FOCUS - Pilot Study of Muscadine Grape Extract to Improve Fatigue Among Older Adult Cancer Survivors
Phase: EARLY_PHASE1
Conditions: Fatigue
Sponsor: Wake Forest University Health Sciences

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
ClinicalTrials.gov:  NCT04495751
1 
Principal Investigator: 
Co-Investigator(s): 
Heidi Klepin, MD, MS 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
Medical Center Boulevard 
Winston-Salem, NC 27157 
Katherine L. Cook, PhD 
Department of Cancer Biology 
Wake Forest School of Medicine 
Patricia Gallagher, PhD 
Hypertension & Vascular Research Center 
Wake Forest School of Medicine 
David R. Soto-Pantoja, PhD 
Department of Cancer Biology 
Wake Forest School of Medicine 
E. Ann Tallant, PhD
Hypertension & Vascular Research Center
Wake Forest School of Medicine
Stacy Wentworth, MD 
Department of Radiation Oncology 
High Point Medical Center 
James Atkins, MD 
Section on Hematology and Oncology 
Wilkes Medical Center 
Bernard Chinnasami, MD 
Section on Hematology and Oncology 
High Point Medical Center 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
2 
 
 
Jason Huff, MD 
Section on Hematology and Oncology 
High Point Medical Center 
 
 
 
Jing Li, MD, PhD 
Section on Hematology and Oncology 
HemOnc Statesville 
 
 
 
George Sanders, MD 
Section on Hematology and Oncology 
High Point Medical Center 
 
 
 
 
Biostatistician: 
Janet A. Tooze, PhD 
Comprehensive Cancer Center of Wake Forest University 
  
  
 
 
WFBCCC Data Manager: 
 
Daphne Witherspoon 
Comprehensive Cancer Center of Wake Forest University 
 
 
 
Study Coordinators:  
 
Katherine Pleasant, BA 
Hypertension & Vascular Research Center 
Wake Forest School of Medicine 
    
  
 
Brittany M. Davis, MDiv 
Hypertension & Vascular Research Center 
Wake Forest School of Medicine 
 
 
 
 
Regulatory Contact:  
 
Emmanuel Vazquez 
Comprehensive Cancer Center of Wake Forest University 
 
  
 
 
 
Participating Institution(s): Wake Forest Baptist Health 
 
 
Version Date: 
4/27/2021 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
3 
 
Table of Contents 
 
2.0 Objectives ..................................................................................................................................................... 11 
2.1 
Primary Objective(s) ............................................................................................................................ 11 
2.2 
Secondary Objective(s) ........................................................................................................................ 11 
3.0 
Patient Selection ..................................................................................................................................... 12 
3.1 
Inclusion Criteria .................................................................................................................................. 12 
3.2 
Exclusion Criteria ................................................................................................................................. 13 
3.3 
Inclusion of Women and Minorities ...................................................................................................... 13 
4.0 
Recruitment and Registration Procedures .............................................................................................. 14 
5.0 
Study Outcomes and Study Measures ................................................................................................... 15 
5.1 
Primary Outcome ................................................................................................................................. 15 
6.0 
Intervention Plan ..................................................................................................................................... 18 
6.1 
Study-Related Activities ....................................................................................................................... 18 
6.2 
Intervention Administration .................................................................................................................. 19 
6.3 
Investigational Agent ............................................................................................................................ 19 
6.5 
General Concomitant Medication and Supportive Care Guidelines ..................................................... 20 
6.6 
Study Assessments ............................................................................................................................. 20 
6.6.1 Vitals signs, performance status .......................................................................................................... 20 
6.7 Duration of Study Intervention ................................................................................................................... 21 
6.8 
Duration of Follow Up .......................................................................................................................... 21 
6.9 
Criteria for Removal from Study Intervention ....................................................................................... 22 
7.0 
Dosing Delays/Dose Modifications ......................................................................................................... 22 
7.1 
Missed doses ....................................................................................................................................... 22 
7.2 
Dose modification ................................................................................................................................ 22 
8.0 
Adverse Events List and Reporting Requirements ................................................................................. 23 
8.1 
Adverse Event List for MGE ................................................................................................................. 23 
8.2 
Adverse Event Characteristics ............................................................................................................. 23 
8.3 
STRC SAE Reporting Requirements ................................................................................................... 23 
8.4 
WFUHS IRB AE Reporting Requirements ........................................................................................... 23 
8.5 
Sponsor Reporting Requirements ........................................................................................................ 24 
9.0 
Pharmaceutical Information .................................................................................................................... 24 
9.1 
Pharmaceutical/Device Accountability ................................................................................................. 24 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
4 
9.2 Muscadine Grape Extract (MGE) .............................................................................................................. 24 
10.0 
Correlative/Special Studies ..................................................................................................................... 25 
11.0 
Data Management .................................................................................................................................. 26 
12.0 
Statistical Considerations ........................................................................................................................ 26 
12.1 
Analysis of Primary Objective .............................................................................................................. 26 
12.2 
Analysis of Secondary Objectives ........................................................................................................ 26 
12.4 
Power and Sample Size ....................................................................................................................... 27 
12.5 
Estimated Accrual Rate ....................................................................................................................... 27 
12.6 
Estimated Study Length ....................................................................................................................... 27 
References ......................................................................................................................................................... 29 
Appendix D - STRC SAE Reporting  v. 7/10/2019 ................................................ ..............................................32 
Appendix L - Frailty Index Calculation Form .......................................................................................................36 
Appendix R: Digit Symbol Substitution Test (DSST) Script ................................................................................ 33 
NOTE:  Appendices/Forms for use by study coordinators or CRU and site nursing staff were moved to the 
Other Documents section of the eIRB application. 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
5 
 
Schema 
Prospective double blind placebo controlled trial of 64 patients age 65 and over with a history of cancer who 
self-report fatigue. After baseline assessment, eligible subjects will be randomized and provided with 
proprietary MGE pills or placebo to take twice daily for 12 weeks (4 capsules twice daily). Subjects will receive 
phone call follow-up for toxicity assessment and adherence review at weeks 2, 4, 8. PROMIS Fatigue will also 
be collected on week 4 and 8 calls. Follow-up assessment will be completed at 12 weeks and a post-treatment 
assessment at 16 weeks. 
 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility, consent and pre-study data collection 
1:1 randomization 
MGE (n= 32)  
MGE provided (12 week supply) 
4 pills twice daily  
Placebo (n= 32) 
Placebo provided (12 week supply) 
4 pills twice daily  
 
Baseline Assessment 
Fatigue, PF, PA, QOL, Biomarkers, 
Safety Labs 
Week 2* 
A, S 
Week 4* 
Fatigue, A,S 
Week 8* 
Fatigue, A,S 
Follow-up Assessment @ Week 12 
Pill count 
Fatigue, PF, PA, QOL, Biomarkers, 
Safety Labs 
Legend: MGE= muscadine grape extract; PF= physical function testing (SPPD, PAT-D, 
6-minute walk); PA=physical activity QOL=quality of life/symptoms; A=adherence, 
S=symptom review; *collected by phone 
End of Treatment Visit 
(30 days post last dose of study drug) 
Fatigue, A 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
7 
 
1.0 Introduction and Background 
 
Fatigue is the most prevalent symptom among cancer survivors and is commonly associated with aging.1 Over 
40% of community dwelling adults over age 65 reported prevalent fatigue in a recent national survey.1 Older 
cancer survivors are therefore at particularly high risk of experiencing persistent fatigue which increases the 
risk of developing disability.  Fatigue is associated with sedentary behavior, decline in physical activity and 
physical function, poor quality of life and contributes to the phenotype of frailty.2-7 This cascade of symptoms 
and behaviors has negative long-term effects including increased risk of cardiovascular events, decreased 
muscle mass, increased health care utilization, and mortality risk.5,8-11  Decreasing fatigue could have a 
significant impact on maintenance of functional independence and overall health for a large proportion of older 
adults.  
 
