Clinical trial NCT03580616
Conditions: Amyotrophic Lateral Sclerosis (ALS)
Sponsor: Elijah W. Stommel
Start date: 2018-10-24; Completion date: 2022-12-20
Enrollment: 43 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of this study is to determine the tolerability of L-Serine oral doses for ALS patients and assess preliminary indications of efficacy

Detailed description:
All patients will receive the same dose of the study treatment over 6 months. For each participant the study will last approximately one year with follow up visits after the treatment period of 6 months is completed. The visits will include blood draws, vital sign checks, neurological and physical exams, pulmonary testing with forced vital capacity (FVC), and questionnaires.

Arms: arm 1: L-Serine 15 grams orally twice a day as tolerated for 6 months
Interventions: intervention 1: L-Serine
Intervention descriptions: intervention 1: L-Serine is a naturally occurring dietary amino acid. It is abundant in soy products, some edible seaweeds, sweet potatoes, eggs and meat.

Protocol documents:
Prot_SAP_000.pdf: 
L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
1
CLINICAL RESEARCH PROTOCOL
                  
Tolerability and Efficacy of L-Serine in Patients with Amyotrophic 
Lateral Sclerosis:  A Phase IIa Study
Regulatory Sponsor:
Elijah W. Stommel, MD, PhD
Dartmouth-Hitchcock Medical Center
Department of Neurology
One Medical Center Drive 
Lebanon, NH 03576
603-650-5104
Funding Sponsor:
Brain Chemistry Labs, Institute for Ethnomedicine
PO Box 3464
Jackson, WY 83001
A not-for-profit 501(c)(3) charity
801-375-6214
Study Product:
L-serine
Initial version:    14Jun2018
Amended:   9Oct2018
Amended:
    9Nov2018
Amended:
    17Jun2019
Amended:                   15APR2020  
Amended:                    02 NOV2020 
Amended:                   27JAN2021
Amended: 
    20AUG2021
Amended:
   29Sep2021
Amended:
    12Nov2021
Amended:
    07Dec2021


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
2
Table of Contents
List of Abbreviations.............................................................................................................4
Study Summary.....................................................................................................................4
1
Introduction....................................................................................................................5
1.1 Background: Prevalence and Clinical Features of ALS................................................5
1.2 Environmental Exposures as Risk Factors for ALS.....................................................6
1.3 L-serine as a Possible Therapy for ALS.........................................................................6
1.4 Investigational Agent.......................................................................................................7
1.5 Preclinical Data.................................................................................................................8
1.6 Phase I Clinical Data......................................................................................................10
1.7 Relationship to Other Clinical Trials............................................................................11
1.8 Study Objectives.............................................................................................................12
1.8.1 Primary Objective..............................................................................................12
1.8.2 Secondary Objective..........................................................................................12
1.8.3 Tertiary Objectives............................................................................................12
2
Study Design..................................................................................................................12
2.1
General Design............................................................................................................12
2.2 Use of Historical Placebo Patient Control Data.........................................................13
2.3 Study assessments.........................................................................................................14
2.4 Blood and Urine Samples.............................................................................................14
2.5 Saliva Samples................................................................................................................15
2.6 Primary Study Endpoints.............................................................................................15
2.7 Secondary Study Endpoints..........................................................................................15
3
Subject Selection and Withdrawal................................................................................16
3.1
Inclusion Criteria........................................................................................................16
3.2
Exclusion Criteria.......................................................................................................16
3.3
Subject Recruitment and Screening.........................................................................16
3.4
Early Withdrawal of Subjects....................................................................................17
3.4.1
When and How to Withdraw Subjects.....................................................17
3.4.2
Data Collection and Follow-up for Withdrawn Subjects.......................17
4
Study Drug.....................................................................................................................17
4.1
Description..................................................................................................................17
4.2 Manufacturer and Manufacturing Details of Bulk Drug Substance........................17
4.3 Stability Data for the Drug Substance.........................................................................18
4.4 Packaging........................................................................................................................18
4.5 Blinding of Study Drug..................................................................................................18
4.6 Treatment Regimen.......................................................................................................18
4.7 Method for Assigning Subjects to Treatment Groups...............................................19
4.8 Preparation and Administration of Study Drug........................................................19
4.9 Subject Compliance Monitoring..................................................................................19
4.10 Prior and Concomitant Therapy................................................................................19
4.11 Receiving, Storage, Dispensing and Return..............................................................19
4.11.1 Receipt of Drug Supplies.................................................................................19
4.11.2 Storage..............................................................................................................19
4.11.3 Dispensing of Study Drug...............................................................................19
4.11.4 Return or Destruction of Study Drug............................................................20
5
Study Procedures..........................................................................................................20


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
3
5.1 Visits and telephone calls..............................................................................................20
5.1.1 Study Timeline and Events........................................................................................23
5.2 Blood Draws and Specimen Processing, Storage, and Shipment............................24
5.3 Optional Consent for Saliva Sample, Biobanking, and Future Analysis of Blood 
Samples..................................................................................................................................24
5.4 Administration of the Columbia Suicide Severity Rating Scale (C-SSRS)..............24
6
Statistical Plan ..............................................................................................................24
6.1 Sample Size Determination...........................................................................................24
6.2 Statistical Methods........................................................................................................25
6.3 Subject Population(s) for Analysis..............................................................................25
6.4 Efficacy/Futility May Result in Early Termination of the Study.............................25
7
Safety and Adverse Events............................................................................................26
7.1
Definitions...................................................................................................................26
7.2
Recording of Adverse Events....................................................................................28
7.3
Reporting of Serious Adverse Events and Unanticipated Problems....................28
7.3.1 Investigator reporting: notifying the Dartmouth IRB...........................................29
7.4
Unblinding Procedures..............................................................................................29
7.5
Medical Monitoring....................................................................................................29
7.6
Internal Data and Safety Monitoring Board...........................................................29
8
Data Handling and Record Keeping ............................................................................30
8.1
Confidentiality.............................................................................................................30
8.2
Source Documents......................................................................................................30
8.3
Case Report Forms.....................................................................................................30
8.4
Records Retention......................................................................................................30
9
Study Monitoring, Auditing, and Inspecting................................................................31
9.1
Study Monitoring Plan...............................................................................................31
9.2
Auditing and Inspecting............................................................................................31
10 Ethical Considerations ..................................................................................................31
10.1 Funding Source...........................................................................................................31
10.2 Conflict of Interest......................................................................................................31
10.3 Subject Stipends or Payments...................................................................................31
10.4 Publication Plan...........................................................................................................32
11 References.....................................................................................................................32
12 Appendices....................................................................................................................36


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
4
List of Abbreviations
ALS: Amyotrophic Lateral Sclerosis
ALSA-Q40: Amyotrophic Lateral Sclerosis Assessment Questionnaire (long form)
ALSFRS-R: Amyotrophic Lateral Sclerosis Functional Rating Scale - Revised
BMAA:  β-N-methylamino-L-alanine
FVC: Forced Vital Capacity
GRAS:  generally recognized as safe
LAR: legally authorized representative
NFT: neurofibrillary tangles 
PI: principal investigator
PHI: protected health information
PRO-ACT: Pooled Resource Open-access ALS Clinical Trials
C-SSRS: Columbia –Suicide Severity Rating Scale
Study Summary
Title
A Phase IIa study of the effects of L-serine in Patients with Amyotrophic 
Lateral Sclerosis: A Phase II Study
Short Title
Phase IIa L-serine trial for ALS patients
Phase
Clinical study phase IIa
Methodology
All patients receive the treatment at the same dose
Study Duration
Expected 4 years
Study Center(s)
Single-center 
Objectives
Determine tolerability of L-serine oral doses for ALS patients and assess 
preliminary indications of efficacy. 
Number of Subjects
50
Diagnosis and Main 
Inclusion Criteria
Patients with a clinical diagnosis of ALS.
Study Product, Dose, 
Route, Regimen
L-serine 15g oral dose twice daily morning and night. 
Duration of 
administration
6 months
Reference therapy
Selected ALS patients in placebo groups in prior treatment trials with 
the same inclusion/exclusion criteria from the PRO-ACT database.
Statistical 
Methodology
Linear mixed effects model.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
5
1
Introduction
This document is a protocol for a human research study. This study is to be 
conductedaccording to US and international standards of Good Clinical Practice (FDA 
Title 21 part 312 and International Conference on Harmonization guidelines), applicable 
government regulations and Institutional research policies and procedures. 
1.1 Background: Prevalence and Clinical Features of ALS
Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s Disease or Motor 
Neuron Disease, is a devastating progressive neurodegenerative disease characterized by 
death of primary motor neurons in the cerebral cortex, brain stem, and spinal cord 
(Brooks et al. 2000). Resultant denervation of the muscles leads to muscle wasting and 
paralysis. Generally, initial indications of ALS are development of a localized weakness, 
often in a peripheral part of the body such as a hand or foot, although sometimes 
problems with breathing, spasticity, and speech/swallowing signal the onset of the 
disease (Miller et al. 2005). Fasciculations are a common sign. Diagnosis of ALS requires 
evidence of both lower and upper motor neuron degeneration with spread of symptoms 
to other regions and evidence of progression (Brooks et al. 2000). 
In Europe, the annual incidence rate of ALS in the general population is 2.16 per 
100,000, with men having a slightly higher annual incidence rate (Logroscino et al. 
2010). In the United States, estimates of annual incidence rates range between 1.6 per 
100,000 (Hirtz et al. 2007) to the most recent estimate of the prevalence of 4.3 per 
100,000 (Mehta et al. 2016). At any one time, roughly 20,000-30,000 Americans are 
living with diagnosis of ALS (Miller et al. 2005) with a prevalence rate of 4.3 per 100,000 
in the U.S. population (Mehta et al. 2016). Thus, ALS fits within the FDA orphan disease 
category.
The burden that ALS places on patients and their families is enormous, with most 
patients dying within three years of diagnosis. Paralysis and loss of motor function is 
progressive, ultimately leading to the inability to walk, to write, to swallow, to speak, and 
to breathe. Although traditionally motor neuron deficits have been emphasized in 
clinical studies of ALS, it is now known that cognitive issues also occur in ALS patients 
with 51% of patients diagnosed with sporadic ALS showing some level of cognitive 
impairment (with 19% showing moderate to severe impairment). The pattern of 
cognitive problems is similar to that seen in familial ALS (fALS) and frontotemporal 
dementia (FTD) (Ringholz et al. 2005, Wheaton et al. 2007). In a study of 160 patients in 
Ireland, 13.8% fulfilled the criteria for FTD (Phukan et al. 2011).
Approximately 8-10% of ALS cases are familial, with 90% of cases being sporadic (sALS). 
Mutations in SOD-1, TDP-43, FUS/TLS, VCP, and TARDBP have all been shown as 
potential causes of ALS with some mutations such as TARDBP also being found in 
individuals with sporadic ALS (Kabashi et al. 2008). Environmental exposures including 
exposures to heavy metals such as lead, mercury, organic solvents, cyanobacteria, and 
pesticides are also being investigated as possible risk factors for ALS (Andrew et al. 2017, 
Caller et al. 2015). 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
6
The current standard of care in the United States is treatment with Riluzole—a glutamate 
antagonist—which was approved in 1995, although in May 2017 the FDA approved the 
use of Edaravone administered as an intravenous infusion for the treatment of ALS. 
Edaravone use in ALS patients in the early stages of their disease resulted in a reduced 
rate of functional decline of ALS patients by about 33% (Abe et al., 2014), and is 
estimated to cost about $145,000 per year for each patient.
1.2 Environmental Exposures as Risk Factors for ALS
Based on ethnomedical studies of a related paralytic disease, Guamanian Amyotrophic 
Lateral Sclerosis / Parkinsonism Dementia Complex (Guamanian ALS/PDC), there has 
been increased interest in chronic exposure to a cyanobacterial toxin, β-N-methylamino-
L-alanine (BMAA) as an additional risk factor for ALS. Produced by endophytic 
cyanobacteria in specialized roots of cycad trees (Cycas micronesica) in Guam, BMAA 
contaminates traditional food items in the Chamorro diet (Cox et al. 2003, Murch et al. 
2004, Banack et al. 2006). The hypothesis that chronic exposure to BMAA triggered an 
ALS-like disease among the Chamorro people of Guam was supported by recent 
replicated experiments with non-human primates in which chronic dietary exposure to 
BMAA triggered a dense tauopathy with sparse amyloid deposits in brain tissues of 
animals fed BMAA, but not in control animals (Cox et al. 2016). The resultant 
neuropathology had some aspects similar to Braak 1 early stage Alzheimer’s disease 
pathology (Braak 1995, Cox et al. 2016) including neurofibrillary tangles (NFT) formed 
from misfolded tau as determined by AT8 immunohistological (IHC) staining and β-
amyloid deposits determined by β-amyloid(1-42) IHC stain; both of these 
neuropathological features were confirmed with Thioflavine-S IHC staining. In addition, 
microglial activation similar to that observed in early stages of ALS was found in animals 
fed BMAA (Davis et al. 2016). 
We used this non-human primate model to test the naturally-occurring dietary amino 
acid L-serine and found that dietary supplementation in non-human primates reduced 
the density of NFT by 35-50% in eight regions of the brain including the amygdala and 
anterior cingulate gyrus (Cox et al. 2016). 
1.3 L-serine as a Possible Therapy for ALS
ALS, like other tangle diseases, is characterized by the presence of misfolded and tangled 
proteins. L-serine was initially investigated as a possible therapy for ALS because L-
serine blocked misincorporation of BMAA into human neuroproteins, reducing protein 
misfolding, aggregation, and subsequent apoptosis (Dunlop et al. 2013). Subsequently, 
the ability of L-serine to alert the unfolded protein response (UPR) has been elucidated 
using in vitro data from human neuronal cell culture and in vivo data from non-human 
primates dosed with L-serine over 140 days compared to controls. Using a UPR real-time 
PCR array with genes from the ER stress and UPR pathways, as well as western blotting, 
it was found that L-serine functions as a small proteostasis regulator, alerting cells to 
quickly respond to oxidative insult (Dunlop, Powell, Guillemin, Cox 2017). This effect of 
L-serine occurs, in part, through upregulation of the ER stress chaperon protein 
disulphide isomerase (PDI), which is involved in refolding misfolded proteins (Dunlop, 
Powell, Metcalf, Guillemin, Cox 2017).


