Conditions: ALS (Amyotrophic Lateral Sclerosis)
Allocation: NON_RANDOMIZED | Intervention model: SINGLE_GROUP
Enrollment: 60
Healthy volunteers: no

Brief summary:
This is a 12-month, widely inclusive, largely virtual, single-center, open-label pilot trial utilizing a historical control group. Participants will receive a Lunasin regimen and will be asked to register for an account of PatientsLikeMe website, where after the initial in-clinic visit, they will be asked to enter specific data.

Detailed description:
The primary hypothesis is that a supplement regimen containing Lunasin (referred to as the Lunasin regimen) can decrease the rate of ALSFRS-S progression by 50% relative to matched historic controls.

ALS is a devastating motor neuron disease that causes rapidly progressive muscle weakness, disability and premature death. In spite of a large number of attempted ALS trials, there are no significant disease-modifying therapies for this condition.

It was recently reported that a patient with a validated diagnosis of ALS, had experienced dramatic objective improvement in speech, swallowing and limb strength while taking a supplement regimen containing Lunasin. Several other patients with ALS have also reported improvement on this Lunasin regiment, though records were not obtainable to validate these.

The Lunasin regimen will consist of:

* LunaRich X Capsules
* Reliv NOW - a mixture of 'vitamins, minerals and super-powered antioxidants'
* Pro-Vantage - a mixture of 'soy protein, medium chain triglycerides, creatine, CoQ10 and supercharged amino acids'

Dosages will be titrated up to target over the first 6 days. The subject will take the highest tolerated dosing for the remainder of the 12-month study.

There are 3 in-clinic visits which occur at screening/baseline, Month 1 and Month 12. For Months 2-11, participants will make "virtual visits" by measuring their own ALSFRS-R score and weight and will record it in the PatientsLikeMe website. They will also self-report any perceived efficacy, compliance, adverse events and changes in concomitant medications.

Arms:
arm 1: The Lunasin regimen consists of:

* LunaRich X Capsules (12 capsules per day)
* Reliv NOW - a mixture of 'vitamins, minerals and super-powered antioxidants' (3 scoops per day)
* Pro-Vantage - a mixture of 'soy protein, medium chain triglycerides, creatine, CoQ10 and supercharged amino acids' (2 scoops per day)

It will be suggested that patients open the LunaRich X capsules and mix the contents of these as well as the other 2 ingredients in water to make a shake. If patients do not tolerate advancing to the next dosage, they will be asked to drop back to the highest dosage they could tolerate. arm 2: For each enrolled participant, matched historical controls will be identified from the PatientsLikeMe database. Participants will be matched according to their ALSFRS-R progression rate before they start on the Lunasin regimen (estimated by assuming their score was normal at 48 on the date of symptom onset).

Interventions: intervention 1: Lunasin Regimen intervention 2: Historical control
Intervention descriptions: intervention 1: LunaRich X Capsules, Reliv Now, ProVantage intervention 2: Matched historical controls will be identified from the PatientsLikeMe database.

Protocol document:
Prot_SAP_000.pdf: 
Page 1 of 22 
 
Full study protocol and statistical analysis plan 
 
NCT02709330 
 
An open-label, single-center, 12-month trial of a Lunasin regimen 
for patients with Amyotrophic Lateral Sclerosis (ALS) 
 
 
IND Number: 128306 
 
Duke IRB Protocol Number: Pro00063754 
 
 
 
 
Protocol Version Date: 
Version 11 
December 26, 2015 
 
 
 
 
 
 
 
Richard Bedlack, MD, PhD 
Associate Professor of Neurology 
DUKE UNIVERSTIY MEDICAL CENTER 
 
 


Page 2 of 22 
 
TABLE OF CONTENTS 
1. 
Purpose/Hypotheses .................................................................................................... 4 
1.1. 
Primary Hypothesis. ............................................................................................. 4 
1.2. 
Secondary Hypotheses. ........................................................................................ 4 
2. 
Background ................................................................................................................. 4 
2.1. 
Amyotrophic Lateral Sclerosis (ALS) and Its Treatment. ................................... 4 
2.2. 
Failure of Previous ALS Trials. ........................................................................... 4 
2.3. 
ALS Reversal on Lunasin. ................................................................................... 5 
2.4. 
How Lunasin Could Influence ALS. .................................................................... 5 
2.5. 
PatientsLikeMe..................................................................................................... 6 
3. 
Design & Procedures: ................................................................................................. 6 
3.1. 
Design Overview. ................................................................................................. 6 
3.2. 
Lunasin Regimen.................................................................................................. 7 
3.3. 
Lunasin Supply. .................................................................................................... 7 
3.4. 
Dose Adjustments and Holidays. ......................................................................... 7 
3.5. 
Compliance........................................................................................................... 8 
3.6. 
Concomitant Medications. ................................................................................... 8 
3.7. 
Use of Riluzole. .................................................................................................... 8 
3.8. 
Outcome Measures ............................................................................................... 8 
3.8.1. 
ALSFRS-R. ................................................................................................... 8 
3.8.2. 
Histone Acetylation. ..................................................................................... 8 
3.8.2.1. 
Histone extractions and Western blots. ..................................................... 9 
3.8.2.2. 
Chromatin immunoprecipitation (ChIP) assay. ......................................... 9 
3.8.2.3. 
RNA extraction and reverse transcription. ................................................ 9 
3.8.3. 
Weight. ........................................................................................................ 10 
3.8.4. 
Enrollment Rate. ......................................................................................... 10 
3.8.5. 
Retention. .................................................................................................... 10 
3.9. 
Study Schedule ................................................................................................... 10 
3.9.1. 
Screening/Baseline Visit. ............................................................................ 10 
3.9.2. 
Week 1, 2 and 3 Calls. ................................................................................ 11 
3.9.3. 
Month 1 and 12 Visits. ................................................................................ 11 
3.9.4. 
Week 1, 2, 3 and Month 2-11 Virtual Visits. .............................................. 11 
3.9.5. 
Schedule of Events Table. ........................................................................... 11 
3.10. 
Participants. .................................................................................................... 12 
3.10.1. 
Inclusion Criteria. .................................................................................... 12 
3.10.2. 
Exclusion Criteria. ................................................................................... 12 
3.11. 
Recruitment & Compensation. ....................................................................... 13 
3.12. 
Consent Process: ............................................................................................. 13 
3.13. 
Capacity to Give Consent. .............................................................................. 13 
3.14. 
Risk Assessment. ............................................................................................ 13 
3.15. 
Costs to Subjects. ............................................................................................ 14 
3.16. 
Data Analysis & Statistical Considerations .................................................... 14 
3.16.1. 
Primary Hypothesis and Sample Size. .................................................... 14 
3.16.2. 
ALS Reversals. ........................................................................................ 14 
3.16.3. 
ALSFRS-R Accuracy. ............................................................................. 15 


