Clinical trial NCT03456882
Conditions: Amyotrophic Lateral Sclerosis
Keywords: AMYOTROPHIC LATERAL SCLEROSIS RNS60 Biomarkers clinical trial therapy
Sponsor: Mario Negri Institute for Pharmacological Research
Start date: 2017-05-30; Completion date: 2021-05-30
Enrollment: 147 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Amyotrophic Lateral Sclerosis (ALS) is a rare lethal neurodegenerative disease involving inflammation. Riluzole, the only drug for ALS, improves median survival by 3 months. This prompts new treatments of ALS. RNS60 is an experimental drug with favorable effects in preclinical studies of neuroinflammation and neurodegeneration. Based on significant efficacy demonstrated in preclinical studies and its excellent clinical safety profile, RNS60 is a promising candidate for a drug to treat ALS. Developing a pharmacodynamic marker will be a first and important step for dose finding and exploration of the mechanism of action in human, and pave the way to trials measuring drug efficacy.

The Investigator propose a multicenter, randomized, double-blind, placebo-controlled, parallel group, Phase II trial. The study centers will be located in Italy and at Massachusetts General Hospital (MGH) in Boston. A total of 142 ALS patients will be randomly assigned to RNS60 or placebo (administered by intravenous infusion once/week and inhaled via nebulization every morning for 24 weeks). All participants will also take riluzole (50-mg tablet twice/day). Blood samples for biomarker analysis (protein, RNA) will be collected in the screening period, on day 1, week 4,12 and 24. Both safety and potential therapeutic effects of RNS60 will be also assessed.

Detailed description:
ALS is a rare neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex. The only drug showing to improve survival in patients with ALS is riluzole. However, the benefits of riluzole only consist in a three-month delay of death while disability and other outcome measures are virtually unaffected. This highlights the need to test novel approaches with documented activity on markers of disease mechanisms and, at the same time, able to slow the progression of the disease.

The major determinants of motor neuron death in ALS remain to be established. Emerging evidence points to an involvement of the adaptive immune response in disease progression. RNS60 is a novel agent with immunomodulatory properties. Adding to previous reports of anti-inflammatory and neuroprotective activities of RNS601,2,3,4, our group showed a protective effect of RNS60 on motor neurons in both in vitro and in vivo models of familial ALS carrying the SOD1G93A mutation (unpublished data). Therefore, RNS60 presents itself as a promising candidate for the treatment of ALS patients. Its exceptional safety profile, demonstrated both in preclinical toxicology studies and FDA-approved clinical phase I studies upon inhaled and IV administration, supports testing of RNS60 in clinical phase II studies in ALS.

The investigators have identified six candidate pharmacodynamic markers of RNS60 that have previously been associated with ALS: 1. T-reg (measured via FOXP3 and CD25 mRNA); 2. Cyp-A; 3. 3-NT; 4. Actin-NT; 5. MCP-1; 6. IL-17.

The investigators have measured and reported the effects on T-reg and IL172 in experimental allergic encephalitis. The investigators also have preliminary unpublished data on MCP1 in allergic asthma.

This background provides the sound rationale for a phase II, biomarker-driven, placebo-controlled, randomized clinical trial.

Primary Objective:

1\. To measure the effect of RNS60 treatment on selected pharmacodynamic biomarkers in ALS patients concurrently treated with riluzole. Candidate markers include 1. T-reg (measured via FOXP3 and CD25 mRNA); 2. Cyp-A; 3. 3-NT; 4. Actin-NT; 5. MCP-1; 6. IL-17.

Secondary Objectives:

1. The preliminary efficacy of RNS60 on functional disability, as measured by the ALSFRS-R scale;
2. The preliminary efficacy of RNS60 in prolonging survival (or time to tracheostomy, whichever comes first);
3. The preliminary efficacy of RNS60 in slowing the decline of forced vital capacity (FVC) from baseline;
4. The tolerability and safety of RNS60 through the identification of unexpected adverse events;
5. The impact of RNS60 on quality of life as measured by ALSAQ-40 scale.

RNS60 has been tested in three Phase I safety studies in the USA (NCT01264783, NCT01057498, and NCT01511302), and a recently completed Phase IIa (NCT02422121) study in UK. Two additional investigator initiated Phase IIa trials are currently ongoing, one at Mass General Hospital (NCT02525471), and one at the University of Zurich (with University of Innsbruck as a second site). The choice of measuring both biological and clinical markers of disease in the same study reflects the attempt to accurately capture the complete clinical impact of RNS60 treatment. If both the biomarker results and clinical measures of the study support the purported efficacy of the drug, a follow-up study (or studies) will be designed to confirm the efficacy of RNS60 in a larger patient population. It is also possible that this study may result in promising biomarker findings but null clinical findings. If this were the case, more dose-finding work would be necessary before ruling out a possible clinical effect. Conversely, positive clinical findings accompanied by negative biomarker findings may necessitate the identification of new biomarkers of target engagement to further guide the drug development process.

Arms: arm 1: RNS60 for injection, i.e. in the IV bags, is produced using 0.9% Sodium Chloride for injection. RNS60 for inhalation, i.e. in the syringes, is produced using 0.9% Sodium Chloride for irrigation. Syringes and IV bags are to remain refrigerated at 2 to 8°C (36 to 46°F) when not in use. RNS60 meets its stability specification for 12 months. arm 2: Normal saline (NS) for injection, i.e. in the IV bags, is packaged 0.9% Sodium Chloride for injection. NS for inhalation, i.e. in the syringes, is packaged 0.9% Sodium Chloride for irrigation. NS does not require refrigerated storage for use. However, for blinding purposes refrigeration is required before distributing to subjects. NS meets stability specifications for 24 months.

RNS60 has been tested in three Phase I safety studies, NCT01264783, NCT01057498, and NCT01511302 in the USA, and a Phase IIa (NCT02422121) study in UK without any safety concern. Two other Investigator initiated Phase IIa trials are currently ongoing, one in Mass General Hospital (NCT02525471), and one in the University of Zurich (with University of Innsbruck as a second site).
Interventions: intervention 1: RNS60
Intervention descriptions: intervention 1: normal saline plus oxigen in nanobubble

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Protocol Title: 
 
The effects of RNS60 on ALS biomarkers 
 
 
 
ACRONYM:  
 
RNS60-ALS 
 
EUDRACT NUMBER 
2016-002382-62 
 
CLINICALTRIALS.GOV NUMBER: NCT03456882 
 
Phrase : 
 
 
II 
 
Promoter:                              Istituto di Ricerche Farmacologiche Mario Negri IRCCS 
 
 
 
 
 
Version N 3.0  
Date:   November 12,   2018 
 
 
 
 
 
 
 
 
 
 
 
 
 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
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TABLE OF CONTENTS 
List of abbreviations ………………………………………………………………………………. 7 
1.0 
INTRODUCTION 
 
1.1 Background and Rationale………………………………………..……………………… 9  
1.2 Pre-clinical Data………………………………………………………………………...... 11 
1.3 Pre-clinical Toxicology Data…………………………………………………………….. 13 
1.4 Clinical Data to Date………………………………………………………………….….. 13 
1.5 Description of RNS60………………………………………………….…………………. 15 
 
1.6 Tolerability…………………………………………………………………………...…… 15 
 
1.7 Dose Rationale……………………………………..……………………………………… 15 
1.8    Pharmacokinetics…………………………………………………………………….…… 16 
 
2.0 
STUDY OBJECTIVES AND OUTCOMES 
 
2.1 Primary Objective………………………………………………………………………... 16 
2.2 Secondary Objectives…………………………………………………………………….. 17 
2.3 Primary Endpoint………………………………………………………………………... 17 
2.4 Secondary Endpoints…………………………………………………………………….. 17 
3.0 
STUDY DESIGN…………………………………………………………………………… 18 
4.0 
STUDY POPULATION 
    4.1    Disease Definition………………………………………………………………………… 18 
 
4.2 Inclusion Criteria…………………………………………………………………...……. 19 
4.3 Exclusion Criteria………………………………………………………………………… 19 
 
4.4 Participating Centers…………………………………………………………….………. 20 
5.0    BIOMARKERS…………………………………………………………………………...... 20 
    5.1   Blood sample collection…………………………………………………………………... 21 
    5.2   Blood sample handling…………………………………………………………………… 21 
    5.3   Sample labels……………………………………………………………………………… 22 
    5.4   Samples numbering……………………………………………………………………… 23 
    5.5   Samples storage…………………………………………………………………….……. 23 
    5.6   Samples shipment………………………………………………………………………… 23 
    5.7   Analytical method………………………………………………………………………… 23 
6.0 
STUDY TREATMENTS 
 
6.1 Study Drug Administration……………………………………………………………… 23 
 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
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6.2 Investigational Product(s)……………………………………………………………….. 24 
 
