Clinical trial NCT05039268
Conditions: Amyotrophic Lateral Sclerosis
Keywords: ALS Amyotrophic Lateral Sclerosis MND Motor Neurone Disease 3K3A-APC
Sponsor: Macquarie University, Australia
Start date: 2021-11-25; Completion date: 2022-09-12
Enrollment: 16 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
Phase 2 open label trial to investigate the safety and potentially efficacy of 3K3A-APC in patients with Amyotrophic Lateral Sclerosis (ALS).

Detailed description:
This Phase 2 open label trial seeks to investigate whether a novel therapy named 3K3A-APC is safe and potentially effective in patients with Amyotrophic Lateral Sclerosis (ALS). A total of 16 patients with ALS will be enrolled into 2 dose cohorts with five doses of 15mg or 30mg doses given 12 hours apart in each cohort. The primary study outcomes are to ensure the safety and tolerability of 3K3AAPC in ALS patients, and to determine whether 3K3A-APC is able to reduce the pathological changes that might possibly cause ALS.

Arms: arm 1: Participants will receive a fixed dose regimen of five doses of 15mg. arm 2: Participants will receive a fixed dose regimen of five doses of 30mg.
Interventions: intervention 1: 3K3A-APC Protein
Intervention descriptions: intervention 1: 3K3A-APC with intravenous dosing of five doses of either 15mg or 30mg at 12 hourly interval. The first 8 patients will receive 15mg dose, and the next 8 patients will receive 30mg dose.

Protocol documents:
Prot_SAP_000.pdf: 
3K3A-APC in ALS 
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Trial No: 
 
 
ZZ-3K3A-202 
Title: 
A Phase 2 Open Label Trial of 3K3A-APC in Amyotrophic Lateral 
Sclerosis. 
Sponsor: 
 
 
Macquarie University 
Principal Investigator: 
Dominic B Rowe PhD FRACP 
 
Date of Protocol: 
 
17 Mar, 2022  
Version: 
 
 
3.3.6 
Commencement of Trial: November, 2021 
 
CONFIDENTIAL 
This document is a confidential communication of Macquarie University.  The recipient agrees 
that no information contained herein will be published or disclosed without written approval.  
This document may be disclosed to appropriate institutional review boards, independent ethics 
committees, or duly authorized representatives of the Therapeutic Goods Administration, U.S. 
Food & Drug Administration, European Medicines Agency, or other regulatory authorities as 
appropriate; under the condition that confidentiality is requested. 
 
 


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Investigator Contact Information 
 
Principal Investigator: 
Dominic Rowe 
 
 
 
 
Professor of Neurology 
 
 
 
 
MQ Health 
 
 
 
 
Suite 204  
 
 
 
 
2 Technology Place 
 
 
 
 
Macquarie University NSW 2109 
 
 
 
 
Ph: (+612) 9812 3720 
 
 
 
 
dominic.rowe@mq.edu.au 
 
Steering Committee: 
Stanley H Appel 
 
 
 
 
Merit Cudkowicz 
 
 
 
 
Berislav Zlokovic 
 
 
 
 
Kent Pryor 
 
 
 
 
Jay Hennock 
 
 
 
 
Bradley Turner 
Co-Investigators: 
 
Gilles Guillemin 
 
 
 
 
Stuart Grieve 
 
 
 
 
John Magnussen 
 
 
 
 
Ian Blair 
 
 
 
 
Nicola Chapman 
 
 
 
 
Balaji Kalband 
 
 
 
 
Victor Villemagne 
 
 
 
 
Glenda Halliday 
 
 
 
 
Sinduja Subramanian 
 
 
 
 
Kristina Barnes 
Trial Coordinator:   
Richard Gan 
Trial Sponsor: 
  
Macquarie University 
Trial Management:  
Macquarie University Clinical Trials Unit 
Data Management:  
McLeod Consulting Group 
Safety Monitoring Committee: 
 
 
 
 
Julian Gold 
 
 
 
 
Howard Levy 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Investigator Agreement 
 
3K3A-APC in Amyotrophic Lateral Sclerosis 
 
 
I have carefully read this protocol including all appendices and agree that it contains all the 
necessary information for conducting the study safely. 
 
I will conduct this study in strict accordance with this protocol and according to the current 
Good Clinical Practice (GCP) regulations and International Conference on Harmonization (ICH) 
guidelines, and local regulatory requirements.   Any changes in procedure will only be made if 
necessary to eliminate immediate hazards and/or to protect the safety, rights or welfare of 
subjects.   
 
I will provide copies of the protocol and all other information relating to this project, which 
were furnished to me, to all physicians and other study personnel responsible to me who 
participate in this study.   I will discuss this information with them to assure that they are 
adequately informed regarding the conduct of the study. 
 
I agree to keep records on all subject information (case report forms, informed consent 
statements and all other information collected during the study) in accordance with the current 
GCP, ICH, local, national and European regulations. 
 
 
 
 
_______________________________________ 
Investigator Name 
 
 
 
______________________________________   
 
______________________ 
Investigator Signature 
 
 
 
Date 
 
 
 


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List of Abbreviations and Definitions 
 
[18F]FEMPA 
 
N-{2-[2-[18F]fluoroethoxy]-5-methoxybenzyl}-N- 
[2-(4-methoxyphenoxy)pyridine-3-yl]acetamide 
 
Beta-HCG 
CI 
Beta-human chorionic gonadotropin 
Confidence Interval 
CRF 
Case Report Form 
CSF 
Cerebrospinal fluid 
CSOC 
Clinical Study Oversight Committee 
DNA 
Deoxyribonucleic acid 
DTI 
ECAS 
Diffusion tensor imaging 
Edinburgh Cognitive and Behavioural ALS Screen 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
EDC 
FVC 
Electronic data capture 
Forced Vital Capacity 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
HREC 
Human Research Ethics Committee 
 
 
 
 
 
 
 
 
 
 


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List of Abbreviations and Definitions (continued) 
 
 
 
ICH 
IUD 
MBq 
MRI 
MGH 
International Conference on Harmonization 
Intrauterine device 
Megabecquerels  
Magnetic Resonance Imaging 
Massachusetts General Hospital  
NOAC 
Novel oral anticoagulants 
PET 
Positron Emission Tomography 
PT 
Preferred Term 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
ST 
TSPO 
ULN 
UMN 
UMNS 
Symptomatic Therapy 
Translocator Protein 
Upper Limit of Normal 
Upper Motor Neuron 
Upper Motor Scale 
 
 
 
 
 
 
 
 
 
 
 


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3K3A-APC in ALS Protocol Synopsis 
 
Protocol 
Number 
ZZ-3K3A-202 
Protocol Title 
A Phase 2 Open Label Trial of 3K3A-APC in Amyotrophic Lateral 
Sclerosis 
Sponsor 
Macquarie University 
Investigator 
Professor Dominic Rowe 
Study Centre 
Macquarie University 
Study Period 
3 months 
Study Objective 
and 
Specific Aims 
The primary objective of this study is to determine the safety and potential 
effective dose of a novel therapy, 3K3A-APC in a small cohort (n=16) of 
patients with Amyotrophic Lateral Sclerosis (ALS).  
 