Page 3 of 22 
 
3.16.4. 
Weight Accuracy. .................................................................................... 15 
3.16.5. 
Enrollment. .............................................................................................. 15 
3.16.6. 
Retention. ................................................................................................ 15 
3.17. 
Adverse Events Monitoring. ........................................................................... 15 
3.18. 
Data & Safety monitoring............................................................................... 17 
3.19. 
Data Collection and Management .................................................................. 17 
3.19.1. 
NeuroBANK™. ....................................................................................... 17 
3.19.2. 
NeuroBANK™ Data Management (DM). .............................................. 17 
3.19.3. 
NeuroBANK Data Entry and Checks...................................................... 17 
3.19.4. 
Data Lock Process. .................................................................................. 18 
3.19.5. 
Data Handling and Record Keeping........................................................ 18 
3.19.6. 
Confidentiality. ........................................................................................ 18 
3.19.7. 
Global Unique Identifier (GUID). ........................................................... 18 
3.20. 
PatientsLikeMe. .............................................................................................. 18 
3.20.1. 
General Technical Information on the PatientsLikeMe Website. ........... 18 
3.20.2. 
Security Information. .............................................................................. 19 
3.21. 
Good Clinical Practices and Human Subjects Protection Training. ............... 19 
4. 
References: ................................................................................................................ 19 
 


Page 4 of 22 
 
1. 
PURPOSE/HYPOTHESES 
1.1. 
Primary Hypothesis   
The primary hypothesis is that a supplement regimen containing Lunasin (hereafter 
referred to as the Lunasin regimen) can decrease the rate of ALSFRS-R progression by 
50% relative to matched historical controls.  
1.2. 
Secondary Hypotheses   
Secondary hypotheses are as follows: 
1. Lunasin regimen can increase the frequency of ALS reversals (defined by an 
improvement of 4 or more points in the ALSFRS-R over the course of 1 year) 
from 1% (observed spontaneously) to at least 2%.   
2. Lunasin regimen can alter histone acetylation in patients with ALS (PALS). 
3. Participants can accurately measure their own ALSFRS-R score and record it on 
PatientsLikeMe relative to data capture by a healthcare professional. 
4. Patients can accurately measure their own weight and record it on PatientsLikeMe 
relative to data capture by a healthcare professional. 
5. The novel features of this pilot trial will be associated with improved participant 
enrollment compared to prior more traditional ALS trials where this is 2 
participants per site per month. 
6. The novel features of this pilot trial will be associated with improved participant 
retention compared to prior more traditional ALS trials where the dropout rate is 
22%. 
2. 
BACKGROUND 
2.1. 
Amyotrophic Lateral Sclerosis (ALS) and Its Treatment   
ALS is a devastating motor neuron disease that causes rapidly progressive muscle 
weakness, disability and premature death.  In spite of a large number of attempted ALS 
trials, there are no significant disease-modifying therapies for this condition (1).  
2.2. 
Failure of Previous ALS Trials   
There are at least 2 reasons that previous ALS trials may have failed.  First, the 
hypotheses could have been wrong.  The hypotheses for many prior trials came from 
observations made in animal models of familial ALS (1).  How well these predict human 
ALS, the vast majority of which is not familial, has been called into question (2).  Second 
the dosing may not have been adequate.  Indeed, most ALS trials have not even 
employed pharmacodynamic biomarkers to test their dosing regimen (3).  In addition to 
these problems, ALS trials are often challenged by slow enrollment (4) and poor retention 
(5).  While some of the enrollment and retention issues may have to do with 
misconceptions on the part of enrolling clinicians and potential participants (4), patients 
with ALS do become more disabled over time, and study burdens including trips to the 
study site may eventually become impossible to bear.  


Page 5 of 22 
 
2.3. 
ALS Reversal on Lunasin   
ALSUntangled is a consortium of ALS clinicians and researchers that reviews alternative 
and off-label treatments being tried by patients with ALS (6). ALSUntangled recently 
reported on a patient with a validated diagnosis of ALS, who experienced dramatic 
objective improvements in speech, swallowing and limb strength while taking a 
supplement regimen containing Lunasin (7).  Several other patients with ALS have also 
reported improvements on this Lunasin regimen, though records were not obtainable to 
validate these (7). 
2.4. 
How Lunasin Could Influence ALS  
Transcriptional dysregulation has been proposed to be part of the pathophysiology of 
ALS (8-11).  Transcriptional dysregulation perturbs cellular function and compromises 
protective mechanisms, leading to neuronal death.  Many proteins, including histones that 
affect the actions of RNA polymerase II on individual genes, regulate transcription. Many 
of these interactions, in turn, are regulated by acetylation, methylation and 
phosphorylation.  Recruitment of histone deacetylases (HDACs) to DNA alters 
nucleosome structure locally and inhibits transcription. Histone acetylation patterns are 
known to be abnormal in patients with ALS and have been the target of previous 
therapeutic trials (12). 
 