6.3 Drug Accountability……………………………………………………………………… 25 
 
6.4 Storage and Handling of RNS60 and Placebo………………………………….………. 25 
6.5 Blinding…………………………………………………………………………….……... 26 
6.6 Randomization…………………………………………………………………...………. 27 
6.7 Treatment Arms…………………………………………………..……………………… 27 
6.8 Treatment Assignment and Subject Numbering………………………..……………… 27 
6.9 Concomitant Medications and Prohibited Treatment…………………………………. 28 
6.10 
Study Drug Discontinuation…………………………………………….……………… 28 
6.11 
Premature Subject Withdrawal………………………………………...……………… 28 
6.12  Modification of Study Intervention/Investigational Product for a Subject ………... 29 
6.13 
Wash-out Period for Eligibility…………………………………………..……………. 29 
6.14 
Treatment Compliance……………………………………………………..………….. 30 
7.0 
STUDY PROCEDURES 
 
7.1 Screening……………………………………………………………………..…………… 30 
7.2    Screening Failure………………………………………………………………………… 31 
7.3    Randomization visit…………………………………………………………………….... 31 
7.4 Infusion Visits………………………………………………………..…………………… 32 
7.5    Blood samples collection visit……………………………………………………………. 33 
7.6    Follow up visits…………………………………………………………………................ 33 
7.7 End of study visit for drop out patient ………………………………………………… 33 
7.8    Study Assessments………………………………………………………….…………….. 33 
7.9 Study Duration………………………………………………………………..………….. 35 
Table 2.  SCHEDULE OF PROCEDURES……………………………………………..……... 36 
8.0 
STUDY ADMINISTRATION 
 
8.1 Resource Utilization……………………………………………………………..……….. 38 
 
8.2 Regulatory and Ethical Compliance…………………………………………………….. 38 
8.3 Responsibilities of the Investigator and IRB/IEC/REB……………………………...... 38 
8.4 Informed Consent………………………………………………………………………... 38 
8.5 Amendments to the Protocol……………………………………………………..……… 39 
8.6 Publication of Study Results…………………………………………………….………. 39 
9.0 
SAFETY MONITORING ………………………………………………………………… 39 
9.1 Emergency Unblinding of Treatment Assignment……………………………...……… 39 


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9.2 Adverse Events………………………………………………………………..………….. 40 
9.3 AE Reporting……………………………………………………………………………... 43 
9.4 Reporting of SAEs and SUSARs……………………………………………..………….. 43 
9.5 Follow up of AE/SAE/DAR/DAE/SUSAR…………………………………..………….. 44 
9.6 Data Monitoring Committee ……………………………………………………………  44 
10.0 QUALITY CONTROL 
 
10.1 Data Review and Data Management……………………………………………………. 45 
10.2 Site Monitoring………………………………………………………………..………….. 45 
10.3 Data Collection……………………………………………………………...…………….. 46 
10.4 Database Management…………………………………………………………………… 46 
11.0 
STATISTICS 
 
11.1 
Sample Size Calculation……………………………………………….……………….. 46 
11.2 
Statistical Analysis Plan………………………………………………………………… 48 
11.3 
Compliance………………………………………………………………..…………….. 49 
11.4 
Dictionary…………………………………………………………….………………….. 49 
12.0 
REFERENCES…………………………………………………………..………………... 50 
PROTOCOL APPROVAL PAGE …………………………………………………………….... 55 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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LIST OF ABBREVIATIONS 
3-NT  
3-nitrotyrosine 
AD      
Alzheimer’s disease 
AE 
 
Adverse event 
Actin-NT 
Nitrated actin 
ALS 
 
Amyotrophic lateral sclerosis 
ALSFRS-R 
ALS Functional Rating Scale-Revised 
ALSAQ-40 
ALS Assessment Questionnaire 
API 
 
Active pharmaceutical ingredient 
BD 
 
Budesonide 
CBC                Complete Blood Count 
CNS  
Central nervous system 
CRF 
 
Case report form 
CRO  
Contract Research Organization 
CSF   
Cerebral Spinal Fluid 
CSN  
Charge-stabilized nanostructures 
Cyp-A  
Cyclophilin A 
DAR  
Drug Adverse Reaction 
DAE  
Drug Adverse Event  
DM 
 
Italian Ministry Decree 
DMC  
Data Monitoring Committee  
EAE                Experimental autoimmune encephalomyelitis 
EKG  
Electrocardiogram 
EMA  
European Medicine Agency 
FADD  
Fas Associated Death Domain 
FasL  
Fas Ligand 
FEV1  
Forced expiratory volume in one second 
FoxP3  
Forkhead box P3 
FVC  
Forced vital capacity 
GCP  
Good Clinical Practice 
ICF 
 
Informed consent form 
ICH 
 
International Conference on Harmonization  
IEC 
 
Independent Ethics Committee 
IFN 
 
Interferon  
IGF-1  
Insulin growth factor 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
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IL 
 
Interleukin 
IMP 
 
Investigational Medical Product 
IND 
 
Investigational new drug 
iNOS  
Inducible nitric oxide synthase 
IRB/IEC/REB Institutional Review Board/Independent Ethics Committee/Research Ethics Board 
IRCCS  
Istituto di Ricerca e Cura a Carattere Scientifico 
IV 
 
Intravenous 
Kg 
 
Kilogram 
LMN  
Lower motor neurons 
LPS 
 
Lipopolysaccharide   
MCP-1 
Monocyte chemoattractant protein -1 
mL 
 
Milliliter 
MND  
Motor neuron disease 
MOG  
Myelin Oligodendrocyte Glycoprotein 
mRNA  
Messenger RNA 
NO 
 
Nitric oxide 
NS 
        
Normal saline 
3-NT  
3-nitrotyrosine 
PBMC  
Peripheral Blood Mononuclear Cells 
PD 
 
Parkinson’s disease 
PP 
 
Per protocol 
QP 
 
Qualified Person 
RNA  
RiboNucleic Acid 
RP 
 
Revalesio Pump 
RRMS  
Relapsing Remitting Multiple Sclerosis 
SAE 
 
Serious adverse event 
SOD1  
Superoxide dismutase 1 
SOP 
 
Standard Operating Procedure 
SUSAR 
Suspected unexpected serious adverse reactions 
Th 
 
