Conditions: Becker Muscular Dystrophy
Keywords: BMD Becker muscular dystrophy epicatechin clinical trial neuromuscular disease
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 2
Healthy volunteers: no

Brief summary:
This is a 48-week open-label extension of our initial proof-of-concept study (UCD0113) in patients with Becker muscular dystrophy who participated in the earlier trial. This single center study will enroll up to 10 adults who will receive the purified nutritional extract (-)-epicatechin 100mg/day orally for 8 weeks. After screening visits, participants will be enrolled in the study if they meet all inclusion criteria. They will be evaluated at screening, baseline, and weeks 4, 8, 12, 24, 16 and 48. The main criterion for success of the study will be presence of one or more biologic or strength and performance outcome measures that yield a response magnitude that allows for sufficient power in a Phase II B study with a sample size of 30 individuals.

Detailed description:

Arms:
arm 1: (-)-epicatechin 50mg twice per day (100mg per day total dose)

Interventions: intervention 1: (-)-Epicatechin
Intervention descriptions: intervention 1: None

Protocol document:
ICF_001.pdf: 
UCD0115B: An Open-label Extension Study of Purified Epicatechin to 
Improve Mitochondrial Function, Strength and Skeletal Muscle Exercise 
Response in Becker Muscular Dystrophy   
NCT03236662  
August 20, 2015 
 


Permission to Take Part in a Human Research Study 
Page 1 of 8 
Initial Version Date 7/7/2015 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: May 27, 2015 
 
Title of research study: UCD0115B:  An open-label extension study of purified epicatechin to improve 
mitochondrial function, strength and skeletal muscle exercise reponse in Becker Muscular Dystrophy. 
Investigator: Craig McDonald, MD 
Why am I being invited to take part in a research study? 
We invite you to take part in this extension research study because you are an adult diagnosed with 
Becker muscular dystrophy and were previously enrolled in the Pilot Epicatechin study.  
What should I know about a research study? 
(Experimental Subject's Bill of Rights)  
• 
Someone will explain this research study to you, including: 
o The nature and purpose of the research study. 
o The procedures to be followed. 
o Any drug or device to be used.  
o Any common or important discomforts and risks. 
o Any benefits you might expect. 
o Other procedures, drugs, or devices that might be helpful, and their risks and benefits 
compared to this study.  
o Medical treatment, if any, that is available for complications.  
• 
Whether or not you take part is up to you. 
• 
You can choose without force, fraud, deceit, duress, coercion, or undue influence. 
• 
You can choose not to take part. 
• 
You can agree to take part now and later change your mind. 
• 
Whatever you decide it will not be held against you. 
• 
You can ask all the questions you want before you decide. 
• 
If you agree to take part, you will be given a signed and dated copy of this document.  
• 
If you agree to take part, you will be given a copy of this document.  
Who can I talk to? 
If you have questions, concerns, or complaints, or think the research has hurt you, talk to the research 
team:   
Craig McDonald, MD   
 
Study PI 
 
at phone number 916-734-2923 
Erik Henricson, MPH   
 
Co-PI  
 
at phone number 916-734-5294 
Candace Aguilar 
 
 
Study Coordinator 
at phone number 916-734-0968 
UC Davis Department of Physical Medicine and Rehabilitation 
4860 Y Street, Suite 3850 
Sacramento, CA 95817 
For emergencies please call the following 24-hour emergency number. Our study doctors are 
available 24 hour a day for these health related emergencies.  
UC Davis Medical Center operator - (916) 734-2011 (Ask for the PM&R Resident on call) 
This research has been reviewed and approved by an Institutional Review Board (“IRB”). Information to 
help you understand research is on-line at http://www.research.ucdavis.edu/policiescompliance/irb-
admin/.You may talk to a IRB staff member at (916) 703-9151, IRBAdmin@ucdmc.ucdavis.edu, or 
2921 Stockton Blvd, Suite 1400, Room 1429, Sacramento, CA 95817 for any of the following: 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
767161
08/20/2015


Permission to Take Part in a Human Research Study 
 
Page 2 of 8 
 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: May 27, 2015 
 
• 
Your questions, concerns, or complaints are not being answered by the research team. 
• 
You cannot reach the research team. 
• 
You want to talk to someone besides the research team. 
• 
You have questions about your rights as a research subject. 
• 
You want to get information or provide input about this research. 
Why is this research being done? 
This research study will test an investigational drug called (-)-Epicatechin. New drugs or biologics that 
have not yet been approved by the FDA or approved drugs that have not yet been approved for a new 
use, and are in the process of being tested for safety and effectiveness. (-)-Epicatechin is purified from 
tea as a nutritional extract. (-)-Epicatechin will be evaluated for the treatment of progressive muscle 
loss and impaired skeletal muscle function in Becker Muscular Dystrophy (BMD) patients.  
(-)-Epicatechin has been shown to improve muscle structure and function in previous studies of animals 
and humans. This study drug is not currently approced by the Food and Drug Administration (FDA) for 
the treatment of BMD.  
The main objective of this study is to test your blood samples for biomarkers and determine whether or 
not the study drug is affecting these molecules and possibly the structure and function of you muscles.  
How long will the research last? 
We expect that you will be in this research study for 48 weeks for a total of 8 visits. Each visit on 
average will be expected to be about a 3 hours induration. 
How many people will be studied? 
We expect about 10 people here will be in this research study at UC Davis. 
What happens if I say yes, I want to be in this research? 
Study Procedures: 
See below for a table of all the study procedures 
Event 
Screening     
Baseline  
PK Week 4 
Week 8 
Week 12 
Week 24 
Week 36 
Week 48 
  
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
  
Day -28 to 0 
Day 1 
Day 28 
Days 53-
59 
Days 81-87 
Days 165-171 
Days 249-255 
Days 333-339 
Informed consent 
X 
  
  
  
  
  
  
  
Medical history 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Event/ 
Medications 
X 
X 
  
X 
X 
X 
X 
X 
ECG 
X 
X 
X 
X 
X 
  
X 
X 
PK 
  
Pre, 2H, 4H 
Pre 
  
Pre 
Pre 
Pre 
Pre 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
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Permission to Take Part in a Human Research Study 
 
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Document Revision Date: May 27, 2015 
 
Complete Blood 
Count/Serum 
Chemistries/Urinalysis 
X 
  
X 
X 
X 
X 
X 
X 
Tbsp. of Blood Volume 
7 tbsp.  
4 tbsp.  
8 tbsp. 
6 tbsp. 
9 tbsp. 
9 tbsp.  
9 tbsp.  
9 tbsp.  
Fasting Insulin, Glucose 
X 
  
  
  
X 
X 
X 
X 
Biomarkers 
  
X 
X 
  
X 
X 
X 
X 
Functional assessments 
  
X 
X 
  
X 
X 
X 
X 
DEXA 
  
X 
X 
  
X 
X 
X 
X 
Dispense Epicatechin 
X 
X 
X 
X 
X 
X 
X 
X 
Questionnaire 
X 
 
 
X 
 
 
 
 
*Multiple blood draws at visits 2, 3, 5, 6, 7, 8 . 
 