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
7
L-serine is a normal part of the human diet, with the average American receiving 3.5 
grams/day from all sources. L-serine is synthesized by neurons and glial cells, but 
deficiencies in endogenous L-serine biosynthetic pathways are associated with a number 
of human neurological conditions (Canu et al, 2014; Metcalf et al. 2017). L-serine has 
shown effectiveness in treating polyneuropathy (Catsman-Berrevoets et al. 1997) and is 
currently being studied as a possible treatment for the hereditary neuropathy HSN-1 
(Garofalo et al. 2011).
Since we have found in replicated studies in non-human primates that supplementing 
the diet with L-serine results in a decreased density of NFT, we now seek to evaluate the 
potential of L-serine in slowing disease progression and functional decline among ALS 
patients. Based on an earlier successful FDA-approved Phase I clinical trial of L-serine in 
20 ALS patients (Levine et al. 2016), we now seek to extend studies of tolerability and 
potential efficacy of L-serine therapy in a Phase IIa trial in a larger number of ALS 
patients. 
1.4 Investigational Agent
L-serine (C3H7NO3; 105.09 g/mol; synonym (S)-2-amino-3-
hydroxypropanoic acid) is a naturally-occurring dietary amino acid. It 
is abundant in soy products, some edible seaweeds, sweet potatoes, 
eggs, and meat. Since some L-serine is produced by astrocytes in the 
brain, it is considered by some authorities to be a non-essential amino 
acid, although as noted below, certain genetic diseases indicate that L-serine is a 
constituent essential amino acid. L-serine is directly involved in the biosynthesis of 
purines, pyrimidines, and other amino acids. L-serine residues in proteins serve, along 
with those of threonine, tyrosine, and histidine, as key sites for phosphorylation 
necessary for proper protein folding and functionality. L-serine is currently prescribed, 
sometimes in combination with glycine, for the treatment of two different genetic 
neurological diseases that result in L-serine deficiency, e.g., 3-phosphoglycerate 
dehydrogenase deficiency and 3-phosphoserine phosphatase deficiency (De Koning 
2006). L-serine has been shown to reduce the production of neurotoxic 
deoxysphingolipids that are responsible for hereditary sensory autonomic neuropathy 
type 1, and has shown encouraging results in a pilot clinical trial (Garofalo et al. 2011).
L-serine is considered GRAS (generally recognized as safe) by the FDA and has been 
approved as a normal food additive under CFR172.320. It is widely sold as a dietary 
supplement. No adverse incidents have been recorded by the FDA from its use. In an 
FDA-approved Phase I human clinical trial of L-serine for ALS patients, sponsored by 
the Brain Chemistry Labs, it was found that patients tolerated doses up to 30 grams/day 
(ClinicalTrials.gov identifier NCT01835782, Levine et al. 2016). Harvard University and 
Massachusetts General Hospital are conducting a two-year study on L-serine 
supplementation to correct biochemistry associated with hereditary sensory neuropathy 
type 1 at doses of 400mg/kg/day (ClinicalTrials.gov identifier NCT01733407).


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
8
1.5 Preclinical Data
In human neuronal cell cultures, L-serine blocks misincorporation of β-N-methylamino-
L-alanine (BMAA) into neuroproteins, which otherwise leads to protein misfolding, 
aggregation, and cell apoptosis (Fig. 1). This protective effect of L-serine was replicated 
in a non-cell synthesis system, where protein incorporation of BMAA increased when L-
serine was removed from the system and when human DNA templates were available in 
comparison with bacterial DNA templates (Glover et al. 2014).  Data on fruit-flies, 
Drosophila melanogaster, reveals that co-feeding with L-serine decreased the amount of 
BMAA incorporated into protein by half (Fig. 2).  In addition, 40% of flies fed BMAA 
died within three days, while co-administration with L-serine saved every fly within this 
time frame (Fig. 2). 
Figure 1:  Inhibition of incorporation or radiolabel into cell proteins by L-serine. Panel 
A, Incorporation of radiolabeled BMAA was inhibited by L-serine in a concentration 
dependent manner. Panel B, D-serine (D-SER) did not significantly impact the 
incorporation of BMAA (NS, P = 0.4419). L-serine significantly inhibited the 
incorporation of BMAA compared to the control cells (CTRL, ***P=0.0002) and D-
serine (D-SER, **P<0.01). Panel C, there was a significantly (****P<0.001) greater 
incorporation of BMAA when all protein amino acids were omitted from the culture 
medium (ALL) compared to when none were (NONE). When only L-serine was omitted 
(L-SERINE) incorporation was restored to approximately 80% (****P=0.0009). 
doi:10.1371/journal.pone.0075376.g004


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
9
Figure 2: Survival of fruit flies fed BMAA and co-administered L-serine
Based on these in vitro and in vivo results suggesting a neuroprotective function of L-
serine, we supplemented the diet of vervets exposed to BMAA with L-serine for 140 days. 
In replicated experiments, we found that co-administration of the dietary amino acid L-
serine significantly reduced neuropathology including the density of NFT, with greater 
than 50% reduction in median NFT densities within the temporal (dorsal and ventral), 
primary motor, and entorhinal (posterior) and insula cortices, and a greater than 35% 
reduction of NFT density in the perirhinal cortex, amygdala, and anterior cingulate 
cortex (Fig. 3).
Figure 3: Reduction of NFT production in vervets fed both BMAA and L-serine 
(pink:high+SER) in comparison with equal amount of BMAA without L-serine (purple: 
high). Data from Cox et al. 2016.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
10
1.6 Phase I Clinical Data 
A phase I human clinical trial for safety of L-serine in ALS patients, funded by the Brain 
Chemistry Labs, was completed and published in 2016 (NCT01835782, Levine et al. 
2016). Patients (n=20) with an ALSFRS-R score > 25 and a FVC score ≥ 60% predicted 
were randomly assigned to four different oral twice-daily dose regimens (0.5, 2.5, 7.5, or 
15g) for six months. Two of the patients withdrew with gastrointestinal problems, but 
otherwise L-serine was well tolerated (Levine et al. 2016). Two ALS patients died during 
the trial plus a third patient who enrolled with a functional vital capacity (FVC) of 45% 
predicted, a protocol violation. The site investigator determined that for all three cases 
“death was due to progression of ALS and was not related to the study drug” (Levine et 
al. 2016, p. 3). No other adverse effects were noted among the remaining study 
participants, and no changes were seen throughout the trial in routine blood studies.
The ALS Functional Rating Scale-Revised (ALSFRS-R), which is strongly correlated with 
functional capacity and survival in ALS patients, was recorded over the course of the trial 
for each patient (Brooks et al. 2000, Cedarbaum et al. 1999, Kaufmann et al. 2005). The 
resultant ALSFRS-R scores for each patient were compared to 430 ALS patients with the 
same inclusion criteria from the placebo arms of five previous failed placebo-controlled 
clinical trials. As noted in the Phase I publication, “L-serine did not appear to accelerate 
functional decline of patients as measured by slope of their ALSFRS-R scores” (Levine et 
al. 2016, p. 1). Statistical analysis of the Phase I clinical data gave a preliminary 
indication that L-serine slowed functional decline of ALS patients in a dose-dependent 
fashion (Figure 4, Table 1). 
Figure 4: Phase I clinical data comparing increasing L-serine doses (groups 2-5) to 430 
historical control patients from placebo arms of previous failed drug trials (group 1).