The specific aims to accomplish the primary objective are: 
a. 
To recruit patients with ALS according to established 
diagnostic criteria with Upper Motor Neuron features. 
b. 
To administer in a hospital environment two dose levels of 
a novel therapy, 3K3A-APC, to patients with ALS 
monitoring for safety. 
c. 
To investigate the biological effect of 3K3A-APC in the 
cohort employing a range of biometric, imaging and 
biological methods. 
d. 
To use the data from this pilot study to inform a larger 
study to assess the effectiveness of 3K3A-APC as a potential 
therapy to slow the progression of ALS. 
Study Design 
Cross sectional, single centre study to assess safety of 3K3A-APC in a small 
cohort of patients with clinically definite ALS, incorporating a range of 
imaging and biologic markers to determine the biological impact of the 
therapy. 
Number of 
Subjects 
16 Subjects Enrolled  
o Clinically Definite ALS according to Awaji Criteria 
o Established Upper and Lower Motor involvement 


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Main Eligibility 
Criteria 
Amyotrophic Lateral Sclerosis (ALS) Subjects: 
Inclusion: 
• Patients must have clinically definite ALS (Awaji Criteria)  
• Male or female age 18 years and less than 75 years at time of ALS 
study. 
• Symptom onset less than 38 months before screening. 
o Participants who fail to meet this criterion may be considered at 
the Principal Investigator’s discretion, provided the participant is 
clinically stable and without any safety concerns. 
• Diagnosis of ALS less than 26 months before screening. 
o Participants who fail to meet this criterion may be considered at 
the Principal Investigator’s discretion, provided the participant is 
clinically stable and without any safety concerns. 
• Clinically definite Upper Motor Neuron signs 
Exclusion: 
• Current treatment with anticoagulants (e.g., warfarin, novel oral 
anticoagulants, heparin) that might preclude safe completion of the 
lumbar puncture. 
• Condition that precludes the safe performance of routine lumbar 
puncture, such as prohibitive lumbar spinal disease, bleeding 
diathesis, or clinically significant coagulopathy or thrombocytopenia.  
• Use of investigational drugs or devices within 60 days prior to 
Baseline (dietary supplements taken outside of a clinical trial are not 
exclusionary, e.g., coenzyme Q10). 
• Prolonged prothrombin time or activated partial thromboplastin 
time >2xULN 
• Severe hypertension or hypotension 
• Glomerular filtration rate (GFR) <35 mL/min 
• Forced vital capacity (FVC) at screening of <50% of predicted.  
o Participants who fail to meet this criterion due to technical 
difficulties may be considered at the Principal Investigator’s 
discretion. 
• Prior exposure to any exogenous form of APC 
• Inability to lie flat for procedures (MRI, PET, LP). 


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• Pregnant or lactating during the study period 
Primary 
Outcome 
The primary study outcomes are: 
To ensure the safety of patients with ALS administered a range of doses of 
3K3A-APC according to a fixed dose regimen of five doses of 15mg or 30mg 
doses given 12 hours apart.  
 
This study also aims to determine whether a novel therapy, 3K3A-APC, is 
able to attenuate microglial activation in the motor cortex of patients with 
amyotrophic lateral sclerosis utilizing serial [18F]FEMPA PET imaging (Day 2 
and Day 9).  In addition, the study design will include two (2) dose cohorts 
with eight patients in each cohort, with the initial cohort to receive 15mg 
and the subsequent cohort to receive 30mg, to determine the dose of 3K3A-
APC that is shown to be most effective in reducing microglial activation.  If 
microglial activation can be attenuated in ALS, then the progression of ALS 
might be slowed. 
Secondary 
Outcomes 
The secondary outcomes are: 
• Comparison between the changes in the mean of 
clinical, imaging and biomic outcomes in various 
subsets between the two dose cohorts. 
• The secondary outcome instruments to be employed 
are: 
• MRI scan of the brain (Day 2 and Day 9), to 
determine whether the blood brain barrier 
integrity can be measured in ALS by Magnetic 
Resonance Imaging, and whether 3K3A-APC is 
able to repair it.  
• Biomic measurements of serum, plasma, urine 
and CSF. This includes a novel method to 
determine whether monocyte activation in the 
peripheral blood can act as a surrogate marker 
of microglial activation state in ALS. 
• These methods will determine whether 3K3A-
APC therapy is able to attenuate the altered 
cytokine and metabolic profiles in ALS. 
Safety 
Assessments 
Incidence of adverse events, proportion of withdrawals due to adverse 
events, vital signs and clinical laboratory assessment changes from baseline. 
Clinical safety laboratory data (haematology, biochemistry, and urine 
chemistry) will be summarised at each protocol scheduled time point, by 


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dose cohort, and overall. Observed values, change from baseline will be 
presented. 
Statistical 
Methods 
Descriptive analyses will be reported for this study. Continuous parameters, 
will be summarised as the number of observations, mean, median, standard 
deviation, minimum, maximum, and if appropriate two-sided 95% CI’s. 
Categorical parameters will be summarised as frequencies, percentages, and 
exact Clopper-Pearson two-sided 95% CI’s. Data will be summarised by dose 
cohort, and overall.  
Primary efficacy variables: 
Adverse events will be coded using the most recent version of the Medical 
Dictionary for Regulatory Activities (MedDRA) coding system to give a 
System Organ Class (SOC) and Preferred Term (PT) for each adverse event 
(AE). 
Treatment emergent AEs will be defined as AEs with an onset date on or 
after the date of administration of study drug. If the onset date is missing, 
the AE will be considered to be treatment emergent. Only treatment-
emergent AEs will be summarized. All reported AEs will be listed. The 
number of AEs, and the number and percentage of patients reporting at least 
one AE will be summarized by PT nested within SOC for required AE types. 
AE data will be summarized by dose cohort, and overall.  
Microglial activation will be summarised at each time point for each dose 
cohort and overall as observed value, change from baseline, and % change 
from baseline. Microglial activation will be summarised with number of 
observations, mean, median, standard deviation, minimum, maximum, and 
two-sided 95% CI’s.  
Secondary efficacy variables: 
The secondary efficacy variables of MRI scan of the brain, and biomic 
measurements of serum, plasma, urine and CSF will be summarized over 
time for each dose cohort, and overall as for the primary efficacy variables. 
Data Access 
Data will be password protected and securely stored at Macquarie 
University and with the contract data management team. All personally 
identifiable information will be removed before it is shared outside the 
study.  
 
 


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Schedule of Activities – ALS Subjects 
 
Visit Number 
SC 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
V11 
Day of the Week 
 
M 
T 
W 
T 
F 
S 
S 
M 
T 
W 
M 
Visit Description (Day) 
-30 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
15 
Written Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion Criteria 
X 
X 
 
 
 
 
 
 
 
 
 
 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical Examination 
X 
X 
 
 
 
 
 
 
X 
 
 
X 
Neurological Examination  
X 
X 
 
 
 
 
 
 
X 
 
 
X 
ECAS 
X 
 
 
 
 
 
 
 
 
 
 
 
FVC 
X 
 
 
 
 
 
 
 
 
 
 
 
ECG 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
Pregnancy Test (Urine) 
X 
X 
 
 
 
 
 
 
X 
 
 
 
Blood Sample for TSPO genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
Clinical Laboratory Assessments 
Xa 
X 
X 
Xa 
Xa 
Xa 
X 
X 
X 
X 
Xa 
Xa 
Research blood and urine samples 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
Lumbar Puncture for CSF examination 
 
 
 
X 
 
 
 
 
 
 
X 
 


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Visit Number 
SC 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
V11 
Day of the Week 
 
M 
T 
W 
T 
F 
S 
S 
M 
T 
W 
M 
Visit Description (Day) 
-30 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
15 
MRI (ALS Protocol) 
 
 
X 
 
 
 
 
 
 
X 
 
 
PET imaging ([18F]FEMPA) 
 
 
X 
 
 
 
 
 
 
X 
 
 
Administration of 3K3A-APC – 5 doses 
(every 12 hours) 
 
 
 
X 
X 
X 
 
 
 
 
 