Informed consent (30 mins) (At Screening) 
Before we begin any procedure we will review this consent form with you and answer any questions 
that you might have. After you sign the consent form we will provide you with a copy. 
Medical history  
We will ask you questions about medical conditions  
Vital signs and physical exam  
We will measure your weight, height, blood pressure and breathing rate. 
Adverse events/ Medications  
We will ask you about medications that you are taking and if anything has happened since the last visit 
that hurt or was out of the ordinary with regard to your health. 
Safety & Drug monitoring/ blood & urine collection  
During each visit we will perform  blood draw(s) and one urine sample collection for safety 
assessments. 
We will collect the following blood samples: 
• 
Complete blood count/Serum chemistries- 6 tablespoons/18 teaspoons 
• 
Biomarker- 1 tablespoon/3 teaspoons 
• 
Fasting insulin/glucose- 1 tablespoon/3 teaspoons 
• 
PK (protein kinase)- 1 tablespoon/3 teaspoons 
 
Electrocardiogram (ECG)  
This test will provide information on how well your heart is working. . 
Strength & Exercise Testing 
You will be asked to perform several exercise tests including the following measures: 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
767161
08/20/2015


Permission to Take Part in a Human Research Study 
 
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Document Revision Date: May 27, 2015 
 
• 
Six minute walk test—you will walk as fast as you can for 6 minutes around 2 cones 
• 
Recumbent cycling test—you will exercise using a recumbent bicycle 
• 
Assisted six minute cycle test---you will cycle as fast as you can for 6 minutes 
• 
Strength test—we will measure the strength of your elbows and knees with a BIODEX 
ergometer (hand and arm operated bicycle) 
• 
Range of motion test—we will assess your range of motion by measuring the extension of your 
knees, ankles, elbows and wrists in front of a motion sensing system 
• 
Breathing tests—you will perform PFTs (Pulmonary Function Tests) to assess how much air you 
can breathe in and out 
• 
Timed Function Testing—we will assess your ability to rise from the floor, climb four standard 
stairs and walk or run for 10 meters 
• 
Upper/Lower Extremity function—we will assess your ability to use your arms and legs 
• 
Anthropometric Measurements—we will measure your waist and hip circumference 
• 
Body Composition by Dual energy X-ray absorptiometry (DEXA)—we will ask you to lie still on 
the bed of the DEXA scanner. The scanning arm will pass over your body one or more times 
and will make a computerized X-ray image of your whole body.You will not feel anything during 
the scan, but you will be asked to lie very still. The purpose of adding this measure to our study 
is to find out your body composition. 
• 
Multifrequency Bioimpedence Assessment (MFBIA)—we will assess the amount of total water 
and fat-free mass in your body. Electrodes are placed on your ankle and wrist. Impedence is 
measured between upper and lower extremity electrodes. You will not feel anything during the 
test. 
Study Medication-Epicatechin 
• 
Medications will be dispensed for the entire period of time between study. The site will dispense 
study medication to you and you will keep a medication log to document all study medication 
dispensed. You will be instructed to take the study medication per dosing schedule, every day 
during the eight-week treatment period.You will be asked to return your empty medication 
container and/or any remaining pills at the next visit. 
• 
You will swallow pills of (-)-epicatechin 50mg twice per day (100mg per day total dose). Study 
medication will be supplied as a clear 25mg gelatin capsule. You will take two capsules in the 
morning at approximately 7:30AM at least 15 minutes before the morning meal and two with the 
evening at approximately 7:30PM at least 1 hour after the evening meal. 
What are my responsibilities if I take part in this research? 
If you take part in this research, you will be responsible to: Tell the doctor if you have any new or 
worsening medical symptoms or problems during the time you receive the study medication as part of 
this study.  It is important that you tell the doctor even if symptoms or problems are mild or you do not 
think they are related to study drug. 
You will need to complete a drug diary and bring your medication to each study visit.  
Please call if you have any questions about study medication or about the use of other drugs during the 
time that you receive study medication. 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
767161
08/20/2015


Permission to Take Part in a Human Research Study 
 
Page 5 of 8 
 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: May 27, 2015 
 
Contact the doctor immediately if an overdose of study drug happens or is suspected.   
What happens if I do not want to be in this research? 
You may decide not to take part in the research and it will not be held against you as participation is on 
a voluntary basis.  
Instead of being in this research study, your choices may include: participating in other available clinical 
trials or choosing not to participate at all.  
What happens if I say yes, but I change my mind later? 
You can leave the research at any time and it will not be held against you. 
If you decide to leave the research, contact the investigator so that the investigator can can recommend 
proper follow up care. 
 
If you stop being in the research, already collected data may not be removed from the study database. 
You will be asked whether the investigator can collect data from your routine medical care. If you agree, 
this data will be handled the same as research data.  
Is there any way being in this study could be bad for me? 
Potential Risks  
Risks of Study Medication (Epicatechin) 
Hypotension (low blood pressure). Given the reported effects of (-)-epicatechin on blood vessels, it a 
potential risk may be associated with hypotension. With cocoa based studies, blood pressure reducing 
effects are only reported in humans that have high blood pressure. There is the possibility that patients 
undergoing drug treatment for high blood pressure if given (-)-epicatechin may develop hypotension.  
Migraines (Headaches). The consumption of cocoa products has been associated with increased 
likelihood of migraine development. (-)-Epicatechin may increase the chances for migraine 
development in certain individuals.  
Bleeding. Anti-clotting like effects have been described in a limited number of reports on the effects of 
cocoa, although no such reactions have been seen to date. It will be important not to take any “blood 
thinning” medications (example: Asprin and Advil) during the study without consulting the study doctor. 
Risks of Blood Tests  
The risks of blood drawing include soreness or bruising at the site of the needle. A local numbing cream 
(EMLA) may be applied to the area. There are no side effects associated with the use of this cream. 
Rarely, a more serious injury, such as hematoma (bleeding under skin) or infection may develop. 
Repeated blood draw may increase the chance of the risks above.  
Risks of Strength Testing and Functional Assessments 
At this time, there are no known risks associated with functional evaluation or muscle strength testing 
methods used in this protocol. However, you may experience mild muscle soreness the day after 
muscle testing.  
Risks of Breathing Tests  
These tests may cause dizziness and lightheadedness during and shortly after the test.  
Risks of Electrocardiogram (ECG) 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
767161
08/20/2015


Permission to Take Part in a Human Research Study 
 
Page 6 of 8 
 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: May 27, 2015 
 
The ECG has no known risks.  
Risks of DEXA  
This study involves a low radiation exposure that is less than other diagnostic tests using ionizing 
radiation. The amount of radiation exposure received in this study is below the levels that are thought to 
result in a significant risk of harmful effects.  
Risks of Multi-Frequency Bioimpedence Assessment (MFBIA)  
There are no known risks of MFBIA testing.  
Risks of Upper Extremity Range of Motion Evaluation  
There are no known risks of upper extremity range of motion evaluation. 
Risk of Pregnancy  
The research may also hurt a pregnancy or fetus in ways that are unknown. The subject should not 
father a baby while in this study, and they should not donate semen/sperm or have vaginal sex 
(intercourse) without using effective birth control.  
Will being in this study help me in any way? 
It is possible the participants will not experience any direct benefit as a result of their study participation. 
However, the data collected during this trial may provide information that will benefit the scientific 
community as well as other individuals with BMD 
What happens to the information collected for the research? 
Efforts will be made to limit use or disclosure of your personal information, including research study and 
medical records, to people who have a need to review this information. We cannot promise complete 
confidentiality. Organizations that may inspect and copy your information include the IRB and other 
University of California representatives responsible for the management or oversight of this study.  
The IRB and the Food and Drug Administration will be granted direct access to your research records 
to conduct and oversee the study. We may publish the results of this research. However, we will keep 
your name and other identifying information confidential. 
 During your participation in this research, data will be collected about you. The de-identified data and 
any specimens, such as blood or tissue, are taken from you for this study, they will become the 
property of the University of California. The specimens may be used in this research, may be used in 
other research, and may be shared with other organizations. The specimens could lead to discoveries 
or inventions that may be of value to the University of California or to other organizations. Under state 
law you do not have any right to money or other compensation stemming from products that may be 
developed from the specimens. 
 
If you agree to share the biological specimen(s) collected from you, please initial here.   
 
