Phase I Study of Muscadine Grape Extract (MGE) in Advanced Malignancy
Phase: PHASE1
Conditions: Advanced Malignant Neoplasm, Metastatic Malignant Neoplasm, Unresectable Malignant Neoplasm
Sponsor: Wake Forest University Health Sciences

Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 1 of 31 
Principal Investigators: 
 
Rhonda L. Bitting, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
Medical Center Boulevard 
Winston-Salem, NC 27157 
 
 
 
 
 
 
Heidi Klepin, MD, MS 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
Medical Center Boulevard 
Winston-Salem, NC 27157 
 
Co-Investigators: 
 
 
 
Patricia Gallagher, PhD 
Hypertension & Vascular Research Center 
Wake Forest School of Medicine 
 
Michael M. Goodman, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Stefan C. Grant, MBBCh, MD, JD, MBA 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Glenn J. Lesser, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Susan Melin, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Boris Pasche, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
W. Jeffrey Petty, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Mercedes Porosnicu, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Jimmy Ruiz, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Paul Savage, MD 

Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 2 of 31 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Steven Sorscher, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Ann Tallant, PhD 
Hypertension & Vascular Research Center 
Wake Forest School of Medicine 
 
Christopher Thomas, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Pierre L. Triozzi, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
George Yacoub, MD 
Section on Hematology and Oncology 
Wake Forest School of Medicine 
 
Biostatistician: 
Janet Tooze, PhD 
Comprehensive Cancer Center of Wake Forest University 
 
Scott Isom, MS 
Comprehensive Cancer Center of Wake Forest University 
 
Study Coordinator:  
 
Shannon Golden, MA 
 
 
 
 
 
Department of Social Sciences & Health Policy 
Wake Forest School of Medicine 
 
Katherine Pleasant 
Department of Epidemiology & Prevention 
Wake Forest School of Medicine 
 
 
Regulatory Contact: 
Megan Farmer, MS 
Comprehensive Cancer Center of Wake Forest University 
 
Alexandra Bolick 
Comprehensive Cancer Center of Wake Forest University 
 
Research Nurses (RN): 
 
Jennifer Thomas 
Comprehensive Cancer Center of Wake Forest University 
 
Data Manager: 
 
 
Deanna Hissim 
Comprehensive Cancer Center of Wake Forest University 
 
Other Team Members: 
 
 

Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 3 of 31 
Jasmine Miller 
Comprehensive Cancer Center of Wake Forest University 
 
Participating Institutions:  
CCCWFU 
Version Date: 
 
 
12/22/15 
Amended: 
 
 
 
10/5/18 (IRB Amendment #26) 
Confidential

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 4 of 31 
 
Table of Contents 
1.0 
Introduction and Background .......................................................................................... 7 
1.1 
Preclinical Studies ...................................................................................................... 7 
1.2 
Preclinical toxicity studies with MGE ..........................................................................11 
1.3 
Clinical Studies ..........................................................................................................13 
2.0 
Objectives .....................................................................................................................14 
2.1 
Primary Objective ......................................................................................................14 
2.2 
Secondary Objectives ................................................................................................14 
3.0 
Patient Selection ...........................................................................................................14 
3.1 
Inclusion Criteria ........................................................................................................14 
3.2 
Exclusion Criteria .......................................................................................................15 
3.3 
Inclusion of Women and Minorities ............................................................................16 
4.0 
Registration Procedures ................................................................................................16 
5.0 
Study Outcomes and Study Measures...........................................................................17 
5.1 
Primary Outcome .......................................................................................................17 
5.2 
Secondary Outcomes ................................................................................................17 
6.0 
Treatment Plan ..............................................................................................................18 
6.1 
Study-Related Activities .............................................................................................18 
6.2 
Treatment Administration ...........................................................................................19 
6.2.1 
Definition of Dose-Limiting Toxicity (DLT) ............................................................19 
6.2.2 
Dose modification in the event of toxicity .............................................................20 
6.3 
Study procedures ......................................................................................................20 
6.3.1 
Recruitment .........................................................................................................20 
6.3.2 
Follow-up Assessments .......................................................................................20 
6.4 
General Concomitant Medication and Supportive Care Guidelines ............................20 
6.5 
Duration of Therapy ...................................................................................................21 
6.5.1    Missed Doses. .......................................................................................................21 
6.6 
Duration of Follow Up ................................................................................................21 
7.0 
Measurement of Effect ..................................................................................................21 
7.1 
Definitions ..................................................................................................................22 
7.2 
Methods for Evaluation of Measurable Disease .........................................................22 
8.0 
Adverse Events List and Reporting Requirements .........................................................23 
8.1 
Adverse Event List for MGE .......................................................................................23 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 5 of 31 
8.2 
Adverse Event Characteristics ...................................................................................23 
8.3 
STRC SAE Reporting Requirements .........................................................................23 
8.4  
WFUHS IRB AE Reporting Requirements .................................................................23 
9.0 
Pharmaceutical Information ...........................................................................................24 
9.1 
Drug Accountability ....................................................................................................24 
9.2 
Muscadine Grape Extract (MGE) ...............................................................................24 
10.0 
Laboratory Studies ........................................................................................................24 
11.0 
Data Management .........................................................................................................25 
12.0 
Statistical Considerations ..............................................................................................25 
12.1 
Analysis of Primary Objective ....................................................................................26 
12.2 
Analysis of Secondary Objective ...............................................................................26 
12.3 
Power and Sample Size ............................................................................................27 
12.4 
Estimated Accrual Rate and Study Length .................................................................27 
12.5 
Interim Analysis Plan .................................................................................................28 
References ...............................................................................................................................28 
Appendices (list) .......................................................................................................................30 
 ~ Apendices are now separate attachments in the eIRB under Protocol Document  
 
 
 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 6 of 31 
 
 
SCHEMA 
 
 
 
 
 
Consent and pre-study data collection 
Assign dose level  
Baseline symptom assessment 
Baseline correlatives  
(phenolic levels, cytokines, FACT-G, 
PROMIS-fatigue SF) 
Muscadine grape extract (MGE) 
administration daily 
Week 4: Symptom assessment for dose 
limiting toxicity (DLT) 
Correlatives (phenolic levels, cytokines, 
FACT-G, PROMIS-fatigue SF, 
adherence) 
Study follow-up: 
Every 4 week symptom assessment on 
MGE if disease stable/responding 
Every 8-12 week imaging on MGE 
Overall survival all subjects 
Week 8: Symptom assessment  
Correlatives (phenolic levels, cytokines, 
FACT-G, PROMIS-fatigue SF, 
adherence) 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 7 of 31 
1.0 
Introduction and Background 
 
It is estimated that one-third of the more than 7 million deaths from cancer worldwide is attributable 
to potentially modifiable risk factors, with 374,000 deaths preventable through diet modification 
alone (not including those attributable to obesity or physical inactivity).1 Diet supplementation for 
the prevention of cancer is an attractive mechanism to control cancer, since it is easy to implement 
to a broad population, even populations with reduced incomes and resources.  Grape extracts or 
active components isolated from grapes have received attention as chemopreventive agents 
based upon their anti-proliferative, anti-inflammatory and anti-oxidant properties.2  
 
The muscadine grape (Vitis rotundifolia), found in the warm, humid climate of southeast United 
States, has a distinct phytochemical composition as compared to other grape varieties.  The 
muscadine grape contains a high concentration of anthocyanin 3,5-diglucosides, ellagic acid, 
ellagic acid precursors, gallic acid, flavan-3-ols and flavonols.3  Several preclinical studies with 
muscadine grape products have revealed anti-tumor activity, including inhibition of tumor cell 
growth and induction of apoptosis.4-7 Evidence from preclinical trials also suggests that muscadine 
grape products may decrease systemic inflammation.8-11  Higher levels of inflammation may not 
only increase cancer recurrence risk,12 but also impair quality of life.  Higher levels of systemic 
inflammation are associated with increased fatigue13, reduced levels of physical activity, 
adiposity14,15 and lower levels of physical function16-18.  By reducing levels of circulating 
inflammatory markers such as CRP and IL-6, muscadine grape products may improve cancer 
outcomes by decreasing symptom burden (particularly fatigue). 
 
