference in days from the Baseline Visit to the date of assessment 
of a given endpoint multiplied by 12 / 365.25. 
The model will include fixed terms for month since the Baseline Visit, treatment group, 
treatment group × month interaction, centered years since ALS symptom onset and centered 
years since ALS symptom onset × month interaction, centered delta-FRS and centered delta-FRS 
× month interaction, centered baseline riluzole use and centered baseline riluzole use × month 
interaction, centered baseline edaravone use and centered baseline edaravone use × month 
interaction, and centered baseline log-transformed serum NfL and centered baseline log-
transformed serum NfL × month interaction. The following equations describe the model with 
regimen random effects: 
𝑌𝑌𝑖𝑖𝑖𝑖= 𝛾𝛾1 + 𝑎𝑎𝑘𝑘(𝑖𝑖)
0
+ 𝑏𝑏𝑖𝑖
0 + 𝛾𝛾2𝑡𝑡𝑖𝑖+ 𝜸𝜸𝟑𝟑
′ 𝒛𝒛𝒊𝒊
+ ൫𝛾𝛾4 + 𝑎𝑎𝑘𝑘(𝑖𝑖)
1
+ 𝑏𝑏𝑖𝑖
1 + 𝛾𝛾5𝑡𝑡𝑖𝑖+ 𝜸𝜸𝟔𝟔
′ 𝒛𝒛𝒊𝒊൯ 𝑚𝑚𝑖𝑖𝑖𝑖+ 𝜖𝜖𝑖𝑖𝑖𝑖 
{𝑎𝑎𝑘𝑘
0, 𝑎𝑎𝑘𝑘
1} ~ 𝑁𝑁(𝟎𝟎, 𝚺𝚺𝒓𝒓), {𝑏𝑏𝑘𝑘
0, 𝑏𝑏𝑘𝑘
1} ~ 𝑁𝑁൫𝟎𝟎, 𝚺𝚺𝒑𝒑൯, 𝜖𝜖𝑖𝑖𝑖𝑖 ~ 𝑁𝑁(0, 𝜎𝜎𝜖𝜖
2) 
Cov(𝒂𝒂𝑘𝑘, 𝒃𝒃𝑘𝑘) = 𝟎𝟎, Cov(𝒂𝒂𝑘𝑘, 𝝐𝝐𝑖𝑖∙) = 𝟎𝟎, and Cov(𝒃𝒃𝑘𝑘, 𝝐𝝐𝑖𝑖∙) = 𝟎𝟎 
(eqn. 2)
where Yij is a given efficacy endpoint measured in participant i at visit j, a0k(i) and a1k(i) are 
random intercept and slope for regimen k to which participant i was assigned, b0i and b1i are 
random intercept and slope for participant i, zi is the vector of covariates (centered time since 
onset, centered delta-FRS, centered baseline riluzole use, centered baseline edaravone use, and 
centered baseline log-transformed serum NfL) for participant i, mij is the time from baseline to 
observation j for participant i in months calculated as days x 12 / 365.25, ti is an indicator 
variable for treatment t to which participant i was assigned, γ1, γ2, γ3, γ4, γ5, and γ6 are estimated 
parameters and vectors of parameters for the fixed effects, and ϵij is the residual for observation j 
for participant i. The regimen-specific random effects are normally distributed with mean 0 and 
unstructured covariance matrix Σr. The participant-specific random effects are normally 
distributed with mean 0 and unstructured covariance matrix Σp. The residuals for a given 
participant are normally distributed with mean 0 and variance σ2ϵ. The regimen-specific random 
effects, participant-specific random effects, and residuals are uncorrelated. 
The following SAS code specifies the model: 
proc mixed data=xxx method=reml; 
  class regimen id trtrnd; 
  model Value = trtrnd|month  
                sx2bl|month dFRS|month rlz|month edv|month NfL|visit  
                / solution cl; 
  random intercept month / subject=regimen type=un; 
  random intercept month / subject=id type=un; 