  
Otherwise, your specimen will be destroyed at the end of this study. Information regarding biomarkers 
and outcome of the study will not be shared.  
The sponsor, monitors, auditors, the IRB, the Food and Drug Administration will be granted direct 
access to your research records to conduct and oversee the study. We may publish the results of this 
research. However, we will keep your name and other identifying information confidential. 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
767161
08/20/2015


Permission to Take Part in a Human Research Study 
 
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For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: May 27, 2015 
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by U.S. 
Law. This Web site will not include information that can identify you. At most, the Web site will include a 
summary of the results. You can search this Web site at any time. 
If we access protected health information (e.g., your medical record), you will be asked to sign a 
separate form to give your permission. Your medical records may become part of the research record.  
If that happens, your research records may be looked at by the sponsor of this study and government 
agencies or other groups associated with the study.  They may not copy or take your personal health 
information from your medical records unless permitted or required by law. 
Federal law provides additional protections of your medical records and related health information. 
These are described in the UC Davis Health System Notice of Privacy Practices 
(http://www.ucdmc.ucdavis.edu/compliance/pdf/notice.pdf) and in an attached document. 
Can I be removed from the research without my OK? 
. The person in charge of the research can remove you from the research study without your approval 
in circumstances when new information regarding safety in this trisl or other trials involving new safety 
concerns with Epicatechin.  We will tell you about any new information that may affect your health, 
welfare, or choice to stay in the research 
What else do I need to know? 
There is no charge for you to participate in this study. Neither you nor your insurance carrier will be 
charged for your taking part in the research. All costs associated with the study will be paid by the 
sponsor/department. 
It is important that you promptly tell the person in charge of the research if you believe that you have 
been injured because of taking part in this study. If you are injured as a result of being in this study, the 
University of California will provide necessary medical treatment. Depending on the circumstances, the 
costs of the treatment may be covered by University or the study sponsor or may be billed to your 
insurance company just like other medical costs. The University does not normally provide any other 
form of compensation for injury.   
You will not be compensated for taking part in this study. 
For more information about compensation, you may call the IRB Administration at (916) 703-9151 or 
email at IRBAdmin@ucdmc.ucdavis.edu . 
 
Your signature documents your permission to take part in this research. 
 
 
 
Signature of subject 
 
Date 
 
 
Printed name of subject 
 
 
 
Signature of person obtaining consent 
 
Date 
 
 
 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
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08/20/2015


Permission to Take Part in a Human Research Study 
 
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For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: May 27, 2015 
 
Signature Block for Capable Adult 
 
 
Printed name of person obtaining consent 
 
 
My signature below documents that the information in the consent document and any other written 
information was accurately explained to, and apparently understood by, the subject, and that consent 
was freely given by the subject. 
 
 
 
Signature of witness to consent process 
 
Date 
 
 
Printed name of person witnessing consent process 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
767161
08/20/2015

Prot_SAP_002.pdf: 
UCD0115B: An Open-label Extension Study of Purified Epicatechin to 
Improve Mitochondrial Function, Strength and Skeletal Muscle Exercise 
Response in Becker Muscular Dystrophy 
NCT03236662 
June 15, 2015 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
UCD0115B:  An open-label extension study of purified epicatechin to 
improve mitochondrial function, strength and skeletal muscle exercise 
reponse in Becker Muscular Dystrophy  
 
Original:  June 15, 2015 
 
 
 
 
 
 
 
 
STUDY COORDINATING CENTER: 
Neuromuscular Research Center 
Department of Physical Medicine and Rehabilitation 
University of California, Davis 
 
STUDY CHAIR: 
 
 
STUDY CO-CHAIR: 
Craig M. McDonald, MD 
University of California, Davis 
 
Erik Henricson, MPH 
University of California, Davis 
 
CO-INVESTIGATOR(S): 
Bjorn Oskarsson, MD 
University of California, Davis 
 
Francisco Villareal, MD, PhD 
University of California, San Diego 
 
Yetrib Hathout, PhD 
Children’s National Medical Center 
 
Nanette C. Joyce, D.O 
University of California, Davis 
 
DATA MANAGER(S): 
Evan de Bie, BS 
University of California, Davis 
 
PROJECT COORDINATOR: 
Erica Goude, MS, CCRC 
University of California, Davis 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
UCD0115B:  An open-label extension study of purified epicatechin to improve mitochondrial function, 
strength and skeletal muscle exercise reponse in Becker Muscular Dystrophy  
 
KEY CONTACTS 
 
STUDY CHAIR 
Craig M. McDonald, MD 
Professor and Chair of Physical Medicine and Rehabilitation 
University of California, Davis 
4860 Y Street, Suite 3850 
Sacramento, CA 95817 
TEL: (916) 734-2923 
FAX: (916) 734-7838 
E-Mail: cmmcdonald@ucdavis.edu 
 
PROJECT COORDINATOR 
Candace Aguilar, BS 
UC Davis Neuromuscular Research Center 
4860 Y Street, Suite 3850 
Sacramento, CA 95817 
TEL: (916) 734-0968 
FAX: (916) 734-7838 
E-Mail: candace.aguilar@ucdmc.ucdavis.edu 
 
PROJECT CENTRAL PHARMACIST 
UC Davis Investigational Drug Pharmacy 
 
INDEPENDENT MEDICAL MONITOR 
Carol VandenAkker, MD 
Department of Physical Medicine and Rehabilitation 
 University of California, Davis 
4860 Y Street, Suite 3850 
Sacramento, CA 95817 
TEL: (916) 734-2923 
FAX: (916) 734-7838 
E-Mail: carol.vandenakker-albanese@ucdmc.ucdavis.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
2 
PROTOCOL SIGNATURE PAGE 
 
UCD0115B:  An open-label extension study of purified epicatechin to improve mitochondrial 
function, strength and skeletal muscle exercise reponse in Becker Muscular Dystrophy  
 
Original:  January 20, 2015 
 
Instructions to Investigators: Please sign and date this signature page, print your name, your title and 
the name of the facility in which the study will be conducted, and return a copy of this page to the 
Project Coordinator. 
 
I confirm that I have read this protocol, I understand it, and I will work according to this protocol and to 
the ethical principles stated in the latest version of the Declaration of Helsinki, the applicable guidelines 
for good clinical practices, and the applicable laws and regulations of the country of the study site for 
which I am responsible, whichever provides the greater protection of the individual.  I will accept the 
monitor’s overseeing of the study. 
 
 
 
 
Signature of Investigator 
Date 
 
 
 
Printed Investigator Name 
Investigator Title 
 
 
 
Name of Facility 
Location of Facility (City, State, Country) 
 
 
 
Signature of Representative of Study 
Sponsor 
Date 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
3 
TABLE OF CONTENTS 
1 
STUDY SUMMARY .................................................................................................................................... 7 
1.1 
BACKGROUND ....................................................................................................................... 7 
1.2 
STUDY PURPOSE ................................................................................................................... 8 
1.3 
SPECIFIC AIMS: ..................................................................................................................... 8 
2 
BACKGROUND AND SIGNIFICANCE ..................................................................................................... 9 
2.1 
THE DYSTROPHINOPATHIES: DUCHENNE AND MUSCULAR DYSTROPHY (DBMD) ....................... 9 
2.2 
EPICATECHIN DIRECTLY TARGETS MULTIPLE POINTS IN DBMD PATHOPHYSIOLOGY.................... 9 
3 
EPICATECHIN OVERVIEW .................................................................................................................... 11 
3.1 
PHARMACOKINETICS / PHARMACODYNAMICS ......................................................................... 11 
3.1.1 
(-)-Epicatechin ............................................................................................................ 11 
4 
PRELIMINARY STUDIES ........................................................................................................................ 13 
4.1 PRECLINICAL DATA .................................................................................................................. 13 
4.1.1 (-)-Epicatechin Increases Mitchondrial Respiration and Biogenesis in Cultured Human Skeletal 
Muscle Cells (unpublished data) ............................................................................................ 13 
4.1.2 (-)-Epicatechin Induces Mitochondrial Biogenesis Pathway in Human Coronary Artery Endothelial 
Cells (unpublished data) ......................................................................................................... 15 
4.1.3 (-)-Epicatechin Induces Expression of Mitochondrial Biogenesis Factor PGC-1α in Aged Mice 
(unpublished data) .................................................................................................................. 15 
4.1.4 (-)-Epicatechin Induces Expression of Mitochondrial Biogenesis Factor PGC-1α and 
Mitochondrial Proteins in δ-sarcoglycan KO Mice (unpublished data) .................................. 16 
4.1.5 (-)-Epicatechin Increases In Vivo Mitchondrial Biogenesis and Muscle Function in Aged 1 Year 
Old Mice 16 
4.1.6 (-)-Epicatechin Increases Mitchondrial Biogenesis and Improves Myocardial Function in Rats
 