Lunasin, as a low potency dietary nutrient, can reportedly affect the expression of a 
number of genes in multiple regulatory and physiological pathways on different cells in 
the body.  It is able to accomplish this by modifying the epigenome and changing gene 
expression through modifications to specific lysine residues in histones H3 and H4. 
Lunasin can turn off disease-causing genes by binding and inhibiting the specific 
acetylation of Lysine 14 on histone H3 (H3K14ac) by the histone acetylase enzyme, 
PCAF (13).  H3 acetylation, which includes H3K14ac, is an epigenetic mark required to 
activate genes involved in cholesterol biosynthesis (13), inflammation (14), hormone 
response (15), metabolic syndrome (16) and stress (17).  By impeding the expression of 
these genes, Lunasin can help prevent formation of age related, degenerative chronic 
diseases. Lunasin can also turn on and increase the expression of health-promoting genes, 
such as those involved with cancer prevention, by enhancing the acetylation of Lysine 16 
on histone H4 (H4K16ac) (18).  H4K16ac is the epigenetic mark associated with the 
opening of condensed chromatin (DNA + histones) to allow easier access of the cellular 
transcriptional machinery to activate and increase gene expression (19).  Loss of 
H4K16ac is a common hallmark of human cancer cells (20)   
 
Thus, if Lunasin can restore normal patterns of histone acetylation in patients with ALS, 
it may help to ameliorate transcriptional dysregulation and importantly slow or reverse 
ALS progression. The novelty of the outlined proposal is that we will be able to measure 
alterations in histone acetylation in the treated patients. This will allow us to correlate 
changes in disease progression with molecular changes. Thus, assessing histone 
acetylation from patient samples can provide us with a “biomarker” for this study.  
 
This study will treat other PALS with the Lunasin regimen used by the above-described 
patient who experienced ALS reversal and determine if anyone else improves.  We will 


Page 6 of 22 
 
look for accompanying changes in histone acetylation.  We will employ a novel design in 
hopes of enhancing enrollment and retention rates. 
2.5. 
How Reliv Now and Provantage Might Influence ALS 
In addition to Lunarich X capsules (the main source of Lunasin in this study) the other 2 
products in the Lunasin regimen are called Reliv Now and Provantage.  These are 
mixtures of more Lunasin as well as various antioxidants and anti-inflammatories.  
Oxidative stress and neuroinflammation are postulated to be part of ALS 
pathophysiology and there have been numerous unsuccessful attempts to treat PALS with 
antioxidants and anti-inflammatories before.  This particular combination has never been 
studied, however. 
2.6. 
PatientsLikeMe  
PatientsLikeMe® (PLM; www.patientslikeme.com) is an online platform that has 
demonstrated an ability to engage patients in learning about their conditions and building 
a large quantitative and qualitative database about patients’ experiences of their 
conditions. PLM is a patient network with over 300,000 members comprising over 2000 
different conditions founded by a family affected by ALS, and to date has conducted over 
30 published studies in ALS including studying patient use of off-label treatments. 
Members who join the site with ALS are asked to complete a number of data fields 
including demographics, disease condition history, treatments, symptoms, weight, 
treatment evaluations and lab measurements (e.g. weight, forced vital capacity). In 
addition, a unique feature of the site is the ability of patients to complete a widely used 
patient reported outcome, the ALS Functional Rating Scale (Revised, ALSFRS-R) which 
allows patients to see their disease progression in context in visual form. Data capture is 
facilitated by a team of PhD scientists, nurses, and pharmacists that incorporate 
information from various medical databases (e.g. RxNorm, ICD-10, MEDDRA). In 
addition the site provides means of social support through a message board forum, private 
messages, and profile commenting. Online methods of data capture have the potential to 
dramatically accelerate trial recruitment, provide more convenient means of data capture 
for patients, and minimize trial complexity by using matched historical controls in lieu of 
a placebo control group. However, as a relatively new technology there are still questions 
of validity which must be addressed to fully realize the potential of such tools. 
3. 
DESIGN & PROCEDURES:  
3.1. 
Design Overview.   
This will be a 12-month, widely inclusive, largely virtual, single-center, open-label pilot 
trial utilizing a historical control group.  Treatment with the Lunasin regimen and all the 
following study outcome measures and labs are being performed exclusively for research 
purposes:  Vital Signs, ALSFRS-R, biomarker testing, adverse events, and concomitant 
medications. Participants will be asked to register for an account on PatientsLikeMe 
(“PLM”) (sign-up process can be found in Appendix H) with the help of the study 
coordinator. User IDs associated with their PatientsLikeMe account will be recorded by 
the study coordinator and shared with study staff at PatientsLikeMe. When registering, 
participants will have the opportunity to review PLM’s terms and conditions as well as 


Page 7 of 22 
 
their privacy policy (Appendix A). After the initial in-clinic visit, participants will be 
asked to enter the following data: weight (Appendix B), Lunasin treatment evaluation 
(Appendix C) and their ALSFRS-R score (Appendix D). Participants will be given the 
following study documents and materials to take home with them: 
 A PatientsLikeMe bag (photo Appendix E) including: 
o Welcome letter (Appendix E) 
o Study One-Sheet (Appendix F) 
o Study Checkup Checklist (Appendix G) 
3.2. 
Lunasin Regimen  
The intervention is the Lunasin regimen used by the patient with the above-described 
ALS reversal (7).  This consists of LunaRich X Capsules (12 capsules per day), a mixture 
of ‘vitamins, minerals and super-powered antioxidants’ called Reliv NOW (three scoops 
per day) and a mixture of ‘ soy protein, medium chain triglycerides, creatine, CoQ10 and 
supercharged amino acids ’ called Pro-Vantage (two scoops per day).  Patients will taper 
up to this target regimen as follows:  Day 1 (Dosage 1): 3 capsules of Lunarich X, ¼ 
scoop of Reliv Now and ¼ scoop of Provantage (do this twice a day for 1 day).  Day 2 
(Dosage 2): 4 capsules of Lunarich X, 1/2 scoop of Reliv Now and 1/2 scoop of 
Provantage (do this twice a day for 2 days).  Day 4 (Dosage 3): 6 capsules of Lunarich X, 
1 scoop of Reliv Now and 1 scoop of Provantage (do this twice a day for 2 days).  Day 6 
(Dosage 4): 6 capsules of Lunarich X, 1 1/2 scoops of Reliv Now and 1 scoop of 
Provantage (do this twice a day for study duration).  It will be suggested that patients 
open the Lunarich X capsules and mix the contents of these as well as the other 2 
ingredients in water to make a shake.  If patients do not tolerate advancing to the next 
Dosage, they will be asked to drop back to the highest Dosage they could tolerate. 
3.3. 
Lunasin Supply  
The products in the Lunasin regimen are being donated to Duke from the Reliv 
Corporation (21) and will be given to patients in the study free of charge for 12 months.   
Products will be stored in Dr. Bedlack’s locked office at Duke.  At the 
Screening/Baseline Visit, participants will receive a 1-month supply.  At the Month 1 
Visit, participants will receive a 2-month supply and thereafter will receive product by 
mail every 2 months for the duration of the study as long as they are continuing to enter 
their online data (see below).  Once this study ends, participants may elect to continue the 
Lunasin regimen by purchasing products from Reliv. 
3.4. 
Dose Adjustments and Holidays  
The investigator may temporarily reduce or stop administering the Lunasin regimen for 
adverse events.  If the adverse event is mild or moderate, the dose may be reduced to the 
prior Dosage (see taper above) until the event improves.  The investigator may then 
choose to resume the dosage escalation or maintain the participant at reduced dose.  The 
dosage can be raised to the previous dose once.  If a related event is serious or life 
threatening, the Lunasin should be stopped and will not be re-challenged.  One drug 
holiday will be allowed; the holiday can last no more than 7 days.  All dose reductions 
and suspensions must be documented in the Neurobank. 