T helper cell 
TLR 
 
Tool like receptor 
TNF 
 
Tumor necrosis factor 
T reg  
regulatory T lymphocytes   
UMN  
Upper motor neurons 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
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1  INTRODUCTION 
1.1  Background and Rationale 
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that 
affects motor neurons in the spinal cord, brainstem and motor cortex. Clinical features include 
fasciculations, muscle wasting and weakness, spasticity, and hyper- or hyporeflexia. Respiratory 
complications usually develop in patients with advanced disease, and the cause of death is generally 
the paralysis of the respiratory muscles and diaphragm (Beghi et al., 2007). Although the exact 
pathophysiological mechanisms underlying neurodegeneration in ALS remain uncertain, a common 
pathological hallmark is the presence of ubiquitin-immunoreactive cytoplasmic inclusions in 
degenerating neurons, followed by an inflammatory reaction (McGeer and McGeer, 2002; Bendotti 
et al. 2012; Malaspina et al., 2015). Prominent neuroinflammation can be observed in 
pathologically affected areas of the central nervous system (CNS) and in spinal cords from both 
human ALS patients and mouse models of the disease (McGeer and McGeer, 2002; Philips and 
Robberecht, 2011). Typically, inflammation in ALS is characterized by gliosis and the 
accumulation of large numbers of activated microglia and astrocytes. Activation of glia is marked 
by elevated production of potentially cytotoxic molecules such as ROS, inflammatory mediators 
such as COX-2, and proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 (McGeer and 
McGeer, 2002). Major histocompatibility complex molecules and complement receptors are highly 
expressed by reactive microglia in the primary motor cortex and in the anterior horn of the spinal 
cords of ALS patients (McGeer and McGeer, 2002; Sta et al., 2011). The major determinants of 
motor neuron death in ALS remain to be established.  
A motor neuron-specific death pathway has been suggested for ALS based on the finding that motor 
neurons isolated from transgenic superoxide dismutase 1 (SOD1) mutant mice were more sensitive 
to Fas- or nitric oxide (NO)-triggered cell death than wild-type motor neurons (Raoul et al., 2002). 
Upon binding of FasL to the Fas receptor, the intracellular portion of Fas recruits the adaptor 
molecule FADD (Fas Associated Death Domain), which then activates a caspase cascade that 
culminates in the death of motor neurons. This pathway was also activated in presymptomatic ALS 
mice (Raoul et al., 2006). Given that astrocytes and microglia produce NO and that astrocytes from 
SOD1 mutant mice produce FasL (Barbeito et al., 2004), glial cells could directly kill motor 
neurons. Another member of the same receptor family, the p75 neurotrophin receptor, has also been 
implicated in ALS-dependent motor neuron death (Pehar et al., 2004). Specifically, nerve growth 
factor secreted by SOD1 mutant astrocytes induced the death of motor neurons expressing p75 by a 
mechanism involving the formation of NO and peroxinitrite (Pehar et al., 2004). Peroxynitrite 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
8 
converts tyrosine to 3-nitrotyrosine (3-NT), leading to protein nitration. Interestingly, high levels of 
nitrated proteins were found in the spinal cord and peripheral blood mononuclear cells (PBMC) of 
mutant SOD1 animal models, already at a presymptomatic stage of the disease, and in PBMC of 
sporadic ALS patients (Casoni et al., 2005; Nardo et al., 2009; Nardo et al., 2011).   
Even though motor neurons are the main cells affected in ALS, increasing evidence points to the 
involvement of neighboring glia cells during pathogenesis (Clement et al., 2003; Yamanaka et al., 
2008a). Cell-specific deletion of mutant SOD1 in astrocytes and microglia reduced disease severity 
and boosted the survival of ALS mice (Boillee et al., 2006; Yamanaka et al., 2008a). Evidence for a 
role for non-neuronal cells has been obtained using chimeric mice selectively expressing SOD1 in 
neurons or in non-neuronal cells (Yamanaka et al., 2008b). In chimeric mice expressing high levels 
of mutant SOD1 in 100% of motor neurons and oligodendrocytes, the presence of wildtype 
neighboring support cells substantially delayed the onset of motor neuron degeneration. Gene 
expression profiling implies that inflammatory cascades are activated before the initiation of motor 
neuron degeneration, suggesting that inflammation could be involved in the presymptomatic phase 
of the disease (Vargas et al., 2008). Mutant SOD1, but not the wild-type protein, has been proposed 
to be secreted into the extracellular space via chromogranin vesicles, causing activation of microglia 
and resulting in motor neuron death in culture (Urushitani et al., 2006). Consistently, mutant SOD1 
is present in the cerebrospinal fluid of ALS patients and is toxic to rodent spinal cord cultures 
(Tikka et al., 2002). Moreover, intracerebral infusion of mutant SOD1 into wild-type mice induced 
microglial activation and cytokine production (Kang and Rivest, 2007).  
Emerging evidence points to an involvement of the adaptive immune response in ALS disease 
progression. In fact, increased levels of CD4+ and CD8+ T lymphocytes and dendritic cells were 
detected in close proximity to dying motor neurons in the spinal cords of SOD1 mutant mice (Chiu 
et al., 2008) and in the brain parenchyma of ALS patients (Kawamata et al., 1992; Henkel et al., 
2004). Infiltrating T cells (Th2, Treg) can be neuroprotective after secreting IL-4, which signals 
reactive microglia to produce neurotrophic factors such as insulin growth factor (IGF-1) (Chiu et 
al., 2008). In the case of ALS, as in Alzheimer’s disease (AD) and Parkinson’s disease (PD) as well 
as other neurodegenerative diseases in which microglia and astrocytes are the primary contributors 
to 
inflammation, 
therapeutic 
approaches 
logically 
would 
aim 
to 
modulate 
the 
sensor/transducer/effector functions of the innate immune system, that is, TLRs, NF-B, and TNF-
α, respectively. These findings suggest that targeting inflammatory pathways might be effective in 
preventing disease progression. 


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The only drug showing to improve survival in patients with ALS is riluzole (Miller et al, 2007). 
However, the benefits consist in a three-month delay of death while disability and other outcome 
measures are virtually unaffected. This highlights the need to test novel approaches to slow the 
progression of the disease. 
1.2  Pre-clinical Data 
RNS60 is a novel, anti-inflammatory and cytoprotective drug that has shown remarkable efficacy in 
animal models of neuroinflammation and neurodegeneration. In multiple in vitro and in vivo models 
of different inflammatory diseases, treatment with RNS60 has shown favorable effects ranging from 
reduced levels of pro-inflammatory cytokines to improved functional outcomes. With direct 
relevance for this study, intraperitoneal and intravenous (IV) injections of RNS60 in multiple rodent 
EAE models showed a remarkable improvement of clinical scores, reduction in the expression of 
inflammatory molecules such as IL-17 and iNOS, reduced gliosis and CNS infiltration by 
inflammatory cells, and finally reduced demyelination.  Moreover, intraperitoneal injection of 
RNS60 caused significant down regulation of proinflammatory molecules in the brain, and neuronal 
protection in mouse models of AD and PD. RNS60 inhibited microglial inflammation via type 1A 
phosphatidylinositol-3 kinase-Akt-CREB-mediated upregulation of IκBα and inhibition of NF-κB 
activation in cultured glial cells (Khasnavis, S. et al,2012). Similarly, RNS60 suppressed NF-κB 
activation through differential modulation of type 1A PI(3)K pathways in a mouse model of PD, 
and thereby protected mice against neurodegeneration (Khasnavis S  et al, 2014). In a rodent EAE 
model, RNS60 treatment achieved a remarkable improvement of clinical scores, reduction in the 
expression of inflammatory molecules such as IL-17 and iNOS, reduced CNS infiltration by 
inflammatory cells, and finally reduced demyelination (Mondal, S. et al. 2012).  
In both, in vitro and in vivo models of familial ALS carrying SOD1G93A mutation, we recently 
observed a reduced up-regulation of iNOS induced by lipopolysaccharide (LPS) in astrocytes (Fig 
1.A) and a protective effect of RNS60 on motor neurons. In an in vitro model of ALS, consisting of 
co-cultures of astrocytes and spinal neurons expressing human SOD1 with G93A mutation, RNS60 
led to a reduced spontaneous and selective loss of large mutant motor neurons (Fig 1.B). In vivo, the 
intraperitoneal treatment of SOD1G93A mice with RNS60 at the onset of the disease (evaluated at 
their body weight peak) caused a significant delay in the appearance of motor dysfunction (Fig.1C) 
and a partial but significant preservation of lumbar spinal cord motor neurons (Fig 1.D) and 
neuromuscular junctions (Fig 1.E). Moreover, RNS60 treatment led to a small but significant 
activation of the neuroprotective PI3K-Akt signaling pathway, as demonstrated by the increased 
phosphorylation of Akt in lumbar spinal cord (Fig 1.F). This demonstrates that the same pathways 
are modulated by RNS60 across preclinical models of different neurodegenerative diseases. 


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Figure 1. Effect of RNS60 in SOD1G93A linked ALS models in vitro and in vivo. A) RNS60 reduces LPS-
induced iNOS in astrocytes cultures. The iNOS mRNA is normalized to SOD2 mRNA levels. Data are 
mean ± SEM (n=4 independent experiments) and analyzed by One Way ANOVA (p value<0.001), Tukey 
Test: ****p<0.001 and **p<0.01 vs. untreated, $$$p<0.005 vs. LPS and §p<0.05 vs. LPS+RNS60. B) RNS60 
protects motor neurons in astrocyte-spinal neuron co-cultures from SOD1G93A mice. Data are mean ± 
SEM (n=5 independent experiments) and analyzed using Two Way ANOVA (int. p <0.04), Tukey Test: 
**p<0.01 vs. NTG untreated, $p<0.05 vs. SOD1G93A untreated and §§p<0.01 vs. NTG NS. C) RNS60 
treatment delays the onset of neuromuscular symptoms in SOD1G93A mice. The age of onset is 
considered as the age at which the mouse fails the grip strength test for the first time (grip latency <90 sec). 
The curves are evaluated by the Log-rank (Mantel-Cox) test (Chi square= 17.10, p <0.001).  D) RNS60 
treatment protects motor neurons in SOD1G93A mice. Motor neuron count from spinal cord L2-L5 
segment (10 sections/mouse). The bar graph represents mean ± SEM (n=3 mice per group). Data are 
analyzed using t-test (p<0.0001). E) RNS60 slightly protects neuromuscular junctions from denervation. 
Data represent denervated plaques as the percentage of total plaques counted in ten frames per section, five 
sections for each mouse, at 20X-magnification (n=5). At symptomatic stage, SOD1G93A mice treated with NS 
show significant denervation (61.76%), which is slightly reduced by the treatment with RNS60  (52.35%). 
Histograms show the mean ± SEM of 5 mice per group. Data are analyzed using t-test (p=0.0237). F) 
RNS60 activates PI3K-Akt signaling in the lumbar spinal cord. Western blot of lumbar spinal cord 
proteins show that the level of phosphorylated Akt (p-Akt) is higher in SOD1G93A mice treated with RNS60 
as compared to NS-treated controls. The bar graph represents mean ± SEM (n=5 mice per group). Data are 
analyzed using t-test (p=0.05). 