Despite reports indicating potential anti-tumor activity,4-6 there are limited clinical studies on the 
efficacy of muscadine grape products in the prevention or treatment of cancer, representing an 
opportunity for novel investigations.  The current study proposes to build upon promising 
preclinical and clinical studies to assess the safety and tolerability of a proprietary muscadine 
grape extract (MGE) formulation, made from muscadine grape skins and seeds and produced by 
Nature’s Pearl, in a Phase 1 clinical trial of patients with metastatic solid tumors who have failed 
prior standard therapies. 
 
1.1 
Preclinical Studies 
 
Literature overview: The anti-tumor properties of muscadine grapes have been 
demonstrated in preclinical models.4-7 Porter et al examined the effects muscadine grape 
extract on lung tumor formation in the fetus following transplacental exposure to a 
polycyclic hydrocarbon. Results indicated that muscadine exposure through drinking 
water reduces both tumor proliferation and angiogenesis, leading to a decrease in tumor 
burden and multiplicity in mouse models.19 Exposure to muscadine grape extract through 
drinking water similarly reduced breast tumor burden and multiplicity in c-neu mice.20  In a 
study by God et al, four varieties of muscadine grape were tested and showed significant 
inhibition of 2-aminoanthracene mutagenesis, high antioxidant activity, and the ability to 
inhibit activities of metalloproteinases, implying that these muscadine products could be 
good inhibitors of carcinogenesis.6 In colon carcinoma cells, muscadine exposure induced 
cell death.4,5  In prostate cancer cell lines, treatment with muscadine grapes induced both 
cell cycle arrest and apoptosis.7 One mechanism by which muscadine grapes cause 
apoptosis is by downregulating the phosphatidylinositol 3-kinase (PI3K)/Akt survival 
pathway.7 The PI3K/Akt pathway is a key oncogenic signaling pathway that has been 
linked to tumorigenesis and resistance to anticancer therapies in a wide variety of tumor 
types.21  A study by Burton et al examined the effects of muscadine grape product on bone 
turnover in prostate and breast cancer cell models overexpressing Snail transcription 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 8 of 31 
factor.22  They show that Snail regulation can be antagonized by muscadine exposure, 
leading to decreased cell invasion, migration and bone turnover, and thus suggesting that 
muscadine grapes could be a promising bioactive treatment for bone metastatic cancer. 
Overview of preliminary data from the investigative team: Preliminary studies from the 
investigative team (Gallagher/Tallant) demonstrate that muscadine grape seeds and skins 
reduced the proliferation of both human breast and prostate cancer cell lines. The 
reduction in breast cancer cell growth was associated with a decrease in activated mitogen 
activated protein (MAP) kinase, a critical enzyme involved in cell proliferation.  In addition, 
muscadine grape liquid extract reduced the tumor burden in a transgenic model of 
spontaneous breast cancer with a concomitant attenuation in proliferation, tumor blood 
vessel 
formation, 
and 
tumor-associated 
fibrosis. These data are described below and 
provide additional rationale to assess the 
efficacy 
of 
Nature’s 
Pearl 
proprietary 
muscadine grape extract (MGE) on cancer 
outcomes in clinical studies. 
Inhibition of Prostate Cancer Cell 
Proliferation by Muscadine Grape 
Components  
Actively growing human LnCaP or PC3 
prostate cancer cells were plated into 
individual wells of 24 well plates and treated 
for 7 days with increasing concentrations of 
extracts from either muscadine grape seeds 
or skins, to determine whether the extracts 
reduced prostate cancer cell proliferation.  As 
shown in Figure 1, extracts from both grape 
seeds and skin reduced the proliferation of 
LnCaP or PC3 human prostate cancer cells.  
The responses were dependent upon the 
dose of extract that was used and similar 
responses were obtained with grape seed 
extract compared to grape skin extract.   
Inhibition of Breast Cancer Cell Proliferation 
by Muscadine Grape Components  
Actively growing human breast cancer cells—
ZR-75-1 ER+ breast cancer cells, MDA-MB-
231 or SKBR3 triple negative breast cancer 
cells--were plated into individual wells of 24 
well plates and treated for 7 days with 
increasing concentrations of extracts from 
either muscadine grape seeds or skins, to 
determine whether the extracts reduced 
breast cancer cell proliferation. As shown in Figure 2, extracts from both grape seeds and 
skin reduced the proliferation of ZR-75-1, MDA-MB-231 and SKBR3 human breast cancer 
cells. The responses were dependent upon the dose of extract that was used and similar 
responses were obtained with grape seed extract compared to grape skin extract. 
Con     
.5
5
50
    
.5
5
50
0
50
100
150
**
**
**
**
   Nature's Pearl
Grape Seed Extract
         (g/mL)
   Nature's Pearl
Grape Skin Extract
         (g/mL)
LnCaP
Cell Growth
PC3
Con
  
.5
5
50
  
.5
5
50
0
50
100
150
**
**
**
   Nature's Pearl
Grape Seed Extract
         (g/mL)
   Nature's Pearl
Grape Skin Extract
         (g/mL)
Cell Growth
Figure 1. LnCaP or PC3 human prostate 
cancer cells were incubated with increasing 
concentrations of either muscadine grape 
seed or grape skin extracts and cell 
proliferation was quantified by counting the 
number of cells per well. The data are 
presented as the percent of the Control 
(Con), which was not treated with either 
extract. n = 12, ** denotes p < 0.01. 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 9 of 31 
 
Inhibition of MAP Kinase Activity by Muscadine Grape Components 
Human MDA-MB-231 or ZR-75-1 breast 
cancer cells were incubated with 50 µg/mL 
muscadine grape seed or grape skin extract 
for increasing periods of time. MAP kinase 
activity was quantified in cell extracts by 
Western blot hybridization using an antibody 
against 
phospho-ERK1/ERK2 
from 
Cell 
Signaling. The decrease in phospho-ERK 
activity was calculated by densitometry using 
β-actin as a loading control. As shown in 
Figure 3, the extracts from both muscadine 
grape seeds and grape skins caused a 
significant 
reduction 
in 
phospho-ERK, 
suggesting that the extracts significantly 
reduced proliferation. There was no significant 
difference in the response to grape seed 
versus grape skin extracts. 
Because treatment with muscadine grape 
seeds versus skins showed no significant difference in effect on cancer cell lines, the 
additional preclinical studies were performed with a muscadine grape liquid extract 
containing both seeds and skins. 
 
Reduction in Tumor Growth by Muscadine Grape Liquid Extract in c-neu Mice 
ZR-75-1
Con     
.5
5
50
    
.5
5
50
0
50
100
150
**
**
Grape Seed Extract
  (g/mL)
Grape Skin Extract
 (g/mL)
Cell Proliferation
MDA-MB-231
Con
  
.5
5
50
  
.5
5
50
0
50
100
Grape Seed Extract
         (g/mL)
Grape Skin Extract
         (g/mL)
**
**
**
** *
Cell Proliferation
SKBR3
Con
  