Page 8 of 22 
 
3.5. 
Compliance   
The method to check compliance for study medication will be to record the amount of 
product dispensed at the Baseline/Screening Visit, and the amount left over at the Month 
1 Visit.  No further compliance checks will be conducted. 
3.6. 
Concomitant Medications   
Throughout the study, investigators may prescribe any other concomitant medications or 
treatments deemed necessary to provide adequate supportive care, provided that they are 
not part of a research study. 
3.7. 
Use of Riluzole   
The use of riluzole will be permitted during the study. Patients taking riluzole must be on 
a stable dose for 30 days prior to screening. 
3.8. 
 Outcome Measures 
3.8.1. ALSFRS-R   
The Revised ALS Functional Rating Scale (ALSFRS-R) will be determined at all phone 
interviews and study visits (Appendix D).  ALSFRS-R is a quickly administered (five 
minute) ordinal rating scale (ratings 0-4) used to determine patients' assessments of their 
capability and independence in 13 functional activities.  All 13 activities are relevant in 
ALS.  Initial validity was established in ALS patients by documenting change in 
ALSFRS-R scores correlated with change in strength over time, was closely associated 
with quality of life measures, and predicted survival (22). The test-retest reliability is 
greater than 0.88 for all 13 items. The ALSFRS-R declines linearly with time over a wide 
range during the course of ALS.  Recent work has shown that the measure can be 
conducted over the phone (23), and it can even be reliably conducted and recorded online 
by patients themselves (24).  The ALSFRS-R will be administered to participants in-
clinic by the study coordinator at Duke on their first visit. Additionally, participants will 
be asked through email from PLM to fill out the ALSFRS-R test on their PatientsLikeMe 
profile once every 30 days.  
3.8.2. Histone Acetylation    
All histone acetylation assays will be carried out in the laboratory of Ghazaleh Sadri-
Vakili, PhD at Massachusetts General Hospital. As in a previous study (12) blood will be 
collected in one 10-ml sodium heparin green top tube and two 5-ml red top tubes (both 
plastic). The 10-ml green top tube will be inverted 8-10 times following collection and 
placed on crushed ice.  The sample will be centrifuged at 8000-9000x g for 20 min within 
1 h of collection. After spinning, the green top tube will be frozen upright on dry ice (-40 
C). The 5-ml red top vacutainer will immediately be frozen upright on dry ice.  All tubes 
will be shipped overnight to Dr. Sadri-Vakili.  In her lab, peripheral blood mononuclear 
cells (PBMC) will be isolated using a standardized Ficoll-Hypaque gradient procedure 
using the Ficoll-Paque Plus (GE Healthcare, Boston, MA) and analyzed for alterations in 
histone acetylation using the following methods.   


Page 9 of 22 
 
3.8.2.1. 
Histone extractions and Western blots   
Histones will be extracted from PBMCs as described previously (25). Briefly, cells will 
be washed with ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold 
radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 
0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl 
fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mM Na33VO4, 1 
mM NaF) supplemented with a protease inhibitor mixture (Roche Applied Sciences, 
Indianapolis, IN, USA) for 10 min. The pellets will be collected by centrifugation at 
10,000 × g for 10 min. The pellets will be washed once in 10 mM Tris-HCl and 37 mM 
EDTA (pH 7.4), and resuspended in 200 µL 0.4 N H2SO4. After overnight incubation on 
ice, the supernatant will be collected by centrifugation at 14,000 × g for 15 min and 
mixed with 2 mL cold acetone and kept at −20°C overnight. The histones will be 
collected by centrifugation at 14,000 × g for 15 min. After one wash with acetone, the 
histones will be air dried and suspended in sterile deionized water. Total histone 
concentration in each sample will be measured using the Bradford assay according to the 
manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA).  Western blot 
analysis will be used to analyze alteration in histone acetylation following treatment as 
described previously (26, 27).  Briefly, 1-5 µg of extracted histone proteins from samples 
will be resuspended in sample buffer. The resuspended samples will be boiled at 95C for 
5 min, and fractionated on a 10-20% tricine gel (Invitrogen, Carlsbad, CA) for 90 min at 
120V.  Proteins were transferred to PVDF membranes in transfer buffer (3% Tris base, 
14.4% glycine, 20% MeOH) at 400 mA x 1 h, and the PVDF will be blocked with 3% 
milk in phosphate-buffered saline before immunodetection with a number of acetylated 
histone antibodies including: anti-di acetyl lysine 9 and lysine 14 histone H3 
(H3K9K14ac2) 1:1500 dilution (Millipore, Billerica, MA) anti-histone H3 1:500 dilution 
(Millipore, Billerica, MA) anti-pan acetylated histone H4 1:1000 (Millipore, Billerica, 
MA) overnight (4C).  Primary antibody incubation will be followed by 4 washes (10 
min, RT) in diH2O before incubation with the secondary antibody (HRP-conjugated goat 
anti-rabbit IgG; Jackson ImmunoResearch Laboratories, West Grove, PA), 4 washes, and 
visualized using the ECL detection system (NEN, Boston, MA). Coomassie gels will be 
used to ensure equal protein loading for western blots.  
3.8.2.2. 
Chromatin immunoprecipitation (ChIP) assay  
ChIP studies will be used to measure alterations of acetylated histones at specific gene 
promoters as described previously (26-29). The same acetylated histone antibodies as 
outlined in the Western Blot methods above will be used for ChIP studies. We will also 
include negative controls such as a no antibody mock and IgG (Jackson, West Grove, 
PA). Input and IP samples will be interrogated with gene promoter-specific primers in 
triplicate reactions using real-time PCR analysis as previously described. Threshold 
amplification cycle numbers (Tc) using iCycler software will be used to calculate IP DNA 
quantities as percentage of corresponding inputs.   
3.8.2.3. 
RNA extraction and reverse transcription   
RNA will be extracted from samples using RNeasy kit (Qiagen, Valencia, CA) according 
to manufacturer’s instructions and as described previously (26,28) to measure changes in 
gene expression.  Reverse transcription reactions will be performed using the Superscript 


