Comprehensive Biomarker Profiling of the IFN-α Pathway in Amyotrophic Lateral Sclerosis Patient Biofluids
Conditions: ALS - Amyotrophic Lateral Sclerosis
Sponsor: Ulysses Neuroscience LTD

 
 
 
 
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CLINICAL STUDY PROTOCOL 
 
 
Study Code: UNL074 
 
Title: Comprehensive Biomarker Profiling of the IFN-α Pathway in Amyotrophic 
Lateral Sclerosis Patient Biofluids 
 
Principal Investigator: Angelo Quattrini 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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CLINICAL STUDY PROTOCOL 
 
Title: Comprehensive Biomarker Profiling of the IFN-α Pathway in Amyotrophic 
Lateral Sclerosis Patient Biofluids 
 
 
 
DISCLAIMER 
This material is strictly private, confidential and property of the Sponsor. Its contents must 
not be disclosed or used except as authorized in writing by the Sponsor.  
 
Study Identifier 
UNL074 
RSO number  
 
Other Protocol Codes 
Not applicable 
Version 
Version 02 
Date 
30 July 2025 
 
 
Sponsor 
Ulysses Neuroscience Ltd 
Pioneer Life Sciences Cherrywood - Building 10 
Cherrywood Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
Authorized Sponsor Representative 
Prof Massimiliano Bianchi  
Ulysses Neuroscience Ltd 
Pioneer Life Sciences Cherrywood - Building 10 
Cherrywood Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
max.bianchi@ulysses-neuro.com 
Funding Source(s) 
Prof Massimiliano Bianchi 
Ulysses Neuroscience Ltd 
Pioneer Life Sciences Cherrywood - Building 10 
Cherrywood Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
max.bianchi@ulysses-neuro.com 
 
Principal Investigator 
Angelo Quattrini, MD. 
UO neurology 
UO Experimental pathology-INSPE 
Tel 02-26435094 
Email: quattrini.angelo@hsr.it 
 

 
 
 
 
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VERSION HISTORY 
 
 
 
 
Protocol 
version n. 
Reason of changes 
Date issued 
01 
First Version 
12/05/2025 
02 
Response to clarifications requested by Ethical Committee 
30/07/2025 

 
 
 
 
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PROTOCOL SIGNATURE PAGE 
 
Study Title: Comprehensive Biomarker Profiling of the IFN-α Pathway in Amyotrophic Lateral Sclerosis 
Patient Biofluids. 
 
Study Identifier: UNL074 
Protocol Version and Date:  
01 – 12/05/2025 
02 – 30/07/2025 
 
The undersigned has read and understood all the aspects of the protocol detailed within this 
document and agrees to supervise and conduct the study in accordance with the protocol, the 
Declaration of Helsinki, and all applicable regulatory requirements. 
 
 
 
     
 
 
Angelo Quattrini  
 
 
 
UO neurology 
UO Experimental 
pathology-INSPE 
 17/07/2025 
Principal Investigator Name 
 
 
Signature 
 
Affiliation 
 
Date 
 
 
 
  
 
 
 
 
Massimiliano Bianchi 
 
 
 
Ulysses Neuroscience 
Ltd 
 
17/07/2025 
Authorized Sponsor 
Representative Name 
 
 
Signature 
 
Affiliation 
 
Date 

 
 
 
 