.5
5
50
  
.5
5
50
0
50
100
Grape Seed Extract
         (g/mL)
Grape Skin Extract
(g/mL)
**
**
**
*
*
Cell Proliferation
Figure 2.  ZR-75-1, MDA-MD-23 or SKBR3 human breast cancer cells were incubated with increasing concentrations of 
either muscadine grape seed or grape skin extracts and cell proliferation was quantified by counting the number of cells 
per well.  The data is presented as the percent of the Control (Con), which was not treated with either extract. n = 9 for 
ZR-75-1 cells, 6 for MDA-MB-231 cells and 18 for SKBR3 cells; * denotes p < 0.05, ** denotes p < 0.01.   
Figure 3.  ZR-75-1 and MBA-MD-231 human 
breast cancer cells were incubated with 50 µg/mL 
muscadine grape seed extract (GSeE) or 
muscadine grape skin extract (GSkE) for 4 h and 
phospho-ERK (both ERK1 and ERK2) were 
quantified by densitometry using β-actin as a 
loading control.  n = 3-7, *** denotes p < 0.001. 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 10 of 31 
The effect of muscadine grape liquid extract 
(MGLE) was tested on female FVB-Tg(MMTV) 
NKAMul/J transgenic mice, in which the 
activated rat Erbb2 (c-neu) oncogene, under the 
direction of the mouse mammary tumor virus 
promoter expressed specifically in the mammary 
gland (c-neu mice), form mammary tumors (the 
equivalent of human breast cancer) by 6 - 8 
months of age.  Mice were treated with MGLE 
beginning at 3 weeks of age (at the time of 
weaning) and were sacrificed at 7½ months of 
age.  The dose of MGLE was approximately 1 
mg of phenolics for a 25 gram mouse (and is 
equivalent to approximately 10 tablespoons or 
148 mL of extract per day for a 70 kg man and 
an average concentration of 20,000 mg of 
phenolics/liter of MGLE).  The muscadine grape 
liquid extract was added to the drinking water. 
Eighteen mice were treated for an average of 
220 days (beginning treatment at 21 days of age, at the time of weaning).  Ten (10) mice 
drank regular drinking water (control) and 8 mice drank water containing the MGLE.  
Although all of the mice developed tumors throughout their mammary tissue, there was a 
significant difference in the weight of the tumor mass, as shown in Figure 4.   
Mammary tissue was fixed in 4% formalin, 
paraffin embedded and sectioned into 5 
micron sections, for immunohistochemical 
analysis. Sections were incubated with an 
antibody to Ki67, as a measure of cell 
proliferation. As shown in Figure 5, tumors 
from mice that drank MGLE had a 
significant decrease in the number of 
proliferating cells. These results indicate 
that 
muscadine 
grape 
liquid 
extract 
reduces cell proliferation, in agreement 
with reduction in cell number and MAP 
kinase activation observed in cultured 
breast cancer cells.   
Tumor sections from c-neu mice drinking 
regular water (control) or MGLE were 
incubated 
with 
an 
antibody 
to 
the 
endothelial cell marker, CD34, and vessels were identified by a combination of 
morphology and positive CD34 immunoreactivity. As shown in Figure 6, tumors from mice 
drinking MGLE had fewer blood vessels than mice drinking regular water, suggesting that 
the grape extract inhibits angiogenesis.   
Interstitial tumoral fibrosis was quantified in breast tumor tissue sections from c-neu mice 
and stained with picrosirius red, a nonspecific collagen stain. Collagen reaction product 
was markedly reduced by MGLE administration as compared to controls (Figure 7).  
Treatment with the MGLE reduced interstitial fibrosis by more than 50%, indicating that 
the extract reduces cancer-associated fibrosis in breast tumors.  
            
C o n tr o l
M G L E
0 .0
2 .5
5 .0
7 .5
1 0 .0
1 2 .5
**
T u m o r W e ig h t (g ra m s )
 
Figure 4.  Mice (c-neu) were given MGLE 
in their drinking water beginning at 3 
weeks of age.  Control mice drank regular 
water.  The mice were euthanized after 
220 days and their mammary tumors 
were removed and weighed.  n = 8-10, ** 
indicates p < 0.01.   
C o n tr o l
M G L E
0
1 0 0
2 0 0
3 0 0
4 0 0
**
K i6 7  p o s itiv e  c e lls
Figure 5.  Sections of tumors from mice drinking 
water (Control) or MGLE were incubated with an 
antibody to the proliferative marker Ki67.  
Representative samples are shown to the left.  n 
= 6–8, ** denotes p < 0.01. 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 11 of 31 
 
 
 
 
 
 
 
 
 
 
1.2 
Preclinical toxicity studies with MGE  
 
The animal studies above were performed with muscadine grape liquid extract from 
Nature’s Pearl (Advance, NC), which was added directly to the rodent drinking water. To 
study the effects of muscadine grape extract in humans, the muscadine grape liquid 
extract is dried, converted to powder form, and encapsulated.  This proprietary product 
produced by Nature’s Pearl is the muscadine grape extract (MGE) used in the preclinical 
toxicity studies below and in the phase 1 clinical study proposed. However, because 
rodents do not swallow capsules, the MGE powder is reconstituted and added to the 
drinking water for the toxicity studies described below.    
 
The primary goal of the rodent toxicity tests on the Nature’s Pearl MGE was to identify the 
range of doses that caused no life-threatening or adverse effects, thereby providing critical 
information for conduct of a Phase I clinical trial to assess toxicity in humans with cancer 
and establishing an estimated clinical margin of safety. Toxicity tests on the proprietary 
MGE that will be used in clinical testing were performed in mice, to assess effects of the 
extract on the general health of the mice as well as their hearts, liver, lungs and kidneys.   
 
C o n tr o l
M G L E
0
2
4
6
8
*
V e s s e l D e n s ity
Figure 6.  Sections of tumors from Control or 
MGLE-treated mice were incubated with an 
antibody to CD34 and blood vessels were identified 
by positive immunoreactivity and morphology.  n = 
8-10, ** denotes p< 0.05. 
C o n tr o l
M G L E
0
1
2
3
4
***
P e rc e n t F ib ro s is /F ie ld
Figure 7.  Sections of tumors from Control or 
MGLE-treated 
mice 
were 
stained 
with 
Picrosirius red to measure tissue fibrosis.  n = 
8-10, *** denotes p < 0.001 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 12 of 31 
Male C57 black mice (8 weeks of age) were randomized into groups to receive drinking 
water alone (control) or MGE in the drinking water at four escalating doses of MGE as 
determined by the measurement of total phenolics. Daily observations of the mice included 
evaluations for weight loss, diarrhea, dehydration, edema, abdominal enlargement or 
ascites, progressive dermatitis, rough hair coat or unkempt appearance due to lack of 
grooming, hunched posture indicative of pain, lethargy or persistent decumbency due to 
loss of appetite, coughing, labored breathing, nasal discharge, jaundice, cyanosis, 
pallor/anemia, neurological signs indicated by inappropriate head carriage or shaking of 
the head, bleeding from any orifice, or any condition interfering with daily activities. Prior 
to sacrifice, the mice were placed in metabolic cages for 24 hours, and food intake and 
fecal and urine output were measured to quantify markers of renal damage. During the 4th 
week of treatment, blood pressure was determined by tail cuff plethysmography in 
conscious mice and cardiac function was assessed in mice anesthetized with isofluorane, 
using the non-invasive, small animal VEVO ultrasound imaging system in the M and B 
modes, to measure or calculated ejection fraction, fractional shortening, stroke volume, 
heart rate and cardiac output were determined.  The mice were sacrificed after one month 
of treatment; tissues (heart, kidney, lung, liver, spleen and brain) were weighed, fixed in 
4% formalin, embedded in paraffin, sectioned at 5 microns and stained with Hematoxylin 
& Eosin (H & E) for analysis by a veterinary pathologist to assess any gross structural 
abnormalities. 
 
Four concentrations of the MGE were tested.  The range of concentrations to be tested 
was based upon previous studies in the Gallagher/Tallant laboratory reported above. In 
those studies, a daily concentration of 1 mg of total phenolics/mouse/day was effective at 
reducing tumor multiplicity.  For the toxicity studies in mice, the four doses of the MGE 
tested were 0.25, 0.5, 1.0 and 2.0 mg total phenolics/mouse/day, administered to the mice 
in their drinking water. Total phenolic content was determined using the Folin-Ciocalteau 
reagent with gallic acid as the standard.  Since an average mouse of this age weighs 
approximately 25 g or 0.025 kg, this corresponds to a dose range of 10, 20, 40 and 80 mg 
total phenolics/kg/day.  Based upon an average mass of 70 kg for a patient, this would 
correspond to 700, 1400, 2800, and 5600 mg phenolics/day. The proprietary Nature’s 
Pearl muscadine grape extract capsule contains approximately 162 mg total 
phenolics/capsule, this would correspond to taking 5, 10, 21 and 42 capsules/day.   
 