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
11
Table 1: ALSFRS-R Slope estimates for different statistical models
Method/Group
Slope (per 
mo decline)
95% Conf Int
p-value for 
L-serine 
effect
ALSFRS-R 
slope 
reduction
Separate Fits
 
 
 
 
 Placebo
1.09
(1.01,1.17)
 L-serine
0.76
(0.32,1.20)
0.14
30%
Linear mixed effects models
 Uncorrected
   Placebo
1.06
(0.98,1.14)
   L-serine
0.73
(0.33,1.13)
0.12
31%
Corrected for initial FVC and symptom duration
   Placebo
1.16
(1.07,1.25)
0.044
34%
   L-serine
0.76
(0.38,1.41)
 Corrected and with dose1
   linear dose eff2
0.06
0.011
   separate dose eff3
 0.5 g
0.16
(-0.60,0.92)
0.68
14%
 2.5 g
0.25
(-0.46,0.96)
0.49
22%
 7.5 g
0.25
(-0.53,1.03)
0.53
22%
15.0 g
0.99
(0.20,1.78)
0.014
85%
1 Dose in grams given twice daily
2 Estimate is per gram reduction in slope of ALSFRS-R
3 Estimates are reductions in slope at the specified dose
The p value of 0.014 shown above refers to the 85% reduction of functional decline for 
patients on the high dose 30g daily using a linear effects model. This model is 
comparable to a similar nonparametric model which demonstrates an increasing effect 
of L-serine with dose. A Jonckheere–Terpstra trend test based on medians of 
uncorrected ALSFRS-R slope estimates ordered by dose (ignoring the varying precision 
of each slope estimate) resulted in the null hypothesis H0 being rejected at the p<0·05 
level; thus, there was a significant relationship between increasing L-serine dose and 
slowing of disease progression as measured by the ALSFRS-R slope.
1.7 Relationship to Other Clinical Trials 
In the Harvard study of 14 subjects with hereditary sensory autonomic neuropathy 
(HSAN1) L-serine was given at either 200 mg/kg or 400 mg/kg (roughly 15 and 30 g/day 
for a 75 kg patient, respectively) and no adverse effects were reported with either dose 
after 10 weeks of therapy (Garolfalo et al. 2011).
There are two additional trials currently reported by clinicaltrials.gov involving L-serine: 
1) NCT01733407, 400mg/kg/d L-serine, L-Serine Supplementation in Hereditary 
Sensory Neuropathy Type 1; 2) NCT02599038, 20 or 200 mg/kg/day, Serine 
Supplementation for Obese Subjects With Fatty Liver Disease.  None of these trials 
currently has reported data, and the fatty liver disease trial was withdrawn prior to 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
12
enrollment due to logistical reasons. It is important to note that trials with L-serine at 
doses of 30 g/day are underway. 
On March 1, 2017, the FDA approved a clinical trial of L-serine for ALS patients funded 
in part by the Brain Chemistry Labs of the Institute for Ethnomedicine, a 501(c)(3) 
charitable research organization, with Drs. Robert Miller, Todd Levine, and Walter 
Bradley serving as co-PIs. This trial (IND 133995) is for a multi-site, nine-month Phase 
II trial for 66 ALS patients using inclusion criteria that select for ALS patients earlier in 
their disease course than in the completed Phase I trial. In this planned larger trial, 
ALSFRS-R scores of the patients in the trial will be compared to 225 subjects selected 
from the PRO-ACT database. 
The complex nature of this planned Phase II trial and the multiplicity of institutions 
involved, caused the estimated costs of this trial to escalate since submission of the 
initial IND application. Additionally, difficulties in obtaining external funding have 
resulted in concomitant delays in the projected date of trial initiation. While remaining 
committed to funding this larger, more complex multi-site trial, Dartmouth-Hitchcock 
Medical Center and Brain Chemistry Labs proposes to initiate a single site Phase IIa trial 
prior to the initiation of the planned larger Phase II trial (IND 13399). Hence, with 
funding from Brain Chemistry Labs, Dartmouth-Hitchcock Medical Center proposes a 
single site Phase IIa trial of the tolerability of L-serine for ALS patients, with the added 
potential of providing additional indications of efficacy, as well as exploratory biomarker 
studies based on measures of the neuroprotective effects of L-serine. 
1.8 Study Objectives
1.8.1 Primary Objective
To assess the tolerability of taking 30g/day of L-serine in ALS patients.
1.8.2 Secondary Objective
To assess the efficacy of L-serine in subjects with probable or definite ALS. 
1.8.3 Tertiary Objectives
To explore the possibility of new ALS progression biomarkers by using neurally-derived 
exosomes from blood samples. Assess effects within subgroups defined by serine 
synthetic gene polymorphisms.
2
Study Design
2.1
General Design
This is a Phase IIa study of the effects of L-serine on ALS patients. Potential candidates 
for participation in this study will be identified by the Investigator as having probable or 
definite ALS by the modified El Escorial criteria (Brooks et al., 2000), an ALSFRS-R 
score >25 and FVC ≥60%. These measures are completed as part of standard practice. 
Patients currently taking an L-Serine supplement at any dose will be required to have a 
30 day washout period prior to the start of study drug.  Up to 50 subjects will be enrolled 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
13
over the course of approximately four years. Subjects participating in the study will 
receive a 15 gram twice daily dose. Study duration will last approximately 48 weeks from 
time of enrollment for each enrolled subject. Study treatment administration will last 
approximately 6 months (24 weeks). After an initial screening/baseline visit, patients 
will be instructed to take one 15 g dose of L-serine per day for a two-week period. If they 
tolerate the drug well, they will be instructed at the end of the second week to increase 
their dose to one 15 g dose of L-serine in the morning and one 15 g dose of L-serine in the 
evening, for a total of 30 g per day for the remainder of the six-month trial.  At any stage 
in the trial, if a patient experiences unacceptable side effects, such as nausea or vomiting, 
the affected patient’s daily dose will be reduced to 15 g/day for the duration of the trial.  
Patients will return at month 3 for assessments and drug dispensing and at month 6 for 
assessments. All remaining study drug will be collected at month 6 and returned to the 
study sponsor. 
2.2 Use of Historical Placebo Patient Control Data
In this Phase IIa trial, we will use historical control data from 430 placebo patients in the 
PRO-ACT database for comparison purposes to test the secondary objective. We will 
assess the efficacy of L-serine in subjects with probable or definite ALS, in the same way 
that occurred in the earlier FDA-approved Phase I clinical trial of L-serine for ALS 
patients (Levine et al, 2016). The use of historical control data in human clinical trials is 
recognized as having “the potential to further streamline the development of drugs” 
(Viele et al, 2014). In this single-arm open label clinical trial the use of historical control 
data presents significant advantages over a more traditional clinical model such as a 
double-blinded, randomized Phase IIa controlled trial (Williams, 2001) of the type we 
are currently conducting at Dartmouth-Hitchcock Medical Center for early stage 
Alzheimer’s patients (ClinicalTrials.gov Identifier NCT03062449).
First, unlike early stage Alzheimer’s disease patients, a rapidly growing segment of the 
ALS patient population take L-serine as a dietary supplement. The primary objective of 
this trial is to assess tolerability of ALS patients taking 30 g/day of L-serine, with a 
secondary objective of assessing the efficacy of L-serine for ALS patients. A commonly 
marketed dietary supplement, L-serine is considered as GRAS and has FDA approval as 
a normal food additive under CFR172.320. It is inexpensive and can be easily purchased 
from the internet. Thus, it is becoming increasingly difficult to recruit ALS patients for 
clinical trials who have not taken, either with the advice of their physician or as a self-
dose regimen, L-serine. At this point, there is a “paucity of willing participants for 
randomization into control and experimental groups” (Desai et al., 2013).
Second, the study outcome of the Phase I clinical trial (Levine et al., 2016) yielded a 
preliminary, but statistically significant, indication that L-serine may slow functional 
decline in ALS patients as measured by the slopes of their ALSFRS-R scores. Thus, there 
are ethical concerns about randomly assigning patients to a placebo group for an 
extended period (in this case six months) if there is a distinct possibility that they could 
benefit from the study drug. Such ethical concerns have been raised in randomly 
assigning patients to other placebo or suboptimal treatments in clinical studies (Desai et 
al., 2013; Polman et al., 2008; Spławiński and Kuźniar, 2004).