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TABLE OF CONTENTS 
Please do not edit the table of contents, only automatic button “Summary update”. 
1. 
KEY STUDY CONTACTS .................................................................................................................... 7 
2. ABBREVIATIONS AND DEFINITIONS .................................................................................................... 8 
2.1 Abbreviations ................................................................................................................................ 8 
2.2 Definitions ..................................................................................................................................... 8 
3. 
STUDY DESCRIPTION AND RATIONALE ........................................................................................... 9 
4. 
OBJECTIVES AND ENDPOINTS ...................................................................................................... 10 
5. 
INCLUSION/EXCLUSION CRITERIA ................................................................................................ 10 
a. 
Inclusion Criteria ...................................................................................................................... 10 
b. 
Exclusion Criteria ..................................................................................................................... 10 
6. INFORMED CONSENT ........................................................................................................................ 11 
7. 
STUDY OBJECT .............................................................................................................................. 11 
8. 
SAMPLE SIZE AND SUBJECT IDENTIFICATION ............................................................................... 12 
Sample Size .................................................................................................................................... 13 
Subject Identification ...................................................................................................................... 14 
9. 
STATISTICAL DESIGN ..................................................................................................................... 14 
10. 
DURATION OF THE STUDY, DISCONTINUATION AND WITHDRAWAL ...................................... 15 
a. 
Duration of the Study............................................................................................................... 15 
b. 
Discontinuation And Withdrawal ............................................................................................. 16 
11. 
SAMPLE HANDLING .................................................................................................................. 16 
12. 
DATA MANAGEMENT ............................................................................................................... 16 
a. 
Definition of source data and source documents .................................................................... 16 
b. 
Documentation of data in Case Report Forms (CRFs) ............................................................. 17 
c. 
Data Recording and Record Keeping ....................................................................................... 17 
d. 
Data Protection ........................................................................................................................ 17 
13. ETHICAL AND REGULATORY CONSIDERATION ................................................................................ 17 
13.1 Responsibilities of the Investigators ......................................................................................... 18 
13.2 Ethics Committee (EC) Approvals ............................................................................................. 18 
14. 
GENDER MEDICINE IN RESEARCH PROTOCOL ......................................................................... 18 
15. 
QUALITY ASSURANCE AND CONTROL ...................................................................................... 18 
16. 
END OF CLINICAL STUDY .......................................................................................................... 18 
17. 
INTELLECTUAL PROPERTY ........................................................................................................ 18 

 
 
 
 
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18. 
PUBLICATION POLICY ............................................................................................................... 19 
19.  
 REFERENCES ............................................................................................................................ 19 
20.  
APPENDIX A:  AMENDMENT HISTORY ..................................................................................... 20 
21. APPENDIX B:  LIST OF CLINICAL SERVICES / LABORATORIES ........................................................... 20 
22. APPENDIX C:  LIST OF ENROLLING SITES ......................................................................................... 20 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
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1. KEY STUDY CONTACTS 
Sponsor 
Ulysses-neuroscience Ltd 
Pioneer Life Sciences Cherrywood - Building 10  
Cherrywood Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
Authorized Sponsor 
Representative 
Prof Massimiliano Bianchi - Ulysses-neuroscience Ltd 
Pioneer Life Sciences Cherrywood - Building 10  
Cherrywood Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
Tel +353 87 600 4092 
max.bianchi@ulysses-neuro.com 
Principal Investigator 
Angelo Quattrini, MD. 
UO neurology 
UO Experimental pathology-INSPE 
Tel 02-26435094 
Email: quattrini.angelo@hsr.it 
Study Clinical Unit  
Angelo Quattrini  
0226435094 – quattrini.angelo@hsr.it  
Unit of Experimental Neuropathology Division of Neuroscience  
 
Funding source(s) 
Prof Massimiliano Bianchi - Ulysses-neuroscience Ltd 
Pioneer Life Sciences Cherrywood - Building 10  
Cherrywood Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
Tel +353 87 600 4092 
max.bianchi@ulysses-neuro.com 
Clinical Trial Center 
Email: ctc.firstcontact@hsr.it; ctc.trialstartup@hsr.it; 
ctc.datamanagement@hsr.it; ctc.quality@hsr.it 

 
 
 
 
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2. ABBREVIATIONS AND DEFINITIONS 
2.1 Abbreviations 
 
AE 
Adverse event 
ALS 
Amyotrophic Lateral Sclerosis 
CRF 
Case Report Form 
CRO  
Contract Research Organization 
CSF 
Cerebrospinal fluid 
DPIA 
Data Protection Impact Assessment 
EC 
Ethics Committee 
ELISA 
Enzyme-Linked Immunosorbent Assay 
GCP 
Good Clinical Practice 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
PI 
Principal Investigator 
SOP 
Standard Operating Procedure 
CTC 
Clinical Trial Center 
OSR 
Ospedale San Raffaele 
 
2.2 Definitions 
 
MESO 
ELISA Meso-scale Discovery Platform 
 
 

 
 
 
 
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3. STUDY DESCRIPTION AND RATIONALE 
 
Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive, fatal, neurodegenerative disorder that 
results in the gradual degeneration of the motor neurons in the motor cortex and spinal cord. ALS is the 
most common type of motor neuron disease with an incidence rate of 2.8 per 100,000 persons/year 
and a mean prevalence rate of 5.4 per 100,000 people in Europe. ALS is characterized by rapidly 
progressive paralysis and death typically occurs from respiratory failure within 2 to 4 years of symptom 
onset, although about 10% of ALS patients manifest a slow form of the disease with a survival of 10 
years or longer (1). Clinical diagnosis of ALS is often a prolonged process due to heterogeneity in 
patient presentation and prognosis as well as a lack of specific diagnostic, prognostic and the diagnostic 
biomarkers. This is a recognized critical bottleneck for both ALS translational research and clinical 
applications. The importance of biomarker discovery for therapeutic advancement is underscored by 
the recent accelerated approval of QALSODY (Tofersen) for treatment of SOD1-ALS patients, a 
decision that was primarily based on reduction in plasma Nf-L in treated patients. Nevertheless, despite 
its value, an increase in circulating Nf-L (2,3) following neuro-axonal damage represents a late 
pathogenic event also found in other neurological disorders besides ALS. The ideal fluid biomarker for 
ALS would be capable to sensitively monitor early disease manifestation, treatment responses and 
dynamic pathogenetic events, features that are not met by Nf-L. Other potential biomarkers of neuronal 
damage in ALS include tau (associated with shorter survival), UCHL1 and TDP-43 (both increased in 
ALS patients). The involvement of microglia and astrocytes to ALS pathogenesis can be studied by 
measuring MCP-1 and GFAP respectively (4-6). Considering the mounting evidence implicating IFN-
alpha signaling in the pathogenesis of ALS (7), we aim to comprehensively profile cytokines, 
neuroinflammatory markers, and neurodegeneration-related analytes in plasma and CSF from clinically 
characterized ALS patients and healthy controls. Biomarker investigation will be performed in a 
retrospective cohort of 31 ALS patients compared with 29 healthy subjects. 
The samples to be analyzed in this study were obtained from a single visit and there is no planned 
longitudinal analysis component as part of this current study. Thus, this project is an initial exploratory 
biomarker study aimed to use established ALS fluid biomarkers as a benchmark to identify further 
potential hypothesis-driven candidate biomarkers within the IFN-alpha pathway. Promising candidate 
biomarkers will not be correlated against disease progression (since there are no follow-ups), but will 
be considered alongside the available clinical data and clinical outcome measures provided by the 
principal physician.  
This study aims to support the initial validation of IFN-alpha pathway activation as a therapeutic target 
and explore its association with disease phenotype to provide insights into potential diagnostic and 
prognostic biomarkers for ALS, facilitating future therapeutic development. 
 
 

 
 
 
 
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4. OBJECTIVES AND ENDPOINTS 
 
Objectives 
Endpoints 
Time point(s)  
Primary Objectives 
To validate established ALS-
related fluid biomarker levels 
within the sample cohort. 
Primary Endpoint 
To evaluate levels of established fluid ALS 
biomarkers such as Nf-L, GFAP, and pTau 
(T181) in plasma and CSF of ALS patients vs. 
healthy controls.   
Blood and CSF 
sampling at day 0 
 
Secondary Objectives 
To identify hypothesis-driven 
novel fluid biomarkers in ALS 
related to the IFN-alpha 
pathway.  
Secondary Endpoints 
To evaluate expression of candidate disease 
biomarkers in plasma in a cohort of ALS patients. 
To evaluate expression of candidate disease 
biomarkers in CSF in a cohort of ALS patients. 
To compare candidate disease biomarkers 
specificity 
and 
sensitivity 
with 
Nf-L, 
the 
benchmark biomarker for ALS 
Blood and CSF 
sampling at day 0 
 
 
5. INCLUSION/EXCLUSION CRITERIA 
a. Inclusion Criteria 
Inclusion criteria for ALS patients:  
• 
Age equal or over 18 years old  
• 
ALS patients, diagnosed accordingly to the revised El Escorial Criteria 
Disease duration <24 months from symptom onset.  
 
Inclusion criteria for controls 
• 
Age equal or over 18 years old 
• 
Subjects without a diagnosis of neurodegenerative disease or neuromuscular disorder. 
b. Exclusion Criteria  
Exclusion criteria for ALS patients:  
• 
FVC <60%;  
• 
nutritional or respiratory failure; Significant hepatic or chronic renal failure or any intervening 
infective (pneumonia, flu-like syndromes, urinary tract infections) or metabolic (acute 
renal failure, hyperosmolar hyperglycaemic state, severe 
hyponatremia) conditions present at the time of assessment that could potentially 
affect biomarker levels.  
• 
ALS patients exhibiting any of these conditions at the time of biofluid sampling as per the 
Case Report From (CRF) will be excluded from sample selection. 
 