18 
4.1.7 (-)-Epicatechin Modulates the Synthesis of Muscle Growth and Differentiation Proteins in 
Cultured Cells and Mice (Aged and MD)(unpublished data) .................................................. 18 
4.1.8  Muscle Regulatory Protein Expression in 6 and 26 month old Mice ............................ 19 
4.1.9 (-)-Epicatechin Stimulates the Expression of MD-Relevant Muscle Structural Proteins in Aged and 
MD Mice (unpublished data) .................................................................................................. 20 
4.1.10 (-)-Epicatechin Reduces Fibrosis in δ-Sarcoglycan KO Mice (unpublished data) ...... 21 
4.1.11 (-)-Epicatechin Reduces Plasma Marker of Muscle Damage and Improves Muscle Strength in 
MDX Mouse Model of MD (unpublished data) ....................................................................... 22 
4.1.12 (-)-Epicatechin Blocks Calcium-Induced Pore Formation in Cardiac Mitochondria (unpublished 
data) 
23 
5.HUMAN STUDIES ....................................................................................................................................... 24 
5.1 CLINICAL STUDY #1:  MITOCHONDRIAL BIOGENESIS AND MUSCLE IMPROVEMENT IN PATIENTS WITH HEART 
FAILURE AND TYPE 2 DIABETES PATIENTS(UCSD) ......................................................................... 24 
5.1.1 Markers of Mitochondrial Biogenesis, Structure and Function...................................... 24 
5.1.2 Markers of Tissue Oxidative Stress .............................................................................. 25 
5.1.3 nNOS Expression .......................................................................................................... 26 
5.2 CLINICAL STUDY #2:  AGE-RELATED CHANGES IN MUSCLE REGULATORY PROTEINS AND EFFECT OF (-)-
EPICATECHIN ON MUSCLE FUNCTION AND PROTEINS IN HEALTHY VOLUNTEERS (UCSD) ................. 28 
5.3 CLINICAL STUDY #3:  PHARMACOKINETICS OF (-)-EPICATECHIN IN HEALTHY VOLUNTEERS (UCSF)29 
5.3.1 Safety and Tolerability ................................................................................................... 30 
5.3.2 Pharmacokinetics .......................................................................................................... 30 
5.3.3 Conclusions ................................................................................................................... 30 
5.4 CLINICAL STUDY #4: EPICATECHIN IN BECKER PHENOTYPE DYSTROPHINOPATHY PATIENTS. ........ 31 
5.4.1 Results........................................................................................................................... 31 
5.4.2 Biomarker outcomes at 8 weeks ................................................................................... 31 
5.4.3 Strength and function and safety outcomes at 8 weeks. .............................................. 34 
5.4.4 Safety outcomes at 8 weeks ......................................................................................... 35 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
4 
6 
RESEARCH DESIGN AND METHODS .................................................................................................. 35 
6.1 SELECTION AND WITHDRAWAL OF PARTICIPANTS ...................................................................... 35 
6.1.1 Criteria for Enrollment ................................................................................................... 35 
6.1.2 
Participant Screening Schedule ................................................................................. 36 
6.1.3   Randomization ............................................................................................................ 37 
6.2 WITHDRAWAL OF PARTICIPANTS ............................................................................................... 37 
6.2.1 Follow-Up of Withdrawn Participants ............................................................................ 37 
6.3 TREATMENT OF PARTICIPANTS ................................................................................................. 37 
6.3.1 Epicatechin administration ............................................................................................ 37 
6.3.2 Criteria for Dose Reductions ......................................................................................... 38 
6.3.3 Study Drug Holiday as Result of Surgical Procedure ................................................... 38 
6.3.4 Concomitant Therapy .................................................................................................... 38 
6.3.5 Patient Care Outside UC Davis ..................................................................................... 38 
7 
PHARMACY ............................................................................................................................................. 39 
7.1 DOSING INFORMATION AND SAFETY STUDIES ............................................................................ 39 
7.1.1 Preclinical Rodent Safety Studies ................................................................................. 39 
7.1.2 Human Safety Studies................................................................................................... 39 
7.2 STUDY DRUG FORMULATION AND PROCUREMENT ..................................................................... 39 
7.2.1 (-)-Epicatechin as a Natural Test Substance (Cardero Therapeutics) .......................... 39 
7.2.2 (-)-Epicatechin as supplies as a natural test product .................................................... 40 
7.3 TREATMENT CYCLES DRUG DISPENSATION .............................................................................. 40 
7.4 INVESTIGATIONAL PRODUCT ACCOUNTING PROCEDURES .......................................................... 41 
7.5 MAINTENANCE OF RANDOMIZATION CODES AND EMERGENCY UNBLINDING ................................ 41 
8 
ASSESSMENT OF EFFICACY ............................................................................................................... 41 
8.1 LABORATORY EFFICACY PARAMETERS ..................................................................................... 41 
8.2 
FUNCTIONAL EFFICACY PARAMETERS ................................................................................... 41 
9 
ASSESSMENT OF SAFETY ................................................................................................................... 43 
9.1 SAFETY PARAMETERS ............................................................................................................. 43 
9.2 
PROCEDURES FOR REPORTING ADVERSE EVENTS ................................................................ 43 
9.3 
DATA AND SAFETY MONITORING BOARD / MEDICAL MONITOR ................................................ 44 
9.4 DURATION OF PARTICIPATION / FOLLOW-UP ............................................................................. 44 
9.5 
TRIAL STOPPING RULES OR DISCONTINUATION CRITERIA....................................................... 44 
10 
HUMAN PARTICIPANTS ...................................................................................................................... 44 
A. 
CHARACTERISTICS OF THE STUDY POPULATION ...................................................................... 44 
B. 
PARTICIPATION OF CHILDREN, WOMEN AND MINORITY POPULATIONS ....................................... 44 
C. 
SOURCES OF RESEARCH MATERIAL FROM LIVING PARTICIPANTS ............................................. 44 
D. 
RECRUITMENT OF PARTICIPANTS ............................................................................................ 44 
i. 
Advertising ........................................................................................................................ 44 
ii. 
Screening ........................................................................................................................ 44 
E. 
INFORMED CONSENT/ASSENT AND ETHICAL CONSIDERATIONS ................................................. 44 
F. 
RETENTION OF PARTICIPANTS ................................................................................................ 45 
G. 
POTENTIAL RISKS .................................................................................................................. 45 
i. 
Risks of Epicatechin ......................................................................................................... 45 
ii. 
Risks of Blood Tests ........................................................................................................ 46 
iii. 
Risks of QMT Strength Testing ...................................................................................... 46 
iv. 
Risks of PFT ................................................................................................................... 46 
v. 
Risks of EKG ................................................................................................................... 46 
vi. 
Risks of Functional Evaluation and Muscle Strength Testing ........................................ 46 
vii. 
Risks of DEXA ............................................................................................................... 46 
viii. 
Risks of Multi-Frequency Bioimpedence Assessment (MFBIA) ................................... 46 
ix. 
Risks of Upper Extremity Range of Motion Evaluation .................................................. 46 
H. 
PROCEDURES FOR MINIMIZING RISKS ..................................................................................... 46 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
5 
I. 
JUSTIFICATION OF RISKS TO PARTICIPANTS .............................................................................. 46 
J. 
BENEFITS ............................................................................................................................... 47 
K. 
FINANCIAL CONSIDERATIONS .................................................................................................. 47 
11  STATISTICS ............................................................................................................................................. 47 
12 
DATA COLLECTION ............................................................................................................................. 47 
12.1 DATA MANAGEMENT SYSTEM ................................................................................................. 47 
12.2 DATA QUALITY CONTROL AND QUALITY ASSURANCE ............................................................... 47 
12.3 SECURITY AND BACKUPS ....................................................................................................... 47 
12.4 DATA MONITORING ................................................................................................................ 48 
13    REFERENCES ....................................................................................................................................... 48 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
6 
 
