A Phase 2 Randomized, Placebo- Controlled, Parallel Group, Double Blinded Single Center Study on Effect of N-acetyl Cysteine Compared to Placebo on Fatigue in Patients With Progressive Multiple Sclerosis
Phase: PHASE2
Conditions: Progressive Multiple Sclerosis, Fatigue
Sponsor: University of California, San Francisco

Confidential – UCSF – NAC Pilot study (Version 4 dated 12 Oct 2016) 
Protocol title: A pilot study of oxidative pathways in MS fatigue 
Document Date: 10/12/2016 
Version: 4 
NCT: 02804594
PI: Emmanuelle Waubant, M.D., PhD 
Professor of Clinical Neurology and Pediatrics 
UCSF MS Center 
Neuroscience Building 
675 Nelson Rising Lane, suite 221 
San Francisco, CA94158 

Confidential – UCSF – NAC Pilot study (Version 4 dated 12 Oct 2016) 
 
 
 
 
 
 
 
 
Protocol title: A pilot study of oxidative pathways in MS fatigue 
 
PI: Emmanuelle Waubant, M.D., PhD 
Professor of Clinical Neurology and Pediatrics 
UCSF MS Center 
Neuroscience Building 
675 Nelson Rising Lane, suite 221 
San Francisco, CA94158 
 
8.Statistical consideration and analysis plan 
8.1 Statistical plan for specific aim 1- To test the tolerability and safety of NAC therapy (1250 
mg three times a day) compared to placebo for 4 weeks to treat progressive MS fatigue. 
 
Study Subjects: Target population- Adult patients with progressive MS and fatigue seen at UCSF. 
 
The safety outcome will be analyzed by including all subjects who have received at least one dose 
of study medication. Participants will be analyzed according to the actual treatment received. The 
assessment of safety will be based on the frequency of adverse events. 
The primary efficacy analysis will be done as an intent-to-treat analysis. Consequently, all 
randomized participants will participate in the analysis as allocated to NAC or placebo. 
8.2 Statistical plan for specific aim 2- To obtain preliminary data on changes in fatigue levels 
and oxidative pathway biomarkers on NAC therapy in subjects with progressive MS fatigue. 
In an intent-to-treat analysis, using Student t-test, the mean change in MFIS score from the 
baseline to week 4 will be compared between the NAC and placebo groups. The per-protocol 
secondary analysis will take into account treatment regimen. We will use similar models to 
compare the changes in brain GSH concentration and blood GSH/GSSG ratio, FSS, NeuroQOL 
fatigue item bank, 9-hole peg test, 25-foot timed ambulation, and Symbol Digit Modalities Test 
(SDMT) at the baseline and week fourth between the NAC and placebo groups. 

Confidential – UCSF – NAC Pilot study (Version 4 dated 12 Oct 2016) 
 
Because there is a possibility for imbalance in the baseline values of potential confounding 
factors (such as EDSS and depression), an a priori secondary analysis is planned. To assess 
the effectiveness of NAC vs. placebo, we will use a linear regression model, comparing the 
mean change in MFIS score from the baseline to week 4, adjusting the models for the 
baseline EDSS and HADS scores. 
We will use the same models to analyze the change in the severity of fatigue as measured 
by MFIS and FSS between week 4 (the end of study drug administration period) and week 
6 (2 weeks after patients have been off study drug). 
Because of the exploratory nature of these analyses, we will not correct our models for 
multiple comparison testing. Statistical analyses will be performed by a biostatistician from 
UCSF biostatistics department blinded to treatment assignment. 
Sample size calculation: There are no available data to base power calculation for this 
aim as such a study has never been performed prior. We will use the data generated for 
this aim to design future studies. 
Primary efficacy endpoint: The primary efficacy analysis will be done as an intent-to-treat 
analysis. Consequently, all randomized participants will participate in the analysis as 
allocated to NAC or placebo. Using Student t-test, the mean change in MFIS score from the 
baseline to week 4 will be compared between the NAC and placebo groups. The per-
protocol secondary analysis will take in account treatment regimen. 
MFIS consists of 21 items with a total score range of 0 to 84 (higher scores indicate worse 
fatigue). As the primary efficacy endpoint of the trial, we will compare the mean change in total 
MFIS score from baseline to week 4 visit between intervention and placebo groups. 
 
Secondary efficacy endpoints (differences between treatment groups): 
- 
Per protocol analysis for changes in MFIS from screening /baseline to week 4. 
- 
MFIS subscales changes between screen/baseline and week 4. 
- 
Change in FSS score from baseline to week 4. 
- 
Change in NeuroQOL fatigue item bank score from baseline to week 4. 
- 
Change in 9-HPT score from baseline to week 4. 
- 
Change in 25-FTW from baseline to week 4. 
- 
Change in SDMT score from baseline to week 4. 
- 
Changes in MFIS and FSS between week 4 and 6. 
 