 
 
 
 
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Exclusion criteria for controls: 
• 
Significant hepatic or chronic renal failure or any intervening infective (pneumonia, flu-like 
syndromes, urinary tract infections) or metabolic (acute renal failure, hyperosmolar 
hyperglycaemic state, severe hyponatremia) conditions present at the time of 
assessment that could potentially affect biomarker levels.  
• 
Controls exhibiting any of these conditions at the time of biofluid sampling as per the Case 
Report From (CRF) will be excluded from sample selection. 
6. INFORMED CONSENT 
Plasma and CSF were collected accordingly to clinical practice indication (GCP). ALS patients whose 
clinical data (demographic, epidemiological, family history of disease and neurological history of the 
patient) and biological samples (plasma and CSF for biomarker dosage) have already been collected, 
after informed consent (protocollo Banca Inspe), from the creation of the INSPE BANK and stored as 
per specific regulation will be included retrospectively. 
This research is aimed at improving the health of people belonging to the same age group or who suffer 
from the same pathology or who are in the same conditions and the research program is subject to a 
motivated favourable opinion from the EC. The research does not involve significant risks to the dignity, 
rights, and fundamental freedoms of the interested party. 
7. STUDY OBJECT 
 
PROCEDURE 
Procedure(s) 
Biomarker analysis 
 
Ulysses Neuroscience Ltd. will perform electrochemiluminescence sandwich 
ELISA using the Meso-Scale Discovery platform (MESO QuickPlex SQ 120 
instrument and analyzed by Discovery Workbench 4.0 software). Samples will 
be analyzed in parallel using the NULISAseq™ proteomic platform (Alamar 
Biosciences) for confirmatory analysis of analyte expression using a secondary 
platform. 
 
Data will be analysed as ALS vs healthy control as raw values of each analyte 
in the samples (pg/mL) and graphed using GraphPad Prism v.9.  
 
Specific analytes to be measured include: 
a) 
U-Plex 
Immuno-Oncology 
Custom: 
IFN-α2a, 
IFN-β, 
IL-12/IL-23p40, 
CXCL10/IP10, CCL2/MCP1. 
b) 
U-Plex Immuno-Oncology Custom: IFN-γ, IL-6, IL-8, IL-13, TNF-α, 
CCL5/RANTES. 
c) 
R-Plex Human TDP-43: TDP-43. 
d) 
S-Plex Neurology Panel 1: GFAP, Neurofilament L, Tau [Total]. 
e) 
S-Plex Human Tau: pTau T181 
f) 
S-Plex Human Tau: pTau T217 
g) 
S-Plex Human Tau: pTau T231 
h) NULISAseq™ CNS Disease Panel 120 
i) 
NULISAseq™ Inflammation Panel 250 AQ 

 
 
 
 
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8. SAMPLE SIZE AND SUBJECT IDENTIFICATION 

 
 
 
 
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Sample Size  This proposed retrospective fluid biomarker study includes a sample size of 29 
control and 31 ALS patient samples. Plasma and CSF samples were selected 
from healthy control individuals in order to ensure that two age-homogeneous 
groups were selected, based on the general age distribution of the ALS patient 
cohort (Healthy Control: 55 ± 12.47 years; ALS: 56.58 ± 13.46 years at time of 
sampling). 
 
Sample sizes were selected based on our previous work and current scientific 
literature. In a prior study completed in collaboration with Dr. Stefano Previtali’s 
group at SRH investigating Charcot-Marie-Tooth disease type 1A (CMT1A), we 
used a comparable number of control and patient samples (n=31 and 45, 
respectively) to analyse neurofilament light chain (Nf-L) levels in plasma. This 
study yielded a highly significant increase in Nf-L levels in CMT1A patient plasma 
compared to healthy controls (p<0.0001), demonstrating the suitability of this 
sample size for detecting biologically meaningful effects in peripheral nervous 
system degenerative disease. 
 