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where month is time in months from the Baseline Visit (assuming 12 months in an average of 
365.25 days per year) and other fields are the same as identified above in Section 6.5.3. 
Between-within denominator degrees of freedom will be used for endpoints with only two 
observations per participant; otherwise, denominator degrees of freedom will be calculated using 
containment rules. The primary estimand will be the treatment-dependent difference in slopes. 
The estimate and its 95% Wald confidence bounds will be obtained by a linear contrast of 
adjusted means. The following SAS code specifies the linear contrast assuming that the sort 
order for treatment group has the active group last: 
  estimate "3|Act vs Plb|Slope (/mn)" month 0 trtrnd*month -1 1 / cl; 
A significant difference in slopes in the direction of greater improvement or less worsening 
among participants randomized to active treatment would support inference of benefit from 
active treatment for the efficacy endpoint being tested. 
The estimand estimated by the primary linear contrast of the random-slopes linear mixed model 
is the difference in mean rate of progression of a given continuous efficacy endpoint in the active 
treatment group relative to the placebo group in the FAS. The estimand is defined by the 
following attributes: 
Treatment: weekly IV infusion of trehalose at a dosage of 0.75 g/kg vs. placebo. 
Population: FAS as defined in Section 6.1. 
Variables: mean rate of change in endpoint from baseline to the Week 24 Visit. 
Intercurrent event: treatment discontinuation: handled via a treatment policy approach, all 
data will be used including data collected during the placebo-controlled period after 
treatment discontinuation. Missing data post-treatment will not be imputed and will be 
handled via a missing at random assumption. 
Population-level summary: difference in conditional mean slopes of active treatment relative 
to placebo. 
Inference from this analysis is supportive of inference from the Bayesian shared-parameter, 
repeated-measures model. 
6.5.5 Survival and Time to Clinical Events 
Survival and time to hospitalizations and clinical events will be analyzed in the FAS, ECC, ERO, 
STF, and SRO analysis sets. Survival analyses that include follow-up beyond the placebo-
controlled period will be analyzed in the ERO analysis set. The summaries and analyses of time 
to death or death equivalent are the same as specified in the M-SAP with the addition of baseline 
age, baseline log-transformed serum NfL level, and baseline TRICALS score as covariates in 
adjusted models, and with the addition that the endpoints of time to death independent of 
occurrence of death equivalents and time to each of the hospitalization and clinical events will be 
separately analyzed using the same models. Analysis of time to King's stage 4a or 4b will 
accommodate interval censoring between ALSFRS-R assessments and will be stratified by 
baseline King's stage. 


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6.5.6 HHD0 and HHD02 
Analyses of HHD0 are the same as specified in the M-SAP with the addition of parallel analyses 
of HHD02, with baseline serum NfL level as an additional covariate, and with the clarification 
that time to zero strength for both analyses is interval censored between HHD assessments. 
6.5.7 Placebo Multiple Imputation 
Placebo multiple imputation analyses are the same as specified in the M-SAP with the addition 
of baseline serum NfL level as a covariate and will be applied to the mixed model repeated-
measures analyses of ALSFRS-R total score and SVC in the FAS. 
The following SAS code specifies the imputation for an endpoint measured every 8 weeks: 
proc mi data=work.outcomes seed=xxx nimpute=50 out=work.out_mi01  
        minimum=. . . . . . x x x x maximum=. . . . . . y y y y  
        minmaxiter=1000; 
  class rlz edv trtrnd; 
  fcs reg(Wk08 = sx2bl dFRS rlz edv NfL Wk00); 
  fcs reg(Wk16 = sx2bl dFRS rlz edv NfL Wk00 Wk08); 
  fcs reg(Wk24 = sx2bl dFRS rlz edv NfL Wk00 Wk08 Wk16); 
  mnar model( Wk08 Wk16 Wk24 / modelobs=(trtrnd="0")); 
  var sx2bl dFRS rlz edv NfL Wk00 Wk08 Wk16 Wk24; 
run; 
where Wk00, Wk08, Wk16, and Wk24 are the values of a given efficacy endpoint at the Baseline, 
Week 8, Week 16, and Week 24 Visits, respectively, trtrnd has a value of zero (0) for 
participants randomized to placebo, and x and y take appropriate values to specify the range of a 
given outcome measure (i.e., 0 and 48 for ALSFRS-R total score; 0 and . [missing value 
indicator] for SVC). 
Inference from these analyses is supportive of inference from the Bayesian shared-parameter, 
repeated-measures model for the primary endpoint and SVC in the FAS. 
6.5.8 Inverse Probability Weights 
To reduce bias from possible treatment-dependent loss to follow-up due to death, intolerance, or 
other causes and possible treatment-dependent initiation of other disease-modifying treatments 
for ALS, the effect of treatment on ALSFRS-R and SVC will be estimated in the mixed model 
repeated-measures analysis using visit-specific stable inverse probability weights (Avagyan et al. 
2021) in the FAS and ERO analysis sets with censoring of observations collected after any post-
baseline initiation of edaravone, riluzole, or sodium phenylbutyrate/taurursodiol. Weights will be 
calculated as the inverse of the predicted probability of loss to follow-up or initiation of other 
disease-modifying treatments, winsorized to the 5th and 95th percentiles. Probability of loss to 
follow-up or initiation other disease-modifying treatments for ALS will be predicted from a 
conditional random forests model (Hothorn et al. 2006) using all baseline characteristics 
specified in Section 6.2 Baseline Characteristics. 
6.5.9 Principal Stratification 
To reduce bias from possible treatment-dependent loss to follow-up due to death, intolerance, or 
other causes and possible treatment-dependent initiation of other disease-modifying treatments 
for ALS, the effect of treatment on ALSFRS-R and SVC will be estimated using the potential 