Despite the common nature of the symptom, there are few pharmacologic interventions shown to improve 
fatigue. Treatments for fatigue after exclusion of modifiable medical causes include physical activity, mind body 
interventions and psychosocial interventions.12 Despite evidence to support these strategies, these 
interventions can be resource intensive and difficult for most adults to maintain, particularly older adults. There 
are no well-tolerated FDA approved medications for treatment of chronic fatigue symptoms. Identifying well-
tolerated, efficacious, and cost effective pharmacologic interventions to reduce fatigue is of high priority for the 
scientific community and of great interest to older adults.13  
 
Nutritional interventions and nutraceuticals represent appealing strategies for treatment of fatigue.14,15 Biologic 
mechanisms associated with fatigue are multi-factorial. Common biologic pathways implicated in fatigue 
symptomatology for both cancer-related and non-cancer related fatigue include systemic inflammation, 
impairment of mitochondrial function, and cardiac dysfunction.6,16,17 Interventions targeting these pathways 
may be of particular relevance to older adults given the association between aging and dysregulation of 
systemic inflammation, mitochondrial function and cardiac health and is in line with the principles of 
geroscience.18-23  
 
Muscadine grape extract (MGE) is a rich dietary source of bioactive phenols, including ellagic acid, gallic acid, 
anthocyanins, and flavan-3-ols, which have been studied with particular interest for their potential anti-
inflammatory and anti-cancer properties.24-39 Preclinical and clinical data from the investigative team show 
effects of MGE on inflammation, mitochondrial bioenergetics and the symptom of fatigue. This pilot study 
proposes translation of these observations to a high need clinical setting to test the effect of MGE 
supplementation on older adults with self-reported fatigue. This study will provide data on the effects of MGE 
on fatigue, function and biologic correlates to evaluate mechanisms.  
 
1.1 Preclinical Studies with Proprietary MGE 
 
Data from the Investigative Team Gallagher/Tallant Lab: 
The MGE administered in the following preclinical studies and Phase 1 clinical trial is a proprietary compound 
that was prepared from the skins and seeds of muscadine grapes by Piedmont Research and Development 
Corporation (PRDC). The skins and seeds have high levels of polyphenols, micronutrients found in plants that 
provide health benefits. The MGE produced by PRDC has a higher concentration of total polyphenols as 
compared to other muscadine grape nutritional supplements on the market (Figure 1). Mass spectrometry 
analysis showed that the MGE from PRDC has a higher content of specific polyphenols as compared to other 
muscadine grape supplements (Figure 2).  It should be noted that the proprietary MGE from PRDC is prepared 
from an aqueous solution of ground seeds and skins; therefore, resveratrol, which has limited solubility in 
water, was not detected. 
 
 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
8 
 
 
1.2 Rationale for testing MGE to improve fatigue 
Several mechanisms to support a potential MGE effect on fatigue have been suggested by pre-clinical studies. 
Data are highlighted below. 
 
A significant effect of MGE on exercise tolerance, an established fatigue 
surrogate, was observed in transgenic (mRen2)27, hypertensive female 
rats administered MGE (0.2 mg 
total phenolics/mL; in the drinking 
water starting at 14 weeks of age after the 
onset of high blood pressure. At 40 weeks 
of age (26 weeks of treatment), exercise 
tolerance was assessed on a treadmill 
(10.2 m x min-1, 5% inclination). Time to fatigue (TTF: min) was increased 
2.3-fold in MGE-treated animals compared to controls (Figure 3). 
Workload (W: g X m) was 2.4-fold higher in MGE-treated animals 
compared to untreated rats (Figure 4). At the end of the study, there were no significant differences in systolic 
blood pressure (166 ± 6 vs 162 ± 3 mm Hg) or body weight (365 ± 10 and 385 ± 8 g) between the control rats 
and the MGE-treated rats. This improvement in exercise tolerance was sustained following 56 weeks of 
treatment in 70 week-old rats (data not shown), suggesting that MGE consumption improves exercise capacity 
in aging adult female hypertensive rats independent of changes in blood pressure or body weight. MGE also 
P R D C
N P
M N
N F
E N
B P
V IT
N C M H
B N
F H N
0
5 0
1 0 0
1 5 0
2 0 0
T o ta l P h e n o lic s  (m g /5 0 0  m g  c a p s u le )
P R D C  - N e w  p ro p rie ta ry
c o m p o u n d
N P  - N a tu re 's  P e a rl
M N  - M u s c a d in e  N a tu ra ls
N F  - N a tu re 's  F o rc e
E N  - E a rth  N a tu ra l
B P  - B io P o w e r
V IT  - V ita c o s t
N C M H  - N C  M u s c a d in e
H e a lth
B N  - B io te c h  N u tritio n
F H N  - F re s h  H e a lth
N u tritio n
****
****
****
********
********
********
Figure 1.  Total phenolic content of 
MGEs determined by Folin-Ciocalteu 
assay using gallic acid as a standard. 
n=3 
separate 
lots, 
****p<0.001 
compared to PRDC. 
0
5
1 0
1 5
2 0
2 5
M u s c a d in e x
N a tu re 's  P e a rl
P re m ie r G o ld
P R D C
C o n c e n tra tio n  [m g /g m ]
C a te c h in -
G a lla te
   P ro -
c y a n id in
E lla g ic
E p ic a te c h in
G a llic
C a te c h in
Figure 2.  Concentration of specific 
phenolic compounds in MGEs were 
determined by ultra-high pressure 
liquid chromatography/mass 
spectrometry.  n=3 separate lots. 
C o n tro l
M G E  T re a te d
0
2 0 0 0
4 0 0 0
6 0 0 0
S e c o n d s
****
Figure 3.  MGE enhanced exercise 
capacity (time to fatigue) in female 
hypertensive rats. n=5; ****<0.0001 
C o n tro l 
M G E  T re a te d
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
W o rk lo a d  (g x m )
****
 
Figure 4.  MGE enhanced exercise 
workload in female hypertensive rats.  
n=5; ****<0.0001 
C o n tr o l 
M G E  tr e a te d
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
8 -O H d G  n g / 2 4 h
*
C o n tro l 
M G E  
A n g  II
A n g  II 
+  M G E
0
1 0
2 0
3 0
4 0
E /e '
* *
#
Figure 6.  Ang II increased left 
ventricular filling pressure (E/e’), 
a measure of diastolic function, 
while 
MGE 
co-administration 
prevented these effects. E/e’ was 
measured by echocardiography 
at week 4.  **p<0.01 compared 
with control, # p<0.05 compared 
to Ang II alone.  
Figure 5.  MGE treated rats had a 
decreased concentration of urinary 
8-OHdG, 
indicating 
reduced 
oxidative stress.  n=5; *p<0.05 
C o n tr o l 
M G E  tr e a te d
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
8 -O H d G  n g / 2 4 h
*

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
9 
 
reduced urinary 8-OHdG (Figure 5), a marker of oxidation, suggesting that oxidative stress may be involved in 
the improvement in exercise capacity.   
 
To assess the effect of MGE on cardiac 
damage, a potential underlying 
mechanism for fatigue, male Sprague-
Dawley rats were pretreated for 1 week 
with MGE in their drinking water (0.2 
mg/mL total phenolics) followed by a 4-
week infusion with Ang II (24 µg/kg/h) 
which increased blood pressure from 114 
mmHg ± 1.9 to 211 mmHg ± 8.5. MGE 
administration had no effect on blood 
pressure or systolic function in 
normotensive or hypertensive rats but 
ameliorated the Ang II-induced decrease 
in diastolic function (Figure 6) and 
increase in interstitial cardiac fibrosis by 
reducing myocardial collagen III and prevented hypertension-induced aortic 
stiffness (Figure 7).  These MGE effects were mediated by attenuating Ang II-
induced dysregulation of inflammation (NF--, IL-6), oxidative stress (Nox, 
NADPH oxidase, catalase), proliferation (MAP kinase), and fibrotic signaling 
pathways (TGF--/Smad, CTGF) (data not shown due to page limitations).  These results suggest that MGE 
may improve patient fatigue by preventing 
cardiac and vascular damage. 
Agents that preserve mitochondrial integrity and 
maintain or increase mitochondrial respiratory 
capacity may prevent cardiac tissue damage due 
to oxidative stress and thereby reduce fatigue. 
Cardiac cells were treated with the redox cycling 
agent, 2,3 dimethoxynaphthoquinone (DMNQ; 15 
µM) which generates both superoxide and 
hydrogen peroxide intracellularly and thus 
mimics pathologies of elevated reactive oxygen 
species. DMNQ increased cardiac cell 
cytotoxicity as measured by LDH release; this 
effect was 
prevented by 
MGE (30 µg 
total phenolics/mL). DMNQ increased the potential 
of the mitochondrial membrane, which was also prevented by MGE, suggesting 
protection of mitochondrial membrane integrity. Cell respirometry measurements 
showed that DMNQ reduced the oxygen consumption rate (OCR) by 50% (both 
basal and maximal respiration, Figure 8), while co-treatment with MGE prevented 
this reduction, suggesting that the protective effect may be mediated by 
preservation of mitochondrial function. MGE prevented the decrease in ATP-linked 
respiration (Figure 9). The basal extracellular acidification rate (ECAR) was 
increased 2-fold with MGE pretreatment when compared to DMNQ alone (13.6 ± 
2.3 DMNQ+MGE vs. 6.8 ±.0.88 DMNQ, n=3, p<0.01), suggesting that MGE may 
also increase glycolysis as a response to stress. These data suggest that treatment 
with MGE enhances mitochondrial function and cellular bioenergetics during 
C o n tro l 
M G E  
A n g  II 
A n g  II 
+  M G E  
0
2
4
6
8
1 0
 (m /s )
**
#
A o rtic  P u ls e   W a v e   V e lo c ity
Figure 7.  Ang II increased 
pulse 
wave 
velocity, 
a 
measure of arterial stiffness, 
while 
co-administration 
of 
MGE prevented these effects. 
**p<0.01 
compared 
with 
control, # P<0.05 compared to 
Ang II alone.  
C o n tro l 
M G E
D M N Q
D M N Q  +  M G E
    