8.3 Statistical plan for specific aim 3- To determine if fatigue severity is associated with 
baseline spectroscopy and oxidative pathway markers in patients with progressive MS. 
 
We will examine the correlation between biomarkers (spectroscopy and blood oxidation 
metabolites) and fatigue (MFIS) at baseline. We will use a linear regression model with MFIS 
score (or its change) as the outcome and the spectroscopy and metabolites as the predictors (in 

Confidential – UCSF – NAC Pilot study (Version 4 dated 12 Oct 2016) 
 
separate models). Using data obtained from the control subjects (patients with progressive MS 
and fatigue severity as measured by MFIS<38) we will also perform a case-control analysis (e.g. 
logistic regression with being fatigued or non-fatigued as the outcome and spectroscopy or blood 
metabolites as predictors. 
 
Study variables 
 
Demographic variables: Age, sex, self-reported race/ethnicity (based on the National Institute 
of Health classification), education (in years) and occupation will be recorded at the screening 
visit. Because the severity of fatigue may fluctuate during the day and based on the ambient 
temperature, the time of the visit and the outside temperature will also be recorded. 
 
Baseline MS-related information: Years since disease onset, type of MS (primary versus 
secondary progressive (per medical records), Expanded Disability Status Scale (EDSS) 
(obtained at screening visit by the examining physician), and 25-foot timed walk (25-FTW), 9-
hole peg test (9-HPT) and Single Digit Modalities test (SDMT) (obtained by a trained technician 
at screening, baseline and week 4). 
 
Fatigue: MFIS and FSS (questionnaires filled by patients after receiving appropriate instructions 
from study staff, see appendix) at screening, baseline, week 4 (last study drug dose), and week 
6 (2 weeks after stopping study drug). 
 
Depression: Hospital Anxiety and Depression Scale (HADS) depression subscale 
(questionnaires filled by patients after receiving appropriate instructions from a study staff, see 
appendix) at screening and baseline. 
 
Quality of life: NINDS NeuroQOL fatigue item bank (questionnaires filled by patients after 
receiving appropriate instructions from study staff, see appendix) at the screening, baseline and 
week 4. 
 
Safety laboratory assessment: At screening, CBC and differential, creatinine, AST, ALT, urine 
analysis and urine pregnancy test will be obtained. At week 4, only creatinine, urine pregnancy 
test, AST and ALT will be measured. 
 
Research laboratory assessment: Plasma level of reduced glutathione (GSH) and oxidized 
glutathione (GSSG) baseline and at 4 week visit for patients who enter treatment phase, and 
baseline research samples will be analyzed for patients who are controls in the study 
 
Conventional MRI sequences: The MR experiments will be performed on the GE 7T research 
scanner in the Surbeck Laboratory using a 32-channel phased array head coil. The 60-minute 
MR protocol will include standard imaging sequences with calibration scans (3-plane localizer, 
coil sensitivity maps), gradient recalled-echo, T1-weighted and T2-weighted images, and 
multiband DWI. Higher order shimming will be performed prior to the spectral data acquisitions. 
The volume of interest for 3D short TE MRSI will be prescribed to have full coverage of the 
thalamus that will enable to measure metabolite levels (such as Cho, Cr, NAA, Glutamate, 

Confidential – UCSF – NAC Pilot study (Version 4 dated 12 Oct 2016) 
 
Glutamine, mI, and GSH) in deep grey matter and surrounding normal appearing white matter. 
GSH-edited MRS will be acquired as well to provide unobstructed detection of 
GSH in the selected of regions of interest. 
 
Data collection: 
 
1) Blood samples from participating patients at the baseline and week 4 visits will be collected 
and analyzed for various metabolites on the oxidative pathway including GSH, GSSG, and 
response ratio for GSH, and GSSG. 
2) 7T MR spectroscopy at the baseline and 4 week visits will be obtained and analyzed for 
concentrations of N-acetyl-aspartate (NAA), glutamate (Glu), glutathione, myo-inositol 
(mI), creatine (Cr), and choline (Cho) over the region of interest (ROI) region and 
concentrations of NAA, Glu, glutathione, mI, Cr, and Cho from normal-appearing white-
matter areas within the ROI region. 
Power considerations: There are no available data to base power calculation for this exploratory 
aim as such a study has never been performed prior. We will use the data generated for this Aim 
to design future studies. 
9.Data collection, management and quality assurance 
 
We will use Redcap (http://project-redcap.org/), a secure web application for building online 
databases, as the trial data management software. The demographic and baseline information 
will be entered directly from the medical records by study coordinator into the study CRF. The 
study manger will review CRFs for completeness and enter into Redcap database. UCSF 
research 
fellow (Dr. Nourbakhsh) will double check the data entered into Redcap for accuracy.