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Therefore, RNS60 presents itself as a promising candidate for the treatment of ALS. Its exceptional 
safety profile, demonstrated both in preclinical toxicology studies and Food and Drug 
Administration (FDA)-approved clinical phase I studies upon inhaled and IV administration, 
supports testing of RNS60 in clinical phase II studies in ALS patients. 
1.3 
Pre-clinical Toxicology Data 
Extensive in vitro and in vivo preclinical testing of RNS60 has been completed at multiple 
institutions and in various models. RNS60 did not show any concerning signs of toxicity in any of 
the in vitro cell culture studies performed to date or in the animal toxicology studies conducted in 
accordance with FDA guidelines for Investigational New Drug (IND) Applications. The 
administration of the maximum deliverable dose of RNS60 by bolus injections (10 mL/kg) or IV 
infusion for 6 (48 mL/kg) or 24 hours (96 mL/kg) daily for 28 days was well tolerated in albino 
rats. There were no RNS60-related mortalities, clinical observations, ocular observations, abnormal 
laboratory results (serum chemistry, hematology and urinalysis), organ weight changes, 
macroscopic or microscopic observations or genotoxic activity. The administration of the maximum 
deliverable dose of RNS60 by bolus injections (10 mL/kg) or IV infusion for 6 (48 mL/kg) or 24 
hours (96 mL/kg) once daily for 28 days was well tolerated and did not reveal toxicity in any target 
organs in cynomolgus monkeys.  
In the bacterial reverse mutation assay (Ames assay) using Salmonella typhimurium strains (TA98, 
TA 100, TA 1535 and TA 1537) and Escherichia coli WP uvrA in the presence and absence of S9 
metabolic activation, RNS60 caused neither an increase in the number of revertant colonies nor a 
dose-related response in any of the tested strains at any dose level. In the mouse lymphocyte assay, 
RNS60 similarly demonstrated minimal cytotoxicity and did not increase the mutation frequency 
relative to the vehicle controls in either the presence or absence of metabolic activation. Therefore, 
it was concluded that RNS60 was not mutagenic. 
1.4 
Clinical Data to Date 
RNS60 has been well tolerated in human safety studies, both after IV infusion and when 
administered by inhalation. In a Phase I safety and tolerability study (Clinicaltrials.gov # 
NCT01264783), RNS60 was administered to 9 healthy subjects as continuous IV infusion at 3 
subsequently increasing rates of 100 mL/h, 150 mL/h, and 200 mL/h over 48 hours at each rate.  No 
serious adverse events (SAEs) were reported in this study and no subjects were discontinued from 
the study due to an adverse event (AE).  Furthermore, no clinically relevant changes were noted in 
clinical laboratory tests, vital signs, physical examinations, oral fluid intake or urine output. The 
most frequently reported AE was mild headache, which occurred most often in the highest infusion 


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12 
rate of 200 mL/h, and was considered possibly related to the treatment.  However, there was an 
increase in overall incidence with the increased infusion rate for both placebo and RNS60-treated 
subjects, with 55% of subjects in the RNS60 group and 33% of subjects in the placebo group 
reporting mild headache.  Since headache is a common symptom of fluid (volume) load it is 
probable that the headaches were primarily a volume-related effect of the infusion.  Other AEs 
reported as treatment related were a single incidence each (11.1%) of mild arthralgia, mild limb 
discomfort, and moderate musculoskeletal pain.  Cardiac monitoring showed only mild, sporadic 
and infrequent Electrocardiogram (EKG) changes from baseline; however these changes were not 
attributed to use of RNS60.   
In a second Phase I study (Clinicaltrials.gov # NCT01057498), RNS60 administered to 16 subjects 
with mild to moderate asthma by inhalation through a nebulizer, at a 4-mL dose twice a day for 28 
days was well tolerated.  No SAEs were reported in this study and no subjects were discontinued 
from the study due to an AE.  Furthermore, no clinically relevant changes were noted in clinical 
laboratory tests, vital signs, or physical examinations.  The most commonly reported treatment 
related AE was mild oropharyngeal pain, reported by 2 subjects (12.5%).  One subject (6.3%) 
reported a single incidence of moderate nasal mucosal disorder.  Other AEs reported as treatment 
related were all mild, and were single incidences (6.3%) of eye allergy, ocular hyperemia, chest 
discomfort, increased blood pressure, increased appetite, headache, somnolence, allergic sinusitis, 
cough, dyspnea, nasal congestion, throat irritation, and pruritus.  Additionally, RNS60 treatment did 
not cause bronchoconstriction, but rather demonstrated positive effects on forced expiratory volume 
in 1 second (FEV1) and peak expiratory flow measurements, as well as improved quality of life 
scores. 
In a third Phase I study (Clinicaltrials.gov # NCT01511302), RNS60 administered by nebulization 
in combination with a budesonide (BD) dose of either 0.25 mg or 0.5 mg for 28 days, to subjects 
with mild to moderate asthma who were previously taking a prescribed corticosteroid, was also well 
tolerated.  57 subjects were treated with the combination of RNS60 and BD (RNS60-BD).  No 
SAEs were reported and no discontinuation of subjects occurred that were related to use of RNS60-
BD.  AEs attributed to the use of RNS60-BD were sporadic, with single incidences (3.4%) of 
moderate migraine, moderate wheezing, moderate asthma prophylaxis, mild chest discomfort, and 
mild increase in hepatic enzymes.  Furthermore, there were no significant findings noted in clinical 
laboratory tests, vital signs, or physical examinations.  Additionally, use of RNS60-BD did not 
cause any incidence of clinically significant bronchoconstriction; rather it demonstrated positive 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
13 
trends in quality of life improvements and reduced use of rescue inhalers. For details of these 
studies refer to the Investigator’s Brochure (IB) for RNS60. 
An ongoing interventional Phase II trial with a weekly IV infusion of RNS60 in relapsing/remitting 
multiple sclerosis (RRMS) patients is currently enrolling subjects at the University hospitals in 
Zurich and Innsbruck. 
An ongoing single center, open label, pilot trial of RNS60 in ALS patients is enrolling at 
Massachusetts General Hospital.  Subjects are receiving weekly IV infusion of RNS60 in addition 
to daily inhalation of RNS60 for six months while being monitored for disease progression and 
microglial activation by PET imaging. 
1.5 
Description of RNS60 
Chemically, RNS60 consists of 0.9% sodium chloride and dissolved oxygen.  RNS60 is 
manufactured by electrokinetic processing in Revalesio’s patented Revalesio Pump (RP).  During 
operation of the RP, the fluid is exposed to very strong hydrodynamic, rotational, vortical and 
longitudinal shear forces, resulting in forceful dispersion of the oxygen.  The combined effect of 
these forces alters the physical relationship between gas and liquid, and has a concomitant effect on 
the electrical properties of the fluid caused by a reorganization of ionic charges and the formation of 
associated electrical double layers.  RNS60 does not contain a chemical Active Pharmaceutical 
Ingredient (API).  
The observed bioactivity of RNS60 is, instead, based on the presence of charge-stabilized 
nanostructures (CSN) generated by the electrokinetic processing in the RP.  The CSNs consist of an 
oxygen core surrounded by layers of separated positive and negative electrical charges.  The 
electrical charges associated with the core oxygen stabilize the layers of separated electrical charges 
and form CSN-associated electrical fields in RNS60.  
1.6 
Tolerability 
As stated in the previous sections referring to preclinical toxicology work, minimal adverse effects 
were seen with the treatment of RNS60.  Section 1.4 explains how RNS60 was safe and well 
tolerated in multiple human safety studies.  RNS60 has demonstrated safety when administered to 
subjects via inhalation as well as when administered intravenously.  
1.7 
Dose Rationale 
The IV and inhaled doses of RNS60 have been selected based on previous experience in clinical 
studies. Because no safety concerns have been identified to date, these doses are expected to be well 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
14 
tolerated by the subjects and provide appropriate exposure to test the hypothesis of the study.  The 
importance of remaining within the margin of safety in dose selection has been taken into account 
while maximizing dosing frequency without infringing on practicality for subjects.   
1.8 Pharmacokinetics 
The observed bioactivity of RNS60 is not based on an API, as RNS60 contains neither a chemical 
drug substance nor a biologic.  Instead, the bioactivity is caused by a physical property of RNS60 
that is associated with the presence of CSNs generated by the electrokinetic processing in the RP.  
The CSNs consist of an oxygen core surrounded by layers of separated positive and negative 
electrical charges.  Preclinical studies have conclusively demonstrated that the oxygen alone is not 
responsible for RNS60’s bioactivity, but that it is the combination of oxygen with the processing in 
the RP that results in a physical quality of the fluid that is able to modulate the stress response of 
cells. As it is currently impossible to directly image CSNs in vivo, traditional pharmacokinetic 
evaluations are not applicable to RNS60. Preclinical data clearly demonstrate, however, that RNS60 
is able to modulate the type 1A PI(3)K signaling pathway within the CNS after systemic 
administration. RNS60 induced the activation of type IA phosphatidylinositol-3 kinase (PI3K) in 
the substantia nigra of healthy as well as MPTP-intoxicated mice within 3 hours post intraperitoneal 
injection, and the effect was sustained until 6 hours post injection (Khasnavis S  et al, 2014). Thus, 
RNS60 is expected to mediate its effects both on peripheral immune cells as well on CNS targets, as 
reported earlier (Modi, KK. et al. 2014; Khasnavis, S. et at. 2014; Mondal, S. et al. 2012). 
  