C o n tro l 
M G E
D M N Q
D M N Q  +  M G E
0
5 0
1 0 0
1 5 0
0
5 0
1 0 0
1 5 0
B a s a l R e s p ira tio n
 (p m o l/m in )
M a x im a l R e s p ira tio n
 (p m o l/m in )
*
*
*
*
Figure 8.  Mitochondrial metabolism was 
measured as the Oxygen Consumption 
Rate (OCR) by Seahorse analysis.  MGE 
prevented the DMNQ-mediated reduction 
in basal and maximal respiration rates. *p 
< 0.05; n=3. 
 
C o n tro l 
M G E
D M N Q
D M N Q  +  M G E
0
1 0
2 0
3 0
4 0
5 0
A T P   L in k e d -re s p ira tio n
 (p m o l/m in )
*
Figure 
9. 
 
Mitochondrial 
metabolism was measured as 
the 
Oxygen 
Consumption 
Rate (OCR).  MGE prevented 
the 
DMNQ-mediated 
reduction in ATP linked to 
respiration. *p < 0.05; n=3. 
 
C o n tr o l 
M G E
3 .5
4 .0
4 .5
5 .0
5 .5
A lp h a  D iv e rs ity
S h a n n o n  In d e x
*
 
Figure 10.  MGE increased 
microbial diversity in mouse fecal 
samples.  
 
C o n tr o l 
M G E
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
F irm ic u te s /B a c te ro id e te s
R a tio
*
Figure 11.  MGE altered the 
ratio of the relative abundance 
of Firmicutes to Bacteroidetes  

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
10 
 
oxidative stress and thus may effectively prevent tissue damage from oxidative stress to reduce fatigue 4T1 Triple 
negative breast cancer cells were injected into the mammary fat pad of 6-week-old female Balb/c mice. After 2 
weeks, tumors were surgically removed and mice received either drinking water or 0.1 mg phenolics/mL of MGE in 
the drinking water. Mice were sacrificed after 4 weeks; tissues and fecal samples were collected for analysis. Alpha 
diversity (Figure 10) and the Firmicutes to Bacteroidetes ratio (Figure 11) were significantly increased in MGE-
treated mice compared to the control, indicating enhanced microbial richness and increased energy harvest by the 
gut microbiome could improve symptoms of fatigue.  Butyrate-producing bacteria, such as Ruminococcus, 
Butyricicoccus and Lachnospiraceae, were increased with MGE (p<0.05) as well as the anti-inflammatory 
compound butyrate compared to other short-chain fatty acids (25.0 ± 2.7% vs 75.3 ± 15.5%; p<0.01).  Of note, the 
MGE supplement is a complex mixture of polyphenols that are further metabolized by the host cells or the gut 
microbiome to secondary and tertiary metabolites, including short chain fatty acids. These metabolites may serve 
as additional energy sources, feeding directly into the TCA cycle and leading to enhanced energy 
production by the mitochondria, to reduce fatigue.  
 
1.3 Clinical Studies with MGE 
The investigative team completed a Phase 1 trial (N=23) 
(ClinicalTrials.gov NCT02583269, IND 128937) in patients with 
advanced stage solid tumors who progressed on prior therapies 
(mean age 72 years) to assess the safety and tolerability of 
proprietary MGE. Five dose levels of the extract from one to five 
pills twice daily (320 to 1600 mg total phenolics/day) were tested 
in a standard 3 + 3 design. No concerning safety events were 
identified; maximum tolerated dose was not reached.40  
 
Median time on therapy was 8 weeks, with 29% of patients 
treated beyond 16 weeks and a median overall survival of 7.2 
months. MGE adherence was high (98%) and the most common 
side effects were flatulence, diarrhea, and constipation. In this 
heavily pre-treated population, among patients on study at 12 
weeks (n=8), higher dose level of MGE was correlated with 
increases in the FACT-G score  (r=0.70, p=0.05) and 
decrease in fatigue (r=-0.70, p=0.05) (Figure 12). In the blood, 
the mean total phenolic level increased from baseline of 2.12 
mg/mL (n = 18, sd = 304) to 2.33 mg/mL at week 8 (n = 16, sd 
=245; p = 0.01). An MGE breakdown component urolithin A 
glucouronidate, a marker for the ingestion/metabolism of MGE, was 
measurable in the urine. At baseline, the mean UAG levels were low 
with a mean of 0.4 µg/mL (n=18, sd=1.5) and increased after 8 weeks of MGE treatment to a mean of 17.4 
µg/mL (n= 16, sd=44.4; p <0.01).   
 
Additional clinical studies: 
The investigative team is currently conducting a double blind randomized trial comparing MGE and placebo 
among men with prostate cancer (N=160) receiving anti-androgen therapy to evaluate effect on fatigue. 
Recruitment is on-going (IND 139288, NCT03496805). 
 
1.4 Rationale for MGE dosing: 
The initial studies looking for an anticancer effect of MGE in both breast and prostate cancer mouse models 
examined 4 different dose levels: 0.25, 0.5, 1 and 2 mg of phenolics per mouse per day. A statistically 
significant effect on tumor growth was seen at all 4 dose levels compared to placebo, and while there was a 
trend toward better efficacy at higher doses, there was no significant difference between the dose levels.  An 
Figure 12 

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
11 
 
average mouse weighs 0.025 kg, therefore this corresponds to a dose range of 10, 20, 40, and 80 mg of 
phenolics/kg/day.   
 
A dose 20 mg of phenolics/kg/day was chosen for additional preclinical studies in mice and rats including those 
reported in 1.2 providing preliminary data for this application. This dose is roughly equivalent to 8 capsules of 
MGE daily (each capsule contains 500mg MGE which corresponds to approximately 165mg of total phenolics). 
The phase 1 dose-escalation study with MGE in advanced cancer patients described above evaluated patients 
at and above this dose level. Based on the preclinical data and the phase 1 study, 8 capsules of MGE per 
day was chosen for the phase 2 prostate study and for this proposed pilot, in which effects on fatigue will be 
assessed in subjects treated for or surviving cancer. 
2.0 Objectives 
 
2.1 
Primary Objective(s) 
 
2.1.1 To evaluate whether administration of MGE supplementation (4 tablets twice daily, 
approximately 1320mg total phenolics) decreases PROMIS Fatigue score from baseline 
to 12 weeks compared to placebo.    
 
Hypothesis: Participants randomized to daily oral MGE administration will experience 
greater decline in self-reported fatigue (PROMIS Fatigue) from baseline to 12 weeks 
(primary outcome) compared to those receiving placebo. 
 
 
2.2 
Secondary Objective(s) 
 
2.2.1 To evaluate whether administration of MGE supplementation (4 tablets twice daily) 
causes changes in physical function (Pepper Assessment Tool for Disability [PAT-D], 
Short Physical Performance Battery), physical fitness (6-minute walk), physical activity 
(Minnesota Leisure Questionnaire), and sedentary behavior (Sedentary Behavior 
Questionnaire) from baseline to 12 weeks compared to placebo. 
 
Hypothesis: Participants on the intervention will have improved physical function, fitness 
and activity while sedentary behavior will be lower at 12 weeks compared to the placebo 
arm.  
 