TABLES 
 
Table 1: Effects of  epicatechin on progressive muscle loss parameters. ..................................................... 11 
Table 2: Selected human oral bioavailability studies of (-)-epicatechin ......................................................... 13 
Table 3: Study procedures. ............................................................................................................................ 37 
Table 4: Dose reduction schedule .................................................................................................................. 38 
Table 5: Contents of the 25mg (-)-epicatechin capsule.................................................................................. 40 
 
Figures 
 
Figure 1: Chemical structures of flavonoid families and (-)-Epicatechin ......... Error! Bookmark not defined. 
Figure 2: Major (-)-epicatechin metabolites in plasma ................................................................................... 12 
Figure 3: (-)-Epicatechin metabolite profile in plasma .................................................................................... 12 
Figure 4: (-)-Epicatechin enhances mitochondrial respiration ........................................................................ 14 
Figure 5: (-)-Epicatechin increases mitochondrial number and cristae density in human skeletal muscle cells 
(unpublished data) ................................................................................................................................... 14 
Figure 6: (-)-Epicatechin induces estrogen-related receptor alpha translocation (activation) into the nucleus 
of HCAEC ................................................................................................................................................ 15 
Figure 7: (-)-Epicatechin increases PGC1-alpha in aged mice. ..................................................................... 16 
Figure 8: (-)-Epicatechin increases PGC1-alpha and mitochondrial proteins in gamma-sarcoglycan knock 
out mice. .................................................................................................................................................. 16 
Figure 9: (-)-Epicatechin increases mitochondrial proteins in 1-year old mice .............................................. 17 
Figure 10: (-)-Epicatechin increases mitochondrial volume density and mitochondrial membrane surface 
area in 1-year old mice. ........................................................................................................................... 17 
Figure 11: Muscle differentiation in time course in C2C12 cells +/- (-)-epicatechin. ...................................... 19 
Figure 12: Muscle regulatory protein expression in 6 and 26 month old mice +/- (-)-Epicatechin ................. 19 
Figure 13: (-)-Epicatechin modulates muscle regulatory proteins in diaphragm muscle in MDX mice. ......... 20 
Figure 14: (-)-Epicatechin increases DAPC proteins in middle aged mice .................................................... 21 
Figure 15: (-)-Epicatechin increases alpha-sarcoglycan protein expression in gamma-sarcoglycan knock out 
mouse muscle. ........................................................................................................................................ 21 
Figure 16: (-)-Epicatechin reduces fibrosis in the gamma-sarcolgycan knock out mouse. ............................ 22 
Figure 17: (-)-Epicatechin reduces plasma CK activity (muscle injury biomarker) in MDX mouse model of 
muscular dystrophy. ................................................................................................................................ 22 
Figure 18: (-)-Epicatechin improves skeletal muscle strength in the MDX mouse model of muscular 
dystrophy. ................................................................................................................................................ 23 
Figure 19: (-)-Epicatechin blocks calcium-induced pore formation in cardiac mitochondria in a dose-
dependent manner. ................................................................................................................................. 23 
Figure 20: (-)-Epicatechin-rich cocoa increases active PGC-1a and decreases acetylated (inactive) PGC1a 
in HF/DM2 patients. ................................................................................................................................. 24 
Figure 21: (-)-Epicatechin-rich cocoa increases mitochondrial complex V protein and cristae abundence in 
HF/DM2 patients. .................................................................................................................................... 25 
Figure 22: (-)-Epicatechin-rich cocoa increases PGC1a and FOX01 association to chromatin from 
quadriceps of HF/DM2 patients. .............................................................................................................. 25 
Figure 23: (-)-Epicatechin-rich cocoa increases antioxidant proteins and total tissue thiols (reduced 
glutathione) in HF/DM2 patients. ............................................................................................................. 26 
Figure 24: (-)-Epicatechin-rich cocoa increases quadriceps muscle nNOS expression in HF/DM2 patients.
 ................................................................................................................................................................. 26 
Figure 25: Muscle growth/differentiation protein expression +/- (-)-epicatechin-rich cocoa in HF/DM2 
patients. ................................................................................................................................................... 27 
Figure 26: Muscle structural proteins induced by  (-)-epicatechin-rich cocoa in HF/DM2 patients. ............... 27 
Figure 27: Changes in sarcomere ultrastructure in representative HF/DM2 patient before and after 
treatment with (-)-epicatechin-rich cocoa. ............................................................................................... 28 
Figure 28: Age-related muscle protein changes in human skeletal muscle samples. ................................... 29 
Figure 29: Changes in muscle strength and follistatin/myostatin ratio following 7 day of (-)-epicatechin 
treatment. ................................................................................................................................................ 29 
Figure 30: Vital signs following (-)-epicatechin administration in healthy subjects ........................................ 30 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
7 
 
1 
STUDY SUMMARY 
1.1 
Background 
(-)-Epicatechin will be evaluated for the treatment of progressive muscle loss and impaired skeletal muscle 
function in Becker Muscular Dystrophy (BMD) patients.   (-)-Epicatechin has been shown to improve muscle 
structure and function in animal and human studies by inducing muscle proteins, mitochondrial biogenesis 
and endogenous anti-oxidant mechanisms.  (-)-Epicatechin and (-)-epicatechin-rich preparations have been 
well-tolerated with no significant safety issues, suggesting that (-)-epicatechin will be compatible with 
chronic administration.  GMP grade (-)-Epicatechin will be provided by Cardero Therapeutics (CT). 
 
(-)-Epicatechin  (-)-Epicatechin is a naturally occurring compound found in many plants and fruits, including 
cocoa seeds, tea and grape[1, 2].   (-)-Epicatechin is a member of the flavanol subfamily of flavonoids,  a 
chemically defined family of plant polyphenols (Figure 1) that have a basic structure of two aromatic rings 
(A and B) linked through three carbons that typically form an oxygenated heterocycle (C ring). The chemical 
characteristics of the C ring define the various subfamilies of flavonoids by providing different arrangements 
of hydroxy, methoxy, and glycosidic groups, and the bonding with other monomers[3].   
 
Figure 1: Chemical structures of flavonoid families and (-)-Epicatechin 
 
The majority of studies of (-)-epicatechin have utilized flavanol-rich cocoa preparations or concentrated tea 
extracts.  Common commercial products that contain 60-90% cocoa can provide a total of ~12 mg of 
flavanols (monomers and multimers)/gram of chocolate. For example, 60% cocoa chocolate by Hershey will 
provide 0.84 mg/gram of (-)-epicatechin and 10.6 mg/gram of total flavanols (9.8 mg as multimers).   
(-)-Epicatechin, monomeric, isolated from tea, will be supplied by Cardero Therapeutics in gelatin capsules, 
each containing 25 mg (-)-epicatechin combined with excipients.   
 