Page 10 of 22 
 
First Strand Synthesis System for RT-PCR reactions (Invitrogen, Carlsbad, CA) using 
specific primers to quantitate the amount of gene expression as compared to a standard 
curve. Quantitative real time-PCR will be performed in an iCycler (Bio-Rad) with the use 
of SYBR-green PCR Master Mix (Applied Biosystems, Foster City, CA) through 50 PCR 
cycles (95°C for 30 sec, 57°C for 60 sec, 72°C for 90 sec). The threshold cycle for each 
sample will be chosen from the linear range and converted to a starting quantity by 
interpolation from a standard curve run on the same plate for each set of primers. The 
mRNA levels will be normalized for each well to the GAPDH mRNA levels.  
3.8.3. Weight 
The study coordinator will measure and record the weight of all participants at the 
Screening/Baseline, Month 1 and Month 12 Visits.  Patients will measure and record their 
own weights at Month 2-11 Visits on their PLM profile (See Appendix B). 
3.8.4. Enrollment Rate   
Enrollment rate will be calculated as the number of participants enrolled per month.  A 
previous meta-analysis showed the mean ALS trial enrollment rate to be 2.2 participants 
per site per month (4). 
3.8.5. Retention    
Retention will be assessed by looking at the dropout rate (the percentage of surviving 
enrolled patients who complete the Month 12 Visit).  A previous meta-analysis showed 
the mean ALS participant dropout rate to be 22% (5). 
3.9. 
Study Schedule 
Participants will be required to make 3 visits to Duke (Screening/Baseline Visit, Month 1 
and Month 12 Visit).  At months 2-11, participants will make “virtual visits” by 
measuring their own ALSFRS-R score and weight, and logging onto the website 
PatientsLikeMe (31) to record it as well as perceived efficacy, compliance, adverse 
events and changes in concomitant medications.  Participants will be reminded to log into 
their PLM profile and record the above-mentioned data points via email reminders from 
PLM (Appendix E). The study coordinator will call participants at weeks 1, 2 and 3 for 
training and any time when participants have not entered data for 6 consecutive weeks. 
The call script for such communications can be found in Appendix E. 
3.9.1. Screening/Baseline Visit   
During the Screening/Baseline Visit, (Time 0) participants will be consented.  Inclusion 
and Exclusion Criteria will be reviewed to ensure the participant qualifies.  Vital signs, 
weight and concomitant medications will be recorded.  Participants will be taught to self-
administer the ALSFRS-R (22) on PLM.  Participants will be taught to register for an 
account on the website PatientsLikeMe (31) (Appendix H) and taught to enter their 
ALSFRS-R, perceived efficacy, compliance, adverse events and concomitant 
medications.  The ALSFRS-R has previously been validated for similar online use by 
patients (24).  Blood will be drawn for baseline biomarker assessments.   Urine will be 
collected for pregnancy testing in at-risk females. 


Page 11 of 22 
 
3.9.2.  Week 1, 2 and 3 Calls   
During the Week 1, 2 and 3 Calls the study coordinator will confirm that participants are 
logging into the PatientsLikeMe site and entering data without difficulty.  The 
coordinator will measure an ALSFRS-R by phone and compare it to the participant-
generated score.  Differences will be reconciled as part of the teaching process. 
 
3.9.3. Month 1 and 12 Visits   
During the Month 1 and 12 Visit, vital signs and weight will be measured, and 
concomitant medications will be reviewed.  Participants will be asked to bring in any 
leftover product as a measure of compliance.  ALSFRS-R’s will be independently 
obtained by the study coordinator and the participant and recorded in the Neurobank. 
Comparison of the coordinator and participant generated ALSFRS-R scores will be used 
to confirm the accuracy of participant scoring at this time point.  Blood will be drawn for 
Month 1 biomarker assessments. 
3.9.4. Week 1, 2, 3 and Month 2-11 Virtual Visits   
During the Week 1, 2, 3 and Month 2-11 Virtual Visits, participants measure their own 
ALSFRS-R and weight and will log into the PatientsLikeMe site to record these as well 
as perceived efficacy, adverse events and concomitant medications.  If participants have 
not entered data within 7 days of a visit, they will receive a reminder email from 
PatientsLikeMe (Appendix E).  If they still have not entered data after another 7 days, 
they will receive a reminder call from the study coordinator. 
3.9.5. Schedule of Events Table   
The following Table shows the schedule of study visits and calls (C=coordinator initiated 
task, P=participant initiated task): 
 
Screening/Baseline 
Visit 
Weeks 1-3 and 
Month 
2-11 
Virtual Visits 
Month 1 and 
12 Visits 
Weeks 1,2,3 
Calls 
Consent 
C 
 
 
 
Inclusion 
and 
Exclusion 
Criteria Review 
C 
 
 
 
Enrolled 
Participants 
Entered into Neurobank 
C 
 
 
 
Vitals, Weight, ALSFRS-R, 
Con Meds, AE Recorded in 
Neurobank 
C 
 
C 
 
Participant 
Registers 
Account on PatientsLikeMe 
P 
 
 
 