2.2.2  To compare changes in health related quality of life (PROMIS Global Health) at 12 
weeks in participants randomized to MGE group vs. placebo.  
 
Hypothesis: Participants on the intervention will report improved quality of life at 12 
weeks compared to the control arm.  
 
2.2.3 To compare changes in the Fried frailty index at 12 weeks in participants randomized to 
MGE vs. placebo. 
 
Hypothesis: Participants on the intervention arm will demonstrate improvement in frailty 
status compared to the placebo arm.  

Pilot study of muscadine grape extract to improve fatigue among older adult cancer survivors (FOCUS) 
Wake Forest Baptist Comprehensive Cancer Center 
WFBCCC #98320 
 
 
 
 
12 
 
 
 
2.3 
Exploratory Objectives 
 
2.3.1  To explore whether MGE administration alters 8-hydroxy-2 deoxyguanosine, peripheral 
blood mitochondrial function, IL-6, TNF-alpha, C-reactive protein, hepatocyte growth 
factor from baseline to 12 weeks compared to the placebo arm. 
 
2.3.2 To explore whether MGE administration alters the microbiome from baseline to 12 
weeks compared to the placebo arm. 
 
2.3.3 To explore whether 8-hydroxy-2 deoxyguanosine, peripheral blood mitochondrial 
function, IL-6, TNF-alpha, C-reactive protein, hepatocyte growth factor and the 
microbiome may mediate changes in self-reported fatigue and physical function, fitness 
and activity from baseline to 12 weeks. 
 
2.3.4 To explore whether MGE administration causes changes in cognitive speed (Digital 
Symbol Substitution Test) from baseline to 12 weeks compared to placebo.  
3.0 
Patient Selection 
3.1 
Inclusion Criteria  
 
3.1.1 Self-reported history of cancer diagnosed > 12 months prior to enrollment excluding non-
melanoma skin cancer with no evidence of disease at enrollment. 
3.1.2 Eligible solid tumor cancer types include Stage 1-3 breast, lung, head and neck, 
colorectal, anal, prostate, melanoma, bladder/ureteral, esophageal, gastric, pancreatic, 
kidney, liver/biliary, uterine, cervical, ovarian, sarcoma. (superficial disease and in situ 
disease only is excluded) 
 
3.1.3 Eligible hematologic malignancies include lymphoma any subtype any stage in 
remission, multiple myeloma in remission, leukemia any subtype in remission.  
 
3.1.4 Eligible prior cancer treatment modalities include surgery, radiation, chemotherapy, 
hormonal therapies, immunotherapy, biologic therapies. 
 
3.1.5 All anti-cancer therapy completed > 12 months prior to enrollment 
 
3.1.6 Age 65 years and older 
3.1.7 Presence of self-reported fatigue defined by a response of “somewhat, quite a bit or very 
much” to the screening question “During the past seven days, did you feel fatigued: Not 
at all, a little bit, somewhat, quite a bit, very much?” 
3.1.8 Ability to walk without requiring assistance from another individual (use of cane or walker 
acceptable)   
3.1.9 Normal organ and marrow function as defined below: 

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-  leukocytes  
 
>3,000/mcL 
-  absolute neutrophil count >1,500/mcL 
-  platelets 
 
 
>100,000/mcL 
-  total bilirubin within normal institutional limits 
-  AST(SGOT)/ALT(SGPT) 
<2.5 X institutional upper limit of normal 
 
-  creatinine clearance 
>30 mL/min 
 
3.1.10 Ability to understand and the willingness to sign an IRB-approved informed consent 
document (either directly or via a legally authorized representative). 
3.2 
Exclusion Criteria  
 
3.2.1 active malignancy or on-going cancer treatment including oral anti-estrogen therapy, 
immunotherapy, biologic therapy. 
 
3.2.2 men receiving androgen deprivation therapy 
 
3.2.3 use of Coumadin or Warfarin 
 
3.2.4 symptomatic congestive heart failure 
  
3.2.5 lung disease requiring oxygen 
 
3.2.6 end stage renal disease requiring dialysis 
 
3.2.7 inability to swallow capsules 
 
3.2.8 chronic nausea or diarrhea defined by a frequency of ≥ once per week 
 
3.2.9 hemoglobin <10 g/dl 
 
3.2.10 diagnosis of dementia 
 
3.2.11 uncontrolled intercurrent illness including, but not limited to ongoing or active infection, 
unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that 
would limit compliance with study requirements. 
 
3.2.12 known untreated hypothyroidism 
 
3.2.13 allergy to muscadine grapes or muscadine grape preparations 
 
3.3 
Inclusion of Women and Minorities 
 
Recruitment to this study is expected to be similar to national data on cancer survivors 
population and the demographics of the WFBCCC. We expect >50% of enrolled participants to 
be women survivors. We will not enroll children (≤ 18 years) as the research pertains to older 
adult survivors. Based on our cancer center data, we expect approximately 3% of participants to 
be Hispanic/Latino (n=2). The breakdown of racial categories is expected to be as follows: 14% 
Black or African American (n=9), 84% White (n=54) and 2% more than 1 race (n=1). Should we 

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not meet or exceed these estimates, the PI will engage the Office of Cancer Health Equity to 
discuss strategies to enhance recruitment in these target populations. Our cancer center has 
policies in place to enhance recruitment of minority participants and will provide translation of 
the protocol and informed consent into Spanish. 
4.0 
Recruitment and Registration Procedures 
 
4.1 
Recruitment 
 
Participants will be recruited from the community via advertisement in the Center for Health 
Aging and Alzheimer’s Prevention Newsletter, newspaper advertisement, postcard mailing, and 
through sharing information with interested community organizations. Subjects will also be 
recruited from the Wake Forest Baptist Cancer Center (WFBCCC) using flyer advertisement 
including but not limited to Survivorship Clinics, mailings to participants, and messages via 
MyWakeHealth, listing the trial in the WFBCCC clinical trial website used by providers and 
monitoring trial recruitment through the Cancer Control and Survivorship Disease Oriented 
Team. Interested participants will contact the MGE Team Clinical Coordinators who will conduct 
prescreening by phone and coordinate in person visits for eligible patients in the Clinical 
Research Unit, or at the High Point,  Wilkes, or Statesville clinic sites.  
 
4.2 
Screening 
 
Study coordinators will pre-screen appropriate patients to assess eligibility via IRB-approved 
HIPAA waiver. Patients who are eligible will be contacted by phone or encountered at a clinic 
visit. Study coordinators will explain the study to potential participants to determine their interest 
and willingness. If a patient is likely eligible, but there is not adequate information available to 
confirm eligibility, the patient will be re-contacted for further assessment.  If the patient has 
documentation of the necessary laboratory studies within the specified window that does not 
appear in the medical record, the study coordinator will obtain and review these documents. If 
the patient needs additional studies in order to assess eligibility, the study coordinator will 
arrange to obtain the necessary studies. Signed consent for eligibility screening will be obtained 
prior to performing the additional studies. If the patient meets eligibility criteria, the study 
coordinator will arrange for the baseline study visit, which will include informed consent, 
registration, randomization, and day 1 study activities. 
 
4.3 
Registration 
 
All participants entered on any WFBCCC trial, whether treatment, companion, or cancer control 
trial, must be linked to the study in EPIC within 24 hours of Informed Consent. Participant must 
be registered prior to the initiation of treatment.   
 