 
 
a – Urine collected for urinalysis  
 


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TABLE OF CONTENTS 
 
Investigator Contact Information ................................................................................ 2 
Investigator Agreement ................................................................................................... 3 
List of Abbreviations and Definitions ......................................................................... 4 
3K3A-APC in ALS Protocol Synopsis ............................................................................ 6 
Schedule of Activities – ALS Subjects .......................................................................... 10 
TABLE OF CONTENTS ..................................................................................................... 12 
1.  
Introduction and Rationale for this Study ................................................ 14 
2. 
Study Design ............................................................................................................ 15 
3. 
Study Objectives ..................................................................................................... 16 
3.1 Primary Objective ............................................................................................................ 16 
3.2 Secondary Objectives ...................................................................................................... 16 
4. 
Study Outcome ........................................................................................................ 17 
5. 
Participants .............................................................................................................. 17 
5.1 Study setting and recruitment ..................................................................................... 17 
5.2 Selection of Study Population ...................................................................................... 17 
6. 
Investigational Plan .............................................................................................. 19 
6.1 Participant Identification Numbers ........................................................................... 19 
6.2 Schedule of Activities ...................................................................................................... 19 
7. 
Study Assessments ................................................................................................ 22 
7.1 Clinical Assessments ....................................................................................................... 22 
7.2 Other Assessments ........................................................................................................... 25 
8. Study Medications ...................................................................................................... 26 
8.1 
Study medication dose, packaging and labelling ............................................ 26 
8.2 
Study medication administration ........................................................................ 26 
8.3 Prohibited Concomitant Medications ........................................................................ 26 
9. Subject Withdrawals ................................................................................................. 26 
10.  Safety/Adverse Events .......................................................................................... 27 
10.1 Adverse Event Definitions .......................................................................................... 27 
10.2  Relationship to Investigational Product ............................................................... 27 
10.3 Intensity/Severity of an Adverse Event ................................................................. 28 
10.4 Recording of Adverse Events ..................................................................................... 29 
10.5 Responsibilities for Reporting Serious Adverse Experiences ........................ 29 
11. Referrals for Unexpected Results of Study Participation .......................... 29 
12.  Potential Risks ......................................................................................................... 29 
12.1 Blood Sampling ............................................................................................................... 29 
12.2 Lumbar Puncture ........................................................................................................... 29 
12.3 MRI – ALS Protocol ........................................................................................................ 30 


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12.4. PET Imaging .................................................................................................................... 30 
12.5 Unknown Risks ............................................................................................................... 30 
13. Regulatory / Ethics .................................................................................................. 30 
13.1. Compliance Statement ................................................................................................ 30 
13.2. Informed Consent ......................................................................................................... 30 
13.2 Human Research Ethics Committee Review ......................................................... 31 
13.3. Protocol Amendments ................................................................................................ 31 
13.4 Subject Confidentiality ................................................................................................. 31 
13.5 COVID-19 Contingency Plan ....................................................................................... 32 
14. Documentation ......................................................................................................... 32 
14.1   Study File and Source notes ...................................................................................... 32 
15. 
Statistical Analysis ............................................................................................ 32 
15.1 
Demographic and Baseline Characteristics ..................................................... 32 
15.2 
Primary efficacy variables ..................................................................................... 33 
15.3 
Secondary efficacy variables: ................................................................................ 33 
15.4 
Clinical Safety Laboratory data ............................................................................ 33 
15.5 
Vital Signs ..................................................................................................................... 33 
15.6 
Physical and Neurological Examinations .......................................................... 33 
16. 
Data Management .............................................................................................. 34 
17. 
Study Monitoring ............................................................................................... 34 
18. Publication of Research Findings ....................................................................... 36 
19. References ................................................................................................................. 36 
20. Samples of Forms ..................................................................................................... 37 
 
 
 


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1.  
Introduction and Rationale for this Study 
 
Multiple mechanisms are suggested to be involved in the cause of sporadic ALS, 
with evidence for many pathogenic processes, none of which clearly lead to 
significant therapies. These include excitotoxicity, oxidative stress, impaired 
cellular defence mechanisms and immune-mediated inflammation, to name just a 
few. There is considerable evidence that inflammation is central in the 
pathogenesis of ALS. Microglia are critical in this inflammatory response, both as 
detectors of motor neuronal injury and in the effector response that ultimately 
produces motor neurone death and progression of disease. The earliest change 
that occurs in the pathology of ALS is activation of microglia, both in post-
mortem human ALS tissue and in animal models of ALS. Activated microglia and 
astrocytes are found in close proximity to dying motor neurons. Their activation 
status and proliferation seemingly increases with disease progression. 
Inflammatory microglia and monocytes have been considered as a therapeutic 
target in ALS, but to date only a few trials exist where attenuation of microglial 
activation is the principal mechanism of action (1). 
It is now possible to measure in vivo activated microglia in the CNS utilizing a 
PET imaging technique targeting the microglial 18kD translocator protein, TSPO.  
In a small trial of 10 patients, Zurcher et al were able to show that [11C]-PBR28 
positron emission tomography revealed increased binding in the motor cortices 
and corticospinal tracts in patients with ALS compared to healthy controls (2). 
This technique may therefore be utilized to measure end organ effectiveness of a 
therapeutic intervention to downregulate microglial activation, and has been 
replicated in further studies, including therapeutic studies in other diseases (3). 
We now have access to an 18F ligand for TSPO termed FEMPA (Piramal) at 
Macquarie University in collaboration with Cyclotek and Piramal (4). 
APC is an endogenous serine protease with systemic anticoagulant, anti-
inflammatory, and anti-apoptotic activities.  3K3A-APC is a 405-residue protein 
expressed via recombinant technology in Chinese hamster ovary (CHO) cells.  Its 
amino acid sequence differs from that of the wild-type human Activated Protein 
C (APC) and of the product drotrecogin alfa (activated) (Xigris®) in that 3 
sequential lysine residues have been replaced with 3 sequential amino-acid 
substitutions (all lysine to alanine); the amino acid substitutions are K191A-
K192A-K193A.  This change retains the cytoprotective effects of native (wild-
type) APC while significantly reducing its anticoagulant effects.  3K3A-APC is an 
APC variant that has been genetically engineered to maximize neuro- and 
cytoprotective activity and minimize anticoagulant activity.  3K3A-APC is 
currently in development for the treatment of moderate to severe acute ischemic 
stroke (ZZ Biotech – see investigational brochure). A Phase 1 trial of 3K3A-APC 
was completed in healthy volunteers (NCT01660230), with demonstrated safety. 
Intravenous administration of 3K3A-APC in multiple dose levels did not result in 
any serious adverse events, severe adverse events or withdrawal from study due 
to an adverse event (5). A Phase 2 trial of 3K3A-APC in Ischemic Stroke 


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(NCT02222714) has also finished (6).  The doses of 3K3A-APC being evaluated in 
the study were 120, 240, 360 and 540 µg/kg.  These doses were based on the 
doses and administration that were tested in the Phase 1 clinical study in normal 
human volunteers during which single- and multiple-dose (q12 hours for 5 
doses) infusions were well tolerated at doses up to 540 μg/kg. Further study has 
demonstrated no relationship between clearance and body weight, so further 
ongoing trials with 3K3A-APC now utilize fixed doses to achieve the same blood 
levels of the investigational product.  
3K3A-APC’s cytoprotective activities are mediated by proteolytic activation of 
protease activated receptor 1. In addition, it has been shown that 3K3A-APC has 
powerful anti-inflammatory action, with reduction of microglial numbers and 
activation state.   In 2009, Zhong et al demonstrated in mutant superoxide 
dismutase-1 (SOD1) mice that administration of 3K3A-APC after disease onset 
slows disease progression and extends survival (7). Furthermore, Winkler et al 
were able to show that blood brain barrier (BBB) breakdown contributes to 
early motor-neuron degeneration in ALS mice and that restoring BBB integrity 
via 3K3A-APC during an early disease phase retards the disease process (8). 
We propose to use two different doses regimens of 3K3A-APC (15mg and 30mg), 
similar to doses demonstrated to be safe in stroke patients, in two cohorts of 
patients with Amyotrophic Lateral Sclerosis. This study will determine safety 
and tolerability in ALS patients.  In addition cerebral [18F]FEMPA PET imaging 
will determine whether microglial activation can be modulated from prior to the 
infusion (Day 2) to after completion of the infusion (Day 9). An MRI method 
recently developed by the team at Macquarie University will also be used to 
assess diffusion kurtosis and integrity of the blood brain barrier (9). Other 
biomarkers of microglial/monocyte activation will be assayed including 
kynurenine metabolites in plasma, serum and urine, monocyte activation state in 
peripheral blood via flow cytometry and proteomics including cytokine and 
chemokine assays, neurofilaments assays and measurement of soluble CD14. 
If microglial activation can be attenuated in ALS, then the progression of ALS 
might be slowed. While this is an acute dosing study over a short period, the 
information derived from this study will inform chronic long-term dosing trials 
for patients with ALS. 
2. Study Design 
ZZ-3K3A-APC-202 is a phase II, single centre, open label, uncontrolled cohort 
trial of 15mg (initial cohort) and 30mg of 3K3A-APC given twice daily 
intravenously for a maximum of 5 doses to patients with ALS. Each patient will 
be admitted to hospital for dosing. For each patient, the trial will run for two 
weeks, including a ten-day hospital stay for 3K3A-APC IV administration and 
study evaluations. The study has an estimated completion time of three months. 
A maximum of 8 patients will participate in each cohort. 
 