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outcome framework and principal stratification (Rubin 2006). We will estimate the visit-specific 
profile of average causal effects in the treatment- and visit-specific principal strata always 
survivors (i.e., the participants who would have lived to a given visit under both the active and 
placebo treatment exposures) and never initiators (i.e., the participants who would have not 
initiated another disease-modifying treatment for ALS prior to a given visit under both the active 
and placebo treatment exposure) in the FAS and ERO analysis sets. Time to loss to follow-up or 
initiation other disease-modifying treatments for ALS will be predicted from a conditional 
survival random forests model (Hothorn et al. 2006) using all baseline characteristics specified in 
Section 6.2 Baseline Characteristics. 
6.5.10 Additional Sensitivity Analyses of Primary and Key Secondary Outcomes 
Sensitivity analyses of primary and key secondary efficacy outcomes are as described in this R-
SAP. 
6.5.11 Subgroup Analyses 
The following subgroups will be analyzed in the random-slopes model (see Section 6.5.4) for 
ALSFRS-R total score and SVC in the FAS by including subgroup, subgroup × month, and 
subgroup × treatment × month terms: 
• El Escorial definite or probable and less than 18 months since onset of weakness (both 
criteria vs. not both),  
• Pre-baseline ALSFRS-R slope (high vs. low by median split),   
• Baseline plasma NfL concentration (high vs. low by median split),  
• Baseline TRICALS score (more negative vs. less negative by median split),  
• Baseline use of riluzole by concomitant medication log (yes vs. no),  
• Baseline use of edaravone by concomitant medication log (yes vs. no), and  
• Baseline use of riluzole and edaravone (neither, riluzole only, edaravone only, both). 
Conditional on findings from initial analyses, the following subgroups will also be analyzed: 
• Age (less than 65 years vs. 65 years or older),  
• Sex (female vs. male),  
• Race (white vs. any minority race with greater than 5% prevalence in the sample),  
• Ethnicity (Hispanic or Latino vs. non-Hispanic or Latino),  
• BMI (less than 25 kg/m2, 25 to less than 30 kg/m2, 30 kg/m2 or more),  
• Chronic kidney disease (CKD) stage (stage 1 or better [eGFR 90 mL/min/1.73m2 or more], 
stage 2 [eGFR 60 to 89 mL/min/1.73m2], stage 3 or worse [eGFR less than 60 
mL/min/1.73m2]),  
• Site (individual sites with at least 5 participants per treatment group and all participants from 
sites with fewer than 5 participants per treatment group pooled). 
For each classification, unknown, not reported, and missing will be considered one group. All 
individuals not included in a specified subgroup will be combined into a mixed, "other" group. 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 33 of 37 
 