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
14
Third, this Phase IIa trial is considered to be preliminary to a larger Phase II trial (IND 
133995) of L-serine for ALS patients (see Section 1.7: Relationship to Other Clinical 
Trials). If the primary objectives of the current trial are not met, and if tolerability of L-
serine at 30 g/day is not demonstrated, or if the secondary objective of assessing efficacy 
of L-serine in subjects with probable or definite ALS does not yield a positive result, this 
could result in redesign or reconsideration of the larger Phase II trial. For these 
purposes, it is important that the proposed Phase IIa trial, if approved by the FDA, be 
quickly initiated and that patient enrollment occur at a rapid pace.
Fourth, the historical control placebo patient data from the PRO-ACT database are 
voluminous. There are 430 control patients in that database who match our inclusion 
criteria for this proposed single-arm trial. This large control patient data base sample has 
the advantage of constrained variability of the range of ALSFRS-R slopes; it is unlikely 
that further increase in control patient numbers will significantly impact either the 
sample mean or sample variance for placebo patients as an estimate of disease 
progression within the ALS patient population. In other words, the large size of the 
comparison group serves to reduce the possibility of a type I error in which the null 
hypothesis would be incorrectly rejected and we would falsely detect an effect that is not 
present.
For these four reasons, we have designed a single-arm open label trial in which all 
patients will receive L-serine at 30 g/day and we will use historical control patient data 
to determine the secondary objective of the study, e.g. possible efficacy.
2.3 Study assessments
See section 5.1 for list of assessments performed at each visit If unable to obtain any of 
the assessment due to restrictions on patient visit by Dartmouth-Hitchcock or the 
participant choice regarding 
Vital signs (blood pressure, heart rate, temperature, respiratory rate, height, weight 
pulse oximetry) 
Neurological exam
Brief physical exam
ALSA-Q40 (Amyotrophic Lateral Sclerosis Assessment Questionnaire (long form))
ALSFRS-R (Amyotrophic Lateral Sclerosis  Functional Rating Scale - Revised)
FVC % predicted (Forced Vital Capacity % predicted)
C-SSRS (Columbia-Suicide Severity Rating Scale)
2.4 Blood and Urine Samples 
If unable to obtain urine or study blood work due to restrictions on patient visit by 
Dartmouth-Hitchcock or the participant choice regarding illness prevention the urine 
and or bloodwork will be obtained at first in person visit after the study visit scheduled 
date.  Study participants that require urine pregnancy testing will be  sent home at 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
15
baseline visit or shipped urine pregnancy tests to be done at home.  The study 
coordinator will contact the participant for the reminder and the results of test.
Urine samples for pregnancy testing will be collected at baseline, 3 months, and 6 
months (Visit 1, Visit 2, Visit 3).  Blood samples will also be collected at baseline, 3 
months, and 6 months (Visit 1, Visit 2, Visit 3).  In addition to safety labs, serum 
pregnancy, and L-serine levels, blood will be used to extract neurally-derived exosomes 
(Goetzl et al., 2015; Winston et al., 2016; Thompson et al, 2016). The RNA content of 
these exosomes will be analyzed for potential biomarkers for L-serine efficacy based on 
analysis of transcripts associated with endoplasmic reticulum stress and the unfolded 
protein response using extraction of mRNA and proteins Exosomes are a class of 
endosome-derived membrane vesicles shed by neural cells (among other cells) and 
transported in blood plasma. We isolate the neurally-derived exosomes using LiCAM 
antibodies, which react with a neural cell adhesion molecule attached to exosome 
membranes. These exosomes provide an accessible source of proteins, RNA, and DNA 
excreted by neuronal cells and have been used to distinguish early stage Alzheimer's 
patients from controls (Fiandaca et al. 2015). We will examine neurally-derived 
exosomes for biomarkers that can temporally associate with ALS progression using 
baseline, mid, and endpoint blood samples. This proof-of-concept study will determine 
the potential utility of exosome biomarkers in future trials of L-serine, or other 
treatments. 
We also study chiral ratios of L- and D-serine in blood samples from patients, comparing 
baseline ratios to endpoint ratios. In combinations with genetic testing, we will 
determine if there is a relationship between human serine synthetic gene deficiency and 
chiral ratios. We will also determine if L-serine therapy alters the ratio of these 
enantiomers (Metcalf et al., 2017). The chiral ratio alterations could be used as a 
potential biomarker of L-serine supplementation biological effect.
2.5 Saliva Samples
In order to look for polymorphisms in key human serine synthetic genes, a saliva sample 
will be taken from each patient enrolled in the clinical trial on a consented voluntary basis. 
Samples will be collected in saliva tubes specific for study of DNA, stabilizing the samples 
for up to 12 months at room temperature. Exon DNA (protein coding DNA) will be 
sequenced for analysis of serine racemase, 3-phosphoglycerate dehydrogenase, 
phosphoserine phosphatase and phosphoserine aminotransferase to determine if positive 
patient responses to administration of L-serine may be due to mutations in their serine 
biosynthetic genes. Responses of individual patients (slope of ALSFRS-R and FVC) to L-
serine treatment will be compared between patients with normal gene sequences for serine 
racemase, 
3-phosphoglycerate 
dehydrogenase, 
phosphoserine 
phosphatase 
and 
phosphoserine aminotransferase and those who have different sequences for those genes. 
2.6 Primary Study Endpoints
The primary endpoint of this study is tolerability.  We will assess the proportion of 
enrolled patients who tolerate and are compliant with the 30g/day dose, and the 
proportion who scale back to 15g/day.  We will also assess reasons for any withdrawals 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
16
from the study. Rates of adverse events will be compared between the treatment and 
historical control groups.
2.7 Secondary Study Endpoints
A secondary endpoint to be analyzed is the possibility that L-serine slows functional 
decline. The slopes of the ALSFRS-R and FVC through time will be used to test for 
treatment effect, comparing L-serine patients with the 430 historical placebo controls 
from the PRO-ACT database.
3
Subject Selection and Withdrawal
3.1
Inclusion Criteria
1.
Valid informed consent by subject.
2.
Diagnosis of probable or definite ALS based on the El Escorial criteria (Brooks et 
al., 2000) within the last three years prior to study enrollment.
3.
ALSFRS-R score > 25 and a FVC score ≥ 60% predicted.
4.
Patients currently taking Riluzole and Edaravone/Radicava will be allowed into 
the trial as long as they have been taking these FDA-approved drugs for three 
months prior to baseline/screening at a stable dose. If the patient has been 
instructed by his or her physician to stop or pause Riluzole or 
Edaravone/Radicava due to an adverse event the requirement for inclusion into 
the study will be off Riluzole or Edaravone/Radicava for seven (7)  days,  This 
also applies if the dosing has not been stable for three (3) months prior to 
enrollment and treatment.  Initiating or restarting these medications after 
enrollment and treatment has begun is allowed 
5.
Age ≥ 18 years old.
3.2
Exclusion Criteria
1.
Diagnosis of probable or definite ALS more than three years prior to study 
enrollment.
2.
Diagnosis or previous history of ischemic stroke, brain tumor, uncontrolled 
diabetes, renal insufficiency (an estimated GFR <60mg/dl)or severe 
hypertension.  Severe hypertension (asymptomatic or hypertensive urgency) is 
defined as severely elevated blood pressure (180 mm Hg or more systolic, or 110 
mmHg or more diastolic) without acute target organ injury.
3.
Diagnosis or previous history of progressive neurodegenerative disease such as 
Alzheimer’s disease, Parkinson’s disease, Lewy Body Disease, Pick’s Disease, 
Huntington’s Disease, Progressive Supranuclear Palsy. However, ALS patients 
diagnosed with frontotemporal dementia will not be excluded from this study.
4.
Diagnosis or previous history of symptomatic peripheral neuropathy. Patients 
with findings of peripheral neuropathy on electrodiagnostic tests only but no 
clinical symptoms at the time of enrollment are eligible.
5.
Undergoing any chemotherapy or radiation therapy for any cancer.
6.
Any medical condition likely to interfere with the conduct of the trial or survival 
of the patient during this study period.
7.
Pregnant women or women who are breast-feeding.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
17
8. If subject has taken an L-Serine supplement less than 30 days prior to start of 
study drug. a 7 day waiting period is required.
9.
In the Investigator’s opinion, subject would be unable to successfully participate 
in the study for any reason.
3.3 Subject Recruitment and Screening
Participation in this research requires informed consent according to Institutional 
Review Board (IRB) guidelines. A signed IRB approved Consent Form is the means of 
documenting this understanding. No one is allowed to participate without having signed 
this statement personally and received an exact copy (at point of signature acquisition). 
Potential recruits are instructed that their participation is completely voluntary and that 
their medical care will not be altered in any way should they elect not to participate. 
Subjects are recruited from patients presenting to or referred to the Department of 
Neurology with a diagnosis of ALS using El Escorial criteria (Brooks et al., 2000).  
Potential subjects that do not present to or are not referred to the Department of 
Neurology may also be prescreened by the principle investigator, sub-investigator, study 
coordinator, or research nurse listed with the IRB for this study to determine potential 
eligibility for participation in this study. PHI may be retrieved from medical records, 
and/or from the subject.  Subjects will be prescreened based on their medical history for 
study inclusion/exclusion criteria. Subjects deemed to be potentially eligible will be 
initially contacted by the investigator and then by the study coordinator or research 
nurse to gather eligibility criteria. Subjects may be given a copy of the consent form and 
subject study information to review prior to enrollment.  
3.4
Early Withdrawal of Subjects
3.4.1
When and How to Withdraw Subjects
Participants who cannot tolerate the study drug, have adverse reactions to the L-serine, 
or would like to withdraw for any other reason can withdraw from the study at any time. 
If a participant experiences an adverse event which is serious or life threatening in 
nature, and deemed to be drug related, the study medication will be discontinued 
immediately. The Investigator may reserve the right to discontinue a subject from study 
treatment if the PI feels it is not in the best interest of the patient to continue.
If a participant discontinues study medication, they will be encouraged to remain in the 
study and complete study related procedures and assessments while not receiving study 
medication. 
If a participant discontinues study medication and does not wish to return for the 
remainder of study visits, this decision will be documented.  They will be asked to return 
to the site for a final study visit. This visit will be conducted with the same study 
procedures and assessments as “Visit 3” listed in Section 6.5. 
3.4.2 Data Collection and Follow-up for Withdrawn Subjects
If a patient is terminated early from the study because of an adverse event, every effort 
will be made to follow the patient until the adverse event resolves and to have the 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
18
ALSFRS-R score recorded, by telephone if necessary, at the 6-month end of study time 
period. 
4
Study Drug 
4.1
Description
L-serine is a non-essential amino acid already present in the human diet.  This drug will 
be presented as a powder in packets containing 15g of L-serine each.  The L-serine will be 
packaged into a foil or Mylar package. Patients will be instructed to begin the trial with a 
two-week dose period where they will take one 15g dose of L-serine in the morning. If the 
15g daily dose is well-tolerated, the patients will be instructed to then take one package 
in the morning and one in the evening, for a total of 30g of L-serine/day for the 
remainder of the trial. Patients will be encouraged to dissolve the L-serine powder in 
water or another beverage or to mix it with their food. 
4.2 Manufacturer and Manufacturing Details of Bulk Drug Substance
Parchem Fine & Specialty Chemicals (415 Huguenot Street, New Rochelle, New York 
10801) is the supplier of L-serine for the trial, and the L-serine was manufactured by 
Wuhan Grand Hoyo Co., Ltd. (No. 1 Industrial Park, Gedian Economy Development 
Zone, E’hou City, Hubei, China). L-serine is produced via fermentation.
The specifications for the drug substance (including the tests performed, analytical 
procedures, and acceptance criteria) are provided by the manufacturer in the 
Investigator Brochure. Brain Chemistry Labs will independently screen L-serine powder 
from lot number 170603 during packaging and throughout the trial using triple 
quadrupole liquid chromatography mass spectrometry in Jackson Hole. Our experience 
in screening L-serine provided by Parchem is that, to date, the lots we have analyzed 
have met the necessary criteria as determined by triple quadrupole mass spectrometry 
for use in human clinical trials and is spectroscopically consistent with an authenticated 
standard provided by Sigma-Aldrich (St. Louis, MO).
4.3 Stability Data for the Drug Substance
L-serine, as a small (105.09 MW) proteinogenic amino acid, is extraordinarily stable. We 
include in the Investigator Brochure long-term stability data from the manufacturer for 
L-serine over 24 months when held at room temperature (<30°) with relative humidity 
<75%. As can be seen, no significant changes were observed in optical rotation or purity 
over this period. We have also performed our own studies on L-serine stability in general 
based on samples from two different manufacturers over a 3.5-year period using triple 
quadrupole mass spectrometry. We were able to independently confirm the stability of 
L-serine powder when maintained at room temperature.
4.4 Packaging
L-serine dose preparation will be conducted in a cGMP FDA-compliant facility by 
Knechtel (7341 Hamlin Avenue, Skokie, IL 60076) and the dose will be placed in 
sealed foil packages. Each package will contain 15 g L-serine powder. The L-serine 
screening and milling procedures by Knechtel are shown in the Investigator 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
19
Brochure. The specification materials used for pouches to package L-serine powder 
are manufactured by ESP Packaging LLC (3001 Red Hill Avenue 1-105, Costa Mesa, 
CA 92626).
Each individual foil pouch containing a single patient dose will be labeled with the lot 
number containing the Julian code date. Each box that is prepared to be delivered to 
the patients will contain multiple pouches designated for use in a specified period of 
time, will include the following statement: “Caution: New Drug—Limited by Federal 
(or United States) law to investigational use.”
Foil packages sufficient for three months will be given to the patients at their first 
visit and also at their three-month visit.  Doses will be sent to Dartmouth-Hitchcock 
Medical Center in periodic shipments over the length of the project. 
4.5 Blinding of Study Drug
All subjects are receiving L-Serine, and no blinding is necessary. 
4.6 Treatment Regimen
All subjects will receive the same dose (15 g twice daily) after the initial two-week dose 
period of 15 g per day, unless they develop unacceptable side effects (such as nausea or 
vomiting). In the event of such intolerable side effects, patients will be instructed to 
reduce the daily dose to 15 g per day, and may increase t0 30g once the intolerable side 
effect has been resolved, according to PI . The study has prepared an optional drug diary 
for the subjects to utilize to assist with accountability. These diaries are optional. 
4.7 Method for Assigning Subjects to Treatment Groups
4.8 Preparation and Administration of Study Drug
Study drug will be dispensed to the subject at study visits or shipped bv the 
Investigational Pharmacy to the participant to arrive at the scheduled visit date  To be 
taken orally by the subject at home. Subjects will be instructed to mix the study drug 
powder with a liquid or food.
4.9 Subject Compliance Monitoring
Subjects  will have the opportunity to use study-provideddrug diaries in effort to improve 
compliance and provide assistance to study staff in compliance monitoring. This is an 
optional task for the subject, and some may choose not to participate. Those who do will 
be asked to bring their drug diaries, and used and unused foil packages with them to 
each study visit for the study coordinator to review and log reported compliance into the 
CRF. For subjects who do not choose to utilize a drug diary, accountability and 
compliance will occur by counting the amount of empty packets returned and the 
comparing it to the number of doses that should have been taken. 
4.10 Prior and Concomitant Therapy
Information regarding a subject’s current medication regimen will be collected to assist 
with safety monitoring and for data collection purposes. Patients will be encouraged to 
follow their physician instructions in taking Riluzole and Earavone/Radicava at the same 
dose if they have already been taking these medications.  If the patient has been 
instructed by his or her physician to stop or pause Riluzole or Edaravone/Radicava due 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
20
to an adverse event the requirement for inclusion into the study will be off Riluzole or 
Edaravone/Radicava for seven (7)  days,  This also applies if the dosing has not been 
stable for three (3) months prior to enrollment and treatment.  Initiating or restarting 
these medications after enrollment and treatment has begun is allowed.  Patients will 
record medication, doses, and any changes to the dosing in their provided diaries. This 
information will be confirmed with the patient by the study coordinator during Visit 1, 2, 
and/or 3 as well as phone visits and then will be entered into the CRF for data collection 
and analysis. 
4.11 Receiving, Storage, Dispensing, and Return
4.11.1 Receipt of Drug Supplies
The L-serine will be shipped directly from Knechtel to Dartmouth-Hitchcock Medical 
Center in batch shipments. Upon receipt of the study treatment supplies by the receiving 
pharmacy, the designated study staff will count and verify that the shipment contains all 
the items noted in the shipment inventory.  Any damaged or unusable study drug in a 
given shipment will be documented in the study files. The investigator will notify the 
Brain Chemistry Labs of any damaged or unusable study treatments that were supplied 
to the site.
4.11.2 Storage
L-serine foil packages should be kept dry location at 10-20 degrees C during storage. 
Patients should be advised to keep their monthly packets in a cool, dry location. 
4.11.3 Dispensing of Study Drug
Each subject will be given foil packages that contain the correct dose (15 g) to be taken 
once a day during the initial two-week period, and twice a day thereafter. Subjects will be 
given at the baseline visit the appropriate number of packages to last three months, plus 
one week (186 packages). At the 3-month visit, any unused drug will be collected, as well 
as the used foil packets to check study drug compliance. Subjects will be given the 
appropriate number of packages to last three months (186 packages). At the 6-month 
visit any unused study drug and used foil packets will be collected by study staff. If the 
subject is unable to attend an in person study visit due to Covid19 or any other 
widespread illness visit restrictions,  the study drug will be shipped to the subject to be 
received at the scheduled study visit date.  The subject will receive notification that the 
study drug will be shipped, and may includea new diary and detailed instructions, 
depending on if the subject is using the study-provided diary  The return of the used 
packets and unused medication will be brought in by the subject at the next in person 
visit or mailed to the study team by the subject.
Patients should return any unused packets and report compliance during study visits. A 
log of study drug issued and study drug returned with date of issue and return will be 
kept by study staff 
4.11.4 Return or Destruction of Study Drug
At the completion of the trial, a final reconciliation of all study drug shipped, consumed 
and remaining will be performed. All remaining drug will be returned to the Brain 
Chemistry Labs, PO Box 3464, Jackson, WY 83001. 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
21
5
Study Procedures
5.1 Visits and telephone calls
The study timeline includes three in-clinic visits as well as five follow-up phone calls, 
with one occurring 6 months after treatment completion (month 12). The study visits 
and timeline are as follows with a -/+ 2 week window  Visits that cannot be attended by 
the subject due to restrictions will be done by telephone by the investigator and the study 
coordinator to obtain information from  the subject.  Assessments that cannot be done 
remotely, will be obtained at the next in person visit.
Visit 1: Baseline: Screening and Enrollment 
Informed consent obtained
Inclusion /Exclusion Criteria
Demographics
Vital signs (blood pressure, heart rate, temperature, respiratory rate, height, weight, 
pulse oximetry)
Brief medical history and neurological and brief physical exam
Concomitant medications
ALSFRS-R
ALSA-Q40
Pulmonary Function - FVC % predicted
Blood draw
Urine pregnancy test
Dispense study drug
Dispense study drug diary (optional)
Dispense instructions for dosing and storage of L-Serine
Saliva sample (optional, and may be obtained at any visit)
C-SSRS
Telephone Call 1: Week 3 (+ or – 2 weeks)
Adverse events
Concomitant medications
L-serine tolerability and dosing
Telephone Call 2: Week 6 (+ or – 2s weeks)
Adverse events
Concomitant medications
L-serine tolerability and dosing
Visit 2: 3 month visit (+ or – 2 weeks)
Vital signs 
Neurological and brief physical exam
ALSFRS-R
ALSA-Q40
FVC % predicted