 
 
Nf-L is increased in the plasma of CMT1A patients 
- Plasma concentrations (pg/mL) of neurofilament 
light chain (Nf-L) in healthy control (n=31) and 
CMT1A 
patients 
(n=45) 
analysed 
by 
MSD 
electrochemiluminescence sandwich ELISA. Plasma 
Nf-L was significantly elevated in CMT1A patients 
compared to healthy controls (p=0.0001). 
 
 
 
Given this experience, as well as data from published ALS biomarkers studies, 
we are confident our current sample size is appropriate for detecting group 
differences in established biomarkers such as Nf-L, GFAP, and pTau (T181) in 
ALS (8-11). Additionally, we are using data from published literature to inform 
expectations for other analytes included in the proposed analysis panel, such as 
inflammatory cytokines, markers of neurological disorder, and different phospho-
tau species. However, we acknowledge that published data on many of these 
biomarkers in ALS are limited or inconsistent, underscoring the importance of 
investigating these analytes within this study. 
 
Furthermore, sample sizes were established based on Cohen’s d power analysis 
performed based on published meta-analyses of case-control studies reporting 
effect sizes, or mean or standardised mean difference (SMD) for the benchmark 
ALS biomarker, Nf-L, in ALS CSF and plasma compared to healthy controls. SMD 
is statistically equivalent to Cohen’s d, in meta-analyses: CSF Nf-L = SMD 1.46, 
Plasma Nf-L = 1.35 (12). With an effect size (Cohen’s d) of 1.06 employed 
combined with an alpha level of 0.002 (considering all 18 biomarkers) and a 
minimum power cutoff of 80%, the minimum sample size required to reach 
statistical power was given to be 30. Considering this power analysis, and that 
dropout rate is not a factor of consideration for these samples because they have 
been already collected, and there is no risk of in blood collection, sample volume, 
or people withdrawing from the study, a sample size of 31 was selected. 

 
 
 
 
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Power Analysis performed 
to inform sample size 
selection for this study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
This study is designed as an exploratory biomarker investigation. Its goals are 
two-fold: (1) to validate established ALS biomarkers using this cohort, and 
(2) to assess a broad panel of additional analytes that may be dysregulated 
in ALS, with a view toward identifying promising candidates for further 
investigation. We believe that this approach is scientifically justified for an initial 
exploratory study and aligns with standard practice in early-phase biomarker 
research. Future studies based on the findings of this work will incorporate 
expanded cohorts and statistical power analyses derived from this foundational 
dataset. 
Subject 
Identification 
 
Pseudonymised: participants are identified by a unique coded identification 
number. This code maintains confidentiality and links the participant to their data 
without revealing their identity. 
 
 
9. STATISTICAL DESIGN 
 
Plasma and CSF samples were selected from healthy control individuals in order to ensure that two 
age-homogeneous groups were selected, based on the general age distribution of the ALS patient 
cohort (Control: 55 ± 12.47 years; ALS: 56.58 ± 13.46 years at time of sampling) to negate both age 
and gender as covariates when analyzing each individual biomarker expression in ALS vs control.  
 

 
 
 
 
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Distribution of ages in across Control and ALS sample cohorts. A) Distribution of age at sampling 
with standard deviation of the total control and ALS patient cohort for this study, as well as breakdown 
of age at sampling with standard deviation for male and female samples. B) Unpaired two-tailed t-test 
with Welch’s correction for age at sampling between control and ALS patient cohorts. No statistically 
significant differences were identified in age at sampling between groups (p=0.7379) or the variance 
within groups (p=0.6887). C) Distribution plot of age at sampling for control and ALS patient samples 
with lines indicating mean group ages. Analyzed by unpaired two-tailed t-test (p=0.7939). D) Distribution 
plot of age at sampling for control and ALS patient samples with lines connecting the nearest age match 
between sample cohorts. Analyzed by paired two-tailed t-test (p=0.3629). 
All statistical analyses will be performed using GraphPad Prism v.9. Each analyte will be analyzed on 
an individual analyte-by-analyte basis by unpaired two-tailed t-test, without multiple comparisons 
performed between analytes as is standard during early-phase exploratory biomarker research. 
While several of the proposed biomarkers to be analyzed show strong correlation with age, selecting 
two age- and gender-homogenous populations for ALS and control samples will mitigate any non-
pathological age-related deviations in specific biomarker levels. 
10. 
DURATION OF THE STUDY, DISCONTINUATION AND 
WITHDRAWAL 
 
a. Duration of the Study 
 
Duration of enrolment: Plasma and CSF are already available in Biobanking-INSPE 

 
 