1. Complete the Eligibility Checklist (Appendix A) 
2. Complete the Protocol Registration Form (Appendix B) 
3. Register the participant in OnCORE – this will alert the cancer center registrar, who will then 
review the signed informed consent and confirm the eligibility.   
Contact Information: 
Protocol Registrar PHONE (336) 713-6767 

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Protocol Registrar FAX (336) 713-6772 
Protocol Registrar E-MAIL (registra@wakehealth.edu)          
4. Participant will not be registered without all required supporting documents. 
Note: For any screening activities performed at an outside institution, the source documentation 
must be provided  
 
To complete the registration process, the study team will:   
1.   Register the participant on the study 
2.   Assign a participant study number 
3.   Randomize the participant 
 
4.4 
Blinding 
 
This is a double-blind study. Only the investigational pharmacists and the statisticians will be 
unblinded. The blind will be maintained until the study is complete. 
5.0 
Study Outcomes and Study Measures 
5.1 
Primary Outcome 
 
5.1.1 Patient Reported Outcomes Measurement System (PROMIS) Fatigue 7a will be used to 
assess fatigue. Fatigue is divided into the experience of fatigue (frequency, duration, and 
intensity) and the impact of fatigue (upon physical, mental, and social activities). Item 
responses are rated on a five-point scale ranging from “never” to “always” and are 
summed for a total score and transformed to a T-score metric, which has a mean of 50 
and a standard deviation of 10. Higher scores indicate more fatigue. The PROMIS 
Fatigue Short-Form 7a has shown robust reliability and validity across multiple 
samples.41-44 
 
5.2 
Secondary Outcomes 
 
5.2.1  The Pepper Assessment Tool for Disability (PAT-D) will used to assess self-reported 
physical function. The (PAT-D) is a 19-item survey designed by Rejeski et al. to assess 
domains of physical function in older adults which contains subscales on mobility, 
instrumental (IADLs), and basic activities of daily living (ADLs).45,46 
 
5.2.2 The Short Form Minnesota Leisure Time Activity Questionnaire (MLTA) will assess 
self-reported physical activity.7  
 
5.2.3  The Short Physical Performance Battery (SPPB) will be used to objectively assess 
lower extremity physical function. This validated measure comprises a short walk, 
repeated chair stands, and balance test. Lower scores on the SPPB have been 
associated with increased risk of disability, hospitalization and worse survival among 
older adults with and without cancer47-49.  
 
5.2.4  The 6-minute walk will be measured to assess physical fitness. The 6-minute walk is 
easy to administer in a clinical setting, accurately assesses submaximal exercise 

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capacity, is an independent predictor of mortality and is correlated with peak V02 
testing.50 
 
5.2.5  The Longitudinal Aging Study Amsterdam (LASA) Sedentary Behavior 
Questionnaire will be used measure sedentary behavior51. 
 
5.2.6  The PROMIS Global Health Short Form (SF) will assess global QOL This is a 10-item 
instrument representing multiple domains inclusive of physical, mental and social health 
in adults52.  
 
5.2.7  Adherence will be measured by pill count at study completion.  
 
5.2.8  The following measures of interest will be collected to allow characterization of frailty 
(Fried Frailty Phenotype), Center for Epidemiologic Studies Depression Scale 
(CES-D), MLTA questionnaire, Grip strength, self-reported weight loss.7,53-55 
 
 
5.3. Exploratory outcomes 
 
5.3.1  Peripheral blood will be collected at the baseline and week 12 study visit,  and will be 
processed to obtain serum for the measurement of cytokines including interleukin 6 (Il-
6), tumor necrosis factor-alpha (TNF-α), C reactive protein (CRP) and HGF and 
polymorphonuclear neutrophils (PMNs) to measure mitochondrial function. Cytokines 
and 8-OHdG will be measured using human ELISA kits (Abcam) and mitochondrial 
function will be assessed using a Seahorse XF Cell Mito Stress Test (Agilent). 
 
5.3.2 Urine will be collected at baseline and 12 weeks to measure the oxidative stress marker 
8-OHdG and urolithin A glucouronidate (UAG), a marker for the ingestion/metabolism of 
MGE. UAG, which is generated from the ingestion of MGE polyphenolic ellagitannins, 
will be measured using an LCMS-8050 triple quadrupole mass spectrometer in the 
Wake Forest School of Medicine Proteomics and Metabolomics Shared Resource.   
 
5.3.4 Fecal specimens will be collected at baseline and at ~12 weeks using an at-home fecal 
sampling kit. Samples will be collected by the patient prior to the initiation of intervention 
and again near the completion of intervention. Patients will be asked to mail samples in 
a pre-paid package to the laboratory. DNA will be isolated from fecal samples using the 
Qiagen DNeasy PowerSoil Pro kit protocol. Metagenomic sequencing will be performed 
by CosmosID Inc. (Rockville, MD). In brief, DNA libraries will be prepared using the 
Illumina Nextera XT library preparation kit, with a modified protocol. Library quantity will 
be assessed with Qubit (ThermoFisher). Libraries will be sequenced on an Illumina 
HiSeq platform 2x150bp. Fecal samples will be read at 12M read-depth. Unassembled 
sequencing reads will be directly analyzed by CosmosID bioinformatics platform for 
multikingdom microbiome analysis and quantification of organism’s relative 
abundance. Fecal microbiomes will be assessed by metagenomic sequencing and 
bioinformatics. A list of all the microbiologic species will be recorded, along with their 
relative abundance recorded as a % relative abundance of the total microbiome.  
 
5.3.5   Digit Symbol Substitution Test (DSST)56,57 is a measure of attention and perceptual 
speed, amenable to change with exercise or fatigue in which participants are given a 

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series of numbered symbols and then asked to draw the appropriate symbols below a 
list of random numbers. The score is the number of correctly made matches in 2 
minutes (120 seconds). 
 
5.4.  Adverse events (AEs) 
AEs will be assessed by the study team at each visit (by phone or in person). AEs of interest are 
flatulence, diarrhea, nausea, dyspepsia, constipation, abdominal pain (see Appendix P). Other 
possibly/probably attributable adverse events will be recorded in addition to any serious adverse 
event. 

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6.0 
Intervention Plan 
6.1 
Study-Related Activities  
 
Measures / Timepoints 
Pre- 
Studya 
Baseline Wk 2 
Wk 4 
Wk 8 Wk 12 
End of 
Trtmt  
Visith 
Informed consent 
X 
 
 
 
 
 
 
Medical and oncologic history   
X 
 
 
 
 
 
 
Concomitant medications assessed by self-report j 
X 
X 
X 
X 
X 
Xg, j 
 
Karnofsky Self-Reported Performance Rating 
Scale 
 
X 
 
 
 
 
 
Vital signs 
 
X 
 
 
 
X j 
 
Adverse event assessment 
 
X 
X 
X 
X 
Xg, j 
X 
Drug dispensingb 
 
X 
 
 
 
 
 
Pill count 
 
 
 
 
 
Xg, j 
 
Telephone follow-upc 
 
 
X 
X 
X 
 
 
Cognitive/Physical function & fitness (DSST, 
SPPB, 6-min walk, grip strength) 
 
X 
 
 
 
X j 
 
CBC with differentiald 
X 
 
 
 
 
X j 
 
Serum chemistriesd 
X 
 
 
 
 
X j 
 
(Partially fasting) labs for phenolic levels (blood 
and urine)e 
 
X 
 
 
 
X j 
 
(Partially fasting) labs for correlative studiese  
(blood and urine) 
 
X 
 
 
 
X j 
 
Stool sample collectionf 
 
X 
 
 
 
X j 
 
PROMIS Fatigue survey 
 
X 
X 
X 
X 
Xg, j 
X 
Other patient reported outcomes (PAT-D, LASA, 
PROMIS Global Health, CES-D, MLTA, Self-
reported weight loss) j 
 
X 
 
 
 
Xg, j 
 
Participant Feedback 
 
 
 
 
 
Xi, j 
 
 
a Labs to assess eligibility must be completed within 30 days prior to the first dose of MGE/placebo.  
b Study drug (MGE or placebo) will be dispensed as a 90-day supply at the baseline visit. 
c Adherence and symptom review will be assessed at each study visit. In addition, participants will be contacted by 
phone as follows, ±3 days: Wk2, Wk4, Wk8.   
d Safety laboratory assessments must be done within 30 days of first dose of MGE/placebo and then on the study visits 
indicated.  These labs include: 
Complete blood count (CBC) with differential: WBC count with differential, platelet count, hemoglobin/hematocrit. 
Serum chemistries (complete metabolic panel, CMP): sodium, potassium, chloride, bicarbonate, blood urea 
nitrogen, creatinine with creatinine clearance, glucose, aspartate aminotransferase (AST), alanine aminotransferase 
(ALT), total bilirubin, alkaline phosphatase. 
e Phenolic levels and other research labs will be obtained at baseline, and 12 weeks (±14 days). Patients are asked to 
avoid coffee, tea, fruit juice, fruit, chocolate or wine after midnight prior to blood draw.  
f   Two kits will be provided to participant at baseline, with detailed instructions for how and when to collect stool at 
home.  
g    If a participant is unable to attend an in person study visit for any reason including COVID-19 related reasons, all 
patient reported measures will be collected in a phone study visit. 