The "other" group will be included in analyses if its prevalence is greater than 5%; otherwise, the 
"other" group will be excluded. 
6.5.12 Comparison of Controls across Regimens 
Comparisons of placebo participants across regimens are the same as specified in the M-SAP 
with baseline serum NfL level as an additional covariate in adjusted analyses plus applicable 
interaction terms as relevant to a given model. 
6.5.13 Pharmacokinetic Analyses 
Serum trehalose concentrations will be summarized by treatment group, time point (pre-dose and 
1, 2, and 4 hours after SOI), and visit in the ERO sample. Concentrations below the limit of 
quantitation (BLQ) will be replaced with one half of the lower limit of quantitation. Summaries 
will include number of observations, number and percentage with concentrations BLQ, 
arithmetic mean, median, standard deviation, minimum, maximum, geometric mean, geometric 
coefficient of variation (calculated as sqrt(exp(variance of log-transformed concentrations) – 1)), 
and 95% confidence bounds for the geometric mean assuming log-normally distributed data. 
Serum trehalose concentration data may be subjected to population pharmacokinetic analysis to 
derive population estimates of pharmacokinetic parameters and test the effect of various 
covariates such as age, weight, and sex. Details of the analysis will be described in a separate 
data analysis plan (DAP). This analysis may be performed by combining data from the current 
study with data from other studies of trehalose, if deemed appropriate. The population 
pharmacokinetic analysis will be performed by Seelos and reported in a separate modelling 
report. 
6.5.14 Pharmacodynamic Biomarker Analyses 
Change in LC3-II levels in CSF will be summarized by treatment group and visit in the ERO. 
Summary statistics will be provided for the values, change from baseline, and percent change 
from baseline at each scheduled assessment time point. The effect of treatment on 
pharmacodynamic response will be estimated in the repeated-measure model (see Section 6.5.3). 
Pearson and Spearman correlations between change in LC3-II levels in the CSF and concurrent 
change in ALSFRS-R total scores and SVC percent-predicted will be estimated among 
participants with complete data. 
6.6 
Safety Analyses 
6.6.1 Treatment-emergent Adverse Events 
Summaries and analyses of treatment-emergent adverse events (TEAE) are the same as specified 
in the M-SAP with the following revisions. 
TEAEs are defined as those adverse events with onset dates in the interval from treatment 
initiation to either the Final Safety Visit if completed, the date of death if the participant dies, 28 
days after last study contact if the participant early terminates or is lost to follow-up, or the date 
of first dose of study drug during participation in the OLE, if so exposed. The interval is 
inclusive except for adverse events that occur on the day of treatment initiation and are known to 
precede first exposure to study drug. 
In addition to summaries specified in the M-SAP, TEAEs will be summarized by Medical 
Dictionary for Regulatory Activities (MedDRA) system organ class and preferred term for fatal 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 34 of 37 
 