Results 
During the 4 week administration of the MGE at the four doses, there were no observations 
of diarrhea, dehydration, edema, abdominal enlargement, loss of hair coat, reduced 
grooming, or decreased level of activity, appetite, drinking or breathing.  There were no 
indications of pallor, jaundice or cyanosis or any neurological signs of discomfort. There 
was no bleeding or discharge. These results suggest that the mice were free from any 
pathology that affected their normal daily activities or health. 
 
The mice were weighed at the end of the treatment period, to determine the effect of the 
MGE on total body weight.  The weight of the mice at the end of the 4-week treatment 
period was similar with the three lowest concentrations of the MGE compared to the 
control group.  Mice treated with the highest concentration of MGE had a small but 
significant decrease in total body weight (from 28.6 ± 1.2 grams to 25.0 ± 1.3 grams, which 
represents less than 15% of their total body weight). However, there was no effect on food 
intake, fecal output or urine volume compared to the untreated mice, measured during the 
last week of treatment, suggesting little effect of the MGE on eating and drinking 
behaviors. 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 13 of 31 
 
At the time of sacrifice, various organs (heart, lung, liver, kidney, spleen and brain) were 
removed and weighed and the organ weights of mice treated with each dose of MGE were 
compared to the untreated control group. A portion of each organ was fixed in formalin, 
sectioned and stained with H & E, for assessment by a veterinary pathologist.  In addition, 
the heart and kidney were assessed by echocardiography and radioimmunoassay of 
urinary proteins, respectively, as a further measure of heart and renal function. There was 
no difference in the weight or structure of the hearts of mice treated with increasing doses 
of MGE. Cardiac parameters including ejection fraction, fractional shortening, cardiac 
output, stroke volume and heart rate were all similar in mice treated with MGE compared 
to untreated Control mice. There was no difference in the weight or structure of the lungs 
of mice treated with MGE compared to the untreated control mice. The weight of the livers 
of mice treated with the two lowest MGE (0.25 and 0.5 mg phenolics/mouse/day) and the 
highest MGE (2.0 mg phenolics/mouse/day) concentrations were no different than the 
weight of the livers of the untreated mice.  However, the weight of the livers of mice in the 
1.0 mg phenolics/mouse/day) were slightly increased compared to the control. Spleen 
weight of mice treated with MGE was not significantly different than untreated control mice 
and the spleens of mice were generally within normal limits with one mouse in the highest 
MGE treatment group having some extra medullary hematopoiesis. The weight of the 
brains of mice treated with MGE was not significantly different than untreated mice. The 
brains were subdivided into four regions—cortex, cerebellum, hypothalamus and 
brainstem. All brain regions examined were within normal limits.  
 
Conclusions from toxicity studies and corresponding clinical doses: 
Little toxicity was observed at any of the doses tested. Therefore, the level of phenolics 
used to treat the mice suggests that this level of phenolics and corresponding numbers of 
capsules will likely be tolerated by patients. However, based upon patient compliance, a 
recommended dose range for patients would include 2, 4, 6, 8 and 10 capsules/day 
(corresponding to approximately 5, 9, 14, 18 and 23 mg phenolics/kg/day). 
 
1.3 
Clinical Studies 
 
Muscadine grape skin extract has been evaluated in clinical trials to a limited extent in the 
setting of cardiovascular disease23 and diabetes.24 In a randomized, double-blind, 
placebo-controlled crossover trial, 50 adults with coronary disease or ≥1 cardiac risk factor 
received muscadine grape seed supplementation (1300 mg daily) and placebo for 4 weeks 
each, with a 4-week washout, and no adverse events were reported.23 Similarly, a 
randomized study of non-diabetic and type 2 diabetic participants using 150 mL of 
muscadine grape juice, muscadine grape wine, and dealcoholized muscadine grape wine 
over a 28-day period had no adverse events reported.24 
 
A muscadine grape product has also been examined in a phase 1/2 trial of men with 
biochemically recurrent prostate cancer.25 The phase 1 portion of the study evaluated 
increasing doses of pulverized muscadine grape skin in a product called MPX (Muscadine 
Naturals Inc, Clemmons NC) in cohorts of two patients, with six patients at the highest 
dose of 4,000 mg (n=14).  Dose selection was based on unpublished preclinical work in 
animal studies.  Four patients experienced adverse gastrointestinal symptoms possibly 
related to the study agent, including grade 1 flatulence, grade 1 soft stools, and grade 1 
burping. No other related adverse events were reported and one patient reported 
improvement of chronic constipation. Six of the 14 patients came off study for disease 
progression (five metastatic, one rising PSA) after exposure for a median of 15 months. 

 
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One patient came off study for myasthenia gravis that was unrelated to treatment. Seven 
patients remained on the study. The lack of dose-limiting toxicities led to the selection of 
4,000 mg/d as the highest dose for further study. Median within-patient PSA doubling time 
increased by 5.3 months (non-significant, P=0.17). Based on the favorable safety profile 
and a possible lengthening of PSA doubling time, the phase 2 portion of this study was 
expanded to 125 patients randomized to placebo, 500 mg/d, or 4000 mg/d MPX arms. 
Recruitment was completed in October 2014 with results pending.  
 
In summary, muscadine grape products have shown anti-cancer activity in preclinical 
models and have demonstrated a favorable safety profile in preclinical and clinical studies. 
This Phase 1 study using MGE represents an opportunity to translate promising preclinical 
data supporting the anti-cancer properties of a natural product into clinical trials. 
 
 
2.0 
Objectives 
 
2.1 
Primary Objective 
 
2.1.1 To determine the safety and MTD of MGE after 4 weeks of administration 
for patients with metastatic solid tumors. 
 
2.2 
Secondary Objectives 
 
2.2.1 To monitor adverse events/toxicity every 4 weeks while on treatment 
 
2.2.2 To evaluate change in phenolic levels (total and component, blood and 
urine) from baseline to 4 and 8 weeks on MGE  
 
2.2.3 To evaluate change in serum cytokines and growth factors from baseline 
to 4 and 8 weeks on MGE 
 
2.2.4 To observe the response rate of MGE in patients with metastatic cancer. 
 
2.2.5 To assess overall and progression-free survival in patients with metastatic 
cancer receiving MGE. 
 
2.2.6 To assess global quality of life (FACT-G) and fatigue (PROMIS-fatigue 
short form) in cancer patients taking MGE. 
 
2.2.7 To assess adherence to MGE treatment. 
 
 
3.0 
Patient Selection 
3.1 
Inclusion Criteria  
 
3.1.1 Patients must have histologically confirmed solid tumor malignancy 
(excluding primary brain tumor) that is metastatic or unresectable and have 
failed standard therapies. Patients are also eligible if patients declined (or 
if their physicians determined them unsuitable for) standard therapy 
options. 

 
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3.1.2 
≥ 18 years of age  
 
3.1.3 
The effects of MGE on the developing human fetus are unknown.  Women 
of child-bearing potential and men must agree to use adequate 
contraception (hormonal or barrier method of birth control; abstinence) prior 
to study entry and for the duration of study participation. Should a woman 
become pregnant or suspect she is pregnant while participating in this 
study, she should inform her treating physician immediately. 
 