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h  End of Treatment Visit will be conducted by phone 30 days (+14 day window) after last dose of study medication. For 
those who end treatment prior to week 12, the End of Treatment Visit can be combined with another existing study 
visit, if that visit occurs 30-44 days after the last dose of study medication.   
I   Participant feedback will be collected in person at Wk12 visit (+30 day window), or by phone at Wk16.  
j Follow-up study visits will occur at 12 weeks and will have a ±14 day allowance window which includes not just the 
visit itself, but any of the activities at that visit. For example, if labs cannot be collected at the actual study visit, the 
±14-day window permits a second attempt at lab collection; all activities, however, must be completed in the ±14-day 
window from the expected visit date. 
 
6.2 
Intervention Administration 
Treatment will be administered on an outpatient basis. Patients will be randomized 1:1 basis to either 
MGE or placebo. The study is double-blind and the patients will take 4 capsules by mouth twice daily.   
6.3 
Investigational Agent 
This study uses a specific formulation of MGE produced by Piedmont Research & Development 
Corporation from a powdered extract obtained from NPC Corporation, formerly known as Nature’s 
Pearl (Advance, NC).  The same product was used for a phase 1 study in advanced cancer patients 
and the on-going Phase 2 study in prostate cancer patients (FDA IND 128937; FDA IND 139288). The 
muscadine grapes are pressed and processed on site per good manufacturing practice (FDA Code of 
Federal Regulations 21). The extract is dissolved in water, dried and converted to powder at 
Pharmachem Laboratories, Inc. (Kearny, New Jersey). The MGE is encapsulated with a vegetable 
capsule of hypromellose, bottled and labeled at Clinical Encapsulation Services (CES) (Schenectady, 
NY). Quality control testing for microbial contamination and phenolic levels occurs numerous times 
during the production process and will continue at regular intervals until study completion. The MGE will 
be tested biannually for total phenolics using the Folin-Ciocalteu method, with gallic acid as the 
standard, and microbes will be assessed annually by American Testing Lab (San Diego, CA). MGE will 
be dispensed by the WFBCCC Investigational Drug Studies Pharmacy which managed the Phase 1 
and on-going Phase 2 study.  
 
6.4 
Placebo Description 
Placebos will be supplied by Clinical Encapsulation Services (CES) (Schenectady, NY), an FDA-
registered facility, registration number 16691092818. The capsules contain microcellulose tinted to 
match MGE and are encapsulated with a vegetable capsule of hypromellose of the same size as the 
MGE capsules. Placebo were bottled and labeled by CES.  Quality control testing for microbial 
contamination (aerobic count, yeast and mold, E.coli/coliform, staph aureus, enterobacteriaceae, and 
salmonella) and phenolic level (to ensure no cross contamination) occurs during the production 
process. Stability testing for microbial contamination and content uniformity will continue at regular 
intervals.   
 
Storage requirements: Capsules should be stored at room temperature. 
Route of administration: Placebo is available for oral administration. 

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6.5 
General Concomitant Medication and Supportive Care Guidelines 
If participants start Coumadin/Warfarin while on study, we will advise them of the potential interaction 
with MGE. Participants who start Coumadin/Warfarin while on study will be asked to stop dosing study 
drug (see section 6.7 regarding indications for discontinuation of intervention). Participants will be 
instructed to inform their prescribing providers of the recommendation to recheck within 7 days of 
starting medication and again within 7 days of completing our study drug. 
6.6 
Study Assessments 
All in-person study-related activities will occur at the Wake Forest Baptist Medical Center Clinical 
Research Unit, or at the High Point, Wilkes, or Statesville clinic sites and do not need to be coupled 
with standard-of-care clinic visits.  
6.6.1 Vitals signs, performance status  
 
Vital sign assessment consists of heart rate, blood pressure, respiration rate, temperature. 
Height and weight will be collected at baseline with weight repeated at follow-up. 
 
Performance status will be assessed at baseline using the Karnofsky Self-Reported 
Performance Rating Scale (KPS).  
 
6.6.2 Laboratory assessments (safety) 
 
The following laboratory studies will be obtained at follow-up: 
• 
Complete blood count (CBC) with differential: Total white blood cell count (WBC) with 
differential, hemoglobin/hematocrit, and platelet count. 
• 
Blood chemistry (CMP): Sodium, Potassium, Chloride, Bicarbonate, Blood Urea 
Nitrogen, Creatinine, Glucose, total bilirubin, alkaline phosphatase, AST (SGOT), ALT 
(SGPT). 
• 
Two 10mL K2 EDTA tubes of blood will be collected for banking at baseline and follow-
up, which is optional and determined at the time of consent 
• 
Critical laboratory values will be reported to the PI who will communicate with the 
participant. 
 
 
 
 
6.6.3 Laboratory assessments (other) 
 
The following laboratory studies will be obtained at specified intervals while on study: 
 
Correlative research labs (refer to section 10.0) 
 
6.6.4  Physical function assessments 
 
Physical performance will be objectively assessed using the Short Physical Performance 
Battery (SPPB). This validated measure comprises a short walk, repeated chair stands, 
and balance test. Submaximal (6-minute walk) exercise capacity will be measured to 
assess physical fitness. The 6-minute walk will be performed to assess fitness. Grip 
strength will be performed to calculate a Fried Frailty Index. These measures will be 
performed by trained research nurses per protocol in the CRU and at participating sites. 

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6.6.5 Cognitive assessment 
Attention and perceptual speed will be measured with the Digital Symbol Substitution 
Test (DSST).  Participants are presented a series of numbered symbols and then asked 
to draw the appropriate symbols below a list of random numbers. This will be conducted 
by trained study coordinators. 
 
6.6.6   Patient reported outcomes 
 
 
 
The PROMIS fatigue survey will be administered at baseline, 2, 4, 8, 12 weeks and the 
End of Treatment Visit (@ week 16).  Other patient-reported outcomes will be 
administered at baseline and 12 weeks as outlined in the study calendar. If unable to 
come in person for follow-up study visit due to COVID-19 or other intercurrent event, 
patient reported data will be collected by phone. 
 
6.7 Duration of Study Intervention 
 
Treatment with MGE will continue for 12 weeks or until one of the following criteria applies:  
• 
Intercurrent illness that prevents further administration of treatment; 
• 
Unacceptable adverse event(s) attributable to MGE; 
• 
Patient misses more than 28 consecutive days of treatment for any reason; 
• 
Participant takes Coumadin or Warfarin while on study 
• 
Changes in the patient's condition render the patient unacceptable for further treatment in 
the judgment of the treating physician; 
• 
Patient decides to withdraw from the study; or 
• 
Patient is noncompliant, determined at the discretion of the principal investigator in 
conjunction with the treating physician 
6.8 
Duration of Follow Up 
 
Patients will be followed for toxicity/adverse events for 30 days after the last dose of the study 
drug.  
 
Patients will be instructed to inform the study staff of any medical concerns that arise during the 
first 30 days after stopping the study drug for any reason. The study team will assess for 
toxicity/adverse events and determine attribution. Of note, a 24-hour washout period was 
adequate in the Phase 1 protocol with the same drug (CCCWFU #01815, manuscript under 
review). Participants will also be called by the study team for the End of Treatment Assessment 
tele-visit 30 days after last study dose to review toxicity and assess resolution/stabilization of 
reported AEs. 
 
Patients removed from the study intervention for unacceptable adverse events will be followed 
until resolution or stabilization of the adverse event. All participants who discontinue taking the 
study drug (see section 6.5) will be asked to continue study visits per protocol. If they are unable 
or unwilling to come to the CRU or participating site as planned, study staff will attempt to collect 

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fatigue and other QOL data by phone in lieu of study visits as long as consent is not withdrawn. 
All participants will be followed for the study outcomes until the end of the study. 
 
 
6.9 
Criteria for Removal from Study Intervention 
 
All patients will be expected to complete their 12-week study visit, unless they withdraw consent 
or are medically unable, even if they have stopped MGE. If they are unable or unwilling, they will 
be contacted by phone and asked to complete the survey-based data. 
 
Patients will be removed from study intervention when any of the criteria listed in section 6.7 
apply. 
7.0 
Dosing Delays/Dose Modifications 
 
7.1 
Missed doses 
 
If a dose of MGE/placebo is missed, the patient will be instructed to take his/her next dose as 
scheduled. Missed doses should not be added to the next dose.  
 
If treatment must be held for any reason, it must be restarted within 28 days, or the patient will 
be removed from the study intervention. Unused pills will be collected at the follow-up study 
visit. 
 
7.2 
Dose modification  
 
There will be no dose-modifications on study. The patient can choose to discontinue 
MGE/placebo at any time. If necessary, MGE/placebo can be held for up to 28 days. 
 