 
 
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Duration of total study period: 1 year 
 
b. Discontinuation And Withdrawal 
 
Not applicable 
 
11. 
SAMPLE HANDLING 
The samples were taken from each participant (accordingly to routine best clinical practice) as part of 
a standard clinical investigation and for future research. Upon collection, samples will be immediately 
processed to preserve their integrity. Blood samples were centrifuged to separate plasma and stored 
at -80C. CSF was collected in sterile conditions and aliquoted to avoid repeated freeze-thaw cycles that 
could degrade the samples. All samples were properly labeled with a unique identifier, devoid of any 
personal identifying information to maintain participant confidentiality. All samples are stored in the 
iNSPE-Biobank under controlled conditions until analysis. 
OSR agrees to provide Ulysses neuroscience with the following biological samples: 
• 
Plasma samples from 31 ALS patients 
• 
CSF samples from 31 ALS patients 
The samples are anonymized and do not contain any patient-identifiable information. 
The samples are stored and transported under appropriate conditions to ensure their integrity. Shipment 
shall be carried out through Ulysses Neuroscience Ltd.’s partner company, Biorep, which specializes 
in the secure transport of biological materials. Biorep shall ensure that all necessary handling and 
storage conditions are met during transit. Ulysses Neuroscience Ltd. shall be responsible for covering 
all costs associated with the shipment of the samples. The samples shall be used by Ulysses 
Neuroscience Ltd. for biomarker research in its own facilities. Ulysses laboratories in Cherrywood 
(Dublin, Republic of Ireland) are fully equipped for analysis of central and peripheral biomarkers related 
to neuroinflammation, synaptic plasticity and cytoskeleton integrity. Samples are also to be shared with 
a collaborator laboratory for further comprehensive protein expression analysis within the scope of the 
study set out in this protocol. The Ulysses Neuroscience Ltd. team has now collected significant data 
and performed several studies that clearly underline the importance of biomarkers in psychiatric and 
rare CNS diseases in bridging preclinical and clinical research.  
Ulysses Neuroscience Limited is an Irish CRO providing clinical and preclinical research. It is committed 
to highest ethical scientific practice for both clinical and preclinical studies. The clinical biomarker 
studies are ethically reviewed and performed by specialised trained staff. 
12. 
DATA MANAGEMENT 
a. Definition of source data and source documents 
Clinical Data: Includes all medical history, treatment administration, collected through case report forms 
(CRFs) 
Biomarker Data: Comprises biochemical and other molecular data collected from blood and CSF. 

 
 
 
 
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b. Documentation of data in Case Report Forms (CRFs)  
All relevant data collected during the study for all the patients enrolled in the study shall be entered in 
the CRF by the principal investigator or someone authorized by the investigator in a timely manner (as 
soon as possible after the information is collected) to ensure that they are clear and legible. The 
physician shall confirm the completeness, correctness, plausibility, and compliance with the ICH 
guidelines and the institutional SOPs of the data by dated signature. An explanation must be provided 
for any and all missing data. The entries shall be made with black ballpoint pen. 
c. Data Recording and Record Keeping 
 
The Principal Investigator must maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified.  
The investigator(s)/institution(s) should permit trial-related monitoring, audits, and regulatory 
inspection(s), providing direct access to source data/documents.  
The investigator must keep the documents on file for at least 25 years after completion or 
discontinuation of the study. After that period, the documents may be destroyed, subject to local 
regulations. Before proceeding to documents’ destruction, sites must inform the Sponsor Investigator 
in writing.  
 
d. Data Protection  
Data Anonymity and Confidentiality 
Anonymity: participants are identified not by name, initials, or birth date but by a unique coded 
identification number. This code helps maintain confidentiality and links the participant to their data 
without revealing their identity. 
Each investigator assures that patients’ anonymity will be maintained and that their identities are 
protected from unauthorized parties. Particular attention should be paid whenever patient data are 
supplied to third parties and may be autonomously processed, or biological samples/materials are taken 
and kept for future research purposes.  
The investigator should keep in a confidential way a patient identification log recording both patient 
code and name. The investigator should also maintain patients’ written consent forms, in strict 
confidence (i.e. not for submission to the Sponsor Investigator). 
 