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3. Study Objectives 
3.1 Primary Objective 
The primary study outcome is the safety of patients with clinically definite ALS 
administered given up to 5 doses of either 15mg or 30mg of 3K3A-APC 12 
hourly. Safety will be evaluated by clinical laboratory testing, physical 
examination and the collection of adverse events.  
In addition, this study aims to determine which dose of 3K3A-APC treatment is 
able to attenuate microglial activation in the motor cortex of patients with 
amyotrophic lateral sclerosis, utilizing [18F]FEMPA PET imaging prior to, and 
after treatment.  In addition, the study will determine the dose of 3K3A-APC that 
is most effective in reducing microglial activation.   
3.2 Secondary Objectives 
The secondary outcomes are the comparison between the changes in the mean of 
clinical, imaging and biomic outcomes in various subsets between the two 
groups. The secondary outcome instruments are: 
MRI scan of the brain (Day 2 and Day 9), to determine whether the blood brain 
barrier integrity can be measured in ALS by Magnetic Resonance Imaging, and 
whether 3K3A-APC is able to repair it. In addition novel methods of measuring 
diffusion kurtosis, a marker of neuronal injury will be used. 
Extensive biomic measurements of serum, plasma, urine and CSF will be 
performed. These will include a novel method to determine whether monocyte 
activation in the peripheral blood can act as a surrogate marker of microglial 
activation state in ALS. In addition multiplex cytokine and chemokine assays will 
determine the biological impact of the therapy. Neurofilament, soluble CD14 and 
kynurenine (and other) measurements will be used to look at acute changes as a 
result of 3K3A-APC. These methods will determine whether 3K3A-APC therapy is 
able to attenuate the altered cytokine and metabolic profiles in ALS. 
It is highly unlikely that such an acute dosing regimen will identify neurological 
changes in the subjects, but accurate clinical and biophysical measurements will 
be performed to detect any such change should they occur. 
 
 
 


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4. Study Outcome 
The mean rates of change and the variability around the mean of clinical, imaging 
and biomic outcomes in the two ALS patient cohorts will be determined over the 
study period. The results will inform potential future studies of the therapeutic 
potential of 3K3A-APC in ALS patients, and guide dosing for chronic studies. 
5. Participants 
5.1 Study setting and recruitment 
Participants will be recruited from the ALS clinic at Macquarie University. 
Participants will be approached regarding the trial where possible, in routine 
follow up clinics. Alternatively potential participants may be approached in 
writing or by email by the investigators to invite them for screening. 
Participants will be hospitalised at Macquarie University Hospital. PET scans and 
MRI scans, and lumbar punctures will be conducted at Macquarie Medical 
Imaging.  
5.2 Selection of Study Population 
5.2.1 Inclusion Criteria 
• 
Age 18-75 years at the time of the screening visit 
• 
Able to provide informed consent in accordance with Good Clinical 
Practice (GCP), International Conference on Harmonization (ICH), and 
local regulations  
• Willing and able to comply with scheduled visits, required study 
procedures and laboratory tests  
• Symptom onset less than 38 months before screening 
o Participants who fail to meet this criterion may be considered at the 
Principal Investigator’s discretion, provided the participant is clinically 
stable and without any safety concerns. 
• Diagnosis of ALS less than 26 months before screening 
o Participants who fail to meet this criterion may be considered at the 
Principal Investigator’s discretion, provided the participant is clinically 
stable and without any safety concerns. 
• 
ALS diagnosed as definite according to the Awaji Criteria. 
• 
Both sporadic and monogenic forms of ALS are to be included.  
• 
Must be on a stable dose of riluzole for at least 30 days prior to the 
screening visit. 


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• 
Clinically definable Upper Motor Neurone signs such as increased tone, 
clonus, hyper-reflexia and/or extensor plantar responses 
• 
Women may not be pregnant, lactating or planning pregnancy during the 
course of the study. 
o 
Includes a negative urine pregnancy test on day of the PET scan 
prior to injection ([18F]FEMPA).   
Women participants and female partners of participating men must be of non-
childbearing potential or be using a highly effective method of birth control 14 
days prior to until at least 24 hours after injection of [18F]FEMPA.  
o Non-child bearing potential is defined as a female that must be 
either postmenopausal (no menses for at least 12 months prior to 
Screening) or a female or male that is surgically sterile (bilateral 
tubal ligation, bilateral oophorectomy or hysterectomy, 
sterilization). 
o Highly effective method of birth control is defined as practicing at 
least one of the following:  A birth control method that results in a 
less than 1% per year failure rate when used consistently and 
correctly, such as oral contraceptives for at least 3 months prior to 
injection, an intrauterine device (IUD) for at least 2 months prior 
to injection, or barrier methods, e.g., diaphragm or combination 
condom and spermicide.  Periodic abstinence (e.g. calendar, 
ovulation, symptothermal, post-ovulation methods) is not 
acceptable. 
 
5.2.2 Exclusion Criteria 
• 
Motor Neurone Disease syndromes that do not have clinically detectable 
Upper Motor Neurone signs (e.g., flail arm or flail leg onset of ALS) 
• 
A clinical diagnosis of dementia as determined by the Investigator. 
• 
Current treatment with anticoagulants (e.g., warfarin, novel oral 
anticoagulants, heparin) that might preclude safe completion of the 
lumbar puncture. 
• 
Condition that precludes the safe performance of routine lumbar 
puncture, such as prohibitive lumbar spinal disease, bleeding diathesis, or 
clinically significant coagulopathy or thrombocytopenia.  
• 
Use of investigational drugs or devices within 60 days prior to baseline 
(dietary supplements taken outside of a clinical trial are not exclusionary, 
e.g., coenzyme Q10). 


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• 
Prolonged prothrombin time or activated partial thromboplastin time 
>2xULN 
• 
Severe hypertension or hypotension 
• 
Glomerular filtration rate (GFR) <35 mL/min 
• 
Prior exposure to any exogenous form of APC 
• 
FVC at screening of <50% of predicted 
o Participants who fail to meet this criterion due to technical difficulties 
may be considered at the Principal Investigator’s discretion. 
• 
Inability to lie flat for investigational procedures (MRI, PET, Lumbar 
Puncture) 
• 
Previously obtained MRI scan with evidence of clinically significant 
neurological disorder (in the opinion of the Investigator). 
• 
Presence of any of the following clinical conditions at the time of 
screening: 
o Drug or alcohol abuse, unstable medical disease (such as unstable 
angina or chronic obstructive pulmonary disease), active infectious 
disease (such as Hepatitis B or C or tuberculosis), current 
malignancy, unstable psychiatric illness (based on a prior 
psychiatric diagnosis that is unstable as determined by the 
subject's treating Psychiatrist) defined as psychosis or untreated 
major depression within 90 days of the screening visit, or any 
other condition that, in the opinion of the Investigator, might 
interfere with study conduct or interpretation of the study results.  
6. Investigational Plan 
6.1 Participant Identification Numbers 
A participant ID Number will be assigned in sequential order to all patients who 
consent to screening. This unique 2-digit participant number for subjects will be 
used to identify the participant on all study forms and lab specimens. 
6.2 Schedule of Activities 
Refer to the protocol synopsis Schedule of Activities that summarizes the 
assessments to be conducted 
6.2.1 Screening Visit 
 All subjects will undergo a screening evaluation prior to the Baseline visit. This 
evaluation will include the following activities and will take about 2 hours to 
complete: 