Many of today’s medications are derived from plant extracts, including such commonly used medications as 
digoxin (foxglove), atropine (belladonna), codeine (poppy), irinotecan (dogwood) and taxol (pacific yew).  
Epicatechin is a flavonoid abundant in cacao that mimics the body’s normal response to exercise training 
and that has demonstrated ability to promote increased mitochondrial growth and quality in multiple tissue 
types including muscle, effectively increasing the body’s energy production capacity and reducing tissue 
oxidative stress.  In populations where vigorous exercise is difficult or impossible and there is a high risk of 
secondary metabolic syndrome / insulin resistance such as individuals with muscular dystrophies, spinal 
cord injury, musculoskeletal or injury-induced mobility limitations, advanced heart disease, diabetes or even 
normal aging, the ability to rapidly produce an exercise training-like effect has wide-ranging benefits.  These 
benefits include improved muscle mass, strength and endurance, insulin sensitivity, lipid profiles, and 
improved exercise capacity / physical activity. Pilot data from a trial of epicatechin in Becker muscular 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
8 
dystrophy patients produced an unprecedented constellation of findings including increased muscle 
dystrophin, eutrophin, and follistatin levels and increased blood follistatin levels and decreased myostatin 
levels  all targets of current Duchenne muscular dystrophy treatment strategies.  Treatment of individuals 
with Duchenne and Becker muscular dystrophies with epicatechin will yield a direct palliative effect, 
targeting multiple aspects of the complex cascade of events that if untreated ultimately leads to life-limiting 
loss of mobility, strength and cardiorespiratory function.   
 
In a proof-of-concept study by our group, we demonstrated that epicatechin use in patients with Becker 
muscular dystrophy 1) improves muscle structure and function, 2) induces production or increased 
persistent content of muscle proteins including dystrophin, utrophin, sarcoglycans, and dysferlin, 3) induces 
mitochondrial biogenesis and 4) improves endogenous muscle growth, repair and regeneration 
mechanisms by increasing follistatin and decreasing myostatin levels.  Epicatechin and epicatechin-rich 
preparations have been well-tolerated with no significant safety issues, suggesting that epicatechin will be 
compatible with chronic administration. 
1.2 
Study Purpose 
This is a 48-week open-label extension of our initial proof-of-concept study (UCD0113) in patients with 
Becker muscular dystrophy who participated in the earlier trial.  This single center study will enroll up to 8 
adults who will receive the purified nutritional extract (-)-epicatechin 100mg/day orally for 8 weeks. After 
screening visits, participants will be enrolled in the study if they meet all inclusion criteria.  They will be 
evaluated at screening, baseline, and weeks 4, 8, 12, 24, 16 and 48.  The main criterion for success of the 
study will be presence of one or more biologic or strength and performance outcome measures (Aims 1 and 
2 below) that yield a response magnitude that allows for sufficient power in a Phase II B study with a 
sample size of 30 individuals.  Endpoints will include: 
• 
Efficacy Primary Biomarker:  Plasma biomarkers of follistatin, myostatin, follistatin:myostatin, BNP, NO 
and fasting insulin/glucose by ELISA and Western blot will be collected at baseline and weeks 12, 24, 
36 and 48. 
• 
Efficacy Secondary Strength and Function:  Strength and exercise performance measures will be 
collected at baseline and weeks 12, 24, 36 and 48 for comparison with baseline measures. 
• 
Primary Safety:  A comprehensive physical/neurological exam, medical and adverse event history, 
hematology, blood chemistry and urinalysis safety profile will be collected at screening, baseline and 
weeks 4, 8, 12, 24, 36 and 48. 
• 
Exploratory Proteomics:  Plasma samples for proteomics analysis by mass spectrometry and 
Somalogic platform will be collected at baseline and weeks 24 and 48 for comparison with baseline 
measures. 
 
1.3 
Specific Aims: 
Aim 1 (Efficacy Primary Endpoints):  Evaluate the effect of epicatechin on circulating blood 
biomarkers of mitochondrial biogenesis and muscle regeneration in children with Duchenne 
muscular dystrophy.  Individuals who receive oral epicatechin daily will show evidence of improved 
mitochondrial biogenesis.  This will be reflected by plasma biomarkers associated with muscle growth and 
repair, vascular response to exercise, inflammation and membrane integrity.  Increased number and quality 
of mitochondria will improve glucose metabolism and insulin response. 
 
Aim 2 (Efficacy Secondary Endpoints):  Evaluate the effect of epicatechin on exercise capacity, 
strength and function in children with Duchenne muscular dystrophy.  Epicatechin will improve 
exercise capacity.  Individuals who receive 24 to 48 weeks of oral epicatechin daily will show improved 
exercise and metabolic performance on the six-minute walk test and graded exercise cycle test that is 
associated with exercise “training-like” responses attributed to increases in mitochondrial biogenesis and 
muscle regeneration, and improvements in skeletal muscle perfusion.  Individuals who receive 24 to 48 
weeks of epicatechin will demonstrate improvements in strength and function as measured by clinical 
exercise testing parameters commonly used in muscular dystrophy clinical trials.  
 
Aim 3 (Cardiac Efficacy Endpoints):  Evaluate the effect of epicatechin on left ventricular strain and 
left ventricular ejection fraction.  Individuals who receive 48 weeks of epicatechin 50-100mg daily will 
show improved measures of LV strain and ejection fraction compared to control subjects.   


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
9 
 
 
Aim 4 (Safety Primary Endpoints):  Evaluate the safety and pharmacokinetic profiles of epicatechin 
in children with Duchenne muscular dystrophy.  Epicatechin will demonstrate an acceptable safety 
profile.  Assessments of safety will include a standard safety panel including hematologic, hepatologic, 
renal and metabolic profiles.  Epicatechin administration to adults with BMD will yield pharmacokinetic 
profiles similar to those previously reported.  Pharmacokinetic studies will include repeat assessments of 
trough, 2-hour post (peak) and 4-hour post epicatechin dose levels. 
 
Aim 5 (Pilot Biomarker Endpoints): Perform pilot evaluation of disease- and epicatechin-specific 
circulating protein profiles as pilot biomarkers for monitoring treatment efficacy.  Epicatechin 
stimulates follistatin expression in tissue and blood. In addition to its effects on muscle regeneration, 
follistatin is known to exert anti-inflammatory and anti-fibrotic effects via antagonism of activin and 
myostatin.  Biomarker discovery studies will yield a panel of circulating proteins that are associated with 
treatment response. 
 
2 
BACKGROUND AND SIGNIFICANCE 
2.1 
The Dystrophinopathies: Duchenne and Muscular Dystrophy (DBMD) 
Muscular dystrophies are a group of diverse genetic diseases featuring progressive muscle weakness, 
degradation of muscle fibers, and loss of function [4].  DBMD is an inherited spectrum disorder primarily 
seen in males, with an incidence of 1:3500-5000 live births [4].  Onset of symptoms can occur over a wide 
age range, with most patients diagnosed between 3 and 15 years of age [5, 6].  DBMD is characterized by 
progressive muscle loss (sarcopenia), with loss of strength, muscle injury, degeneration, atrophy, and 
eventually fibrosis and fatty replacement [7]. Patients with the more severe Duchenne (DMD) phenotype 
typically begin to lose ambulatory ability by 9-11 years of age and most are wheelchair dependent by 12-14 
years.  Most Becker (BMD) patients are more mildly affected and lose ambulation across a much wider age 
range from the late teenage years to late adulthood if at all.  Both groups experience secondary 
complications of weakness including lung (e.g., breathing problems, infections) and heart (e.g., 
cardiomyopathy) complications that significantly impair mobility and participation and reduce lifespan[7, 8].  
 