2 
STUDY OBJECTIVES AND OUTCOMES 
2.1 
Primary Objective 
To measure the effect of RNS60 treatment on selected pharmacodynamic biomarkers in ALS 
patients concurrently treated with riluzole. Candidate markers include: 
1. T-reg (measured via FOXP3 and CD25 mRNA);  
2. Cyp-A;  
3. 3-NT;  
4. Actin-NT;  
5. MCP-1;  
6. IL-17. 
 
 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
15 
2.2 
Secondary Objectives 
 
1. The preliminary efficacy of RNS60 on functional disability, as measured by the ALSFRS-R 
scale; 
2. The preliminary efficacy of RNS60 in prolonging survival (or time to tracheostomy, 
whichever comes first); 
3. The preliminary efficacy of RNS60 in slowing the decline of forced vital capacity (FVC) 
from baseline; 
4. The tolerability and safety of RNS60 through the identification of unexpected adverse 
events; 
5. The impact of RNS60 on quality of life as measured by ALSAQ-40 scale. 
 
2.3 
Primary Endpoint 
The mean change over time in the proportion of T-reg (measured via FOXP3 and CD25 mRNA); 
the mean change over time in the plasma concentrations of Cyp-A,  3-NT, Actin-NT, MCP-1, IL-17 
in the two treatment arms, during the entire treatment period (from baseline to 24 weeks). 
 
2.4 
Secondary Endpoints 
1.  The mean change in the proportion of T-reg (measured via FOXP3 and CD25 mRNA); the 
mean change over time in the plasma concentrations of Cyp-A, 3-NT, Actin-NT, MCP-1, 
IL-17  during the follow up period (from 24 to 48 weeks); 
2. The cumulative proportion of subjects becoming no longer self-sufficient at 4, 12, 24, 36 
and 48 weeks in the two treatment arms (defined as those scoring 2 or lower on at least one 
of the ALSFRS-R items for swallowing, cutting food and handling utensils, or walking); 
3. The mean change of ALSFRS-R total score in the two treatment arms during the treatment 
and follow up period; 
4. The cumulative proportion of deaths or tracheostomies in the two treatment arms during the 
entire study including follow-up; 
5. The mean change of FVC score in the two treatment arms during the treatment and follow 
up period; 
6. The total number of subjects in the two treatment arms experiencing at least one AE leading 
to treatment discontinuation at 4, 12 and 24 weeks; 
7. The mean number of AEs per treatment arm at 4, 12, 24 and 48 weeks; 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
16 
8. The mean change of ALSAQ-40 score measuring quality of life at the completion of the 
treatment and follow up period. 
3 
STUDY DESIGN 
The study is a multicenter, randomized, double-blind, placebo-controlled, parallel group, add-on 
pilot trial in patients taking riluzole.  Patients diagnosed with ALS who meet the study’s inclusion 
and exclusion criteria and sign the Informed Consent Form (ICF) will be enrolled.  There will be 
two groups making up a total of 142 subjects:  Group A (71 subjects) and Group B (71 subjects).  
Subjects will be randomly assigned to receive treatment with either RNS60 or placebo while 
concomitantly taking riluzole (50 mg twice a day).  RNS60 or placebo will be administered 
intravenously once a week as well as inhaled via nebulization every morning (6 days a week on 
non-infusion days) for 24 weeks.  All visit windows (± 1 or + 4 days according to study procedure) 
are consecutive calendar days and are calculated from the day the participant starts study treatment 
(the day of the Randomization-Baseline Visit). 
Blood samples will be collected five times for enrolled subjects to measure biomarkers. The first 
blood sample collection will be at baseline before the first infusion dose. Safety and efficacy will be 
assessed by way of physical exam, vital signs and AEs.  Changes in disability and quality of life 
will be assessed using the ALSFRS-R scale, FVC and ALSAQ-40 scale.  The overall treatment 
duration will be 24 weeks. Each subject will be followed up for a maximum period of 48 weeks 
(24-week treatment + 24-week follow up) or until death or tracheotomy, whichever occurs first.  
4 
STUDY POPULATION 
4.1  Disease definition 
[….] 
Included in the study may be cases of bulbar-onset and spinal-onset ALS.  Cases with only bulbar 
signs or symptoms for the first three months from onset, with spinal symptoms or signs occurring 
later, are considered bulbar onset ALS.  Cases in which any spinal symptoms or signs appeared in 
the first three months are considered spinal onset ALS.  Bulbar onset and spinal onset ALS will be 
considered separately at the analysis stage. 
Patients not fulfilling the diagnostic criteria as indicated in the protocol cannot be enrolled. 
4.2 
Inclusion Criteria 
1) Age 18 through 80 years inclusive;  


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
17 
2) Geographically accessible to the site and able to come to the site center once a week for 24 
weeks; 
3) “Definite” or “Probable” or “Probable laboratory-supported” ALS diagnosis according to 
the revised El Escorial criteria; 
4) Disease duration 6 to 24 months from symptom onset to study entry;  
5) Self sufficiency: Satisfactory bulbar and spinal function (score 3+ on the ALSFRS-R for 
swallowing, cutting food and handling utensils, and walking);  
6) Satisfactory respiratory function (FVC ≥80% of predicted);  
7) Documented progression of symptoms in the last three months as measured by the 
ALSFRS-R scale (decrease of at least one point);  
8) Ability to understand and comply with the study requirements; 
9) Ability to give written informed consent personally or, as an alternative, via a legally 
authorized representative; 
10) Treatment with riluzole 50 mg twice/day for at least 1 month prior to screening visit; 
4.3 
Exclusion Criteria 
1) History of HIV, clinically significant chronic hepatitis, antecedent polio infection, or other 
active infection;  
2) Motor neuron disease (MND) other than ALS; 
3) Involvement of other systems possibly determining a functional impairment (as measured by 
the end-points) for the entire duration of the study;  
4) Other severe clinical conditions (e.g., cardiovascular disorders, neoplasms) with impact on 
survival or functional disability in the next 12 months;  
5) Renal insufficiency as defined by a serum creatinine > 1.5 times the upper limit of normal 
6) Poor compliance with previous treatments;  
7) Other experimental treatments in the preceding 3 months prior to screening visit;  
8) Women who are lactating or able to become pregnant (e.g. who are not post menopausal, 
surgically sterile, or using inadequate birth control) and men unable to practice 
contraception for the duration of the treatment and 3 months after its completion;  
9) Unwillingness or inability to take riluzole;  
10) Poor compliance with an inhalation device; 
11) Abnormal liver function defined as AST and/or ALT > 3 times the upper limit of the 
normal. 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
18 
4.4 
Participating Centers 
Patients will be recruited by tertiary ALS centers representative of all Italian regions and by the 
Massachusetts General Hospital (MGH) in Boston (USA). 
 
5. 
BIOMARKERS 
Six candidate pharmacodynamic markers of treatment efficacy with RNS60 have been selected 
because they have been previously associated with ALS, they are modulated by neuroinflammation, 
and because some of them have been shown to be modified by RNS60 or by other compounds with 
anti-inflammatory properties in in vitro and in vivo models of different inflammatory conditions 
(see Table 1). 
Table 1. Pharmacokinetic biomarkers used in this study 
Type 
Marker 
In ALS Patients 
Effect of RNS60 
RNA 
FoxP3 
/CD25 (T-
reg) 
T-regs are lower in rapidly 
progressing ALS patients and 
inversely 
correlate 
with 
progression rates (Henkel et al., 
2013). 
 
RNS60 upregulates FoxP3 and 
enriches T-regs in experimental 
autoimmune 
encephalomyelitis  
(EAE), mouse model of multiple 
sclerosis (Mondal et al., 2012). 
Protein 
Cyp-A 
Increased levels in PBMC of ALS 
patients 
and 
associated 
with 
disease progression (Nardo et al., 
2011) 
Not known 
Amino 
acid 
3-NT 
Increased levels in PBMC of ALS 
patients (Nardo et al., 2009) 
Not 
known, 
decreased 
by 
compounds 
with 
anti-
inflammatory properties (Hooper 
et al., 2000; Yip et al., 2013) 
Protein 
Actin-NT 
Increased levels in PBMC of ALS 
patients (Nardo et al., 2011) 
Not known 
Protein 
IL17 
Increased serum and CSF levels 
of ALS patients (Rentzos et al., 
2010) 
Reduced in RNS60 treated MOG 
and T cell transfer induced 
models of EAE in rats and mice 
(Mondal et al., 2012). Reduced in 
combination 
with 
IFN- 
in 
asthma patients after 4 weeks of 
treatment 
(Revalesio, 
unpublished data). 
Protein 
MCP1 
High levels in CSF and serum of 
ALS patients (Kuhle et al., 2009; 
Baron et al., 2005).  
Reduced in allergen challenge 
model of asthma in rats after 
RNS60 treatment (Ghosh et al., 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
19 
2011). 
 