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
22
Concomitant medications
Adverse events
Blood draw
Urine pregnancy test
Collection of unused study drug and diary (if applicable)
Dispense study drug
Dispense study drug diary (optional)
Saliva sample (optional, and may be obtained at any visit)
C-SSRS
Telephone Call 3: Week 18 (+ or – 2 weeks)
Adverse events
Concomitant medications
L-serine tolerability and dosing
Visit 3: 6 month visit (+ or – 2weeks)
Vital signs
Neurological and brief physical exam
ALSFRS-R
ALSA-Q40
FVC % predicted
Concomitant medications
Adverse events
Blood draw
Urine pregnancy test
Collection of unused study drug and diary (if applicable)
Saliva sample (optional, and may be obtained at any visit)
C-SSRS
Telephone Call 4: Week 28 (+ or – 2 weeks)
Adverse events
Concomitant medications
Continued supplementation status
Telephone Call 5: Week 48 (+ or – 2 week)
Adverse events
Concomitant medications
Continued supplementation status
ALSFRS-R 
Overall study impression


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
23
5.1.1Study Timeline and Events (+ / - 2 week for each visit/phone call)
Visit / Telephone
Visit 1
Telephone 
1
Telephone
2
Visit 2
3 
Months
Telephone 
3
Visit 3
6 
Months 
Treatment 
ends
Telephone 
4
Telephone 
5
Week
Week 
0
Week 3
Week 6
Week12
Week 18
Week 24
Week 28
Week 48
Informed Consent
X
Inclusion/ 
Exclusion 
Criteria 
X
Demographics
X
Brief Medical History
X
Concomitant 
Medications 
X
X
X
X
X
X
X
X
Adverse Events
X
X
X
X
X
X
X
Brief Physical Exam
X
X
X
Neurological Exam
X
X
X
Vital 
Signs- 
weight, 
height, 
temperature, 
blood pressure, pulse, 
respiratory rate, pulse 
oximetry 
X
X
X
Pulmonary 
Function 
(FVC % predicted) 
X
X
X
ALSFRS-R
X
X
X
X
ALSA-Q40
X
X
X
C-SSRS
X
X
X
Optional Saliva Sample
X
X
X
Blood samples drawn
X
X
X
Safety labs (CBC, CMP)
X
X
X
Serum pregnancy test
X
Urine pregnancy test
X
X
X
Saliva Sample (optional)
X
X
X
Dispense study drug 
X
X
Dispense 
study 
drug 
diary 
(optional) 
and 
information 
X
X
Collection of diary and 
unused 
study 
drug 
(optional)
X
X
Study drug compliance 
X
X
L-serine tolerability
X
X
X
X
X
Continued 
Supplementation Status 
X
X
Study Impression 
X


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
24
5.2 Blood Draws and Specimen Processing, Storage, and Shipment
Safety labs will be drawn at three time points: Visit 1 (baseline: screening/enrollment), 
Visit 2, and Visit 3.  Safety labs, sent to DHMC Pathology laboratory will include:
Complete Blood Count
Comprehensive Metabolic Panel
Safety labs may be repeated at the discretion of the investigator
Serum pregnancy test to be done at Visit 1 (screening)
Urine pregnancy testing will be done at Visit 1 (screening), Visit 2 and Visit 3.  Blood 
draw and urine pregnancy tests will be done at next in person visit if the subject is unable 
to attend the visit due to restrictions.  Urine pregnancy testing, if required, will be 
performed at home by the subject with study dispensed urine pregnancy tests.  The 
coordinator will call the subject as a reminder to do the test at  the visit date, and will 
also document in source the result as reported by the subject.
In addition, concurrent with the safety labs, blood samples will be drawn and sent to the 
Brain Chemistry Labs for analysis of L-serine concentrations and neurally-derived 
exosomes at three time points (Visit 1 (baseline/enrollment), Visit 2, and Visit 3).
Optional saliva samples can be obtained once during the study at any visit, and will be 
sent to the Brain Chemistry Labs where they will be stored for batch genetic sequencing 
as indicated in Section 2.5. 
5.3 Optional Consent for Saliva Sample, Biobanking, and Future Analysis of 
Blood Samples
There will be an option in the informed consent document for a saliva sample to be 
obtained to look for polymorphisms in key human serine synthetic genes and for patients 
to have a portion of their blood sample drawn to be de-identified, saved, and used for 
future research by the Brain Chemistry Labs.  The researchers at Brain Chemistry Labs 
wish to use the optional donations for future ALS research. This may help researchers 
learn about factors that can help prevent or promote disease in the future. No results will 
be reported back the patient.  No extra blood is required for the blood donation.  Opting 
in is not required for study participation. 
5.4 Administration of the Columbia Suicide Severity Rating Scale (C-SSRS)
The Columbia Suicide Severity Rating Scale (C-SSRS) will be administered at Visits 1, 2, 
and 3.  The investigator will review answers given by the patient before the end of the 
visit in order to identify risk as presented with this evidence based tool.  Any score 
greater than 0 may indicate the need for mental health intervention as decided by the 
investigator.  A score of 4 (active suicidal ideation with some intent to act) or 5 (active 
suicide ideation with specific plan and intent) will be used to trigger further evaluation or 
immediate contact with a prompt referral to a mental health professional and/or 
possibly the emergency department.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
25
6
Statistical Plan
6.1 Sample Size Determination
Anticipating that patients may drop out of the study before the six month visit for 
various reasons, which we conservatively estimate at 20%, or 9 patients, enrollment of 
n=50 patients will be adequate to provide the needed statistical power for data analysis. 
With n=36 participants completing the full six month study visit in the L-serine group, 
we expect to have 80% power to detect a meaningful difference in functional decline, 
defined as a difference in mean ALSFRS-R slope of 0.43 between the L-serine treatment 
and historical placebo control groups (ratio 10 placebo patients: 1 L-serine patient, 
standard deviation (SD) = 0.88, alpha = 0.05). However, as noted below in 6.4, we 
reserve the right to terminate the trial at n = 30 participants completing the full six-
month trial in the L-serine group if it is statistically determined that the trial is either 
futile or efficacious. 
6.2 Statistical Methods
The primary outcome of the study is tolerability of the L-serine oral formulation. We will 
assess the proportion of patients in the treatment group who: report experiencing GI 
symptoms on the 15 g/day dose, report experiencing GI symptoms on the 30 g/day dose, 
or withdraw prior to the 6-month visit.  
 
