The Effect of RNS60 on ALS Biomarkers (RNS60)

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.

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effect of RNS60 on motor neurons in both in vitro and in vivo models of familial ALS carrying the SOD1G93A mutation (unpublished data).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: The preliminary efficacy of RNS60 on functional disability, as measured by the ALSFRS-R scale; The preliminary efficacy of RNS60 in prolonging survival (or time to tracheostomy, whichever comes first); The preliminary efficacy of RNS60 in slowing the decline of forced vital capacity (FVC) from baseline; The tolerability and safety of RNS60 through the identification of unexpected adverse events; 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.

Multicenter, randomized, double-blind, placebo-controlled, parallel group, add-on phase II trial. ALS Patients who meet the study's inclusion/exclusion criteria and sign the Informed Consent Form will be enrolled. A total of 142 subjects will be randomly assigned to receive treatment with either RNS60 or placebo while concomitantly taking riluzole (50 mg tablet t.i.d.). RNS60 or placebo will be administered intravenously once a week as well as inhaled via nebulization every morning in the remaining six days of each week for 24 weeks. Blood samples for biomarker analysis will be collected on day 1, week 4, w12, and w24. Safety and preliminary 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. Each patient will be followed up for a maximum period of 48 weeks (24-week treatment + 24-week follow up) or until death or tracheostomy, whichever occurs first.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

**ALSFRS-R = ALS Functional Rating Scale - Revised** The mean change of ALSFRS-R (**min score 0 corresponding to maximum functional impairment - max score 48 corresponding to no functional impairment; higher score = better outcome**) over the on-treatment period and the off-treatment follow-up period. Measured at weeks 0, 4, 12, 24, 36, 48.

24 weeks on-treatment period (week 0 - week 24) + 24 weeks off-treatment follow-up period (week 24 - week 48)

24 weeks on-treatment period (week 0 - week 24) + 24 weeks off-treatment follow-up period (week 24 - week 48)

The mean change of Forced Vital Capacity percent value (FVC%) over the on-treatment period and the off-treatment follow-up period. Measured at weeks 0, 4, 12, 24, 36, 48.

24 weeks on-treatment period (week 0 - week 24) + 24 weeks off-treatment follow-up period (week 24 - week 48)

The total number of subjects in the two treatment arms experiencing at least one adverse event (AE) leading to treatment discontinuation at 4, 12 and 24 weeks

**ALSAQ-40=ALS Assessment Questionnaire - 40 items** The mean change of ALSAQ-40 over the on-treatment period and the off-treatment follow-up period. Measured at weeks 0, 24, 48. The ALSAQ-40 is a 40-item questionnaire measuring health status and health related quality of life in ALS patients. It is divided in 5 domains: the physical mobility (it addresses problems of mobility); the ADL (activities of daily living) and independence (it addresses a variety of limitations in ADL); the eating and drinking (it adresses problems eating solid foods, swallowing and drinking liquids); the communication (it addresses a variety of problems in communicating with others); the emotional reactions (it addresses various emotional problems). ** For each domain the score has the following range: Min score =0 corresponding to the better condition - max score =100 corresponding to the worse condition. (A higher score corresponds to a better outcome ) **

24 weeks on-treatment period (week 0 - week 24) + 24 weeks off-treatment follow-up period (week 24 - week 48)

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.

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.

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.

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.

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.

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.

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.

24 weeks on-treatment period (week 0 - week 24) + 24 weeks off-treatment follow-up period (week 24 - week 48)

Inclusion Criteria: Age 18 through 80 years inclusive; Geographically accessible to the site and able to come to the site once a week for 24 weeks; Definite, probable, probable laboratory supported ALS diagnosis according to the revised El Escorial criteria; 4) Disease duration 6 to 24 months from symptom onset; 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; 8) Ability to understand and comply with the study requirements and to give written informed consent personally or via a legally authorized representative; 9) Treatment with riluzole 50 mg twice/day for at least 1 month prior to screening visit. Self sufficiency: this term reflect independence in daily living activities. It is an intuitive parameter to indicate preservation of key functional activities, and - not least - it has shown to be a valid and reliable measure Exclusion Criteria: History of HIV, clinically significant chronic hepatitis, antecedent polio infection, or other active infection; Motor neuron disease (MND) other than ALS; Involvement of systems other than motor possibly determining a functional impairment (as measured by the end-points) for the entire duration of the study; Other severe clinical conditions (e.g., cardiovascular disorders, neoplasms) with impact on survival or functional disability in the next 12 months; Renal insufficiency as defined by a serum creatinine > 1.5 times the upper limit of normal; Poor compliance with previous treatments; Other experimental treatments in the preceding 3 months; 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; Unwillingness or inability to take riluzole; 10) Poor capability to use an inhalation device; Abnormal liver function defined as AST and/or ALT > 3 times the upper limit of the normal.