TEAEs, TEAEs that occurred during a participant's COVID-19 infection (defined as 5 days prior 
to symptom onset to end of COVID-19 symptoms or end of double-blind follow-up, if ongoing), 
and TEAEs and serious TEAEs stratified by the following subgroups: baseline riluzole use, 
baseline edaravone use, age, sex, race, ethnicity, weight, BMI, and CKD stage. Subgroup 
classifications will be the same as described in Section 6.5.12 Subgroup Analyses except that the 
"other" group will be retained regardless of prevalence. 
Treatment-dependent differences in the proportion of participants experiencing a given type of 
TEAE will not be tested.  
6.6.2 Safety Labs 
Summaries and analyses of clinical safety labs are the same as specified in the M-SAP with the 
revision that lab results collected more than 28 days after last dose of study drug will not be 
tabulated, that abnormal levels will be classified to a toxicity grade based on quantitative grading 
using National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events 
(CTCAE) version 5.0, and with the addition that maximum toxicity over all post-baseline visits 
that occur within 28 days or fewer after last dose of study drug will be included in shift tables 
along with visit-specific shifts. 
6.6.3 ECG Results 
Summaries of ECG parameters and findings are the same as specified in the M-SAP. 
6.6.4 Vital Signs and Weight 
Summaries and analyses of vital signs and weight are the same as specified in the M-SAP. 
6.6.5 Suicidality 
Summaries of suicidality are the same as specified in the M-SAP. 
6.7 
Other Analyses 
6.7.1 Participant Disposition 
All participants consented to the Master Protocol between the time of the first and last consent of 
a participant assigned to a regimen included in the FAS will be summarized as a single set for the 
following events: consented to the Master Protocol, failed screening for the Master Protocol, 
other reasons not assigned to a regimen (including timing out of the screening window, death, 
withdrawal of consent, early termination, and administrative termination), and assigned to a 
regimen. Reasons for Master Protocol screen failure will be summarized. 
All participants in the above sample assigned to a regimen will be summarized as two sets (final 
screening for RGE vs. final screening for a non-RGE contributing regimen) for the following 
events: consented to a regimen, failed screening for a regimen, other reasons not randomized 
within a regimen (including timing out of the screening window, death, withdrawal of consent, 
early termination, and administrative termination), and randomized within a regimen. If a given 
individual is screened multiple times prior to randomization within a regimen, then the final 
screening experience of that individual will be summarized. Reasons for RGE screen failure will 
be summarized separately for all participants screened for RGE whether that was their final 
screening experience or not. 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 35 of 37 
 
All participants in the FAS will be summarized as two sets (randomization to active study drug 
vs. randomization to placebo) for the following events: initiated regimen-specific study drug, 
prematurely terminated study participation due to death, withdrawal of consent, early 
termination, loss to follow-up, or administrative termination, completed 24-week follow up, and 
completed a safety follow-up visit vs. continued into the OLE. Reasons for withdrawal of 
consent or early termination after randomization will be summarized. 
6.7.2 Study Drug Compliance and Tolerance 
Summaries of study drug compliance and tolerance are the same as specified in the M-SAP with 
the clarification that summaries will be reported for the ERO and SRO analysis sets and that date 
of permanent discontinuation of study drug is the date of last use of double-blind study drug 
among all participants in a given analysis set. The number of days of exposure to study drug will 
be calculated as the number of days from dose initiation to drug withdrawal, inclusive, less any 
day(s) during which use of study drug was interrupted. The proportion of participants who 
interrupted study drug or reduced study drug dosage and the time to first study drug interruption 
or dosage reduction will be summarized. The number of days of exposure to a reduced dosage of 
study drug will be summarized. 
6.7.3 Concomitant Medication Use 
Summaries of concomitant medication use are the same as specified in the M-SAP with the 
clarification that medications taken at baseline and those initiated after first dose of study drug 
that were not taken at baseline will be separately summarized and will be classified by ATC 
Therapeutic class and WHODrug Preferred base name. 
Time to post-baseline initiation of edaravone, riluzole, or sodium phenylbutyrate/taurursodiol 
individually and in combination will be summarized by Kaplan-Meier product-limit estimates 
among the subset of participants who were not on a given drug or combination of drugs at 
baseline. 
6.7.4 Medical History 
Medical histories will be summarized by MedDRA system organ class, high level term, and 
preferred term in the STF and SRO analysis sets. 
6.7.5 Blindedness 
The proportions of participants and site investigators who report on the Exit Questionnaire a 
guess of active vs. placebo treatment assignment, each level of surety of that guess, and each of 
five pre-specified reasons for making a treatment assignment will be summarized by treatment 
group in the FAS and ERO. Treatment-dependent differences in the proportion guessing active 
treatment assignment will be tested among all respondents and among those stating they are at 
least somewhat sure of their guess by Fisher's exact test and the difference in proportion guessing 
active treatment assignment will be estimated with confidence bounds. 
6.7.6 Impact of COVID-19 Pandemic 
The proportions of planned assessments missed due to COVID-19 restrictions or disruptions will 
be summarized by treatment group, visit, and type of assessment in the FAS and ERO . Protocol 
deviations that resulted from COVID-19 restrictions or disruptions will be summarized by 
treatment group and type of deviation in the FAS and ERO and listed. 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 36 of 37 
 
