The Effect of RNS60 on ALS Biomarkers

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ALS carrying the SOD1G93A mutation (unpublished data).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 The investigators also have preliminary unpublished data on MCP1 in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.

Inclusion Criteria:

1. Age 18 through 70 years inclusive;

2. Geographically accessible to the site and able to come to the site once a week for 24
weeks;

3. Definite or probable 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:

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 systems other than motor 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;

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 capability to use an inhalation
device;

10. Abnormal liver function defined as AST and/or ALT > 3 times the upper limit of the
normal.