DBMD results from mutations in the gene encoding dystrophin, a key subsarcolemmal protein in the 
dystrophin-associated protein complex (DAPC) at the muscle cell membrane [9].   The DAPC links the 
muscle cytoskeleton with the extracellular matrix and appears to play a role in muscle stabilization during 
contraction as well as nNOS signaling [10, 11]. Loss of dystrophin destabilizes the DAPC and results in 
abnormal muscle membrane permeability, detectable as large elevations in plasma creatine kinase at birth 
and before the appearance of physical symptoms [5].   BMD is attributed to quantitative or qualitative 
decrements in dystrophin expression, while patients with DMD experience complete or near-complete loss 
of dystrophin.   
2.2 
Epicatechin directly targets multiple points in DBMD pathophysiology 
The process of progressive strength loss in individuals with dystrophinopathies is due to a complex 
pathophysiologic cascade that occurs as a secondary effect of the loss of the dystrophin protein.  
Progressive damage to muscle tissue occurs due to changes in the structure of the contractile apparatus 
due to partial or complete loss of the dystrophin associated protein complex (DAPC), and results in 
secondary damage to and depletion of mitochondria.  The mitochondrial injury itself is attributed to oxidation 
injury, calcium accumulation and excessive metabolic demands of regenerating muscle, where cellular 
energy demands are not sufficient to compensate.  Alteration of expression profiles associated with 
improved oxidative phosphorylation in normal exercise response are decreased in DMD and reduce 
effectiveness of tissue remodeling [12, 13].  Activity or exercise-induced mitochondrial upregulation is also 
associated with improvements in insulin resistance and reduced incidence of diabetes, and decreases in 
mitochondrial capacity seen in dystrophinopathy patients are also seen in diabetic patients, suggesting that 
reduced activity or capacity in these pathways more accurately reflects physical activity status rather than 
any specific disease process.  Thus, it is expected that drugs targeting mitochondrial biogenesis will have 
wide-ranging applications outside of the dystrophinopathies, and may also include groups such as patients 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
10
with diabetes and obesity, age-related muscle wasting (sarcopenia), and sarcopenias that occur secondary 
to other conditions such as spinal cord injury and stroke  
 
Epicatechins represent a novel small molecule approach to intervention directly upregulating this 
mitochondrial pathway.  Treatment with epicatechins increases mitochondrial synthesis of ATP in response 
to metabolic demand within 48 hours, and concurrently activates the transcription pathway for muscle 
growth and regeneration by upregulating the protein PGC1a, which in turn upregulates follistatin, which is a 
key regulator of the growth antagonist myostatin.  This has been demonstrated in neonatal mice, where 
PGC1a over expression resulted in increased expression of key muscle proteins including utrophin and 
type-1 myosin heavy chain as well as key mitochondrial proteins [14].  Overexpression of PGC1a also 
drives oxidative gene expression, aiding damaged mitochondria and supporting ATP production [15-20].  
Selsby et al demonstrated that the increase in production of muscle proteins secondary to PGC1a 
overexpression increased Type 1 muscle fiber count, improved resistance to eccentric contraction-induced 
injury and fatigue and reduced evidence of muscle tissue necrosis in the mdx model of dystrophinopathies, 
and provides evidence that the approach improves dystrophic muscle resistance to structural damage 
secondary to activity and exercise [14].   
 
Further evidence suggests that NF-kB, a protein which contributes to healthy tissue remodeling and 
adaptation in response to exercise, paradoxically reduces activation of PGC1a pathways in diseased 
conditions such as dystrophinopathies, diabetes and cachexia, contributing to increased metabolic and 
oxidative stress to muscle tissue and subsequent loss of mitochondria, increased inflammation, and tissue 
degeneration [21].   
 
In addition to decreased resistance to mechanical stressors, patients with dystrophinopathies also under 
express nitric oxide (NO) and show evidence of a subsequent lack of normal vascular response to 
exercise[22].  This reduces oxygen transport capacity to muscles during exercise, creating an environment 
where the tissue is chronically oxygen-starved during activity.  Oxygen depravation leads to increased 
oxidative stress as the remaining mitochondria are forced to utilize relatively greater proportions of 
anaerobic mechanisms to create energy for the muscle tissue.  The by products of this anaerobic process 
include damaging pro-inflammatory reactive oxygen species (ROS) that have been shown to trigger chronic 
activation of the NF-kB pathway discussed above. 
 
The combination of reduced resistance to mechanical stress and reduced oxygen supply and resulting 
chronic inflammation creates a “double hit” that promotes a vicious cycle of tissue damage, inflammation 
and impaired remodeling that ultimately results in gradual and eventually almost complete replacement of 
muscle with non-functional fibrotic and fatty tissue. 
 
In the absence of genetic repair, there is a great unmet clinical need for palliative therapies that can 
slow the progressive loss of muscle function due to secondary damage and/or improve skeletal 
muscle function.  The epicatechin signaling pathway represents a direct intervention that addresses many 
of the defects that occur secondary to the loss of dystrophin that are currently under investigation.  
Treatment with the drug directly increases expression of follistatin resulting in reduction in production of 
myostatin and improved muscle growth and regeneration.  Indirect immune-mediated reductions in 
myostatin using targeted antibodies has become a key therapeutic approach in dystrophinopathies [23].  
Epicatechin use also results in upregulation of PGC1a which increases the number and quality of 
mitochondria in muscle tissue that leads to reduced oxidative stress, improved muscle function and 
improved insulin resistance.  Indirect approaches to reduce oxidative stress using CoenzymeQ10 and 
idebenone are currently in development in DMD and have shown positive results [24, 25].   Indirect 
approaches to reducing inflammation and tissue remodeling that occur secondary to oxidative stress 
include ongoing study of anti-fibrotic drugs such as pentoxifylline and halofuginon [26-28].   Treatment with 
epicatechin directly results in increased NO production, improves vascular response to exercise and 
reduces tissue hypoxia and oxidative stress.  Studies of the ability of PDE5 inhibitors (sildenafil, tadalafil) to 
block downstream degradation of of NO-derived vasodilators is currently being explored in DMD patients to 
improve oxygen supply to muscle tissue[29].  A summary of published and unpublished preclinical and 
clinical data supporting the evaluation of epicatechin in DBMD is presented in Table 1.   
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
11
Table 1: Effects of epicatechin on progressive muscle loss parameters. 
Progression of MD 
due to: 
 Epicatechin Effects 
Loss of 
Dystroglycan 
Proteins 
Stimulated the expression of multiple components of the dystroglycan protein assembly in mice and 
humans, including compensatory expression in a MD mouse model 
Mitochondrial 
Depletion 
Increased expression of transcription factor PGC1a and stimulated mitochondrial biogenesis, 
increased expression of all electron transport complex proteins and increased mitochondrial density 
and mitochondria cristae density in mouse and human muscle 
nNOS Deficiency 
In a pilot clinical trial in patients with diabetes and heart failure,  epicatechin-treated patients showed a 
statistically significant increase in quadriceps muscle nNOS expression 
Oxidative injury 
Increased the expression of endogenous anti-oxidant enzymes superoxide dismutase and catalase 
and increased total thiols in skeletal muscle of aged patients with diabetes and heart failure 
Muscle 
Degeneration 
Reduced creatine kinase activity (plasma marker for muscle membrane injury); improved histological 
appearance in sarcomere morphology in aged patients with diabetes and heart failure  
Impaired Muscle 
Regeneration 
Increased markers of muscle regeneration in skeletal muscle of mdx mice and aged patients with 
diabetes and heart failure 
Muscle Weakness 
Improved muscle strength in mdx mouse model of MD and physical activity in the delta Sarcoglycan 
KO mouse model of limb girdle dystrophy; increased grip strength in middle-aged human volunteers 
Inflammation And 
Fibrosis 
Prevented the myocardial fibrosis associated with the delta-sarcoglycan KO mouse model of MD 
 