7. Validation 
7.1 
Primary Efficacy Analysis 
Validation of the primary efficacy analysis is as specified in the M-SAP. In addition, Seelos will 
perform a replication of the primary efficacy results (including any sensitivity analyses) using an 
independent programmer. 
7.2 
Secondary, Exploratory, and Safety Analyses 
Validation of secondary, exploratory, and safety analyses are the same as specified in the M-SAP 
with the following clarifications. 
Three levels of validation will be used: 
Level 1: Replication of results by an independent programmer. 
Level 2: When a macro is used to generate multiple results, one result will be replicated by 
an independent programmer. 
Level 3: Log files will be inspected for error messages and / or relevant warnings. 
A separate validation document will be created in which the validation level applied for each 
analysis is specified. The level of validation will be based on whether the result is considered 
core or supplementary. Core results will be based on validation level 1 or level 2. Supplementary 
results will be based on level 3 validation. Examples of core results include primary and key 
secondary efficacy and safety endpoints. Examples of supplementary results include exploratory 
efficacy endpoints, selected sensitivity analyses, and selected sensitivity analysis data sets for 
secondary efficacy endpoints. Any analyses that fail due to small sample size or lack of 
convergence will be omitted with comment rather than programmed separately to find a post-
hoc, work-around solution. 
8. References 
The following references are cited in addition to those specified in the M-SAP: 
Avagyan V, Vansteelandt S. Stable inverse probability weighting estimation for longitudinal 
studies. Scand J Statist. 2021 Sep;48(3):1046–67. 
Balendra R, Jones A, Jivraj N, Knights C, Ellis CM, Burman R, Turner MR, Leigh PN, Shaw 
CE, Al-Chalabi A. Estimating clinical stage of amyotrophic lateral sclerosis from the ALS 
Functional Rating Scale. Amyotroph Lateral Scler Frontotemporal Degener. 2014 Jun;15(3-
4):279-84. 
Hothorn T, Hornik K, Zeileis A. Unbiased recursive partitioning: a conditional inference 
framework. J Comp Graph Stat. 2006;15(3):651–674. 
Roche JC, Rojas-Garcia R, Scott KM, Scotton W, Ellis CE, Burman R, Wijesekera L, Turner 
MR, Leigh PN, Shaw CE, Al-Chalabi A. A proposed staging system for amyotrophic lateral 
sclerosis. Brain. 2012 Mar;135(Pt 3):847-52. 
Rubin DB. Causal inference through potential outcomes and principal stratification: Application 
to studies with "censoring" due to death. Statist. Sci. 2006 Aug;21(3):299-309. 


Cudkowicz (PI) HEALEY ALS Platform Trial: RGE Statistical Analysis Plan 
Version 2.0, 10 Oct 2023 
 
Page 37 of 37 
 
Westeneng HJ, Debray TPA, Visser AE, van Eijk RPA, Rooney JPK, Calvo A, Martin S, 
McDermott CJ, Thompson AG, Pinto S, Kobeleva X, Rosenbohm A, Stubendorff B, Sommer 
H, Middelkoop BM, Dekker AM, van Vugt JJFA, van Rheenen W, Vajda A, Heverin M, 
Kazoka M, Hollinger H, Gromicho M, Körner S, Ringer TM, Rödiger A, Gunkel A, Shaw 
CE, Bredenoord AL, van Es MA, Corcia P, Couratier P, Weber M, Grosskreutz J, Ludolph 
AC, Petri S, de Carvalho M, Van Damme P, Talbot K, Turner MR, Shaw PJ, Al-Chalabi A, 
Chiò A, Hardiman O, Moons KGM, Veldink JH, van den Berg LH. Prognosis for patients 
with amyotrophic lateral sclerosis: development and validation of a personalised prediction 
model. Lancet Neurol. 2018 May;17(5):423-433. 
 


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