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• An explanation of the purpose, procedures, potential risks and 
benefits of this study and informed consent will be obtained 
• Demographics  
• Review of the subject’s medical and family history 
• Review of concomitant medications 
• Vital signs (blood pressure, heart rate and temperature) 
• General physical examination 
• Neurological examination (ALSFRS-R, MGH Upper Motor 
Neuron scale assessment (MGH UMN)) 
• Edinburgh Cognitive and Behavioural ALS Screen (ECAS) 
•  Collect blood sample for DNA for TSPO genotype 
• Blood and urine collection for clinical laboratory assessments  
• Blood and urine collection for research samples 
• Urine pregnancy test for women of childbearing potential. 
• Electrocardiogram (ECG)  
• Forced Vital Capacity (FVC) 
• A review of the inclusion/exclusion criteria to confirm that the 
subject is eligible to continue to the Baseline visit 
6.2.2 Baseline Assessments (Day 1, 2 and 3): 
The activities for the Baseline Assessments will be completed within 30 days of 
completing the Screening visit. Screening and Day 1 can occur on the same day. 
 Baseline assessments will include the following activities and will take Day 1-3 
to complete, as listed in the schedule of activities. These assessments and 
subsequent activities will occur as an inpatient of Macquarie University Hospital.  
All assessments listed below must be completed prior to enrollment of the 
subject into the treatment phase of the study. 
• Review of continuing ability to consent 
• Vital signs (blood pressure, heart rate and temperature), height 
and weight 
• Physical examination 
• ECG  


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• Blood collection for clinical laboratory assessments (daily) 
• Urinalysis (day 3) 
• Blood and urine collection for research samples (daily) 
• Urine pregnancy test (Day 1) 
• Neurological exam (incl. ALSFRS-R and UMN Index) 
• Brain MRI (Day 2) 
• Brain PET Imaging (Day 2) 
• Lumbar puncture for collection of CSF (Day 3) 
• Review of concomitant medications 
• Review of current medical conditions 
• Review of adverse events related to lumbar puncture 
• Repeat review of the inclusion/exclusion criteria 
Once all Visit 1 activities have been completed and the Investigator determines 
that all eligibility criteria have been met, the subject may be enrolled into the 
study. Once enrolled, the subject will be enrolled in one of two dose levels of 
3K3A-APC (15mg or 30mg). 
 
6.2.3 Inpatient Daily Assessments (Visit 4-10, Days 4-10) 
Administration of 3K3A-APC will occur as an inpatient of Macquarie University 
Hospital at 12 hourly intervals commencing Day 3 and continuing to Day 5. 
Subjects will be assessed daily while inpatients in Macquarie University Hospital.  
The assessments are outlined in the schedule above and will include: 
• Vital signs (Daily) 
• ECG (Daily, excluding day 6 and 7)  
• Clinical laboratory assessments (EUC, LFT, FBC, APTT, PT) 
• Urinalysis (Day 4, 5,10) 
• Research blood and urine samples (Daily, excluding day 6 
and 7) 
• Urine pregnancy test (Day 8) 
• Physical examination (Day 8) 
• Neurological examination (ALSFRS-R, MGH UMN scale) 
(Day 8) 
• Repeat MRI Brain (Day 9) 
• Repeat PET Imaging (Day 9)  


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• Repeat Lumbar puncture (Day 10) 
• Assessment of adverse events (Daily) 
 
Following completion of Day 10 assessments, the subject will be discharged 
home for follow up as an outpatient for the end of study visit (Visit 11). 
6.2.4 End of Study Assessment (Visit 11 Day 15) 
The end of study visit will be completed as an outpatient. End of study 
assessments will include: 
• Vital signs  
• ECG 
• Physical examination 
• Neurological examination (ALSFRS-R, MGH UMN Scale)  
• Blood and urine collection for clinical laboratory assessments 
• Blood and urine collection for research samples 
• Assessment of adverse event 
6.2.5 Early termination visit 
All patients who discontinue early from the study should return to the clinic to 
complete the procedures for Visit 11. Study personnel should make every effort 
to conduct all protocol-specified procedures to complete the study.  
7. Study Assessments 
7.1 Clinical Assessments 
7.1.1 Vital Signs 
 
Blood pressure, heart rate and temperature will be measured at every 
visit via standard methods. Height and weight will be recorded at baseline. 
7.1.2 General Physical Examination 
 
At screening and baseline (Day 1), Visit 8 and Visit 11, a general physical 
examination will be recorded by the investigator including cardiac, respiratory, 
abdominal examination. 
7.1.3 Neurological Examination 
 
Standardised neurological examination will be performed by the principal 
investigator at screening, Visit 1, Visit 8 and Visit 11. This will include 
standardised assessments of UMN features such as tone, reflexes and primitive 
reflexes (plantar response).  Assessment of motor function will be determined in 
standardised fashion (MRC grade, pinch grip and grip strength). ALSFRS-R and 
UMN Scale will be assessed and calculated. 


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7.1.3 Electrocardiograph 
 
A standard 12 lead ECG will be recorded at screening, baseline and each 
visit of the study (excluding day 6 and 7) via standard methods. 
7.1.4 Forced Vital Capacity 
 
Forced vital capacity in the standing or seated position will be obtained 
via standard methods at screening. 
7.1.5 Clinical Laboratory Tests 
Routine clinical laboratory tests indicated in the table below will be performed at 
screening and follow up visits according to the visit schedule. A central 
laboratory (Sonic) will be implemented in order to guarantee identical analysis 
methods, consistent normal ranges and thus common interpretation of 
laboratory changes.  If not stated otherwise, venous whole blood will be collected 
in blood collection tubes (vacutainers).   
The coagulation panel (PT/APTT) will be reviewed by the Investigator prior to 
lumbar puncture.  
No more than 40 mL will be drawn at any visit, including both clinical and 
research blood samples (see 7.2.4 below). 


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As part of the clinical laboratory tests, urine will be collected for urinalysis at 
screening, day 3-5., day 10 and day 15.  
A urine pregnancy test will be conducted at screening and prior to [18F]FEMPA 
PET scan on Visit 1 and Visit 8. 
All samples for laboratory analysis must be collected, prepared, labelled, and 
shipped according to the laboratory’s requirements.   
7.1.6 Adverse Events 
During each visit participants will be asked about adverse events.  AEs will be 
recorded on an ongoing adverse event log.  
7.1.7  Concomitant Medications 
All concomitant medications during hospitalization will be recorded on a log. 
CLINICAL LABORATORY TESTS 
METABOLIC PANEL 
BLOOD COUNT 
Sodium (Na) 
Potassium (K) 
Chloride (Cl) 
Bicarbonate (HCO3) 
Urea 
Glucose  
Calcium (Ca)  
Creatinine (Cr) 
Bilirubin Total 
Albumin 
Total Protein 
Aspartate aminotransferase (AST) 
Alanine aminotransferase  (ALT) 
Alkaline Phosphatase (ALP) 
Creatinephosphokinase (CPK) 
Iron studies (Fe, Ferritin) 
Prothrombin time (PT) 
Activated Partial Thromboplastin Time (APTT)  
White Blood Cell Count (WBC) 
Red Blood Cell Count (RBC) 
Hemoglobin (Hb) 
Hematocrit (HCT) 
Platelet Count (PLT) 
 


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7.2 Other Assessments 
7.2.1 TSPO Genotyping 
A sample of venous blood will be collected at screening for DNA extraction to be 
used for TSPO genotyping to determine whether subjects have high affinity, 
mixed affinity or low affinity binding for the TSPO ligand according to 
established methods (10).  
7.2.2 Microglial PET Imaging 
Subjects will undergo [18F] FEMPA PET imaging to assess the activation state of 
microglia in the brain. On Day 2 and Day 9 subjects will receive approximately 
200 MBq of [18F] FEMPA with dynamic brain scanning from time=0 to 50 
minutes according to previously established protocols. Scanning time will be 50 
minutes in total. 
7.2.3 Brain MRI (ALS protocol) 
Subjects will undergo a structural MRI brain scan on Day 2 and on Day 9 
according to established protocols (9). At the discretion of the investigator and 
imaging staff, subjects who have presence of pacemakers, aneurysm clips, 
artificial heart valves, cochlear implants, metal fragments or foreign objects in 
the eyes, skin or body or any other known contra-indication to MRI may be 
advised not to complete a baseline (or follow-up) MRI scan, but these subjects 
may still participate in the study.  MRI is being conducted to assess the structure 
of the brain, for co-registration with PET images as well as assessing the integrity 
of the blood brain barrier. 
7.2.4 Biomic Blood and Urine samples 
Whole blood (8 mL), serum (about 10 mL) and plasma (about 10 mL) will be 
collected to conduct proteomic, metabolomic and other analyses. Urine (about 
10 mL) will be collected to conduct analyte analyses.  Where possible the 
research blood samples will be collected in a fasted state (i.e., minimum of 8 
hours since last meal/food intake) to ensure the quality of samples for future 
analyses.  All research samples will be stored indefinitely for research purposes. 
7.2.5 Lumbar Puncture for CSF Examination 
The lumbar puncture (LP) will be performed under image intensifier by a 
qualified radiologist appointed by the investigator.  A lumbar puncture for the 
collection of 10 mL of CSF will be conducted for all subjects at Visit 3 and Visit 10 
unless there is evidence of clinically significant coagulopathy or 
thrombocytopenia that would interfere with the safe conduct of the procedure. 
The first 2 mL of CSF will be processed at the local laboratory facility to conduct 
standard analyses on cell count, protein and glucose levels.  Subjects will be 
closely monitored during the procedure and following the procedure. The CSF 
samples will be stored indefinitely for research purposes for analytes relevant to 
inflammation and monocyte activation.  