We will begin by assessing the comparability of the baseline functional status of the 
patients by comparing symptom duration, ALSFRS-R scores, and FVC measures for the 
historical placebo group vs. the L-serine treatment group at time zero using a t-test. If 
the data are not normally distributed, we will transform the data or use a non-parametric 
statistical analysis.
 
We will then longitudinally assess the declining slopes of the ALSFRS-R scores 
throughout the study period by constructing linear mixed-models to analyze repeated 
measures over the ALSFRS-R assessment time points. We will assess the between-
patient effect of treatment. Our null hypothesis is that there is no difference between the 
mean ALSFRS-R slopes for the fixed-effect of the historical placebo vs. L-serine 
treatment. The models will include individual random-effects to account for time-
invariant factors related to genetic susceptibility, and other unobservable individual 
confounders. Models will be adjusted for differences in baseline ALSFRS-R or FVC and 
symptom duration, as needed. In addition, we will assess trends in the within-patient 
effect of L-serine treatment on ALSFRS-R scores, FVC%, as well as the exosomal 
biomarkers compared to the baseline visit.   
6.3 Subject Population(s) for Analysis
The primary study analysis and any applicable secondary analyses will include any 
subject enrolled into the study who has received at least one treatment dose.
6.4 Efficacy/Futility May Result in Early Termination of the Study
In 2010, the FDA issued a draft Guidance for Industry entitled, “Adaptive Design Clinical 
Trials for Drugs and Biologics.” While carefully considering the limitations of adaptive 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
26
designs, including potential to increase the chance of erroneous positive conclusions, 
and loss of study power in blinded interim analyses, the FDA noted in Section V.D. that 
“Methods have been developed that allow valid analyses of interim data and provide 
well-recognized alpha spending approaches to address the control of the Type I error 
rate . . . If the drug is more effective than expected, the accumulating data can offer 
strong statistical evidence of the therapy’s success well in advance of the planned 
completion of the study.” 
One of the obvious benefits of an adaptive design is that not only can a trial be stopped 
on early indications of efficacy or futility, but less patients are needed to complete the 
trial as compared to a fixed sample design. Although there has been some consideration 
given to sequential monitoring of patients in a single-arm trial (Thall et al., 1995), with 
the benefit of data from the Phase I clinical trial of L-serine for ALS patients, we have 
been able to estimate the minimal sample size necessary to replicate preliminary 
indications of efficacy if we have in the Phase IIa trial n ≥ 27 patients, based on the 
differences between slopes of the ALSFRS-R scores of the low-dose patients (1 g/day) 
and the high-dose patients (30 g/day) in the Phase I trial. Since we propose an open 
label single-arm Phase IIa trial using data from historical placebo control patients, we 
will make an initial analysis of the secondary objective after the 30th patient finishes the 
six-month trial. If the magnitude of efficacy observed in the Phase I trial is replicated, or 
if no efficacy whatsoever is detected, we may choose to terminate the Phase IIa trial 
without continuing to the planned 50 patients. However, if there are preliminary 
indications of efficacy that do not rise to the level of replication of the possible efficacy 
detected in the Phase I trial, or if the analysis of biomarkers which constitutes the 
tertiary objective appears productive, we may choose to continue the trial to the full n = 
50 patient design.
7
Safety and Adverse Events
Information obtained from any standard of care visits as well as study visits and 
telephone calls will be reviewed for adverse events.  Adverse events will be recorded in 
the participants study file and CRF, along with date of occurrence, date of resolution, 
severity and any actions taken. 
7.1
Definitions
Unanticipated Problems Involving Risk to Subjects or Others
Any incident, experience, or outcome that meets all of the following criteria: 
Unexpected in nature, severity, or frequency (i.e. not described in study-related 
documents such as the IRB-approved protocol or consent form, the investigators 
brochure, etc.)
Related or possibly related to participation in the research (i.e. possibly related 
means there is a reasonable possibility that the incident experience, or outcome 
may have been caused by the procedures involved in the research)
Suggests that the research places subjects or others at greater risk of harm 
(including physical, psychological, economic, or social harm).


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
27
Adverse Event
An adverse event (AE) is any symptom, sign, illness or experience that develops or 
worsens in severity during the course of the study.  Intercurrent illnesses or injuries 
should be regarded as adverse events.  Abnormal results of diagnostic procedures are 
considered to be adverse events if the abnormality:
results in study withdrawal
is associated with a serious adverse event
is associated with clinical signs or symptoms
leads to additional treatment or to further diagnostic tests
is considered by the investigator to be of clinical significance
Serious Adverse Event
Adverse events are classified as serious or non-serious.  A serious adverse event is 
any AE that is: 
fatal
life-threatening
requires or prolongs hospital stay
results in persistent or significant disability or incapacity
a congenital anomaly or birth defect
an important medical event
Important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance.   They may jeopardize the subject, and may require 
intervention to prevent one of the other serious outcomes noted above.  For example, 
drug overdose or abuse, a seizure that did not result in in-patient hospitalization or 
intensive treatment of bronchospasm in an emergency department would typically be 
considered serious. 
All adverse events that do not meet any of the criteria for serious should be regarded as 
non-serious adverse events. 
Adverse Event Reporting Period
The study period during which adverse events must be reported is normally defined as 
the period from the initiation of any study procedures to the end of the study treatment 
follow-up.  For this study, the study treatment follow-up is defined as 30 days following 
the last administration of study treatment.
Preexisting Condition
A preexisting condition is one that is present at the start of the study.  A preexisting 
condition should be recorded as an adverse event if the frequency, intensity, or the 
character of the condition worsens during the study period. 
General Physical Examination Findings
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.  


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
28
Post-study Adverse Event
At the last scheduled visit, the investigator should instruct each subject to report any 
subsequent event(s) that the subject, or the subject’s personal physician, believes might 
reasonably be related to participation in this study.  The investigator should notify the 
study sponsor of any death or adverse event occurring at any time after a subject has 
discontinued or terminated study participation that may reasonably be related to this 
study.  The sponsor should also be notified if the investigator should become aware of 
the development of cancer or of a congenital anomaly in a subsequently conceived 
offspring of a subject that has participated in this study. 
Abnormal Laboratory Values
The PI will make the determination of clinical significance of lab values that are out of 
normal range. Those of clinical significance will be reported as adverse events.
Hospitalization, Prolonged Hospitalization or Surgery
Any adverse event that results in hospitalization or prolonged hospitalization should be 
documented and reported as a serious adverse event unless specifically instructed 
otherwise in this protocol.  Any condition responsible for surgery should be documented 
as an adverse event if the condition meets the criteria for an adverse event.
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are 
reported as an adverse event in the following circumstances:
Hospitalization or prolonged hospitalization for diagnostic or elective surgical 
procedures for a preexisting condition.  Surgery should not be reported as an 
outcome of an adverse event if the purpose of the surgery was elective or 
diagnostic and the outcome was uneventful.
Hospitalization or prolonged hospitalization required to allow efficacy 
measurement for the study.
Hospitalization or prolonged hospitalization for therapy of the target disease of 
the study, unless it is a worsening or increase in frequency of hospital admissions 
as judged by the clinical investigator.
7.2
Recording of Adverse Events
At each contact with the subject, the investigator must seek information on adverse 
events by specific questioning and, as appropriate, by examination.  Information on all 
adverse events should be recorded immediately in the source document, and also in the 
appropriate adverse event module of the case report form (CRF).  All clearly related 
signs, symptoms, and abnormal diagnostic procedures results should recorded in the 
source document, though should be grouped under one diagnosis.
7.3
Reporting of Serious Adverse Events and Unanticipated Problems
Investigators and the protocol sponsor must conform to the adverse event reporting 
timelines, formats and requirements of the various entities to which they are 
responsible, but at a minimum those events that must be reported are those that are:


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
29
related to study participation,
unexpected, and 
serious or involve risks to subjects or others 
If the report is supplied as a narrative, the minimum necessary information to be 
provided at the time of the initial report includes:
Study identifier
Study Center
Subject number
A description of the event
Date of onset
Current status
Whether study treatment was discontinued
The reason why the event is classified as 
serious
Investigator assessment of the association 
between the event and study treatment
7.3.1
Investigator reporting: notifying the Dartmouth - HitchcockIRB
Adverse events and unanticipated problems posing risks to subjects or will be reported to 
the Dartmouth- Hitchcock IRB as required by the DHH- IRB, within the required 
timeframe and using the appropriate form or as a written report of the event. 
Copies of each report and documentation of IRB notification and receipt will be kept in 
the Clinical Investigator’s study file.
7.4
Unblinding Procedures
All patients receive the same dose, therefore no blinding is necessary.
7.5
Medical Monitoring
It is the responsibility of the Principal Investigator to oversee the safety of the study at 
his/her site.  This safety monitoring will include careful assessment and appropriate 
reporting of adverse events. In addition to PI oversight, a medical monitor external to 
the study will be identified. Medical monitoring will include a regular assessment of the 
number and type of serious adverse events.
Currently, the Medical Monitor is Dr. Victoria Lawson, MD, Neurologist at 
Dartmouth Hitchcock Medical Center. Dr. Lawson is not involved in the 
study in any way other than as a medical monitor. 
This monitoring will take place once every year, and will include a thorough review of the 
annual report, which includes enrollment numbers, withdrawals, unexpected and 
expected adverse events, and deaths. Each annual report will also include a passage 
about the study, including length and procedures performed. 
After completion of the monitoring review, the monitor will sign off on her review, and 
signal if a follow-up discussion is required. This form will be signed by both the medical 
monitor and by the study PI. 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
30
8
Data Handling and Record Keeping
8.1
Confidentiality
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 
(HIPAA).  Information obtained during prescreening also applies to the Health 
Insurance Portability and Accounatabily Act of 1996.  No PHI used for prescreening will 
be released for presentation or publication. 
Those regulations require a signed subject authorization informing the subject of the 
following: 
What protected health information (PHI) will be collected from subjects in this study
Who will have access to that information and why
Who will use or disclose that information
The rights of a research subject to revoke their authorization for use of their PHI. 
In the event that a subject revokes authorization to collect or use PHI, the investigator, 
by regulation, retains the ability to use all information collected prior to the revocation of 
subject authorization.  For subjects that have revoked authorization to collect or use PHI, 
attempts will be made to obtain permission to collect at least vital status (i.e. that the 
subject is alive) at the end of their scheduled study period.
8.2
Source Documents
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  
Source data are contained in source documents.  Examples of these original documents, 
and data records include: hospital records, clinical and office charts, laboratory notes, 
memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing records, 
recorded data from automated instruments, copies or transcriptions certified after 
verification as being accurate and complete, microfiches, photographic negatives, 
microfilm or magnetic media, x-rays, subject files, and records kept at the pharmacy, at 
the laboratories, and at medico-technical departments involved in the clinical trial.
8.3
Case Report Forms
The study case report form (CRF) is the primary data collection instrument for this 
study.  All data requested on the CRF must be recorded.  All missing data must be 
explained.  If a space on the CRF is left blank because the procedure was not done or the 
question was not asked a written notation will be made. If an item is not applicable to the 
individual case, written notation will be made.  
8.4
Records Retention
It is the investigator’s responsibility to retain study essential documents for at least 2 
years after the last approval of a marketing application in their country and until there 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
31
are no pending or contemplated marketing applications in their country or at least 2 
years have elapsed since the formal discontinuation of clinical development of the 
investigational product.  These documents should be retained for a longer period if 
required by an agreement with the sponsor.  In such an instance, it is the responsibility 
of the sponsor to inform the investigator/institution as to when these documents no 
longer need to be retained. 
9
Study Monitoring, Auditing, and Inspecting
9.1
Study Monitoring Plan
The Clinical Trials Office will be monitoring this study.  The investigator will allocate 
adequate time for monitoring activities which includes but is not limited to reviewing 
source documents and case report forms; checking for study medication accountability 
and study drug dispensation documentation; reviewing regulatory documents and the 
protocol.  The Investigator will also ensure that the monitor or other compliance or 
quality assurance reviewer is given access to all the above noted study-related documents 
and study related facilities (e.g. pharmacy, diagnostic laboratory, etc.), and has adequate 
space to conduct the monitoring visit.
9.2
Auditing and Inspecting
The study investigator will permit study-related monitoring, audits, and inspections by 
the IRB, the sponsor, government regulatory bodies, and Dartmouth compliance and 
quality assurance groups of all study related documents (e.g. source documents, 
regulatory documents, data collection instruments, study data etc.).  The study 
investigator will ensure the capability for inspections of applicable study-related facilities 
(e.g. pharmacy, diagnostic laboratory, etc.).
Participation as an investigator in this study implies acceptance of potential inspection 
by government regulatory authorities and applicable Dartmouth compliance and quality 
assurance offices.
10 Ethical Considerations
This study is to be conducted according to US and international standards of Good 
Clinical Practice (FDA Title 21 part 312 and International Conference on Harmonization 
guidelines), applicable government regulations and Institutional research policies and 
procedures.
10.1 Funding Source
This study will be funded by the Brain Chemistry Labs at the Institute for 
Ethnomedicine, a not-for-profit 501(c)(3) charity in Jackson Hole, Wyoming. 
10.2 Conflict of Interest
All Dartmouth investigators will follow the Dartmouth conflict of interest policy. The 
Brain Chemistry Labs has applied for patents for the use of L-serine to treat 
neurodegenerative illness (US 13/683,821).