5.1   Blood sample collection  
Blood samples of a total volume of 21 ml will be collected from each subject five times during the 
entire trial. Fasting is not needed, but the patient is encouraged to have a light breakfast before 
blood collection. Blood for biomarker assessments will be collected in two 4-ml EDTA vacutainers 
(BD cat. no. 367861) for plasma samples, two 2.5-ml PAXgene tubes (BD cat. no.762165) for RNA 
samples and two 4-ml Vacutainer® CPT™ Cell Preparation Tube (BD cat. no. 362781) for PBMC 
samples at scheduled visits:  
 At the day 1 visit (baseline - randomization), blood samples will be collected prior to first 
IV RNS60/placebo dose (pre-dose, off drug blood sample).  
 At the week 4, 12 and 24 study visits, the blood sample will be collected just prior to drug 
administration.  
 An additional blood sample will be obtained at the week 48 visit (last study visit). 
5.2    Blood sample handling 
The time of sample collection and the start time of the processing for plasma and PBMC samples 
will be recorded. Samples should be processed as soon as possible and within 2 hours after 
collection.  
Plasma samples: Invert EDTA vacutainers several times (at least 5 times) after blood draw. 
Centrifuge the blood samples in the vacutainers at 3000xg for 20 minutes at room temperature (18 
to 25°C). Aliquot the plasma into two 1.8-ml Nunc cryo tubes labeled per vacutainer (4 samples). 
Freeze plasma samples at -70°C as soon as possible. 
Blood for RNA samples: Invert PAXgene tubes several times (at least 5 times) after blood draw. 
Samples must stay upright at room temperature for a minimum of 24 hours and no more than 72 
hours before being frozen. Freeze the whole blood sample at -20°C as soon as possible. 
PBMC: Invert the Vacutainer® CPT™ Cell Preparation tubes gently 10 times and centrifuge at 
room temperature (18 to 25°C) for 30 minutes at 1500xg (RCF).  After centrifugation, mononuclear 
cells and platelets will be in a whitish layer just under the plasma layer (see Figure).  


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
20 
 
For recovering the separated mononuclear cells, resuspend the cells into the plasma by inverting the 
un-opened BD Vacutainer® CPT™ Tube gently 5 to 10 times. To collect the cells, open the BD 
Vacutainer® CPT™ Tube and pipette the entire contents of the tube above the gel into a separate 
vessel. Add PBS to bring volume to 15 mL. Cap tube. Mix cells by inverting the tube 5 times 
gently. Centrifuge for 15 minutes at 300xg (RCF). Aspirate as much supernatant as possible without 
disturbing cell pellet. Freeze plasma samples at -70°C as soon as possible. 
A specific procedure will be adopted by the US Center in keeping with the study’s laboratory 
manual. 
5.3  Sample labels 
[…..] 
5.4  Sample numbering 
[….] 
5.5 
Sample Storage 
In each study center as well as at the “Mario Negri” IRCCS the samples will be stored in a 
dedicated room to be accessed only by authorized persons. For each sample the storage, usage and 
transfers will be performed according to the requirements aimed at preserving quality, integrity, 
accessibility and availability of the sample. 
5.6  Sample shipment 
[…] 
5.7  Analytical method 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
21 
Analysis of RNA, Cyp-A, 3-NT, Actin-NT in PBMC samples are described in the study’s 
laboratory manual.  
 
6 
STUDY TREATMENTS 
6.1    Study Drug Administration 
RNS60 or placebo supply will be sent to site by ALMAC group and should be kept refrigerated in 
the Hospital Pharmacy with access limited to those directly involved in the study.  
RNS60 or placebo will be administered weekly intravenously (starting on Day 1) at the clinic site 
by properly trained staff designated by the investigator.  Subjects will receive 375 mL RNS60 or 
placebo infused at a rate of 700 mL/hr. Infusion documentation should include recording infusion 
start and stop times, volume infused, and any interruptions to the infusion using an ad-hoc form.    
During the remaining days of the week, treatment with nebulized RNS60 or placebo will be 
performed at home in the morning on non-infusion days.  The subject will remove the kit box of 2 
syringes from refrigeration, and visually inspect the syringe to ensure that the plunger and tip cap 
have remained intact during storage.  If either the plunger or the tip cap is loose, the faulty syringe 
shall be set aside and returned to study staff on the next infusion.  Any faulty syringes will be sent 
back to Revalesio for investigation.  Subjects will receive a number of syringes that will guarantee a 
sufficient number of doses between visits.  To dispense a syringe, subjects should first remove the 
tip cap of the syringe, then gently push the plunger down to dispense the full 2 mL of 
RNS60/placebo from the syringe into the nebulizer dosing cup. The procedure is then repeated with 
a second syringe to get to the full daily dose of 4 mL.  The dose should be nebulized immediately 
thereafter until the contents of the nebulizer cup are empty (approximately 15 minutes).  Subjects 
should try to dose at a similar time each morning.  Subjects will be provided with and trained to use 
a Pari Mini system and LC Sprint nebulizers (Italian subjects) or Pari Trek system and LC Sprint 
nebulizers (US patients). Dosing records for nebulization will be completed at home at the time of 
each daily nebulization.  The dosing record will include the date and time of nebulization as well as 
the number of syringes used. 
All subjects will concurrently take 50-mg riluzole twice a day during the entire treatment period. 
According to the European Medicine Agency (EMA) guidelines, riluzole can be discontinued when 
the plasma transaminase levels exceed by fivefold the baseline levels.  
The first dose of riluzole should be taken each day at the end of infusion or nebulization. The 
second dose should be taken after 12 ± 2 hours. 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
22 
6.2    Investigational Product(s) 
[…] 
RNS60 
RNS60 for infusion, i.e. in the IV bags, is produced using 0.9% Sodium Chloride for injection. 
RNS60 for inhalation, i.e. in the syringes, is produced using 0.9% Sodium Chloride for irrigation.  
Both RNS60 for infusion and RNS60 for inhalation are processed in the Revalesio Pump with 
elevated levels of oxygen.  RNS60 is supplied for single-use only and contains no preservatives.   
Syringes and IV bags are to remain refrigerated at 2 to 8°C (36 to 46°F) when not in use. RNS60 
meets its stability specification for 12 months. 
Placebo 
Normal saline (NS) for infusion, i.e. in the IV bags, is packaged 0.9% Sodium Chloride for 
injection. NS for inhalation, i.e. in the syringes, is packaged 0.9% Sodium Chloride for irrigation.  
NS does not require refrigerated storage for use.  However, for blinding purposes refrigeration is 
required before distributing to subjects.  NS meets stability specifications for 24 months. 
6.3 
  Drug Accountability 
[…] 
6.4 
  Storage and Handling of RNS60 and Placebo 
[…] 
6.5 
Blinding 
[…] 
6.6 
Randomization 
All eligible subjects will be randomized to receive either RNS60 or placebo (in both IV and 
inhalation modes of administration) for 24 weeks.  Treatment allocation will be centrally managed 
by the study statistician using a computer-generated, permuted block (with a block size of 4), 1:1 
randomization scheme. A computer-generated randomization list will be provided to each 
investigational site.   
Individual treatment disclosure envelopes (code break) will also be provided to the investigative 
site.   


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
23 
6.7 
Treatment Arms 
142 patients with ALS will be randomized in 2 parallel groups, in a ratio of 1:1. One group will be 
treated with RNS60 and one group will be treated with placebo.  
Group A 
RNS60 375 mL IV once a week 
+ 
RNS60 4 mL inhaled once a day (six days/week) 
+ 
Riluzole 50 mg twice a day 
Group B 
NS 375 mL IV once a week 
+ 
NS 4 mL inhaled once a day (six days/week) 
+ 
Riluzole 50 mg twice a day 
 
6.8 
Treatment Assignment and Subject Numbering 
[…] 
6.9 
Concomitant Medications and Prohibited Treatment 
Any concomitant treatment can be continued as per medical indication. 
Except for other experimental drugs, there are no restrictions for concomitantly used medications.  
However, the subjects and investigators must keep records of the drugs on the concomitant 
medication section of the e-CRF along with daily dose and duration of use.  
 
6.10  Study Drug Discontinuation 
If study treatment is discontinued before the defined treatment duration period ends, the subject will 
be considered to have prematurely withdrawn from the study. Subjects lost to follow up should be 
recorded as such on the e-CRF. Subjects who discontinue study drug before completing the study 
should be scheduled for a visit as soon as possible, at which time all the assessments listed for the 
study completion visit will be performed for safety evaluations during the 30 days following the last 
dose of study drug. Subjects who discontinue the study drug should be considered withdrawn from 
the study after the final visit assessments are performed or when it is clear that the subject will not 
return for assessment. 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
24 
6.11 
Premature Subject Withdrawal 
The duration of the study includes 24 weeks of treatment plus 24 weeks of follow up. Subjects may 
voluntarily withdraw from the study or can be dropped at the discretion of the investigator at any 
time. Subjects must be withdrawn from the study if any of the following occurs: 
- Unwillingness to comply with procedures as outlined in the study protocol; 
- Withdrawal of consent; 
- Loss to follow-up; 
- Repeated non-compliance to dosing regimen or visit schedule (intake of 50% of 
recommended dose or less in three consecutive intervals between visits); 
- Intolerable adverse events which may or may not be related to study medication and, if 
deemed drug-related, non remitting with down titration;  
- Death/Tracheostomy; 
- Requirement of medication doses greater than those allowable in the study; 
- Abnormal laboratory value(s) deemed of medical concern; 
- Administrative problems; 
- Female becoming pregnant; 
- Severe protocol deviations (at the Study Coordinator judgment).  
If a subject withdraws or fails to return for visits, the investigator must determine the primary 
reason for the subject’s premature withdrawal from the study, and record this information on the 
study-completion visit. Subjects must return all unused/remaining study drug to the investigative 
site. For subjects who are lost to follow up, the investigator should show “due diligence” by 
documenting in the source documents the steps taken to contact the subject, e.g., dates of telephone 
calls, registered letters, etc. Subjects who are prematurely withdrawn from the study will not be 
replaced. 
Discontinued use of riluzole during the treatment period implies the discontinuation of the 
experimental drug. The subject will undergo a final safety evaluation and data collected up to the 
time of drug discontinuation will be used in the data analysis. 
 