Authorization and Data Entry 
Authorized Personnel: Access to data is restricted to authorized personnel only. This access control is 
crucial for maintaining the integrity and confidentiality of the data. 
13. ETHICAL AND REGULATORY CONSIDERATION  
The Principal Investigator ensures that the study is conducted in agreement with this protocol, the Good 
Clinical Practice, the current version of Declaration of Helsinki and the applicable regulations. 
The protocol and any amendments are subject to review and approval by the concerned Ethics 
Committee(s) and Health Authority(ies) 
 

 
 
 
 
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13.1 Responsibilities of the Investigators 
 
The Investigator(s) undertake(s) the responsibility to perform the study in accordance with this Protocol, 
Good Clinical Practice, and the applicable regulatory requirements. The Investigator is required to 
ensure compliance with the investigational product schedule, visits schedule, and procedures required 
by the protocol. The Investigator agrees to provide all information requested in the Case Report Form 
(CRF) in an accurate and legible manner. The investigator may implement a deviation from, or a change 
of, the protocol to eliminate an immediate hazard(s) to study subjects without prior EC 
approval/favorable opinion. As soon as possible, the implemented deviation or change, the reasons for 
it, and, if appropriate, the proposed protocol amendment(s) should be submitted. The investigator must 
have available an adequate number of qualified staff and adequate facilities for the foreseen duration 
of the study to conduct the study properly and safely. 
13.2 Ethics Committee (EC) Approvals  
This clinical study protocol as well as the Informed Consent are to be submitted to the appropriate 
Ethics Committee for only notification or approval, in this case it is mandatory to obtain the written and 
dated approval, signed by the chairman with Ethics Committee(s) composition. The clinical study the 
documents reviewed, the list of voting members and their qualifications, and the date of the review 
should be clearly stated on the written Ethics Committee approval. 
 
14. 
GENDER MEDICINE IN RESEARCH PROTOCOL  
ALS predominantly affects males, with a male/female incidence rate ratio of 1.4, and people in their late 
adulthood, with median age at diagnosis of 65.2 years for men and 67.0 years for women. To avoid 
bias in the gender dimension we will implement appropriate protocols for treating male and female 
biological samples (equal proportion and indistinguishability with respect to gender). 
 
15. 
QUALITY ASSURANCE AND CONTROL 
 
Quality Assurance and Quality Control systems based on written SOPs are in place at the Sponsor 
Investigator site.  
 
16. 
END OF CLINICAL STUDY  
In accordance with applicable regulation, ICH GCP and SOPs, the PI shall notify the end of the 
clinical study within 15 days from the end of the clinical study and the reasons for such action. 
17. 
INTELLECTUAL PROPERTY 
 
A clinical observational study agreement will be stipulated with an ad hoc contract. 

 
 
 
 
Pioneer Life Sciences Cherrywood - Building 10 Cherrywood 
Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
Republic of Ireland 
Registered in the Republic of Ireland No 646226 
https://ulysses-neuro.ie 
 
+353 87 600 4092 
18. 
PUBLICATION POLICY 
 
After completion of the study, the Sponsor Investigator prepares a draft manuscript containing final 
results of the study on the basis of the statistical analysis. The manuscript is delivered to the co-authors 
for comments and then sent to a scientific journal for publication. 
All publications, abstracts, presentations, manuscripts and slides - issued by the Investigators of the 
collaborative sites and including data from the present study- should be submitted to and reviewed by 
the Sponsor Investigator at least 3 (three) weeks in advance the planned date for the submission to the 
scientific journal.  
19.   
REFERENCES 
 