3.1.4 Ability to understand and the willingness to sign an IRB-approved informed 
consent document. 
 
3.1.5 ECOG performance status ≤2. 
 
3.1.6 Patients must have adequate organ and marrow function as defined below: 
-   absolute neutrophil count >1000/mcL 
-   platelets 
>50,000/mcL 
-   total bilirubin 
within normal institutional limits 
-   AST(SGOT)/ALT(SGPT) <2.5 X institutional upper limit of normal 
-   creatinine clearance 
>40 mL/min 
 
3.1.7 Stable supplement usage for >2 weeks prior to starting and agrees not to 
change while on this study.   
 
3.1.8 Life expectancy ≥ 3 months 
 
3.1.9 Patients who declined standard therapies or whose physicians determined 
they were not suitable for standard therapy options are eligible.  
3.2 
Exclusion Criteria  
 
3.2.1 Patients who have had chemotherapy or radiotherapy within 2 weeks (6 
weeks for nitrosoureas or mitomycin C) prior to entering the study or those 
who have not recovered from adverse events due to agents administered 
more than 2 weeks earlier. 
 
3.2.2 Patients may not be receiving any other investigational cancer-directed 
agents. 
 
3.2.3 History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to MGE. 
 
3.2.4 Patients unable to take oral medications or those with history of 
malabsorption due to bowel resection or gastrointestinal disease. 
 
3.3.5   Patients with uncontrolled diarrhea or persistent nausea/vomiting requiring 
daily antiemetic therapy for symptom management within the past 21 days 
 
3.2.6 Uncontrolled intercurrent illness including, but not limited to ongoing or 
active infection, symptomatic congestive heart failure, unstable angina 

 
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pectoris, cardiac arrhythmia, or psychiatric illness/social situations that 
would limit compliance with study requirements. 
 
3.2.7 Pregnant women are excluded from this study because of potential for 
teratogenic or abortifacient effects with using MGE. Because there is an 
unknown but potential risk for adverse events in nursing infants secondary 
to treatment of the mother with MGE, breastfeeding should be 
discontinued. 
 
3.2.8 Patients with primary brain tumors are excluded due to lack of data on MGE 
crossing the blood brain barrier. Patients with metastatic brain tumors may 
be enrolled. 
 
3.3 
Inclusion of Women and Minorities 
 
Men and women of all races and ethnicities who meet the above-described 
eligibility criteria are eligible to participate in this study.  
 
The study consent form will also be provided in Spanish for Spanish-speaking 
participants. We anticipate enrolling between 2 and 30 participants on this study. 
Based on CCCWFU population estimates, we expect approximately 40% of 
participants to be women. Translating this to our maximum sample size estimate 
of 30, we plan to enroll at least 12 women if we reach our maximum accrual. We 
do not expect the percentage of Hispanic/Latino or racial minority cancer patients 
eligible for this study to be higher than the percentage of Hispanic or racial minority 
new cancer patients seen at CCCWFU (1.7% and 14.4%, respectively); therefore, 
we plan to enroll at least 4 racial minority and 1 Hispanic/Latino patient if we reach 
our maximum accrual. 
 
Should we not meet or exceed these estimates, the PI will engage the Cancer 
Center Health Equity Advisory Group to discuss strategies to enhance recruitment 
in these target populations. 
 
 
4.0 
Registration Procedures 
 
Patients must be registered prior to the initiation of treatment.   
 
You must perform the following steps in order to ensure prompt registration of your patient: 
 
1. Complete the Eligibility Checklist (Appendix B) 
2. Complete the Protocol Registration Form (Appendix A) 
3. Alert the Cancer Center registrar by phone, then send the signed Informed Consent Form, 
Eligibility Checklist and Protocol Registration Form to the registrar, either by fax or e-mail. 
Protocol Registration is open from 8:30 AM - 4:00 PM, Monday-Friday. 
Contact Information: 
Protocol Registrar PHONE (336) 713-6767 
Protocol Registrar FAX (336) 713-6772 
Protocol Registrar E-MAIL (registra@wakehealth.edu)      
4. Fax/e-mail ALL eligibility source documents with registration. Patients will not be registered 
without all required supporting documents. 

 
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Note: If labs were performed at an outside institution, provide a printout of the results. 
Ensure that the most recent lab values are sent. 
 
To complete the registration process, the Registrar will:   
 
 
assign a patient study number 
 
assign the patient a dose 
 
register the patient on the study 
 
 
5.0 
Study Outcomes and Study Measures 
 
5.1 
Primary Outcome 
 
5.1.1 The primary outcome measure will be dose limiting toxicity (DLT) assessed 
by SAEs in patients with metastatic cancer receiving MGE under the 
proposed regimen at 4 weeks.  
 
5.2 
Secondary Outcomes 
 
5.2.1 Adverse events/toxicity at weeks 4, 8 and every 4 weeks thereafter if 
remain on treatment 
 
5.2.2 Identification of measured phenolic levels (total and component phenolics) 
at baseline,4, and 8 weeks 
 
5.2.3 Evaluate change in systemic cytokines, growth factors from baseline to 4 
and 8 weeks including but not limited to: TNFα, IL6, IL6R, IL8, IL1α, IL1β, 
IGF-1, VEGF  
 
5.2.4 Overall response rate (CR, PR, and SD) at 8 weeks. 
 
5.2.5 PFS and OS in patients with metastatic cancer receiving MGE under the 
proposed regimen. 
 
5.2.6 Quality of life in cancer patients taking MGE using the Functional 
Assessment of Cancer Therapy–General (FACT-G) measure and fatigue, 
as measured by the PROMIS Fatigue-Short Form. 
 
5.2.7 Adherence to MGE treatment, as assessed by patient-reported medication 
logs and pill count every 4 weeks while on treatment. 
 
 
 

 
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6.0 
Treatment Plan 
6.1 
Study-Related Activities  
 
Measures / Forms 
Pre- 
Studya Baseline Week 
2h 
Week 
4j 
Week 
8k 
Every 4 
weeks k Off-study 
Informed consent 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Medical history 
X 
 
 
X 
X 
X 
 
Concurrent meds 
X 
 
 
X 
X 
X 
 
Physical exam 
X 
 
 
X 
X 
X 
 
Vital signs 
X 
 
 
X 
X 
X 
 
Weight 
 
X 
 
 
 
 
 
Height 
 
X 
 
 
 
 
 
ECOG Performance Status 
X 
 
 
X 
X 
X 
 
Pill Diary 
 
X 
 
X 
X 
Xi 
 
Adherence assessment 
 
 
X 
X 
X 
 
 
Quality of life and fatigue 
assessments 
 
X 
 
X 
X 
X 
 
Tumor measurement by imagingb 
X 
 
 
 
X 
Xb 
 
Tumor markers (if applicable)c 
 
X 
 
X 
X 
 
 
CBC w/diff, platelets 
X 
X 
 
X 
X 
X 
 
Serum chemistriesd 
X 
X 
 
X 
X 
X 
 
B-HCGe 
X 
 
 
 
 
 
 
INR checkf 
 
 
X 
 
 
 
 
Phenolic levels (blood and urine) 
 
X 
 
X 
X 
 
 
Research labs (cytokines and 
growth factors) 
 
X 
 
X 
X 
 
 
Adverse event evaluation 
 
X 
X 
X 
X 
X 
 
Vital statusg 
 
 
 
 
 
 
X 
a Pre-study imaging listed in table must be completed within 42 days prior to registration. Additional 
pre-study activities need not be repeated if performed within 10 days of starting study drug. 
b CT imaging or other imaging modality will be obtained at baseline (within 42 days of registration) 
and at 8 weeks (± 7 days).  Imaging will then be q12 weeks (± 28 days) for as long as the patient 
remains on study. CT, PET/CT, or MRI are acceptable imaging modalities and all imaging will be 
considered per standard of care for patients on active cancer treatments. 
c Tumor-specific markers may include but are not limited to testosterone level and PSA for prostate 
cancer patients, CEA for colorectal patients, and CA19-9 for pancreatic cancer patients as performed 
per standard of care.  
 
d Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, 
glucose, potassium, total protein, SGOT[AST], SGPT[ALT], sodium. 
 
e Serum pregnancy test (women of childbearing potential). 
f Patients taking warfarin will have INR checked within 2 weeks of starting MGE. This can be 
performed locally. 
      g Vital status will be assessed by chart review or phone every 6 weeks (± 14 days) after  
      patient comes off study. 
      h Phone contact will occur 2 weeks after each clinic visit (± 3 days) until the Week 8 visit is complete. 
i Patients will not be asked to complete a pill diary after 6 cycles (24 weeks). 
j Week 4 study appointments will be scheduled at day 28 ±3 days. 
k Study appointments at Week 8 and after will be scheduled 28 days after the prior appointment, ± 7 
days  
 
 
 
 

 
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6.2 
Treatment Administration 
Treatment will be administered on an outpatient basis. Reported adverse events 
and potential risks are described in Section 8.0. No investigational or commercial 
agents or therapies other than those described below may be administered with 
the intent to treat the patient's malignancy. 
 