6.12  Modification of Study Intervention/Investigational Product for a Subject 
Any dosage adjustment, including the reason for and dates of adjustment, will be documented in the 
CRF for each subject requiring this manipulation. The PIs or licensed physician Sub-Investigator  
may reduce the dosage of study drug or discontinue the study drug in its entirety for AEs thought to 
be related to the study drug or for other reason during the trial (the reason for, and dates of 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
25 
suspension or dose reduction must be documented).  If the AE is mild or moderate, the dosage may 
be reduced until the event improves.  The PIs may then choose to resume the higher dosage or 
maintain the subject at a reduced dosage. The option to continue with administration by IV only or 
inhalation only is allowable. 
If the event is serious or life threatening, and deemed to be definitely drug related, the study drug 
will be discontinued immediately. Study subjects must remain off the study drug permanently. 
Subjects may not resume study drug.  All AEs will be followed to resolution.  
 
6.13  Wash-out Period for Eligibility 
If a subject is receiving treatment with another experimental drug, this subject requires a 3-month 
wash-out period before participating in the screening visit.  
6.14  Treatment Compliance 
[…] 
 
7 
STUDY PROCEDURES 
[…] 
 
7.9  Study Duration 
The overall duration of the study will be approximately 36 months as indicated below: 
- 
Administrative and ethical procedures: 6 months  
- 
Duration of subject enrolment: 12 months 
- 
Study drug administration: 24 weeks/subject 
- 
Follow-up period: 24 weeks/subject 
- 
Close-out monitoring visits and statistical analysis: 4 months 
- 
Final report and data publication: 2 months 
 
 
 


RNS60-ALS V3.0 Dated on November  12, 2018 
 
 
26 
 


RNS60-ALS V3.0 Dated on November 12,   2018 
 
 
27 
Table 2.  SCHEDULE OF PROCEDURES 
  
  
Treatment Period 
Follow-up 
Period 
 
  
Screening  
(-28 days) 
Random
ization –
baseline 
Day 1 
 
Weeks 
1 - 3 
Week 
4 
Weeks  
5 - 11 
Week 
12 
Weeks  
13-23 
Week 
24 
Week 
36 
 Week 
48 
End 
of 
study 
(only 
for 
drop outs) 
Informed consent 
x 
 
 
 
 
 
 
 
 
 
 
Patient’s numbering assignment 
x 
 
 
 
 
 
 
 
 
 
 
Date of visit 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Demographic data 
x 
 
 
 
 
 
 
 
 
 
 
Blood sample collection 
(biomarkers) 
 
x 
 
x 
 
x 
 
x 
 
x 
x 
Personal and clinical data 
x 
 
 
 
 
 
 
 
 
 
 
Physical exam 
x 
x 
 
 
 
 
 
 
 
 
 
Neurological exam 
x 
x 
 
x 
 
x 
 
x 
x 
x 
x 
Medical 
history/ 
Concomitant 
medical disorders 
x 
x 
 
 
 
 
 
 
 
 
 
Concomitant medications 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Inclusion/exclusion criteria 
x 
x* 
 
 
 
 
 
 
 
 
 
Randomization 
 
x 
 
 
 
 
 
 
 
 
 
Study drug infusion 
 
x 
x 
x 
x 
x 
x 
 
 
 
 
Study drug management for 
nebulization 
 
x 
x 
x 
x 
x 
x 
x 
 
 
x 
Study drug dispensing 
 
x 
x 
x 
x 
x 
x 
 
 
 
 
Vital signs, weight 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Height 
x 
x 
 
 
 
 
 
 
 
 
 
ALSFRS-R scale 
x 
x 
 
x 
 
x 
 
x 
x 
x 
x 
FVC 
x 
x 
 
x 
 
x 
 
x 
x 
x 
x 
ALSAQ-40 scale 
 
x 
 
 
 
 
 
x 
 
x 
x 


RNS60-ALS V3.0 Dated on November 12,   2018 
 
 
28 
Clinical lab tests** 
x 
x 
 
x 
 
x 
 
x 
 
 
x 
Pregnancy test 
 
x 
 
 
 
 
 
x 
 
 
 
x 
Compliance check (nebulization) 
 
 
x 
x 
x 
x 
x 
x 
 
 
x 
AE assessment 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Reason of discontinuation 
 
 
 
 
 
 
 
 
 
 
x 
 
* Certain eligibility criteria are assessed at Day 1, prior to randomization 
** According to the physician judgment. Clinical lab test needed for inclusion criteria could be valid if performed up to 6 months before 
randomization visit 
 


RNS60-ALS V3.0 Dated on November 12,   2018 
 
 
29 
7 
STUDY ADMINISTRATION 
8.1 
Resource Utilization 
[…] 
8.2 
Regulatory and Ethical Compliance 
This study was planned and shall be performed according to the principles of Good Clinical 
Practice (ICH-GCP), the declaration of Helsinki, and the national laws and regulations about 
clinical studies. The study will not start without written approval of an Independent Ethics 
Committee (IEC) and the written informed consent of the patients. This study was designed and 
shall be implemented and reported in accordance with the protocol. 
8.3 
Responsibilities of the Investigator and IRB/IEC/REB 
[…] 
8.4 
Informed Consent  
Eligible patients can be included in the study only after proving written  the IRB/IEC/REB -
approved informed consent, or, if incapable of doing so, after such consent has been provided by a 
legally acceptable representative. In cases where the patient’s representative gives consent, the patient 
should be informed about the study to the extent possible given his/her understanding. The physician 
will obtain a signed informed consent form (ICF) from the subject prior to any procedures. The 
signed and completed ICF will be accessible in the patient’s records on site. A copy of the signed 
ICF will be given to each subject. 
The process of obtaining informed consent should be documented in the patient source documents. 
Women of child bearing potential should be informed that taking the study treatment may involve 
unknown risks to the fetus if pregnancy were to occur during the study and agree that in order to 
participate in the study they must adhere to the contraception requirement for the duration of the study. 
8.5 
Amendments to the Protocol 
Any change or addition to the protocol can only be made in a written protocol amendment. Only 
amendments that are required for patient safety may be implemented prior to IRB/IEC/REB 
approval in accordance with the Italian legislation. Notwithstanding the need for approval of formal 
protocol amendments, the investigator is expected to take any immediate action required for the 
safety of any patient included in this study, even if this action represents a deviation from the 


RNS60-ALS V3.0 Dated on November 12,   2018 
 
 
30 
protocol. In accordance with the GCP rules and the Italian legislation, the acceptance of an 
amendment of the study protocol by each participating institution is subjected to the local 
IRB/IEC/REB approval or notification. 
8.6 
Publication of Study Results 
The results of the study will be published whether or not the experimental drug is considered 
superior to placebo. 
 
9 
SAFETY MONITORING 
Safety monitoring for all patients enrolled in the study will include laboratory safety assessments 
and clinical evaluations, as shown in Table 2.  All AEs and SAEs will be recorded. Subject diaries 
about AEs and/or concomitant medication will not be copied but will be considered source 
documents. AE documentation by the investigator will include the date of onset and duration of the 
AE, as well as the severity and causality of each AE, and the actions taken, including the 
discontinuation of the experimental drug, where required.  
9.1 
Emergency Unblinding of Treatment Assignment 
[…] 
9.2 
Adverse Events 
The Investigator is responsible for recording all AEs observed during the study.  
For expected (listed) AEs of RNS60, see the current version of the IB. 
All AEs will be collected starting from the signing of informed consent through the infusion visits 
and the follow up. During this period the investigator is responsible for reporting to the sponsor all 
non-serious AEs, SAEs occurring in subjects treated by the investigator in the clinical trial. 
AEs (but not SAEs) occurring before starting study treatment but after signing the informed consent 
form are recorded on the Medical History/Current Medical Conditions Case Report Form. 
 
[…] 
Investigators must code the grade of all AEs occurring during the trial. For more detail about grade 
criteria, refer to “Common Terminology Criteria for Adverse Events (CTCAE)” Version 4.0 
Published: May 28, 2009 (v4.03: June 14, 2010) from the U.S. DEPARTMENT OF HEALTH 
AND HUMAN SERVICES National Institutes of Health National Cancer Institute. 