Subjects who notify the study team of potential grade 2 or higher study drug-related toxicity 
concerns will be contacted by the study PI for further information. Subjects will permanently 
discontinue the study drug for any attributable grade 4 toxicity. If participants experience grade 
2 or 3 toxicity potentially attributable to study drug, dosing will be held and appropriate 
supportive care recommendations made. Subjects will be re-assessed weekly until toxicity has 
resolved to grade 0-1. If the subject continues to experience grade 2 or 3 toxicity, he/she will 
hold the study drug for up to 27 days, and the study team will again follow up with the patient to 
ensure toxicity has resolved to grade 0-1. If the toxicity persists beyond 27 days and is possibly 
attributable to the study drug, the study drug will be permanently discontinued. If the toxicity 
resolves to grade 0-1 within 27 days, the subject will restart the study drug. If the same grade 2 
or higher possibly-attributable toxicity returns despite appropriate supportive care measures (ie 
diarrhea despite use of anti-diarrheal medication), the subject will permanently discontinue the 
study drug.  
 
Guidelines for study drug discontinuation include 1) any attributable grade 4 toxicity, 2) any 
attributable grade 2 or 3 toxicity that does not resolve within 27 days or recurs upon re-initiation 
of study drug, 3)  study drug not taken for >28 consecutive days for any reason. 
 

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Guidelines for holding MGE/placebo: 1) any attributable grade 2 or 3 toxicity; 2) participant 
experiences medical or personal concern that is not attributable to study medication but 
prohibits study drug administration (i.e. inability to swallow, hospitalization). 
 
8.0 
Adverse Events List and Reporting Requirements 
 
8.1 
Adverse Event List for MGE 
 
Expected: Based on previous studies, minimal AEs are expected, which may include flatulence, 
diarrhea, nausea, dyspepsia, constipation, and abdominal pain. 
8.2 
Adverse Event Characteristics 
 
• 
CTCAE term (AE description) and grade:  The descriptions and grading scales found in 
the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will 
be utilized for AE reporting. All appropriate treatment areas should have access to a copy of 
the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the 
CTEP web site (http://ctep.cancer.gov). 
• 
‘Expectedness’: AEs can be ‘Unexpected’ or ‘Expected’ (see Section 7.1 above) for 
expedited reporting purposes only.   
• 
Attribution of the AE: 
- 
Definite – The AE is clearly related to the study intervention. 
- 
Probable – The AE is likely related to the study intervention. 
- 
Possible – The AE may be related to the study intervention. 
- 
Unlikely – The AE is doubtfully related to the study intervention. 
- 
Unrelated – The AE is clearly NOT related to the study intervention. 
8.3 
STRC SAE Reporting Requirements 
 
The Safety and Toxicity Reporting Committee (STRC) is responsible for reviewing SAEs for WFBCCC 
Institutional studies as outlined in Appendix D. All Adverse Events that occur during protocol 
intervention and are coded as either 1) unexpected grade 4, 2) unplanned inpatient hospitalization 
> 24 hours (regardless of grade), or grade 5 (death) must be reported to the STRC using the using 
the SAE console in WISER. All WFBCCC Clinical Protocol and Data Management (CPDM) staff 
members assisting a Principal Investigator in investigating, documenting and reporting an SAE 
qualifying for STRC reporting are responsible for informing a clinical member of the STRC as well as 
the entire committee via the email notification procedure of the occurrence of an SAE.  
8.4 
WFUHS IRB AE Reporting Requirements 
 
Any unanticipated problems involving risks to subjects or others and adverse events shall be promptly 
reported to the IRB, according to institutional policy. Reporting to the IRB is required regardless of the 
funding source, study sponsor, or whether the event involves an investigational or marketed drug, 
biologic or device. Reportable events are not limited to physical injury, but include psychological, 
economic and social harm. Reportable events may arise as a result of drugs, biological agents, 

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devices, procedures or other interventions, or as a result of questionnaires, surveys, observations or 
other interactions with research subjects. 
 
All members of the research team are responsible for the appropriate reporting to the IRB and other 
applicable parties of unanticipated problems involving risk to subjects or others. The Principal 
Investigator, however, is ultimately responsible for ensuring the prompt reporting of unanticipated 
problems involving risk to subjects or others to the IRB. The Principal Investigator is also responsible 
for ensuring that all reported unanticipated risks to subjects and others which they receive are reviewed 
to determine whether the report represents a change in the risks and/or benefits to study participants, 
and whether any changes in the informed consent, protocol or other study-related documents are 
required. 
 
Any unanticipated problems involving risks to subjects or others occurring at a site where the study has 
been approved by the WFUHS IRB (internal events) must be reported to the WFUHS IRB within 7 
calendar days of the investigator or other members of the study team becoming aware of the event. 
 
Any unanticipated problems involving risks to subjects or others occurring at another site conducting 
the same study that has been approved by the WFUHS IRB (external events) must be reported to the 
WFUHS IRB within 7 calendar days of the investigator or other members of the study team becoming 
aware of the event. 
 
Any event, incident, experience, or outcome that alters the risk versus potential benefit of the research 
and as a result warrants a substantive change in the research protocol or informed consent 
process/document in order to insure the safety, rights or welfare of research subjects. 
8.5 
Sponsor Reporting Requirements  
 
This study has no third part sponsor. This study is funded by a CTSI pilot grant. 
9.0 
Pharmaceutical Information 
 
Muscadine grape extract is a nutraceutical compound. 
  
A list of the adverse events and potential risks associated with the investigational agents administered 
in this study can be found in Section 8. 
9.1 
Pharmaceutical/Device Accountability  
 
Patients will be instructed to return their pill bottles and remaining pills at each study visit.  Drug 
accountability logs will be maintained by the Wake Forest Investigational Drug Services for the 
investigative agent (MGE) used under this protocol. These logs shall record quantities of study 
drug received and quantities dispensed to patients, including lot number, date dispensed, 
patient identifier number, protocol number, dose, quantity returned, balance remaining, and the 
initials of the person dispensing the medication. 
9.2 Muscadine Grape Extract (MGE) 
 
 

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Product description: This pilot study uses a specific formulation of muscadine grape extract (MGE) 
produced by Piedmont Research & Development Corporation from a powdered extract obtained from 
NPC Corporation, formerly known as Nature’s Pearl (Advance, NC). The same product was used for a 
phase 1 study in participants with advanced cancer (FDA IND 128937; clinicaltrials.gov NCT02583269) 
and a phase 2 study in participants with prostate cancer (FDA IND 139288; clinicaltrials.gov 
NCT03496805). 
 
The muscadine grapes are pressed and processed on site per good manufacturing practice (FDA Code 
of Federal Regulations 21). The extract is dried and converted to powder at Pharmachem Laboratories, 
Inc. (Kearny, New Jersey). The MGE is encapsulated with a vegetable capsule of hypromellose, 
bottled, and labeled at Clinical Encapsulation Services (CES) (Schenectady, NY). Quality control 
testing for microbial contamination (aerobic count, yeast and mold, E.coli/coliform, staph aureus, 
enterobacteriaceae, and salmonella) and phenolic levels occurs numerous times during the production 
process. Stability testing for both microbial contamination and phenolic levels will continue at regular 
intervals until the study is complete.   
 
Storage requirements: Capsules should be stored at room temperature. 
 
Route of administration: MGE is available for oral administration. 
10.0 Correlative/Special Studies 
 
Blood: To be collected at baseline, and 12 weeks.  Approximately 20 mL of blood will be collected into 
an EDTA tube to measure cytokines and growth factors, including interleukin 6 (Il-6), tumor necrosis 
factor-alpha (TNF-α), C reactive protein (CRP), HGF, and polymorphonuclear neutrophils (PMNs) to 
measure mitochondrial function. Samples will be collected by CRU or participating site nursing staff 
according to a protocol they establish with the study team. Samples will then be transported on ice to 
the Cell & Molecular Biology Core in the Hypertension Center at BioTech Place (575 Patterson Ave, 
Winston-Salem, NC 27101), using a commercial courier service.  Samples will be stored at -80°C in the 
Hypertension Center (3rd Floor, BioTech Place) indefinitely until batched analysis. Analysis will take 
place in the Cell & Molecular Biology Core. 
 
Two 10mL  K2 EDTA tubes of blood will be collected for banking at baseline and follow-up, which is 
optional and determined at the time of consent. 
 