Teach 
Participant 
to 
Measure and Record Con 
Meds 
and 
AE 
in 
PatientsLikeMe Database 
C 
 
 
C 
Participant Measures and 
Records ALSFRS-R, Weight, 
P 
P 
P 
 


Page 12 of 22 
 
Compliance, 
Perceived 
Efficacy, Con Meds and AE 
in PatientsLikeMe Database 
Blood Draw for Biomarkers 
C 
 
C 
 
Urine 
for 
Pregnancy 
Testing 
C 
 
 
 
Confirm that patients are 
logging into PatientsLikeMe 
and 
entering 
ALSFRS-R, 
Weight, Perceived Efficacy, 
Compliance, Con Meds and 
AE 
 
 
 
C 
Objectively 
check 
compliance with Lunasin 
regimen 
 
 
C 
 
 
3.10. Participants   
Participants will be patients with ALS (PALS) who are cared for by Dr. Bedlack, or who 
call our site inquiring about this study.  Approximately 50 participants will be enrolled at 
Duke. 
3.10.1. 
Inclusion Criteria   
Each participant must meet all of the following criteria at screening and baseline (unless 
otherwise specified) to participate in the study: 
1. Male or female, aged at least 18 years. 
2. Sporadic or familial ALS diagnosed as possible, laboratory-supported 
probable, probable, or definite as defined by revised El Escorial criteria.  
3. Patient is able to understand and express informed consent (in the opinion 
of the site investigator). 
4. Patient has access to the Internet on a desktop computer, laptop, or tablet 
and has a working email address.  
5. Patient or caregiver is willing and able to use a computer and enter data on 
a secure website. 
6. Patient is able to read and write English.  
7. Women must not be able to become pregnant (e.g., post-menopausal, 
surgically sterile, or using adequate birth control methods) for the duration 
of the study and three months after study completion. Adequate 
contraception 
includes: 
abstinence, 
hormonal 
contraception 
(oral 
contraception, implanted contraception, injected contraception or other 
hormonal contraception, for example patch or contraceptive ring), 
intrauterine device (IUD) in place for ≥ 3 months, barrier method in 
conjunction with spermicide, or another adequate method.  
3.10.2. 
Exclusion Criteria   
Participants will be excluded for any of the following: 


Page 13 of 22 
 
1. Patient is taking other experimental treatments for ALS. 
2. Prior side effects from Lunasin. 
3. Known soy allergy. 
4. Patient has a medical or psychiatric illness that could in the investigator’s 
opinion interfere with the patient’s ability to participate in this study. 
5. Pregnant women or women currently breastfeeding. 
3.11. Recruitment & Compensation   
Patients will be mainly recruited from Dr. Bedlack’s Duke ALS Clinic or will have called 
our center to inquire about the study.  Since Dr. Bedlack’s clinic and our phone lines are 
open to all relevant demographic groups, all groups will have access to this study.  In 
addition we will post the following on the Duke ALS Clinic website and Twitter feed: 
“Duke Lunasin Study is now open.  Call Karen Grace at 919 668-2844 for details.”  No 
compensation will be provided. 
3.12. Consent Process:  
Consent will be obtained by Dr. Bedlack, or by his study coordinator.  Patients will be 
given all the time they need to review the written consent and ask questions about it.  No 
study procedures will occur prior to the consent form being signed. 
3.13. Capacity to Give Consent   
Only patients with appropriate capacity to provide informed consent will be offered a 
consent form. 
3.14. Risk Assessment   
The above-described Lunasin regimen appears reasonably safe.  A recent review found 
only rare patients reporting any side effects from it (7).  These included swelling, 
lightheadedness, rash and stomachache.  One website cautions patients with soy allergies 
against taking Lunasin (31).  While Reliv says this is safe (32), we will exclude patients 
with known soy allergies from this study. 
 
The only measures associated with any risk are the blood draws (pain, bleeding, bruising, 
infection). 
 
Risk associated with participation in the Duke or PatientsLikeMe database include loss of 
confidentiality.  Risks of logging into Internet for any website including PatientsLikeMe 
include inadvertently downloading malicious software such as viruses that could put 
personal information or computer integrity at risk.  These risks are minimized by the 
properties of the Neurobank and the PatientsLikeMe website (see below).   
 
Participants will be provided with contact numbers for the research staff. All sites will 
have an appropriate staff member available 24 hours a day, 7 days a week in the event of 
an emergency.  Participation in this research study is purely for research purposes, and is 
not intended to provide any direct benefit to the volunteers other than altruism 
(knowledge that they are helping investigators develop better treatments, and potentially 
helping other patients down the road).   No at risk populations such as minors, prisoners, 


Page 14 of 22 
 
pregnant women, or cognitively impaired adults will be included in this study.  Patients 
who elect not to participate will have standard of care options for their ALS including 
participation in Dr. Bedlack’s multi-disciplinary ALS clinic and the drug riluzole.   
3.15. Costs to Subjects   
There is no cost to subjects for participating in this study.    
3.16. Data Analysis & Statistical Considerations   
3.16.1. 
Historical Controls 
For each enrolled participant, matched historical controls will be identified from the 
PatientsLikeMe database.  Participants will be matched according to their ALSFRS-R 
progression rate before they start on the Lunasin regimen (estimated by assuming their 
score was normal at 48 on the date of symptom onset).  Full details of the matching 
process are described online (33). 
3.16.2. 
Primary Hypothesis and Sample Size   
For the primary hypothesis, we will run a t-test comparing the ALSFRS-R progression 
rate in our participants to a group of matched historical controls identified from the 
PatientsLikeMe community (30).  This type of analysis has previously been used to look 
for treatment effects in patients with ALS taking lithium (33), as well as dexpramipexole 
and various forms of sodium chlorite (34).  We made the following assumptions to 
calculate a sample size requirement for this analysis: 
  
1. The mean ALS-FRS progression in 12 months is 11 points (rate of ~0.9 
points/month) and the standard deviation at 12 months is ~8 ALS-FRS points 
(estimates based on unpublished data from PatientsLikeMe. 
2. The effect size we are looking for is to halve the mean progression rate 
3. The treatment is not having a harmful effect (allows us to use a one-tailed 
comparison) 
4. There is a 5% chance of seeing an effect this big by chance alone 
5. We desire a 90% chance of finding an effect this big, assuming it is real 
6. There will be a dropout rate of 15% (less than the 22% rate seen in more 
traditional ALS trials (5). 
 