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31 
9.3 
AE Reporting 
All AEs, regardless of severity and whether or not they occurred during the treatment or follow-up 
period, are to be recorded on the appropriate AE pages in the e-CRF.  The investigator will include 
the date of onset and duration of the AE, as well as the severity and causality of each AE, and the 
actions taken.  Each event should be recorded separately. 
[…] 
In case of patients needing a Non Invasive Ventilation (NIV), it is necessary to record the procedure 
into the AE form. 
The investigator shall report all AEs and DAEs into the e-CRF, without reporting to “Mario Negri” 
IRCCS. 
9.4 
Reporting of SAEs and SUSARs 
[…] 
The following serious events should not be recorded as SAE due to the usual progression of the 
disease and the investigator judgment: 
- 
Death (only if caused by the disease progression); 
- 
Tracheotomy; 
- 
PEG/RIG placement. 
If these events occur, the Investigator must record them into the AE form. 
 
9.5 
Follow up of AE/SAE/DAR/DAE/SUSAR 
Any AE/SAE/DAR/DAE/SUSAR observed from randomization up to the end of the study will be 
followed up to resolution.  Resolution means that the subject has returned to a baseline state of 
health or the Investigator does not expect any further improvement or worsening of the AE.  
9.6 
Data Monitoring Committee 
According to the EMEA GUIDELINE ON DATA MONITORING COMMITTEES (January 2006) 
a Data Monitoring Committee (DMC) is an external group of independent experts assessing the 
progress, safety data and efficacy endpoints of this clinical study. […] 
 


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32 
10 
QUALITY CONTROL 
[…] 
 
11 
STATISTICS 
11.1 Sample Size Calculation 
We analyzed preliminary data on Cyp-A which showed an increase of 0.27 over six months (Nardo, 
G. et al. 2011). With 142 patients enrolled, we will have 80% power to detect a 44% decrease in the 
rate of progression in Cyp-A at a two sided p=0.05 significance level.  This assumes a drop-out rate 
of 1.75% per month (10% over the study).  The calculation is based on the method shown in 
(http://hedwig.mgh.harvard.edu/biostatistics/node/42). For the other biomarkers we only had data 
on the between patient variation and not on the within patient variation which could be used for 
calculating sample size for the proposed study. Usually the within patient variation is less than the 
between patient variation, so power calculated using the between patient variation is conservative. 
The T-reg values were assessed in slowly progressing vs. rapidly progressing ALS43 and the 
estimated difference was used as the difference expected to slow the progression of the disease in 
patients receiving RNS60. A total of 128 patients is needed to detect a difference in means of 0.25 
units between the two treatment arms at 24 weeks with a standard deviation (SD) of 0.5. Similar 
assumptions were made for the other biomarkers for which, except for Actin-NT (that would 
require 1542 patients), numbers range from 8 to 68 (Nardo, G. et al. 2009; Nardo, G. et al. 2011; 
Henkel, J. et al. 2013; Chiò A. et al., 2011): 
- MCP-1: a total of 16 patients is needed to detect a difference in means of 220 units between 
the two treatment arms at 24 weeks with a standard deviation (SD) of 140; 
- Actin-NT: a total of 1542 patients is needed to detect a difference in means of 0.1 units 
between the two treatment arms at 24 weeks with a standard deviation (SD) of 0.7; 
- 3-NT: a total of 24 patients is needed to detect a difference in means of 50 units between the 
two treatment arms at 24 weeks with a standard deviation (SD) of 40; 
- Cyp-A: a total of 68 patients is needed to detect a difference in means of 1.4 units between the 
two treatment arms at 24 weeks with a standard deviation (SD) of 2.0. 
 
For ALSFRS-R, the software http://hedwig.mgh.harvard.edu/biostatistics/node/42 shows the 
variance parameters which are from the Celebrex Study.  
The variance  of the random effects is the matrix 31.8 (intercept), 0 .6687 (slope) with a covariance 
of 0.8527. The error has a variance of 2.7. 


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33 
With 142 patients enrolled, we will have 80% power to detect a 43% decrease in the rate of 
progression in ALSFRS over 6 months, at a two sided p=0.05 significance level.  The study is also 
powered (in this case p=0.05 two sided) to show an absolute 25% reduction in the proportion of 
patients becoming non self-sufficient at 24 weeks, using a validated outcome measure with strong 
clinical significance (Marin, B. et al. 2015). In two studies from our group (Beghi, E. et al 2013; 
Chiò, A. et al. 2010) the average proportion of patients becoming non self-sufficient after 24 weeks 
in the placebo arm was 85%. 
11.2 Statistical Analysis Plan 
The study will use a modified intent to treat approach with all patients included who took at least on 
dose of the study medication or placebo.  The primary analysis, described below, is valid as long as 
the probability of non-observation of a data point is independent of the value that would have been 
observed conditional on the previous observed data. In the case of ALSFRS this method has been 
shown to be robust  (Ref http://www.ncbi.nlm.nih.gov/pubmed/22987690). Separate analyses will 
be performed in: 1. All randomized subjects receiving at least one dose of study medication 
(Intention-to-treat population); 2. Study completers: subjects completing the 12-month follow-up; 3. 
Study completers and compliers: subjects completing the 12-month follow-up and taking at least 
75% of assigned dose. 4. All randomized subjects excluding protocol deviations (Per protocol, PP 
population). Descriptive statistics will be performed comparing the two treatment groups and safety 
of RNS60 and placebo. Continuous variables will be described using mean and standard deviation 
or median and interquartile range; categorical variables will be described as counts and percentages.  
Biomarkers analysis: 
The primary analysis will be a repeated measures analysis of variance (ANOVA) with a treatment 
variable that is the same for all treatments at baseline and then differs at later observation times. We 
will use an unstructured variance covariance matrix.  The contrast of interest will depend on the 
outcome. Note that this is more general than a random effects model in situations where the 
observation times are fixed. For biomarkers that progress over of time such as Cyp-A, the primary 
analysis will compare the rate of change in the biomarker over time in the placebo and active 
treatment groups.  In biomarkers that do not progress but rather are elevated in patients, the primary 
analysis will be a comparison in the treatment effect.  Both of these analyses are contrasts in a 
repeated measures ANOVA, the former by treatment X time, and the latter being treatment X 
(time>0).  Each biomarker will be analyzed separately.  
Efficacy analysis: 


RNS60-ALS V3.0 Dated on November 12,   2018 
 
 
34 
The numerical change of FVC, ALSFRS-R and ALSAQ-40 during the treatment and follow-up 
period will be assessed using repeated measures ANOVA, with an unspecified variance covariance 
matrix. The hypothesis will be tested using the time*treatment interaction which measures 
differences in the slope of the ALSFRS-R. Survival (time to death or tracheostomy) and time to 
functional disability (i.e., to reach a non self-supporting status or death or tracheostomy) will be 
assessed using actuarial methods with death and non-self supporting status as end-points. Survival 
will be described with the Kaplan-Meier method. Differences between the two treatment groups 
will be compared using the log-rank test. The statistical significance will be set at the 0.05 level for 
a two-tailed test.   
The repeated measures ANOVA will use all available data, using Maximum Likelihood methods as 
suggested by (http://www.nap.edu/read/12955/chapter/1).  
 
Safety analysis will be performed in all subjects receiving at least one dose of the experimental 
drug. All AEs, SAEs and AEs leading to treatment discontinuation will be recorded, listed and 
compared in the two treatment arms at any follow-up visit and at the end of the study. The number 
of patients reporting at least one AE or SAE will be compared between the treatment arms using the 
fisher exact test. The total number of AE and SAE will be compared between the treatment arms 
using a poisson model. MedDRA code system will be used for all recorded AE/SAE.  
Given the exploratory value of this study, multiple comparison adjustments for Type I errors will 
not be considered as a more conservative significance level would lead to miss any potential 
treatment effect.  Furthermore, if there is a suggestive benefit (p<0.1) in the clinical measures and 
several of the biomarkers, this will be considered sufficient evidence for further study. 
If at the end of the patient enrolment period less than 50% of patients are enrolled, an interim-
analysis will be performed to decide whether or not to continue the trial. 
11.3 Compliance 
The compliance with the assigned at-home treatment will be measured by counting the number of 
used/unused syringes returned by the patient at each visit. A subject will be considered non-
compliant if the amount of unused drug exceeds 25% of the assigned drug. The intake of less than 
50% of the total amount of nebulized drug for 3 consecutive visits or in the event that the infusion is 
not performed for 2 consecutive visits implies withdrawal from the study. 


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35 
11.4 Dictionary 
Concomitant medications entered into the database will be coded using the World Health 
Organization drug reference list (http://www.whocc.no), which employs the anatomical therapeutic 
chemical classification system. Medical history/current medical conditions and adverse events will 
be coded using the medical dictionary for regulatory activities (MedRA) terminology (MedRA, 
2011). 
 
12 
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