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
32
10.3 Subject Stipends or Payments
Patients will not be paid, but will be given a travel stipend and/or allowance, and as 
needed, overnight accommodations near Dartmouth-Hitchcock to participate in clinical 
visits.
10.4 Publication Plan
Publication of results will be the responsibility of the Brain Chemistry Labs in 
conjunction with Dartmouth-Hitchcock Medical Center. 
11
References
Abe K, Itoyama Y, Sobue G, Tsuji S, Aoki M, Doyu M, Hamada C, Kondo K, Yoneoka T, 
Akimoto M, Yoshino H. Confirmatory double-blind, parallel-group, placebo-
controlled study of efficacy and safety of edaravone (MCI-186) in amyotrophic lateral 
sclerosis patients. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration. 
2014 Dec 1;15(7-8):610-7.
Andrew AS, Caller TA, Tandan R, Duell EJ, Henegan PL, Field NC, Bradley WG, 
Stommel EW. Environmental and Occupational Exposures and Amyotrophic Lateral 
Sclerosis in New England. Neurodegenerative Diseases. 2017;17(2-3):110-6.
Anonymous. Guidance FD. Adaptive design clinical trials for drugs and biologics. 
Biotechnol Law Rep. 2010;29(2):173.
Banack SA, Murch SJ, Cox PA. Neurotoxic flying foxes as dietary items for the Chamorro 
people, Marianas Islands. Journal of ethnopharmacology. 2006;106(1):97-104.
Braak H, Braak E. Staging of Alzheimer's disease-related neurofibrillary changes. 
Neurobiol. Aging, 1995. 16, 271-278.  
Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the 
diagnosis of amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis and other 
motor neuron disorders. 2000 Jan 1;1(5):293-9.
Caller TA, Andrews A, Field NC, Henegan PL, Stommel EW. The epidemiology of 
amyotrophic lateral sclerosis in New Hampshire, USA, 2004-2007. 
Neurodegenerative Diseases. 2015;15(4):202-6.
Canu N, Ciotti MT, Pollegioni L (2014) Serine racemase: a key player in apoptosis and 
necrosis. Front Synaptic Neurosci 6:1–15.
Catsman-Berrevoets C, deKlerk J, Huymans J et al (1997) Inborn error of serine 
biosynthesis, a new phenotype. Eur J Paediatr Neurol 1:A43.
Cedarbaum JM, Stambler N, Malta E, Fuller C, Hilt D, Thurmond B, Nakanishi A, Bdnf 
Als Study Group, 1A complete listing of the BDNF Study Group. The ALSFRS-R: a 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
33
revised ALS functional rating scale that incorporates assessments of respiratory 
function. Journal of the neurological sciences. 1999 Oct 31;169(1):13-21.
Cox PA, Banack SA, Murch SJ. Biomagnification of cyanobacterial neurotoxins and 
neurodegenerative disease among the Chamorro people of Guam. Proceedings of the 
National Academy of Sciences. 2003 Nov 11;100(23):13380-3.
Cox PA, Davis DA, Mash DC, Metcalf JS, Banack SA. Dietary exposure to an 
environmental toxin triggers neurofibrillary tangles and amyloid deposits in the 
brain. Proceedings of the Royal Society B. 2016. 283: 20152397. 
Davis DA, Donne AD, Banack SA, Palmour R, Bradley WG, Cox PA, Mash DC, 
Neuroinflammation in vervet brains following chronic dietary exposures to a 
cyanobacterial toxin. Neuroscience 2016: Poster 46.19 / Q5, November 12, 2016.
De Koning TJ. Treatment with amino acids in serine deficiency disorders. J Inherit 
Metab Dis 2006;29:347–351.
Desai JR, Bowen EA, Danielson MM, Allam RR, Cantor MN. Creation and 
implementation of a historical controls database from randomized clinical trials. 
Journal of the American Medical Informatics Association. 2013 Feb 28;20(e1):e162-
8.
Dunlop RA, Cox PA, Banack SA, Rodgers KJ. The Non-Protein Amino Acid BMAA Is 
Misincorporated into Human Proteins in Place of L-Serine Causing Protein 
Misfolding and Aggregation. PLOS ONE 2013.8, e75376. 
Dunlop RA, Powell J, Guillemin GJ, Cox PA. 2017. Mechanisms of L-Serine 
Neuroprotection in vitro Include ER Proteostasis Regulation. Neurotoxicity 
Research. 2017 Nov 2:1-0.
Dunlop RA, Powell JT, Metcalf JS, Guillemin GJ, Cox, PA. 2017. L-Serine-Mediated 
Neuroprotection Includes the Upregulation of the ER Stress Chaperone Protein 
Disulfide Isomerase (PDI). Neurotoxicity Research DOI: 10.1007/s12640-017-9817-7
Fiandaca, MS, Kapogiannis D, Mapstone M, Boxer A, Eitan E, Schwarz JB, Abner EL, 
Petersen RC, Fegeroff HJ, Miller BL, Goetzl EJ. Identification of prelincial 
Alzheimer's disease by a profile of pathogenic proteins in neurally derived blood 
exosomes: A case-control study. Alzheimer's & Dementia 2015. 11:600-607.
Garofalo K, Penno A, Schmidt BP, Lee HJ, Frosch MP, von Eckardstein A, Brown RH, 
Hornemann T, Eichler FS. Oral L-serine supplementation reduces production of 
neurotoxic deoxysphingolipids in mice and humans with hereditary sensory 
autonomic neuropathy type 1. The Journal of clinical investigation. 2011 Dec 
1;121(12):4735.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
34
Goetzl EJ, Boxer A, Schwartz JB, Abner EL, Petersen RC, Miller BL, Kapogiannis D. 
Altered lysosomal proteins in neural-derived plasma exosomes in preclinical 
Alzheimer disease. Neurology. 2015 Jul 7;85(1):40-7.
Hirtz D, Thurman DJ, Gwinn-Hardy K, Mohamed M, Chaudhuri AR, Zalutsky R. How 
common are the “common” neurologic disorders? Neurology. 2007 Jan 
30;68(5):326-37.
Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, Velde CV, Bouchard JP, 
Lacomblez L, Pochigaeva K, Salachas F, Pradat PF. TARDBP mutations in 
individuals with sporadic and familial amyotrophic lateral sclerosis. Nature genetics. 
2008 May 1;40(5):572-4.
Kaufmann P, Levy G, Thompson JL, Delbene ML, Battista V, Gordon PH, Rowland LP, 
Levin B, Mitsumoto H. The ALSFRSr predicts survival time in an ALS clinic 
population. Neurology. 2005 Jan 11;64(1):38-43.
Levine TD, Miller RG, Bradley WG, Moore DH, Saperstein DS, Flynn LE, Katz JS, 
Forshew DA, Metcalf JS, Banack SA, Cox PA. Phase I clinical trial of safety of L-
serine for ALS patients. Amyotrophic Lateral Sclerosis and Frontotemporal 
Degeneration. 2017 Jan 2;18(1-2):107-11.
Logroscino G, Traynor BJ, Hardiman O, Chiò A, Mitchell D, Swingler RJ, Millul A, Benn 
E, Beghi E. Incidence of amyotrophic lateral sclerosis in Europe. Journal of 
Neurology, Neurosurgery & Psychiatry. 2010 Apr 1;81(4):385-90.
Mehta P, Kaye W, Bryan L, Larson TC, Copeland T, Wu J, Muravov O, Horton K. 
Prevalence of amyotrophic lateral sclerosis—United States, 2012–2013. Surveillance 
Summaries 65(8).
Metcalf JS, Dunlop RA, Powell JT, Banack SA, Cox PA. 2017. L-Serine: a Naturally-
Occurring Amino Acid with Therapeutic Potential. Neurotoxicity Research, Sep 19:1-
9.
Metcalf JS, Lobner D, Banack SA, Cox GA, Nunn PB, Wyatt PB, Cox PA. 2017. Analysis of 
BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and 
toxicity of D-BMAA. Amino Acids 2017: 1-3. DOI: 10.1007/x00726-017-2445-y.
Miller RG, Gelinas D, O’Connor P. Amyotrophic Lateral Sclerosis. American Academy of 
Neurology, Demos Medical Publishing: New York, NY, 2005.
Murch SJ, Cox PA, Banack SA. A mechanism for slow release of biomagnified 
cyanobacterial neurotoxins and neurodegenerative disease in Guam. Proceedings of 
the National Academy of Sciences of the United States of America. 2004 Aug 
17;101(33):12228-31.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
35
Phukan J, Elamin M, Bede P, Jordan N, Gallagher L, Byrne S, Lynch C, Pender N, 
Hardiman O. The syndrome of cognitive impairment in amyotrophic lateral sclerosis: 
a population-based study. Journal of Neurology, Neurosurgery & Psychiatry. 2011 
Jan 1:jnnp-2011.
Polman CH, Reingold SC, Barkhof F, Calabresi PA, Clanet M, Cohen JA, Cutter GR, 
Freedman MS, Kappos L, Lublin FD, McFarland HF. Ethics of placebo-controlled 
clinical trials in multiple sclerosis A reassessment. Neurology. 2008 Mar 25;70(13 
Part 2):1134-40.
Ringholz GM, Appel SH, Bradshaw M, Cooke NA, Mosnik DM, Schulz PE. Prevalence 
and patterns of cognitive impairment in sporadic ALS. Neurology. 2005 Aug 
23;65(4):586-90.
Spławiński J, Kuźniar J. Clinical trials: active control vs placebo—what is ethical? 
Science and engineering ethics. 2004 Mar 1;10(1):73-9.
Thall PF, Simon RM, Estey EH. Bayesian sequential monitoring designs for single‐arm 
clinical trials with multiple outcomes. Statistics in medicine. 1995 Feb 28;14(4):357-
79.
Thompson AG, Gray E, Heman-Ackah SM, Mäger I, Talbot K, El Andaloussi S, Wood 
MJ, Turner MR. Extracellular vesicles in neurodegenerative disease—pathogenesis to 
biomarkers. Nature reviews. Neurology. 2016 Jun 1;12(6):346.
Viele K, Berry S, Neuenschwander B, Amzal B, Chen F, Enas N, Hobbs B, Ibrahim JG, 
Kinnersley N, Lindborg S, Micallef S. Use of historical control data for assessing 
treatment effects in clinical trials. Pharmaceutical statistics. 2014 Jan 1;13(1):41-54.
Wheaton MW, Salamone AR, Mosnik DM, McDonald RO, Appel SH, Schmolck HI, 
Ringholz GM, Schulz PE. Cognitive impairment in familial ALS. Neurology. 2007 Oct 
2;69(14):1411-7.
Williams LS. Randomized controlled trials: methodology, outcomes, and interpretation. 
In: Biller J and Bogousslavsky J, eds, Clinical Trials in Neurological Practice, Blue 
Books of Practical Neurology. 2001 Dec 31;25:1-26.
Winston CN, Goetzl EJ, Akers JC, Carter BS, Rockenstein EM, Galasko D, Masliah E, 
Rissman RA. Prediction of conversion from mild cognitive impairment to dementia 
with neuronally derived blood exosome protein profile. Alzheimer's & Dementia: 
Diagnosis, Assessment & Disease Monitoring. 2016 Dec 31;3:63-72.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
36
12
Appendices
Appendix 1. ALS Functional Rating Scale - Revised (ALSFRS-R)
Appendix 2. ALSA – Q40
Appendix 3. C-SSRS