Based on these assumptions we will again need to enroll 50 participants (and compare 
them to 50 matched historical controls). 
3.16.3. 
ALS Reversals   
To look for an increase in the frequency of ALS reversals, we will count the number of 
participants who have an ALSFRS-R score that improves by 4 points or more over 12 
months.  The observed frequency of spontaneous ALS reversals defined in this way is 1% 
(35).   We will look for an increase in this frequency to at least 2% (1 in 50). 


Page 15 of 22 
 
3.16.4. 
ALSFRS-R Accuracy   
To confirm that our participants can accurately measure their own ALSFRS-R, we will 
compare the ALSFRS-R obtained by the coordinator with that obtained by the 
participants themselves at the Month 1 Visit.  Correlational analysis between these 2 
scores will be performed with Spearman's rho, as was done in a previous study (24), 
because of the ordinal nature of the ALSFRS-R. 
3.16.5. 
Weight Accuracy   
To confirm that our participants can accurately measure their own weight, even as they 
become more disabled by ALS, we will compare the participant-generated weight with 
the weight obtained by the study coordinator at the Month 1 and Month 12 visits.  A 
simple description of the accuracy (percent agreement between the weights) will be used. 
3.16.6. 
Enrollment   
To determine whether the participant enrollment rate in this study is greater than in 
studies with more traditional designs, we will compare our enrollment rate (participants 
enrolled per month) to the mean ALS trial enrollment rate of 2 (4) using a chi-squared 
test. 
3.16.7. 
Retention   
To determine whether participant retention in this study is greater than in studies with 
more traditional designs we will compare our dropout rate (percentage of living enrolled 
participants that complete the Month 12 Visit) to the mean ALS participant dropout rate 
of 22% (28) using a chi-squared test. 
3.17. Adverse Events Monitoring   
All adverse events (AEs), whether observed by the Investigator, elicited from the 
participant or volunteered by the participant, and whether ascribed to the drug or not, will 
be recorded.  This will include the following: a brief description of the event, the date of 
onset, the date of resolution, the duration and type of the event, the severity, contributing 
factors and any action taken with respect to the study drug. This recording will 
commence with the institution of protocol-specific procedures (including any 
pretreatment procedures) and continue at each study visit or telephone contact until 4 
weeks following the last study related visit. 
 
For each adverse event, the relationship to the study drug will be recorded as one of the 
choices on the following scale: 
DEFINITE 
Causal relationship is certain (i.e., the temporal relationship 
between drug exposure and the adverse event onset/course is reasonable, there is a 
clinically compatible response to de-challenge, other causes have been eliminated 
and the event must be definitive pharmacologically or phenomenologically using 
a satisfactory re-challenge procedure if necessary). 
PROBABLE High degree of certainty for causal relationship (i.e., the temporal 
relationship between drug exposure and the adverse event onset/course is 


Page 16 of 22 
 
reasonable, there is a clinically compatible response to de-challenge [re-challenge 
is not required] and other causes have been eliminated or are unlikely). 
POSSIBLE 
Causal relationship is uncertain (i.e., the temporal relationship 
between drug exposure and the adverse event onset/course is reasonable or 
unknown, de-challenge/re-challenge information is either unknown or equivocal 
and while other potential causes may or may not exist, a causal relationship to the 
study drug does not appear probable). 
UNLIKELY Not reasonably related, although a causal relationship cannot be 
ruled out (i.e., while the temporal relationship between drug exposure and the 
adverse event onset/course does not preclude causality, there is a clear alternate 
cause that is more likely to have caused the adverse event than the study drug). 
NOT RELATED 
No possible relationship (i.e., the temporal relationship 
between drug exposure and the adverse event onset/course is unreasonable or 
incompatible, or a causal relationship to study drug is implausible). 
 
The severity of each adverse event must be recorded as one of the choices on the 
following scale: 
MILD 
 
No limitation of usual activities 
MODERATE 
Some limitation of usual activities 
SEVERE  
Inability to carry out usual activities 
 
The expectedness of an AE must be indicated when reporting adverse events.  An 
unexpected adverse event is any adverse experience for which the specificity or 
severity of the event is not consistent with the current investigator brochure.   
A serious adverse drug event (SAE) is defined as any adverse event that occurs 
during the study that results in any of the following outcomes: death, a life-
threatening adverse event (i.e., the participant was at immediate risk of death from 
the event as it occurred; does not include an event, that had it occurred in a more 
severe form, might have caused death), inpatient hospitalization or prolongation 
of existing hospitalization (hospitalizations scheduled before enrollment for an 
elective procedure or treatment of a pre-existing condition which has not 
worsened during participation in the study will not be considered a serious 
adverse event), a persistent or significant disability/incapacity (substantial 
disruption of one's ability to conduct normal life functions), a congenital 
anomaly/birth defect, a medically important event or required medical 
intervention to avoid one of the above outcomes.  In addition to the above 
procedures for AEs, all SAEs will be reported to the IRB within 24 hours of 
recording.  All serious adverse event information will be followed until resolution 
or an appropriate end point is reached. This may involve contacting other 
clinicians responsible for the participant’s care to obtain information on 
diagnoses, investigations performed and treatment given 
 
Fatal or life-threatening, unexpected adverse events will be reported to the FDA 
by telephone, facsimile, or in writing as soon as possible, but no later than 7 
calendar days after first knowledge by the Sponsor-investigator. Serious, 


Page 17 of 22 
 
unexpected adverse events that are not fatal or life-threatening will be reported to 
the FDA by telephone, facsimile, or in writing as soon as possible, but no later 
than 15 calendar days after first knowledge by the sponsor-investigator. 
3.18. Data & Safety monitoring  
Adverse events will be tracked throughout the study as described above and below.  
There is no formal safety monitoring plan or DSMB, nor are there any formal stopping 
rules.  PI will review and sign off on all adverse events and promptly report these to the 
IRB. 
3.19. Data Collection and Management 
3.19.1. 
NeuroBANK™   
NeuroBANK is a collaboration and data repository platform maintained by the 
Massachusetts General Hospital (MGH) Neurological Clinical Research Institute (NCRI).  
This platform facilitates: 
1. Capture of clinical and research data from neurologic patients for 
individual projects in a structured and secure system; 
2. Aggregating and sharing uniform, deidentified and/or anonymized 
datasets for secondary analyses. 
3.19.2. 
NeuroBANK™ Data Management (DM)   
DM is responsible for the development, execution and supervision of plans, policies, 
programs, and practices that control, protect, deliver, and enhance the value of data and 
information assets. 
 