Dose-Escalation Schedule 
Dose Level 
Dose of MGE 
Level 1 
1 pill po BID 
Level 2 
2 pills po BID 
Level 3 
3 pills po BID 
Level 4 
4 pills po BID 
Level 5 
5 pills po BID 
 
6.2.1 Definition of Dose-Limiting Toxicity (DLT) 
Dose-limiting toxicity (DLT) will be defined as grade 3 gastrointestinal side 
effects attributable to treatment or any grade 4 or 5 adverse event 
attributable to treatment as per the Common Terminology Criteria for 
Adverse Events (CTCAE) 4.0 criteria. Any adverse event attributable to 
treatment and resulting in permanent discontinuation of MGE will be 
considered a DLT. Toxicity will be assessed at 4 weeks. Any DLTs that 
occur 1 day (day 29) after the 4 week period will be evaluated and 
considered as potentially attributable to the administration of MGE during 
the first 4 weeks. The washout period for this drug is 24 hours. Dose 
escalation will proceed within each cohort as follows:  
 
Number of Patients 
with DLT at a Given 
Dose Level 
Escalation Decision Rule 
0 out of 3 
Enter 3 patients at the next dose level. 
> 2 
Dose escalation will be stopped. This dose level will be 
declared the maximally administered dose (highest dose 
administered). Three (3) additional patients will be entered 
at the next lowest dose level if only 3 patients were treated 
previously at that dose. 
1 out of 3 
Enter at least 3 more patients at this dose level. 
 
If 0 of these 3 patients experiences DLT, proceed to 
the next dose level. 
 
If 1 or more of this group suffer DLT, then dose 
escalation is stopped, and this dose is declared the 
maximally administered dose. Three (3) additional 
patients will be entered at the next lowest dose level if 
only 3 patients were treated previously at that dose. 
<1 out of 6 at highest 
dose level below the 
maximally 
administered dose 
This is generally the recommended phase 2 dose 
(Maximum Tolerated Dose [MTD]). At least 6 patients 
must be entered at the recommended phase 2 dose. 

 
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6.2.2 Dose modification in the event of toxicity 
In the event of dose-limiting toxicity, the patient will be removed from the 
study and the escalation decision rules defined above will be followed.  
There will be no dose-modifications on study. Either the patient or the 
treating physician can chose to discontinue MGE at any time. If necessary, 
MGE can be held for up to 7 days in the first month and for up to 21 days 
subsequently, as described in Section 6.5.1. 
 
6.3 
Study procedures 
 
6.3.1 Recruitment 
 
6.3.1.1 
Potentially eligible patients will be recruited from the Wake 
Forest Comprehensive Cancer Center 
6.3.1.2 
The research nurse (RN) will be contacted to review eligibility 
and to consent eligible patients in person. 
6.3.1.3 
The RN will obtain baseline surveys, clinical information and 
ensure research labs are drawn and processed per protocol. Pill 
dairy will be provided at each visit. 
6.3.1.4 
Study drug (30-day supply) will be provided to participant by the 
RN. 
 
6.3.2 Follow-up Assessments 
 
6.3.2.1 
Study coordinator will contact participants 2 weeks after each 
clinic visit until week 8 to assess medication adherence and 
ascertain any potential adverse events. 
6.3.2.2 
The RN will review AE with the patient at each follow-up visit in 
clinic, perform pill count, obtain clinical information per protocol 
and administer surveys at 4 and 8 weeks per protocol. RN will 
obtain study labs are each visit and provide 30-day supply of 
MGE at prescribed dose level. 
 
6.4 
General Concomitant Medication and Supportive Care Guidelines 
 
Patients should receive full supportive care, including transfusions of blood and 
blood products, antibiotics, antiemetics, etc., as clinically indicated. Anti-
inflammatory or narcotic analgesics may be offered as needed. Medications 
considered necessary for the patient's well-being may be given at the discretion of 
the managing physician, i.e., chronic treatments for concomitant medical 
conditions, as well as agents required for life-threatening medical problems, etc. 
The reason(s) for treatment, dosage, and dates of treatment should be recorded 
on the flow sheets. 
 
Participants on warfarin should have their INR checked within 2 weeks of starting 
MGE to ensure there is no clinically significant interaction. Further blood work to 
monitor warfarin levels are at the discretion of the treating physician per usual care. 
 
 

 
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6.5 
Duration of Therapy 
 
In the absence of treatment delays due to adverse events, treatment may continue 
until one of the following criteria applies: 
 
 
Disease progression, either by standard imaging or clinical parameters per the 
judgment of the treating physician, 
 
Intercurrent illness that prevents further administration of treatment, 
 
Unacceptable adverse event(s), 
 
Patient decides to withdraw from the study, or 
 
General or specific changes in the patient's condition render the patient 
unacceptable for further treatment in the judgment of the treating physician. 
 
6.5.1 Missed doses 
 
If a dose of MGE is missed, the patient will be instructed to take their next 
dose as scheduled. Missed doses should not be added to the next dose.  
 
 
6.5.1.1 Missed doses during first 28 days 
 
If the patient is unable to take their dose of MGE for more than 7 
consecutive days due to unforeseen circumstances unrelated to 
drug toxicity, then the subject will not be evaluable for the primary 
outcome and will be removed from the study. Participants who 
come off study prior to 28 days for reasons other than toxicity will 
be evaluated for DLT during the washout period (24 hours, per 
section 6.2.1). 
 
 
6.5.1.2 Missed doses after first 28 days 
 
If treatment must be held for any reason, it must be restarted within 
21 days or the patient will be removed from the study.   
 
6.6 
Duration of Follow Up 
 
Patients removed from the study for unacceptable adverse events will be followed 
until resolution or stabilization of the adverse event. Patients who are removed or 
withdraw from the study for any reason other than toxicity will be followed for vital 
status only after the 24 hour washout period. Patients will be followed every 6 ± 2 
weeks until death for monitoring survival study endpoints. 
 
 
7.0 
Measurement of Effect 
 
Although response is not the primary endpoint of this trial, patients with measurable disease will 
be assessed by the treating physician, based on the clinical radiologic report and clinical status, 
per usual care.  For the purposes of this study, patients will be reevaluated with imaging 8 weeks 
(± 7 days) after study initiation and then every 12 weeks (± 28 days) while on MGE.   
 

 
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7.1 
Definitions  
 
 
 
Evaluable for toxicity (primary outcome): All patients will be evaluable for toxicity 
from the time of their first treatment with MGE. Per section 6.5.1, patients who miss 
7 consecutive days of treatment in the first 28 days for reasons other than toxicity 
are not evaluable and will be removed from the study and replaced. 
 
Inevaluable for objective response: When no imaging/measurement is done at all 
at a particular time point, the patient is not evaluable (NE) at that time point.   
 
Measurable disease: Measurable lesions are defined as those that can be 
accurately measured in at least one dimension (longest diameter in the plane of 
measurement is to be recorded) with a minimum size of 10 mm by CT scan, 10 
mm caliper measurement by clinical exam, or 20 mm by chest X-ray. 
 