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
37
L-serine IIa in 
ALS
Patient Number: 
_________
Visit: __________
Date: _________
ALS FUNCTIONAL RATING SCALE (ALSFRS-R)
4
Normal Speech processes
3
Detectable speech with disturbances
2
Intelligible with repeating
1
Speech combined with nonvocal communication
1. Speech
0
Loss of useful speech
4
Normal
3
Slight but definite excess of saliva in mouth; may have nighttime 
drooling
2
Moderately excessive saliva; may have minimal drooling
1
Marked excess of saliva with some drooling
2. Salivation
0
Marked drooling; requires constant tissue or handkerchief
4
Normal eating habits
3
Early eating problems – occasional choking
2
Dietary consistency changes
1
Needs supplemental tube feeding 
3. Swallowing
0
NPO (exclusively parenteral or enteral feeding)
4
Normal 
3
Slow or sloppy; all words are legible
2
Not all words are legible
1
Able to grip pen but unable to write
4. Handwriting
0
Unable to grip pen
4
Normal
3
Somewhat slow and clumsy, but no help needed
2
Can cut most foods, although clumsy and slow; some help 
needed
1
Food must be cut by someone, but can still feed slowly
5a. Cutting Food and 
Handling 
Utensils 
(patients 
without 
gastrostomy)
0
Needs to be fed
4
Normal 
3
Clumsy but able to perform all manipulations independently
2
Some help needed with closures and fasteners
1
Provides minimal assistance to caregivers
5b. Cutting Food and 
Handling 
Utensils 
(patients 
with 
gastrostomy)
0
Unable to perform any aspect of task 
4
Normal function
3
Independent and complete self-care with effort or decreased 
efficiency
2
Intermittent assistance or substitute methods
1
Needs attendant for self-care
6. 
Dressing 
and 
Hygiene
0
Total dependence 


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
38
L-serine IIa in 
ALS
Patient Number: 
_________
Visit: __________
Date: _________
4
Normal 
3
Someone slow and clumsy, but no help needed
2
Can turn alone or adjust sheets, but with great difficulty 
1
Can initiate, but not turn or adjust sheets alone 
7. Turning in bed and 
adjusting bed clothes
0
Helpless
4
Normal 
3
Early ambulation difficulties
2
Walks with assistance
1
Nonambulatory functional movement only
8. Walking
0
No purposeful leg movement 
4
Normal 
3
Slow 
2
Mild unsteadiness or fatigue 
1
Needs assistance 
9. Climbing Stairs
0
Cannot do 
4
None 
3
Occurs when walking 
2
Occurs with one or more of the following: eating, bathing, 
dressing 
1
Occurs at rest, difficulty breathing when either sitting or lying 
10. Dyspnea
0
Significant difficulty, considering using mechanical respiratory 
support 
4
None
3
Some difficulty sleeping at night due to shortness of breath, does 
not routinely use more than two pillows 
2
Needs extra pillow in order to sleep (more than two) 
1
Can only sleep sitting up 
11. Orthopnea
0
Unable to sleep 
4
None
3
Intermittent use of NIPPV
2
Continuous use of NIPPV during the night 
1
Continuous use of NIPPV during the night and day 
12.Respiratory   
Insufficiency
0
Invasive mechanical ventilation by intubation or tracheostomy 
Total Score for ASLFRS-R: ____ / 48    
L-serine IIa in 
ALS
Patient Number: 
_________
Visit: __________
Date: _________


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
39
ALSA-Q40
Please complete this questionnaire as soon as possible. If you have any 
difficulties filling in this questionnaire by yourself, please have someone to help 
you. However it is your responses that we are interested in.
The questionnaire consists of a number of statements about difficulties that you 
may have experienced during the last 2 weeks. There are no right or wrong 
answers: your first response is likely to be the most accurate for you. Please check 
the box that best describes your own experience or feelings.
Please answer every question even though some may seem very similar to 
others, or may not seem relevant to you.
All the information you provide is confidential.
The following statements all refer to difficulties that you may have had during 
the last 2 weeks.
Please indicate, by checking the appropriate box, how often the following 
statements have been true for you.
The following statements all refer to certain difficulties that you may 
have had during the last 2 weeks. Please indicate, by checking the 
appropriate box, how often the following statements have been true for 
you.


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
40
If you cannot walk at all
please check Always/cannot walk at all.
How often during the last 2 weeks have the following been true?
Please check one box for each question
Never
Rarely
Sometime
s
Often
Always/ 
cannot 
walk at all
1. Have found it 
difficult 
to 
walk 
short distances, e.g. 
around the house 
☐
☐
☐
☐
☐
2. I have fallen over 
while walking. 
☐
☐
☐
☐
☐
3. I have stumbled or 
tripped 
while 
walking. 
☐
☐
☐
☐
☐
4. I have lost my 
balance 
while 
walking. 
☐
☐
☐
☐
☐
5. I have had to 
concentrate 
while 
walking. 
☐
☐
☐
☐
☐
6. Walking has worn 
me out. 
☐
☐
☐
☐
☐
7. I have had pains in 
my 
legs 
while 
walking. 
☐
☐
☐
☐
☐
8. I have found it 
difficult to go up and 
down the stairs. 
☐
☐
☐
☐
☐
Never
Rarely
Sometimes
Often
Always/ 
cannot 
walk at all
9. I have found it 
difficult to stand up. 
☐
☐
☐
☐
☐


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
41
10. I have found it 
difficult 
to 
move 
from sitting in a 
chair 
to 
standing 
upright. 
☐
☐
☐
☐
☐
11. 
I 
have 
had 
difficulty using my 
arms and hands. 
☐
☐
☐
☐
☐
12. I have found 
turning and moving 
in bed difficult. 
☐
☐
☐
☐
☐
13. 
I 
have 
had 
difficulty 
picking 
things up. 
☐
☐
☐
☐
☐
14. 
I 
have 
had 
difficulty 
holding 
books 
or 
newspapers, 
or 
turning pages. 
☐
☐
☐
☐
☐
15. 
I 
have 
had 
difficulty 
writing 
clearly. 
☐
☐
☐
☐
☐
16. I have found it 
difficult to do jobs 
around the house.
☐
☐
☐
☐
☐
17. I have found it 
difficult 
to 
feed 
myself. 
☐
☐
☐
☐
☐
18. 
I 
have 
had 
difficulty 
combing 
my hair or brushing 
and/or flossing my 
teeth. 
☐
☐
☐
☐
☐
Never
Rarely
Sometimes
Often
Always/ 
cannot 
walk at all
19. 
I 
have 
had 
difficulty 
getting 
dressed. 
☐
☐
☐
☐
☐
20. 
I 
have 
had 
difficulty washing at 
the bathroom sink. 
☐
☐
☐
☐
☐


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
42
21. 
I 
have 
had 
difficulty 
swallowing. 
☐
☐
☐
☐
☐
22. 
I 
have 
had 
difficulty eating solid 
food. 
☐
☐
☐
☐
☐
23. 
I 
have 
had 
difficulty 
drinking 
liquids. 
☐
☐
☐
☐
☐
24. 
I 
have 
had 
difficulty 
participating 
in 
conversations. 
☐
☐
☐
☐
☐
25. I have felt that 
my speech has not 
been 
easy 
to 
understand.
☐
☐
☐
☐
☐
26. I have stuttered 
or 
slurred 
my 
speech. 
☐
☐
☐
☐
☐
27. I have had to talk 
very slowly. 
☐
☐
☐
☐
☐
28. I have talked less 
than I used to do. 
☐
☐
☐
☐
☐
29. 
I 
have 
been 
frustrated with my 
speech. 
☐
☐
☐
☐
☐
30. I have felt self-
conscious about my 
speech. 
☐
☐
☐
☐
☐
31. I have felt lonely. 
☐
☐
☐
☐
☐
Never
Rarely
Sometimes
Often
Always/ 
cannot 
walk at all 
32. 
I 
have 
been 
bored. 
☐
☐
☐
☐
☐
33. 
I 
have 
felt 
embarrassed 
in 
social situations. 
☐
☐
☐
☐
☐
34. 
I 
have 
felt 
hopeless about the 
future. 
☐
☐
☐
☐
☐


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
43
35. I have worried 
that I am a burden to 
other people. 
☐
☐
☐
☐
☐
36. I have wondered 
why I keep going. 
☐
☐
☐
☐
☐
37. I have felt angry 
because 
of 
the 
disease. 
☐
☐
☐
☐
☐
38. 
I 
have 
felt 
depressed. 
☐
☐
☐
☐
☐
39. I have worried 
about 
how 
the 
disease will affect me 
in the future. 
☐
☐
☐
☐
☐
40. I have felt as if I 
have 
lost 
my 
independence. 
☐
☐
☐
☐
☐
Please make sure that you have checked one box for each question
Thank you for completing the questionnaire


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
44


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
45


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
46


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
47


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
48


L-Serine Phase IIa ALS Version: 12Nov2021
CONFIDENTIAL
49