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8. Study Medications 
8.1 
Study medication dose, packaging and labelling 
3K3A-APC is supplied frozen in vials of 5mg at a concentration of 1.0mg/mL.  
Upon thawing, the sterile solution  is clear and  requires  dilution in 0.9% sodium 
chloride for  intravenous infusion. 3K3A-APC will be prepared for infusion by 
clinical trials pharmacist using aseptic technique. For the first treatment cohort, 
3 vials of 3K3A-APC will be diluted to 100mL. For the second cohort, 6 vials of 
3K3A-APC will be diluted to 100mL. See pharmacy manual for more information 
regarding study medication handling.  
Infusion bags will be labelled by the clinical trials pharmacist with study name 
(3K3A-APC in ALS) and protocol number (ZZ-3K3A-202), patient name and 
study number, and medication name (3K3A-APC) and concentration. 
8.2 
Study medication administration 
The study medication must be infused within 8 hours of preparation. 
The 100mL of study medication will be administered by IV infusion over 15 
minutes (by pump set to 400mL/hr). Five doses of study medication will be 
administered at 12 hourly intervals ± 1 hour starting on day 3 of hospitalization 
and finishing on day 5 of hospitalisation. Study drug should be flushed from 
infusion line with 0.9% sodium chloride. 
If a hypersensitivity reaction develops, infusion is stopped immediately and no 
further doses of study medication will be administered. 
8.3 Prohibited Concomitant Medications 
Anticoagulant medications are prohibited during the treatment period, and for 7 
days prior and 7 days post study drug administration. Anticoagulant medications 
include heparins, low molecular weight heparins, novel oral anticoagulants (e.g. 
dabigatran, rivaroxaban and apixaban), warfarin. 
Medications that might interfere with monocyte activation such as systemic 
corticosteroids or immune suppressants are prohibited from screening visit until 
follow up at day 15.  
It is preferred that subjects do not participate in clinical trials of other 
investigational interventions during the entire study. 
All concomitant medications reported at the time of the screening visit and for 
the duration of the subject’s participation should be recorded on the 
Concomitant Medication Log. 
9. Subject Withdrawals 
Subjects will be advised in the written informed consent forms that they have the 
right to withdraw from the study at any time without prejudice, and may be 
withdrawn at the investigator’s or sponsor’s discretion at any time. A subject 


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should be withdrawn from the study if the investigator considers it to be 
medically necessary, or if the subject withdraws consent.  All reasons for subject 
withdrawals from the study will be recorded in the source documentation and 
appropriate eCRF.   
10.  Safety/Adverse Events 
Site investigators and coordinators will be instructed to assess for adverse 
events at in-person study visits. 
10.1 Adverse Event Definitions 
Adverse Events (AE) 
An AE is any undesirable experience occurring to a subject during a clinical trial, 
whether or not considered related to the investigational product. 
Pre-existing conditions (i.e., undesirable experiences, signs or symptoms that 
begin prior to the Screening Visit) will be recorded on the medical history CRF 
page. These conditions will not be reported as an AE unless they worsen in 
intensity or frequency after the Screening Visit. 
Treatment-emergent AEs are undesirable experiences, signs, or symptoms that 
begin or worsen in intensity or frequency at the time of or after the 
administration of study drug. 
Serious Adverse Event (SAE) 
An SAE is an AE that is fatal or life-threatening, or results in prolongation of 
hospitalization, persistent or significant disability/incapacity. A life-threatening 
AE is an AE that, in the view of the investigator, places the subject at immediate 
risk of death from the event, as it occurred.  Important medical events that may 
not result in death, be life-threatening, or require hospitalization may be 
considered an SAE when, based upon appropriate medical judgment, they may 
jeopardize the subject and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition. 
Unexpected Adverse Event 
For FDA reporting purposes, an unexpected AE is an AE not previously reported 
or an AE that occurs with specificity, severity or frequency that is not consistent 
with the current investigator’s brochure. 
10.2  Relationship to Investigational Product 
The assessment of the relationship of an AE to the administration of the study 
drug is a clinical decision based on all available information at the time the event 
is being documented.  The following definitions of the relationship between the 
study drug and the AE (including SAEs) should be considered: 
• Unrelated - No possible relationship 


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The temporal relationship between drug exposure and the adverse event 
onset/course is unreasonable or incompatible, or a causal relationship to study 
drug is implausible. 
• Unlikely - Not reasonably related, although a causal relationship 
cannot be ruled out 
While the temporal relationship between drug exposure and the adverse event 
onset/course does not preclude causality, there is a clear alternate cause that is 
more likely to have caused the adverse event than the study drug. 
• Possible - Causal relationship is uncertain 
The temporal relationship between drug exposure and the adverse event 
onset/course is reasonable or unknown, de-challenge or re-challenge 
information is either unknown or equivocal, and while other potential causes 
may not exist, a causal relationship to the study drug does not appear probable. 
• Probable - High degree of certainty for causal relationship 
The temporal relationship between drug exposure and the adverse event 
onset/course is reasonable.  There is a clinically compatible response to de-
challenge (re-challenge is not required), and other causes have been eliminated 
or are unlikely. 
• Definite - Causal relationship is certain 
The temporal relationship between drug exposure and the adverse event 
onset/course is reasonable, there is a clinically compatible response to de-
challenge, other causes have been eliminated, and the event must be definitive 
pharmacologically or phenomenologically, using a satisfactory re-challenge 
procedure if necessary. 
10.3 Intensity/Severity of an Adverse Event 
In addition to assessing the relationship of the administration of the 
investigational product to AEs, an assessment is required of the intensity 
(severity) of the event. The following classifications should be used: 
• Mild:   
A mild AE is an AE, usually transient in nature and generally not 
interfering with normal activities. 
• Moderate:   
A moderate AE is an AE that is sufficiently discomforting to 
interfere with normal activities. 
• Severe:   
A severe AE is an AE that incapacitates the subject and prevents 
normal activities.   Note that a severe event is not necessarily a 
serious event.  Nor must a serious event necessarily be severe. 


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10.4 Recording of Adverse Events 
Adverse events including clinical significant biological sample test 
abnormalities,, whether observed by the investigator, elicited from or 
volunteered by the subject, should be recorded on the Adverse Event Log.  This 
will include a brief description of the experience, the date of onset, the date of 
resolution, the severity, and seriousness and whether the event was related to 
participation in the research study. 
10.5 Responsibilities for Reporting Serious Adverse Experiences 
• 
The Investigator should notify the sponsor within 24 hours of his/her 
becoming aware of the occurrence of a serious adverse experience or 
suspected unexpected serious adverse reaction (SUSAR). 
• 
The following information should be supplied if available at the time of the 
email or telephone call: subject number, subject age and gender, date of onset 
of event, event description, whether event required treatment, death and 
autopsy report, an identification of which criteria for a serious experience 
have been met, the Investigator’s current opinion of the relationship between 
the event and study participation. 
• 
The Investigator will comply with Macquarie University HREC regulations 
regarding the reporting of adverse experiences. 
11. Referrals for Unexpected Results of Study Participation 
If a research assessment, lab or MRI result reveals a clinically significant 
abnormality (e.g., renal impairment on metabolic profile) the subject should be 
informed of this result and the Investigator should provide the subject with the 
appropriate referral as necessary.  
12.  Potential Risks 
12.1 Blood Sampling 
Risks associated with venous blood draw include pain and bruising at the site 
where the blood is taken.  Sometimes people can feel lightheaded or even faint 
after having blood drawn.   
12.2 Lumbar Puncture 
The most common risks of a lumbar puncture are pain at the site and a 
temporary headache usually due to a small amount of CSF leakage around the 
needle insertion site.  Lying down for a few hours after the test can make a 
headache less likely to occur. There is a slight risk of infection because the needle 
breaks the skin’s surface, providing a possible portal of entry for bacteria.  A 
temporary numbness to the legs or lower back pain may be experienced.  There 
is a small risk of bleeding in the spinal canal.  Subjects will have blood drawn at 
Screening to test for coagulopathies.   