Non-measurable lesions: Non-measurable lesions include: bony metastases, 
leptomeningeal disease, ascites, pleural or pericardial effusion, inflammatory 
breast disease, lymphangitic involvement of skin or lung, abdominal masses or 
organomegaly identified by physical exam that is not measurable by reproducible 
imaging techniques. 
 
Progression-Free Survival is defined as the duration of time from the start of 
treatment to the time of progression or death. Patients will be followed until disease 
progression and death. 
 
Overall Survival is defined as the duration of time from the start of treatment to 
date of death. 
 
7.2 
Methods for Evaluation of Measurable Disease 
 
 
The same method of assessment and the same technique should be used to 
characterize each identified and reported lesion at baseline and during follow-up 
whenever possible. Measurements will be obtained from usual care imaging and 
assessed by the PI(s) in confirmation with the physician of record. 
 
 
CT is the best currently available and reproducible method to measure lesions 
selected for response assessment. MRI and PET/CT are also acceptable. 
 
Lesions on a chest X-ray may be considered measurable lesions if they are 
clearly defined and surrounded by aerated lung. However, CT is preferable. 
 
Clinical lesions will only be considered measurable when they are superficial 
and ≥10 mm in diameter as assessed using calipers. For the case of skin 
lesions, documentation by color photography, including a ruler to estimate the 
size of the lesion, is recommended. 
 
Tumor markers alone will not typically be used to define response. If markers 
are initially above the upper normal limit, they must normalize for a patient to 
be considered in complete response. 
 
Cytology and histology can be used in rare cases (e.g., for evaluation of 
residual masses to differentiate between Partial Response and Complete 
Response or evaluation of new or enlarging effusions to differentiate between 
Progressive Disease and Response/Stable Disease). 

 
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8.0 
Adverse Events List and Reporting Requirements 
 
8.1 
Adverse Event List for MGE 
 
Expected: Based on previous studies, only minimal AEs are expected, including 
flatulence, diarrhea, nausea, dyspepsia, and abdominal cramping. 
 
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 4.0 will be utilized for AE reporting. All appropriate 
treatment areas should have access to a copy of the CTCAE version 4.0. A 
copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov). 
 
‘Expectedness’: AEs can be ‘Unexpected’ or ‘Expected’ (see Section 8.1 
above) for expedited reporting purposes only.   
 
Attribution of the AE: 
- 
Definite: The AE is clearly related to the study treatment. 
- 
Probable: The AE is likely related to the study treatment. 
- 
Possible: The AE may be related to the study treatment. 
- 
Unlikely: The AE is doubtfully related to the study treatment. 
- 
Unrelated: The AE is clearly NOT related to the study treatment. 
 
8.3 
STRC SAE Reporting Requirements 
 
The Safety and Toxicity Reporting Committee (STRC) is responsible for reviewing SAEs 
for CCCWFU Institutional studies as outlined in Appendix C. STRC currently requires that 
all unexpected 4 and all grade 5 SAEs on these trials be reported to them for review. All 
CCCWFU Clinical Research Management (CRM) 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, 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 

 
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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 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. 
 
 
9.0 
Pharmaceutical Information 
 
Muscadine grape extract is a nutraceutical compound. 
   
A list of the adverse events and potential risks associated with the investigational agent 
administered in this study can be found in Section 8.0. 
 
9.1 
Drug Accountability 
 
Drug accountability logs will be provided and 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)  
 
 
Product description: MGE will be supplied by Nature’s Pearl (Advance, North 
Carolina). 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 obtained from 
CapsCanada (Pompano Beach, Florida), bottled, and labeled at Nature’s Pearl. 
Quality control testing for antimicrobial contamination (aerobic count, yeast and 
mold, E.coli/coliform, staph aureus, enterobacteriaceae, and salmonella) and 
phenolic levels occurs numerous times during the production process.   
 
Storage requirements: Capsules should be stored at room temperature. 
 
Route of administration: MGE is available for oral administration. 
 
 
10.0 
Laboratory Studies 
 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 25 of 31 
10.1 
Phenolic assays 
  
Total and component phenolics will be measured from blood and urine at baseline, 4 
weeks, and 8 weeks if the patient remains on MGE. Blood assays will require 
approximately 10ml of blood. For the polyphenolic analysis26 one tube of blood at each 
time point will be collected in tubes containing EDTA and ascorbic acid and stored at -
80°C for an indefinite period of time. The blood sample will be thawed on ice and used to 
measure polyphenols by ultra-high performance liquid chromatography (UPLC) coupled 
to mass spectroscopy detection (UPLC-MS). 
 
A spot urine collection will be collected at baseline, 4 weeks, and 8 weeks if the patient 
remains on treatment with MGE. The urine will be collected into a urine collection cup and 
mixed with ascorbic acid; 20 mL aliquots will be stored at -80°C for an indefinite period of 
time. The urine will be thawed on ice and used to measure polyphenols by UPLC-MS.   
 
10.2 Systemic cytokines and growth factor assays 
 
Approximately 10ml of blood will be collected in a tube that does not contain EDTA 
(silicone-coated, red-top tube) at baseline, 4 weeks, and 8 weeks (if the patient remains 
on MGE treatment) to measure cytokines and growth factors (including but not limited to 
TNFα, IL6, IL6R, IL8, IL1α, IL1β, IGF-1, VEGF), using the Cira multiplex solid phase 
immunoassay platform from Aushon and the Human Cytokine 2 and Angiogenesis Factor 
1 Arrays27-29, as well as individual ELISA’s for CRP, TGFβ1, IGF-1. The blood will be 
allowed to clot. Serum will be collected and samples will be stored at -80°C for an indefinite 
period of time.  
  
11.0 
Data Management 
 
Forms/Data collected 
Collection Time Point 
Data Management 
Informed consent document 
Pre-study 
ORIS/WakeOne 
Protocol Registration & Race Verification Forms  
Pre-study 
ORIS 
Adverse Event Log (Toxicity including CBC with 
differential, platelets, serum chemistries) 
Each monthly clinic visit 
ORIS (labs to be 
pulled from EPIC) 
Labs (CBC with differential, platelets, serum 
chemistries) 
Baseline and monthly 
clinic visit 
ORIS (labs to be 
pulled from EPIC) 
Treatment Response Evaluation  
Monthly clinic visit 
REDCap 
End of Treatment Evaluation of Best Response 
Off-study 
REDCap 
Baseline Data Collection Form 
Baseline 
REDCap 
Follow-up Data Collection Form 
Monthly clinic visit 
REDCap 
Correlative Research Labs Data Collection Form 
Baseline, week 4, week 8 REDCap 
Telephone Follow-up Form 
Weeks 2 & 6 
REDCap 
FACT-G 
Baseline and monthly 
REDCap 
PROMIS-Fatigue 
Baseline and monthly  
REDCap 
Daily Pill Dairy 
Monthly clinic visit 
REDCap 
Off Study Documentation Form 
Off-study 
REDCap 
Follow-Up Form for Survival 
Every 6 weeks 
ORIS 
 
12.0 
Statistical Considerations 
 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 26 of 31 
12.1 
Analysis of Primary Objective 
 
The primary objective of determining the safety and MTD of MGE will be completed 
by monitoring toxicities and using the dose escalation plan detailed above.   
 
To determine the maximum tolerated dose (MTD) of MGE in patients with 
metastatic cancer, we will use a standard 3+3 Phase 1 dose escalation statistical 
design. All MGE dose escalations conducted will be escalated according to the 
dose escalation scheme (see Section 6.2). The number of patients in each cohort 
will be 3. If no patients in any cohort develop a dose-limiting toxicity (DLT), dose 
escalation will continue in cohorts of 3 patients up to the maximum dose. However, 
if a DLT is observed in 1 of 3 patients at any dose level, the cohort of that dose 
level will be expanded to a maximum of 6 patients. If 1 of the 6 patients has a DLT, 
the dose escalation procedure will continue in 3 patients for each subsequent 
cohort. However, once a DLT is observed in a total of 2 patients at any dose level, 
dosing of MGE in patients at that dose level will stop immediately, even though the 
total number of patients at the last cohort may be as few as 2.  Dose escalation is 
considered to be complete. The dose level that induces a DLT in 2 or more patients 
is considered to be above MTD, and the dose level immediately below the dose 
level that induced a DLT in ≥2 patients is considered the MTD for MGE if ≤1 
participants of 6 patients at that level have a DLT. 
 