[1] Riva N, Domi T, Pozzi L, Lunetta C, Schito P, Spinelli EG, Cabras S, Matteoni E, Consonni M, Bella 
ED, Agosta F, Filippi M, Calvo A, Quattrini A. Update on recent advances in amyotrophic lateral 
sclerosis. J Neurol. 2024 Jul;271(7):4693-4723. 
[2] Sferruzza G, Bosco L, Falzone YM, Russo T, Domi T, Quattrini A, Filippi M, Riva N. Neurofilament 
light chain as a biological marker for amyotrophic lateral sclerosis: a meta-analysis study. Amyotroph 
Lateral Scler Frontotemporal Degener. 2022 Aug;23(5-6):446-457. 
[3] Falzone YM, Russo T, Domi T, Pozzi L, Quattrini A, Filippi M, Riva N. Current application of 
neurofilaments in amyotrophic lateral sclerosis and future perspectives. Neural Regen Res. 2021 
Oct;16(10):1985-1991. 
[4] Turco A, Primiceri E, Chiriacò MS, La Pesa V, Ferrara F, Riva N, Quattrini A, Romano A, Maruccio 
G. Advancing amyotrophic lateral sclerosis disease diagnosis: A lab-on-chip electrochemical 
immunosensor for ultra-sensitive TDP-43 protein detection and monitoring in serum patients'. Talanta. 
2024 Jun 1;273:125866. 
[5] Schito P, Russo T, Domi T, Mandelli A, Pozzi L, Del Carro U, Carrera P, Agosta F, Quattrini A, 
Furlan R, Filippi M, Riva N. Clinical Features and Biomarkers to Differentiate Primary and Amyotrophic 
Lateral Sclerosis in Patients With an Upper Motor Neuron Syndrome. Neurology. 2023 Aug 
22;101(8):352-356.  
[6] Falzone YM, Domi T, Mandelli A, Pozzi L, Schito P, Russo T, Barbieri A, Fazio R, Volontè MA, 
Magnani G, Del Carro U, Carrera P, Malaspina A, Agosta F, Quattrini A, Furlan R, Filippi M, Riva N. 
Integrated evaluation of a panel of neurochemical biomarkers to optimize diagnosis and prognosis in 
amyotrophic lateral sclerosis. Eur J Neurol. 2022 Jul;29(7):1930-1939. 
[7] Hui BSM, Zhi LR, Retinasamy T, Arulsamy A, Law CSW, Shaikh MF, Yeong KY. The Role of 
Interferon-α in Neurodegenerative Diseases: A Systematic Review. J Alzheimers Dis. 2023;94(s1):S45-
S66. 
8] Brodovitch, A., Boucraut, J., Delmont, E. et al. Combination of serum and CSF neurofilament-light 
and neuroinflammatory biomarkers to evaluate ALS. Sci Rep 11, 703 (2021).  
[9] Halbgebauer S, Steinacker P, Verde F, et al. Comparison of CSF and serum neurofilament light and 
heavy chain as differential diagnostic biomarkers for ALS. Journal of Neurology, Neurosurgery & 
Psychiatry (2022); 93:68-74. 

 
 
 
 
Pioneer Life Sciences Cherrywood - Building 10 Cherrywood 
Business Park  
Loughlinstown  
Co. Dublin, Republic of Ireland 
Republic of Ireland 
Registered in the Republic of Ireland No 646226 
https://ulysses-neuro.ie 
 
+353 87 600 4092 
[10] Benninger F, Glat MJ, Offen D, Steiner I. Glial fibrillary acidic protein as a marker of astrocytic 
activation in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis. J Clin Neurosci. 2016 
Apr;26:75-8.  
[11] Mastrangelo, A.; Vacchiano, V.; Zenesini, C.; Ruggeri, E.; Baiardi, S.; Cherici, A.; Avoni, P.; 
Polischi, B.; Santoro, F.; Capellari, S.; et al. Amyloid-Beta Co-Pathology Is a Major Determinant of the 
Elevated Plasma GFAP Values in Amyotrophic Lateral Sclerosis. Int. J. Mol. Sci. 2023, 24, 13976. 
[12] Agah E, Mojtabavi H, Behkar A, Heidari A, Ajdari A, Shaka Z, Mousavi SV, Firoozeh N, Tafakhori 
A, Rezaei N. CSF and blood levels of Neurofilaments, T-Tau, P-Tau, and Abeta-42 in amyotrophic 
lateral sclerosis: a systematic review and meta-analysis. J Transl Med. 2024 Oct 21;22(1):953. 
 
20.  APPENDIX A:  AMENDMENT HISTORY 
List details of all protocol amendments here whenever a new version of the protocol is produced. 
Amendment 
No. 
Protocol 
Version 
No. 
Date 
issued 
Rationale 
Study 
status 
Details of Changes  
 
 
 
 
 
 
 
21. APPENDIX B:  LIST OF CLINICAL SERVICES / LABORATORIES 
Fill in form the form CTC 021B.  
22. APPENDIX C:  LIST OF ENROLLING SITES 
Fill in the form CTC 021C.