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12.3 MRI – ALS Protocol 
Subjects should notify the study doctor if they suffer from claustrophobia 
because they may become anxious while in the magnetic resonance scanner.  
There may be loud noises such as knocking or hammering that occur while the 
MRI is being conducted.  Subjects should also inform the study doctor if they 
have a pacemaker or metal implants (screws, plates or clips) because this may 
preclude MR evaluation. The imaging will take 60 minutes. 
12.4. PET Imaging 
Specific potential risks for PET imaging are as follows: 
1) radiation exposure from [18F]FEMPA 
2)  (see companion protocol), 
3) having an intravenous injection. 
 
12.5 Unknown Risks 
In addition to the known risks listed above, the imaging procedures in this study 
may cause unknown risks to the participant, or a developing embryo or fetus or 
possible risks to the future offspring of male participants.  Female subjects or a 
female partner of a male subject who report a pregnancy within 30 days of 
[18F]FEMPA injection will be asked to have a urine pregnancy test. 
13. Regulatory / Ethics 
13.1. Compliance Statement 
This study will be conducted in accordance with the Good Clinical Practice (GCP) 
and the International Conference on Harmonization (ICH) guidelines and any 
applicable national and local regulations.  
All procedures not described in this protocol will be performed according to the 
study Operation Manuals unless otherwise stated.  Laboratory tests/evaluations 
described in this protocol will be conducted in accordance with quality 
laboratory standards as described in the central laboratory manual unless 
otherwise stated. 
13.2. Informed Consent 
In accordance with relevant regulations, an informed consent agreement 
explaining the procedures and requirements of the study, together with any 
potential hazards/risks must be read by and explained to each subject.  Each 
subject will sign such an informed consent form.  The subject must be assured of 
the freedom to withdraw from participation in the study at any time.  
It is the Investigator’s responsibility to make sure that the subject understands 
what she/he is agreeing to and that written informed consent is obtained before 


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the subject is involved in any protocol-defined procedures including screening 
procedures.  It is also the Investigator’s responsibility to retain the original 
signed consent form and provide each subject with a copy of the signed consent 
form.  
The Project Management Team must be given an opportunity to review the 
consent forms prior to site Human Research Ethics Committee (HREC) 
submission and before it is used in the study. 
13.2 Human Research Ethics Committee Review 
All necessary information will be supplied by the Investigator including the 
protocol and consent forms to the HREC for review and approval.  The 
Investigator agrees to provide the HREC all appropriate material.  The trial study 
will not begin until the Investigator has obtained appropriate HREC approval.  A 
copy of the approval letter and approved consent form must be submitted to the 
CRO. 
The Investigator will request from the HREC a composition of the members 
reviewing the protocol and informed consents.  Appropriate reports on the 
progress of this study by the Investigator will be made to the HREC and the CRO 
in accordance with institutional and government regulations.  The CRO will 
notify the site when the HREC may be notified of study completion. It is the 
Investigator’s responsibility to notify the HREC when the study ends.  This 
includes study discontinuation, whether it is permanent or temporary. A copy of 
the site HREC acknowledgement of study completion must be submitted to the 
Project Management Team. 
13.3. Protocol Amendments 
Changes to the protocol should only be made via an approved protocol 
amendment.  Protocol amendments must be approved by the study’s Steering 
Committee and the HREC prior to implementation, except when necessary to 
eliminate hazards and/or to protect the safety, rights or welfare of subjects. 
13.4 Subject Confidentiality 
The site Investigator must assure that the confidentiality of subjects, including 
their personal identity and personal medical information, will be maintained at 
all times.  Subjects will be identified by consecutive ID numbers issued by the 
data management team on case report forms and other study materials 
submitted to data management, the pathology laboratory, and bio-repository. 
After a subject signs an informed consent form, it is required that the site 
Investigator permit regulatory agency personnel to review the signed informed 
consent(s) and that portion of the subject’s medical record that is directly related 
to the study.  This shall include all study relevant documentation including 
subject medical history to verify eligibility, laboratory test result reports, 
admission/discharge summaries for hospital admissions occurring while the 
subject is in the study, and autopsy reports for deaths occurring during the study 
(when available).   


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13.5 COVID-19 Contingency Plan 
 
The COVID-19 pandemic and the restrictions imposed by the NSW government 
present an unprecedented challenge to clinical trials. However, compliance and 
adherence to these regulations and the recommendations of Macquarie 
University is a priority in order to maintain the safety of staff and trial 
participants.  
The following protocol modifications will be applied as deemed appropriate by 
the Principal Investigator in accordance with COVID-19 regulations: 
• The exclusion criteria of forced vital capacity (FVC) at screening of <50% of 
predicted will not be assessed as part of eligibility for trial participation. 
• The inclusion criteria of: 
– symptom onset less than 36 months before screening 
– diagnosis of ALS less than 24 months before screening 
will be re-assessed by the Principal Investigator in the event that a trial 
participant’s eligibility is affected by delays to their randomization date.  
14. Documentation 
14.1   Study File and Source notes 
The Investigator shall maintain and retain site documentation consistent with the 
requirements of the TGA annotated ICH-GCP. Source notes will include all 
printouts/reports of tests/procedures, as specified in the protocol, for each 
subject. This documentation, together with the subject’s hospital/medical records, 
and the participant’s original signed consent form is the subject’s source 
information for the study. 
15. Statistical Analysis 
15.1 Demographic and Baseline Characteristics 
Descriptive analyses will be reported for this study. Continuous parameters, will 
be summarised as the number of observations, mean, median, standard 
deviation, minimum, maximum, and if appropriate two-sided 95% CI’s. 
Categorical parameters will be summarised as frequencies, percentages, and 
exact Clopper-Pearson two-sided 95% CI’s. Data will be summarised by dose 
cohort, and overall.  


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15.2 Primary efficacy variables 
Adverse events will be coded using the most recent version of the Medical 
Dictionary for Regulatory Activities (MedDRA) coding system to give a System 
Organ Class (SOC) and Preferred Term (PT) for each adverse event (AE). 
Treatment emergent AEs will be defined as AEs with an onset date on or after the 
date of administration of study drug. If the onset date is missing, the AE will be 
considered to be treatment emergent. Only treatment-emergent AEs will be 
summarized. All reported AEs will be listed. The number of AEs, and the number 
and percentage of patients reporting at least one AE will be summarized by PT 
nested within SOC for required AE types. AE data will be summarized by dose 
cohort, and overall.  
Microglial activation as measured by [18F] FEMPA will be summarised at each 
time point for each dose cohort and overall as observed value, change from 
baseline, and % change from baseline. Microglial activation will be summarised 
with number of observations, mean, median, standard deviation, minimum, 
maximum, and two-sided 95% CI’s.  
15.3 Secondary efficacy variables: 
The secondary efficacy variables of MRI scan of the brain, and biomic 
measurements of serum, plasma, urine and CSF will be summarized over time for 
each dose cohort, and overall as for the primary efficacy variables. 
15.4 Clinical Safety Laboratory data 
Clinical safety laboratory data (haematology, biochemistry, and urine chemistry) 
will be summarised at each protocol scheduled time point, by dose cohort, and 
overall. Actual values and actual changes from baseline will be presented. 
Urinalysis results evaluation (normal, abnormal not clinically significant or 
abnormal clinically significant) will be summarised at each protocol scheduled 
time point, by dose cohort with frequency tabulations. 
15.5 Vital Signs 
Vital sign measurements will include height, weight, body mass index (BMI), 
body temperature, respiratory rate, heart rate and systolic and diastolic blood 
pressure. Vital signs will be collected at the times stipulated in the Protocol 
Schedule of Assessments. 
Vital sign results will be summarised at each protocol scheduled time point, by 
dose cohort and overall. Actual values and actual changes from baseline will be 
presented. 
15.6 Physical and Neurological Examinations 
A physical examination and neurological examinations will be performed at 
times stipulated in the Protocol Schedule of Assessments. Physical examination 
and neurological findings will be listed by Visit and Participant. Clinically 
significant changes will be captured as adverse events. 