In the event that the MTD is not observed after the 5 dose levels listed in Section 
6.2 have been tested, the investigators will consider stopping additional dose 
escalation. The decision will be based on the actual dose level tested, the extent 
of anti-tumor efficacy observed, and the type of toxicity observed, etc. 
 
12.2 
Analysis of Secondary Objective 
 
12.2.1 To monitor toxicity at 4, 8, and every 4 weeks thereafter on MGE 
 
To analyze objective 2.1.1, we will summarize any expected toxicities, any 
laboratory based toxicities, and any grade 3 or higher gastrointestinal 
toxicities and any grade 4 or higher toxicities using frequency tables overall 
and by week. 
 
12.2.2 To evaluate change in phenolic levels (total and component, blood and 
urine) from baseline to 4 and 8 weeks 
 
To analyze objective 2.2.2, we will fit a mixed model ANOVA model with 
phenolic level as the outcome and time (baseline, 4, and 8 weeks) as a 
fixed effect and subject as a random effect. We will use contrasts to 
estimate the changes from baseline to week 4 and week 8. 
 
12.2.3 To evaluate change in serum cytokines and growth factors from baseline 
to 4 and 8 weeks on MGE 
 
To analyze objective 2.2.3, we will fit a mixed model ANOVA model with 
the outcome of interest (cytokine or growth factor) as the outcome and time 
(baseline, 4, and 8 weeks) as a fixed effect and subject as a random effect. 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 27 of 31 
We will use contrasts to estimate the changes from baseline to week 4 and 
week 8. 
 
12.2.4 To observe the response rate of MGE in patients with metastatic cancer. 
 
To analyze objective 2.2.4, we will characterize response at 8 weeks using 
frequency table.  We will also characterize the best response at the end of 
treatment with MGE using a frequency table. 
 
12.2.5 To assess overall and progression-free survival in patients with metastatic 
cancer receiving MGE. 
 
To analyze objective 2.2.5, we will summarize overall survival and 
progression-free survival using the Kaplan-Meier method.  We will calculate 
median survival rates and associated 95% confidence intervals. Patients 
will be censored at date of last contact. 
 
12.2.6 To assess global quality of life and fatigue in cancer patients taking MGE. 
 
Quality of Life and fatigue will be monitored every 4 weeks until the end of 
the study. To analyze objective 2.2.6, we will fit a mixed model ANOVA 
model with phenolic level as the outcome and time (baseline, every 4 
weeks) as a fixed effect and subject as a random effect. We will use 
contrasts to estimate the changes from baseline to each follow-up time 
period. 
 
12.2.7 To assess adherence to MGE treatment. 
 
To analyze objective 2.2.7, we will summarize the percent of pills taken at 
the end of every 4 week period i using the formula: 
 
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑜𝑓 𝑝𝑖𝑙𝑙𝑠 𝑡𝑎𝑘𝑒𝑛𝑖= (𝐷𝑜𝑠𝑒 𝐿𝑒𝑣𝑒𝑙 × 7 × 4)𝑖−𝑃𝑖𝑙𝑙 𝐶𝑜𝑢𝑛𝑡𝑖
(𝐷𝑜𝑠𝑒 𝐿𝑒𝑣𝑒𝑙 × 7 × 4)𝑖
. 
 
In the formula above, pill count will be calculated from counting the number 
of pills returned as well as by summarizing the patient’s pill diary. The 
percent of pills taken will be summarized by dose level using the mean and 
95% confidence interval. 
 
12.3 
Power and Sample Size 
 
The study will enroll a minimum of 2 participants and a maximum of 30. 
 
 
12.4 
Estimated Accrual Rate and Study Length 
 
We anticipate accruing at least 2 patients per month on this protocol and thus 
expect that accrual for this Phase 1 trial will be completed in less than 16 months, 
and that the MTD can be determined within 1 month of the last accrual. Patients 
will be followed until death. 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 28 of 31 
 
 
12.5 
Interim Analysis Plan 
 
This is a standard 3+3 design with monitoring throughout the study and stopping 
rules as defined above. 
 
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Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
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Appendix A – Protocol Registration Form 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 30 of 31 
See separate attachment in the eIRB under Protocol Document. 
 
Appendix B – Subject Eligibility Checklist 
See separate attachment in the eIRB under Protocol Document 
 
Appendix C – Mandatory STRC SAE Reporting Guidelines 
See separate attachment in the eIRB under Protocol Document 
 
Appendix D – Treatment Response Evaluation 
See separate attachment in the eIRB under Protocol Document. 
 
Appendix E – End of Treatment Evaluation of Best Overall Response 
See separate attachment in the eIRB under Protocol Document. 
 
Appendix F – FACT-G 
See separate attachment in the eIRB under Protocol Document. 
 
Appendix G – PROMIS-Fatigue SF 
See separate attachment in the eIRB under Protocol Document. 
 
Appendix H – Daily Pill Diary 
See separate attachment in the eIRB under Protocol Document 
 
Appendix I – Baseline Data Collection Form 
See separate attachment in the eIRB under Protocol Document 
 
CBC results will be drawn directly into the database from ORIS. Do not abstract them from the 
medical record.  Results will include WBC, RBC, Hemoglobin, Hematocrit, MCV, Neutrophils, 
Lymphocytes, Platelets. 
 
Serum chemistry results will be drawn directly into the database from ORIS. Do not abstract 
them from the medical record. Results will include Albumin, Alkaline phosphatase, Total 
bilirubin, Bicarbonate, BUN, Calcium, Chloride, Creatinine, Glucose, Potassium, Total protein, 
SGOT[AST], SGPT[ALT], Sodium. 
 
Cancer-specific tumor marker values will be drawn directly into the database from ORIS. Do not 
abstract them from the medical record. 
 
Appendix J – Follow-Up Data Collection Form 
See separate attachment in the eIRB under Protocol Document 
 
CBC results will be drawn directly into the database from ORIS. Do not abstract them from the 
medical record.  Results will include WBC, RBC, Hemoglobin, Hematocrit, MCV, Neutrophils, 
Lymphocytes, Platelets. 
 
Serum chemistry results will be drawn directly into the database from ORIS. Do not abstract 
them from the medical record. Results will include Albumin, Alkaline phosphatase, Total 
bilirubin, Bicarbonate, BUN, Calcium, Chloride, Creatinine, Glucose, Potassium, Total protein, 
SGOT[AST], SGPT[ALT], Sodium. 
 

 
Phase 1 Study of Muscadine Grape Extract (MGE) in Advanced Malignancy 
Comprehensive Cancer Center of Wake Forest University (CCCWFU) 
CCCWFU # 01815 
Protocol version date 10/5/2018 
 
 
 
 
 
 
 
      Page 31 of 31 
Cancer-specific tumor marker values will be drawn directly into the database from ORIS. Do not 
abstract them from the medical record. 
 
Appendix K – Correlative Research Labs Data Collection Form 
See separate attachment in the eIRB under Protocol Document 
 
Appendix L – Telephone Follow-Up Form 
See separate attachment in the eIRB under Protocol Document 
 
Appendix M – Off Study Documentation Form 
See separate attachment in the eIRB under Protocol Document 
 
Appendix N – Follow-Up Form for Survival 
See separate attachment in the eIRB under Protocol Document 
 
Appendix O – Adverse Event Log 
See separate attachment in the eIRB under Protocol Document 
 
Appendix P – Physician Order Form for Study Drug 
See separate attachment in the eIRB under Protocol Document