All data will be managed in compliance with applicable Sponsor and regulatory 
requirements. Site personnel will collect, transcribe, correct, and transmit the data onto 
source documents, Case Report Forms (CRFs), and/or other forms used to report, track 
and record clinical research data.  DM is responsible for developing, testing, and 
managing clinical data management activities. 
3.19.3. 
NeuroBANK Data Entry and Checks   
Site personnel are instructed to enter information into the NeuroBANK™ Electronic Data 
Capture (EDC) System.  Data capture is the responsibility of the staff at the site under the 
supervision of the Site Investigator (SI).  During the study, the Site investigator must 
maintain complete and accurate documentation for the study. 
 
The NeuroBANK™ platform provides password protection.  An edit checking and data 
clarification process will be put in place to ensure accuracy of the data.  Logic and range 
checks as well as more sophisticated rules may be built into the eCRFs to provide 
immediate error checking of the data entered.  The system has the capability to 
automatically create electronic queries for forms that contain data that are out of range, 
out of window, missing or not calculated correctly.  The sites will only have access to the 
queries concerning their subjects.   


Page 18 of 22 
 
3.19.4. 
Data Lock Process   
The platform will have the ability to lock the project-specific visits to prevent any 
modification of data once the project is closed.  Once this option is activated, every user 
will have Read-Only access to the data. 
3.19.5. 
Data Handling and Record Keeping   
The Site Investigator (SI) is responsible to ensure the accuracy, completeness, legibility, 
and timeliness of the data reported.  Data reported in the eCRF derived from source 
documents should be consistent with the source documents and discrepancies should be 
explained. 
3.19.6. 
 Confidentiality   
The NeuroBANK™ software and patient data reside on servers located in the Partners 
Healthcare Systems (Partners) server farm.  Physical and software access to the servers 
and security is provided by the Partners IT department.  Members of the NeuroBANK™ 
management team will do everything, within reason, to keep a participant’s identity 
protected. 
3.19.7. 
Global Unique Identifier (GUID)   
A patient Global Unique Identifier (GUID) will be used as the identifier for individuals 
participating in the study in NeuroBANK™. The GUID is an 11-character string that is 
generated using encryption technology and algorithms licensed by the NCRI from the 
National Institutes of Health (NIH). 
 
The GUID is generated on a secure website that utilizes 128-bit Secure Socket Layer 
(SSL).  Of note, this website is not linked to NeuroBANK™.  The GUID is generated 
using an irreversible encryption algorithm – it accepts twelve identifying data elements, 
(e.g. last name at birth, first name at birth, gender at birth, day, month and year of birth, 
city and country of birth, etc.), and produces a unique random-generated character string, 
or GUID.  No identifying information is stored in the system; it is simply used to 
generate the GUID.  If the same information is entered again, the same GUID will be 
returned.  
 
The GUID is entered into NeuroBANK™ when the patient is being created in the system. 
As the same patient may participate in multiple studies, NeuroBANK™ will also allow 
capturing a study-specific ID for the patient.  Participants will be given the option to opt 
out of sharing their de-dentified information across studies in Neurobank.  For more 
information about NeuroBANK™ or the GUID, please go to: www.neurobank.org. 
3.20. PatientsLikeMe  
3.20.1. 
General Technical Information on the PatientsLikeMe Website   
The PatientsLikeMe website is one of the oldest and most complex (in terms of lines of 
code) Ruby on Rails commercial website in the world. We manage over 300,000 users, 
2,300 conditions, and over 20 million medical datapoints of information.  Our published 
uptime target is 99.97% uptime; we have beaten this benchmark for the past three years. 


Page 19 of 22 
 
For 2014, our uptime number was 99.998%. We count unplanned downtime against our 
uptime number, but actual planned downtime is very small. We push out releases almost 
on a daily basis, only a handful of which require any downtime at all, and the average 
downtime is under 2 minutes.  
3.20.2. 
Security Information   
We require that patient data be stored on physical servers with clearly documented 
physical and network security protocols. We minimize accounts and access to servers 
with patient data on them. All individual machines that handle patient or industry partner 
data are encrypted and subject to audit according to our SOP’s. In addition, all 
PatientsLikeMe employees undergo background checks as a condition of employment. 
On the topic of physical security, our servers are hosted by RackSpace, a recognized 
leader in data center security. Physical access to the servers is tightly controlled and 
monitored. At PatientsLikeMe’s offices, all means of entry to the building are locked and 
controlled. Team members must use a key, key card, or input a door code to enter the 
building. To date, we are unaware of any unauthorized access to our building and have 
not experienced any issues with break-in, theft, and so forth. Our building is monitored 
by security personnel 24 hours a day, 7 days a week. 
 
On the topic of network security, we maintain strict firewall rules. Virtual private 
network access is required to access any part of our server infrastructure. We maintain 
separate virtual networks for production, testing, and client access environments to 
further 
enhance 
our 
network 
security.  
 
Please refer to the whitepaper covering Security and Privacy Policies included as 
Appendix J within this document.  
3.21. Good Clinical Practices and Human Subjects Protection Training  
The investigator and coordinator involved with the conduct of this study will be certified 
in Good Clinical Practices (GCP) and Human Subjects Protection training. Human 
Subjects Protection training certification will be obtained by completing approved 
training, such as the online computer based training offered by the NIH Office of Human 
Subjects Research (http://ohsr.od.nih.gov).  
4. 
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