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16. Data Management 
An Internet accessible Electronic Data Capture (EDC) system for data 
management will be utilized for this study, designed by the data management 
team.  This system will be protected by 128-bit server certificates and will utilize 
authenticated, password-protected accounts for each investigator.  The EDC 
system will be designed to ensure timeliness and accuracy of data as well as the 
prompt reporting of data from the study.  The system will be compliant with 
relevant FDA regulatory requirements per 21 CFR Part 11 and will be stored on 
an Australian based server. 
. 
Data review, coding and query processing will be done through interaction with 
the data management team, site personnel and the Study Monitor.  Queries will 
be generated in real-time as data are entered.  Once the data are submitted to the 
EDC system, they are immediately stored in the central study database located at 
McLeod Consulting Group and will be accessible for review by data management 
staff.  Any changes to the data will be fully captured in an electronic audit trail.  
As data recorded by sites in eCRFs are received, narrative text of adverse 
experiences and concomitant medications will be periodically coded using 
established coding mechanisms.   
The cycle of electronic data entry, review, query identification/resolution, and 
correction occurs over the course of the study period until all subjects have 
completed the study. 
Data will be securely maintained by the data management team.  Once the data 
management team, in conjunction with the principal investigator agree that all 
queries have been adequately resolved and the database has been deemed 
“clean.” The database will be officially signed off and deemed locked.  All 
permissions to make changes (append, delete, modify or update) the database 
are removed at this time. 
All data obtained during the conduct of the study, will be stored indefinitely for 
research purposes.  Research data will be made available to researchers to 
conduct analyses if requested and if required for publication in peer-reviewed 
journals. 
17. Study Monitoring 
In accordance with TGA annotated ICH Guidelines for Good Clinical Practice. The 
study will be monitored by an independent Clinical Trial Monitor reporting to 
the Macquarie University Clinical Trial Unit delegated responsibility by the study 
sponsor (Macquarie University) to verify that: 


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(a) 
The rights and well-being of human subjects are protected. 
(b) 
The reported trial data are accurate, complete, and verifiable from source 
documents. 
(c) 
The conduct of the trial is in compliance with the currently approved 
protocol/amendment(s), with GCP, and with the applicable regulatory 
requirement(s). 
The monitoring activities will include: 
• 
Verifying that the site investigators and coordinators have adequate 
qualifications, that resources remain adequate throughout the trial period, 
and that facilities, equipment, and staff are adequate to safely and properly 
conduct the trial. 
• 
Verifying that the site investigators follow the approved protocol and all 
approved amendment(s), if any. 
• 
Verifying that written consent was obtained for each subject participating in 
the trial. 
• 
Verifying that the investigators are enrolling only eligible subjects. 
• 
Verifying that source documents and other trial records are accurate, 
complete, kept up-to-date and maintained. 
• 
Verifying that the investigator provides all the required reports, notifications, 
applications, and submissions to the HREC, and that these documents are 
accurate, complete, timely, legible, dated, and identify the trial. 
• 
Monitoring adverse events, concomitant medications and intercurrent 
illnesses. Determining whether all adverse events (AEs) are appropriately 
reported within the time periods required by GCP, the protocol, and the 
HREC. 
• 
Communicating deviations from the protocol, Standard Operating 
Procedures, GCP, and the applicable regulatory requirements to the 
investigator and taking appropriate action designed to prevent recurrence of 
the detected deviations. 
• 
The HREC shall conduct continuing review of research covered by these 
regulations at intervals appropriate to the degree of risk, but not less than 
once per year, and shall have authority to observe or have a third party 
observe the consent process and the research. Continuing review by the 
HREC routinely includes interim progress reports, as directed by the Board, 
review of proposed changes to research, adverse event reports, review of any 
protocol deviations, visits to the research site, and annual review of the 
research. 


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18. Publication of Research Findings 
Publication of results of this study will be governed by the Steering Committee in 
accordance with the International Committee of Medical Journal Editors (ICJME) 
Uniform Requirements for Manuscripts Submitted to Biomedical Journals.  
19. References 
 
1. 
Miller RG, Zhang R, Block G, Katz J, Barohn R, Kasarskis E, et al. NP001 
regulation of macrophage activation markers in ALS: a phase I clinical and 
biomarker study. Amyotroph Lateral Scler Frontotemporal Degener. 2014;15(7-
8):601-9. 
2. 
Zurcher NR, Loggia ML, Lawson R, Chonde DB, Izquierdo-Garcia D, Yasek 
JE, et al. Increased in vivo glial activation in patients with amyotrophic lateral 
sclerosis: assessed with [11C]-PBR28. Neuroimage Clin. 2015;7:409-14. 
3. 
Jucaite A, Svenningsson P, Rinne JO, Cselenyi Z, Varnas K, Johnstrom P, et 
al. Effect of the myeloperoxidase inhibitor AZD3241 on microglia: a PET study in 
Parkinson's disease. Brain. 2015;138(Pt 9):2687-700. 
4. 
Varrone A, Oikonen V, Forsberg A, Joutsa J, Takano A, Solin O, et al. 
Positron emission tomography imaging of the 18-kDa translocator protein 
(TSPO) with [18F]FEMPA in Alzheimer's disease patients and control subjects. 
Eur J Nucl Med Mol Imaging. 2015;42(3):438-46. 
5. 
Lyden P, Levy H, Weymer S, Pryor K, Kramer W, Griffin JH, et al. Phase 1 
safety, tolerability and pharmacokinetics of 3K3A-APC in healthy adult 
volunteers. Curr Pharm Des. 2013;19(42):7479-85. 
6. 
Lyden P, Pryor KE, Coffey CS, Cudkowicz M, Conwit R, Jadhav A, et al. Final 
Results of the RHAPSODY Trial: A Multi-Center, Phase 2 Trial Using a Continual 
Reassessment Method to Determine the Safety and Tolerability of 3K3A-APC, A 
Recombinant Variant of Human Activated Protein C, in Combination with Tissue 
Plasminogen Activator, Mechanical Thrombectomy or both in Moderate to 
Severe Acute Ischemic Stroke. Ann Neurol. 2019;85(1):125-36. 
7. 
Zhong Z, Ilieva H, Hallagan L, Bell R, Singh I, Paquette N, et al. Activated 
protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting 
SOD1 in motor neurons and microglia cells. J Clin Invest. 2009;119(11):3437-49. 
8. 
Winkler EA, Sengillo JD, Sagare AP, Zhao Z, Ma Q, Zuniga E, et al. Blood-
spinal cord barrier disruption contributes to early motor-neuron degeneration in 
ALS-model mice. Proc Natl Acad Sci U S A. 2014;111(11):E1035-42. 
9. 
Welton T, Maller JJ, Lebel RM, Tan ET, Rowe DB, Grieve SM. Diffusion 
kurtosis and quantitative susceptibility mapping MRI are sensitive to structural 
abnormalities in amyotrophic lateral sclerosis. Neuroimage Clin. 
2019;24:101953. 
10. 
Owen DR, Yeo AJ, Gunn RN, Song K, Wadsworth G, Lewis A, et al. An 18-
kDa translocator protein (TSPO) polymorphism explains differences in binding 
affinity of the PET radioligand PBR28. J Cereb Blood Flow Metab. 2012;32(1):1-5. 
 


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20. Samples of Forms 
 
ALSFRS-R 
MGH UMN Scale 
Edinburgh Cognitive and Behavioural ALS Screen (ECAS)