Urine: At least 20 mL of urine will also be collected at baseline and follow-up for 8-OH deoxyguanosine 
levels and urolithin A glucouronidate (UAG), stored at -80°C indefinitely until batched analysis. Samples 
will be transported on ice using commercial courier service to the Cell & Molecular Biology Core in the 
Hypertension Center at BioTech Place (575 Patterson Ave, Winston-Salem, NC 27101). 
 
Stool samples: Fecal specimens will be collected at baseline and at ~12 weeks using an at-home fecal 
sampling kit. Two kits will be provided to participant at baseline, with detailed instructions for how and 
when to collect stool at home. Participants will be asked to mail samples in a pre-paid package to the 
laboratory. DNA will be isolated from fecal samples using the Qiagen DNeasy PowerSoil Pro kit 
protocol. Metagenomic sequencing will be performed by CosmosID Inc. (Rockville, MD). In brief, DNA 
libraries will be prepared using the Illumina Nextera XT library preparation kit, with a modified protocol. 
Library quantity will be assessed with Qubit (ThermoFisher). Libraries will be sequenced on an Illumina 
HiSeq platform 2x150bp. Fecal samples will be read at 12M read-depth. Unassembled sequencing 

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reads will be directly analyzed by CosmosID bioinformatics platform for multikingdom microbiome 
analysis and quantification of organism’s relative abundance. Fecal microbiomes will be assessed by 
metagenomic sequencing and bioinformatics. A list of all the microbiologic species will be recorded, 
along with their relative abundance recorded as a % relative abundance of the total microbiome. 
11.0 Data Management 
 
Item/Form  
Location 
Informed consent document 
WakeOne 
Protocol registration & race/ethnicity verification 
OnCore 
Study visit data collection forms: Baseline, week 12 
REDCap* 
Medications and supplements 
OnCore 
Labs 
REDCap* 
Correlative labs 
REDCap* 
Adverse event ascertainment 
REDCap* 
Pre-toxicity and adverse events log 
REDCap* 
Patient surveys 
REDCap* 
Cognitive/Physical function & fitness forms  
REDCap* 
Telephone surveys 
REDCap* 
Off-treatment/Off-study form 
REDCap*/OnCore 
*This project will utilize REDCap Clinical Data Interoperability Services. This is a special feature for importing data into 
REDCap from WakeOne. It provides an adjudication process whereby REDCap users can approve all incoming data from 
WakeOne before it is officially saved in their REDCap project. REDCap Clinical Data Interoperability Services can only be 
enabled by a REDCap administrator who serves as an honest broker to PHI. REDCap’s Clinical Data Interoperability Services 
can only be accessed by users with valid WakeOne credentials. Using the Clinical Data Interoperability Service requires using 
the Medical Record Number (MRN) as a key to automatically gather demographics and laboratory data and reduces data entry 
errors. 
12.0 Statistical Considerations 
 
12.1 Analysis of Primary Objective 
 
 
To address the primary objective, we will use a mixed effects model with a constraint of a common 
baseline mean across treatment groups and an unstructured covariance matrix to model all fatigue 
measures over time, and use linear contrasts to estimate the difference in change and corresponding 
90% confidence interval between the two groups from baseline to follow-up.58 All outcomes will be 
measured even if the participants discontinue the intervention, to provide an estimate of effectiveness. 
12.2 Analysis of Secondary Objectives 
 
For secondary outcomes listed in Objective 2.2.1, 2.2.2, and 2.2.3, which are based on baseline and 
follow-up measures, we will estimate 95% confidence intervals for the differences between the two 

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groups for the secondary outcomes at 12 weeks.  We will also fit a ANCOVA model adjusted for the 
baseline value of the outcome to each secondary outcome. 
 
12.3 Analysis of Exploratory Objectives 
 
To analyze the biomarker outcomes in Objective 2.3.1, which are measured continuously, we will also 
use the ANCOVA approach described for the secondary objectives, and will construct 95% for the 
differences in the change between the two groups at 12 weeks. 
 
To meet Objective 2.3.2, we will examine the proportion of different bacteria taxa at each time point, 
and will use a marginalized two-part beta regression model to account for the compositional nature of 
the data.59 If we are unable to fit this model, we will examine changes using McNemar-Bowker tests for 
within-person changes and for mammary and gut composition at 12 weeks.   
 
To meet Objective 2.3.3, we will include the biomarker data in the models of the primary and secondary 
outcomes described above, and evaluate if the effects of the intervention on each outcome is 
attenuated in the presence of the biomarker.  Attenuation of 10% or more in the intervention parameter 
estimate will indicate potential mediation in the presence of a clinically meaningful change of both the 
primary or secondary outcome and the biomarker of interest. 
 
For Objective 2.3.4, we will compare the change in the DSST score between the 2 groups at 12 weeks, 
using a mixed model with linear contrasts. 
 
12.4 Power and Sample Size 
 
The minimally important difference (MID) established for the fatigue scale is 3-5, and the SD of change 
in our pilot study was 6.4 over 8 weeks in 12 participants. Using the upper limit of the one-sided 90% CI 
for this SD (9.0) to account for potential bias, indicates an effect size of ≥0.33 would be meaningful. For 
pilot studies, rather than calculating power based on hypothesis testing, use of 80% or 90% one-sided 
confidence intervals are frequently suggested.61-63 With N=30 participants per group, for a difference 
of 0 the upper bound of the 90% CI is 0.33; i.e., if we observe no difference between the groups, it 
would be unlikely that the true effect size is greater than 0.33.To adjust for 5% drop out we will inflate 
our sample to 32/group. For secondary objectives, which are based on baseline and follow-up 
measures, we will estimate 90% confidence intervals for the differences between the two groups for the 
secondary measures of interest. Adherence rates will be estimated using pill counts and examined 
using a mean and 95% confidence interval. Subgroup analyses of the outcomes will be explored 
among those that achieved ≥80% adherence vs. those that did not. 
12.5 Estimated Accrual Rate 
 
The expected accrual is 64 participants. We anticipate accruing an average of 3 participants per week. 
12.6 Estimated Study Length 
 
Participants will be followed for a total of 12 weeks for study procedures. A 30-day phone visit will be 
conducted after the last dose of the study drug thus the study will be completed 12-16 weeks after the 
last participant is accrued. 
 
 

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Appendix L - Frailty Index Calculation Form  
 
Score will be programmed to calculate automatically, after other forms are entered into the database. 
 
Study team to review weight loss collected on baseline form: 
1. Have you had more than 10 pounds of unintentional weight loss in the past year? (Baseline form) 
Yes ___ No ___ 
 
 
Study team to record the following: 
2. Review the response to question #7 on the CES-D survey which states "everything I did was an effort". Did 
the participant answer either "3-4 days /week" or "5-7 days per week"?  Yes ___ No ___ 
 
3. Review the response to question #20 on the CES-D survey which states "I could not get going". Did the 
participant answer either "3-4 days /week" or "5-7 days per week"?  Yes ___ No ___ 
 
 
Study team to review data from 4-meter walk trials from SPPB are recorded 
 
4-meter walk test, faster of 2 trials  (seconds, with up to 2 decimal places) ___________ 
 
 
Study team to review data from from grip strength testing are recorded: 
 
 
Strongest trial for right hand: (kg) _________ 
 
 
Strongest trial for left hand:  (kg)  _________ 
 
 
Study team to review MLTQ was collected and recorded. 
 
 
 

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Appendix R: Digit Symbol Substitution Test (DSST) Script  
 
 
***This is a sample of the test. Permission was received to obtain and use the full test.***  
 
 
 
Next I am going to have you copy some symbols. Take a look at these boxes up top. Notice that they have a 
number up top and a symbol below. Each number has its own symbol. 
 
Now look at the boxes down here. The boxes have numbers in the top but the bottom is empty. What I am 
going to have you do is put the symbol in bottom boxes that should go there. Let me demonstrate. 
 
Here is the 6, the 6 has this symbol, so you would put in the box under the 6.  Here is the 8, the 8 has this 
symbol, so you would put in the box under the 8.  Here is the 3, the 3 has this symbol, so you would put in the 
box under the 3. 
 
Now I am going to have you fill in the boxes up to the heavy black line here. 
 
Good! Now that you know how to do them, when I say BEGIN, I am going to have you start here and fill in as 
many boxes as you can without skipping any. Work as quickly as you can without making any mistakes. 
Complete each row before going to the next. Keeping working until I tell you to stop.   
 
Ready? BEGIN.