It must be stressed that these muscle-related therapeutic activities are unique to epicatechin and do not 
represent general flavonoid properties, as other flavonoids such as quercetin fail to exert similar effects.  
Epicatechin biology is chirally specific, as its diastereomer, catechin, is not only inactive in stimulating 
mitochondrial biogenesis, it antagonizes epicatechin’s effects, possibly due to steric competition at a 
binding site. Nor is the effect due to anti-oxidation, as catechin and quercetin are anti-oxidants equal in 
potency to epicatechin but are inactive with respect to mitochondria. 
3 
EPICATECHIN OVERVIEW 
3.1 
Pharmacokinetics / Pharmacodynamics 
3.1.1 
(-)-Epicatechin 
(-)-Epicatechin is orally bioavailable, as the consumption of (-)-epicatechin or cocoa products can result in 
pharmacologically relevant levels of (-)-epicatechin and its metabolites in blood. In addition to the literature 
reviewed below, the results of a recent human pharmacokinetic study are presented in Section 6.3. 
The absorption of flavonoids occurs mainly in the small intestine and takes place within minutes. In the 
small intestine, flavanols are extensively glucuronidated and partially methylated [30, 31], allowing 
negligible amounts of native (-)-epicatechin in the mesenteric circulation.  
 
In the liver, further glucuronidation, methylation, and sulfation can take place [30, 32, 33]. Several studies 
have determined the presence of these conjugates in the plasma and urine of rodents and humans [33-38], 
as well as in rat bile [32] and brain [39]. In plasma, (-)-epicatechin is present almost exclusively as 
conjugated metabolites[37, 38].    
 
Total plasma concentrations of (-)-epicatechin plus its metabolites are found in the low-micromolar range as 
soon as 1 h after the consumption of a flavanol-rich food (cocoa), with Tmax ~2 hours [34, 37-46]. The most 
abundant (-)-epicatechin metabolites detected in plasma after dark chocolate ingestion were (-)-
epicatechin-3’-β-D-glucur- onide,(-)-epicatechin3’-sulfate,and 3’-O-methyl-(-)-epicatechin-5-sulfate[45].  A 
schematic representation of these metabolites is shown in Figure 2.   In addition, significant levels of 3’-O-
methyl-(-)- epicatechin sulfates substituted in the 4’ and 7 positions were identified [45].   
 
Relative amounts of (-)-epicatechin metabolites identified in plasma in two separate studies are shown in 
Figure 3.   
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
12
Figure 2: Major (-)-epicatechin metabolites in plasma. 
 
 
Schematic representation of the primary structurally-related (-)-epicatechin metabolites found in plasma after injestion of (-)-
epicatechin-containing test beverage [38].  UGT,  UDP-glucuronosyltransferase; SULT, sulfotransferase; COMT, catechol O-
methyltransferase. 
 
Figure 3: (-)-Epicatechin metabolite profile in plasma 
 
A.  (-)-Epicatechin metabolite profile in plasma 2 hours after ingestion of 1.8 mg/kg body weight (-)-epicatechin [38]. Unmodified (-)-
epicatechin represented <0.5%.      B.  Profile of (-)-epicatechin metabolites in plasma after ingestion of 100g of dark chocolate ((79 
mg (-)-epicatechin)) [37].  Data are expressed as the percentage of total plasma AUC0–24 h. Unmodified (-)-epicatechin not detected.  
EC-3’-Gluc, (-)-epicatechin-3’-β-D-glucuronide; EC-4’-Gluc, (-)-epicatechin-4’- β-D-glucuronide; EC-7-Gluc,(-)-epicatechin-7-β-D-
glucuronide; EC-3’-S, (-)-epicatechin-3’-sulfate; 3’Me-EC-5-S, 3’-O-methyl-(-)-epicatechin 5-sulfate; 3’Me-EC-4’-S, 3’-O-methyl-(-)-
epicatechin 4’-sulfate; 3’Me-EC-7-S, 3--O-methyl- (-)-epicatechin 7-sulfate; and 4’Me-EC-5-S, 4’-O-methyl-(-)-epicatechin 5-sulfate. 
 
The Tmax (time to maximum plasma concentration) of (-)-epicatechin and most of its metabolites is 1-2 
hours, elimination half-time (t1/2) from plasma is ~2-2.5 hours, and over 90% of the urinary excretion of 
these compounds was complete by 8 hours[38, 46, 47].  Oral bioavailability (peak plasma concentration/mg 
ingested) for (-)-epicatechin in cocoa/chocolate is ~10-20 nM/mg injested (Table 2).  Assuming ~3L plasma 
in a 75 kg man, oral bioavailability of (-)-epicatechin (% ingested dose in plasma at Cmax, total epicatechin 
and metabolites), can be estimated as 1-2%.  A value of 1.1% was reported for a 45 mg (-)-epicatechin 
dose in a green tea catechin mixture [47]. 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
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Table 2: Selected human oral bioavailability studies of (-)-epicatechin  
Reference 
Test Article 
(-)-Epicatechin 
(mg) 
Peak (-)-Epicatechin 
Species  in Plasma (nM) 
Peak (-)-Epicatechin 
Exposure (nM/mg) 
Loke, W.M., et al., 
2008  [48] 
(-)-Epicatechin 
200 
3570 
17.85 
Ottaviani, J.I., et al., 
2011 [43] 
 (-)-epicatechin added to 
cocoa vehicle 
125 (mean) 
~900 
~7.2 
Ottaviani, J.I., et al., 
2012 [38] 
Cocoa drink 
135 (mean) 
1245 
9.3 
Actis-Goretta, L., et 
al., 2012 [37] 
Dark Chocolate 
79 
873 
11.1 
Donovan, J.L., et al., 
2006 [49] 
Cocoa drink 
55 
630 
11.5 
Schroeter, H., et al., 
2006 [42] 
Cocoa flavanols: 
 917  mg  
 
174 
 
~1950 
 
~11.2 
Baba, S., et al., 2000 
[35] 
Dark chocolate 
220 
4770 
21.7 
 
Oral doses of (-)-epicatechin between 25 and 200 mg have been shown to be pharmacologically relevant.   
A 200 mg oral dose increased measures of plasma NO and decreased the vasoconstrictor endothelin-1 
[48], while administration of an ~75 mg dose (1 mg/kg body weight) resulted in significant improvement in 
vascular function (FMD) [42].   A recent meta-analysis of clinical data reported that an intake of 25 mg (-)-
epicatechin per day was associated with significant reductions in SBP and DBP [50]. 
 
4 
PRELIMINARY STUDIES  
4.1 Preclinical Data 
Collaborators of Cardero Therapeutics at the University of California, San Diego (UCSD) have conducted 
cell culture and animal studies examining the ability of (-)-epicatechin to induce mitochondrial biogenesis 
and SkM protein synthesis, resulting in improved muscle structure and function.      
4.1.1 (-)-Epicatechin Increases Mitchondrial Respiration and Biogenesis in Cultured 
Human Skeletal Muscle Cells (unpublished data) 
The effect of (-)-epicatechin on mitochondrial respiration, biogenesis and structure in human skeletal 
muscle cells in vitro was examined.   Human skeletal muscle cells from a healthy subject were treated with 
0.1 µM of (-)-epicatechin (in cell culture) for 48 hours.   The treated muscle cells manifested a markedly 
enhanced capacity for oxidative phosphorylation (Figure 4).  The ability of epicatechin to stimulate the 
capacity for oxidative phosphorylation in human muscle fibers treated with epicatechin allows the muscle 
cell to substantially increase its rate of oxidative phosphorylation in response to the metabolic demand 
placed on the cells by FCCP, which decreases the mitochondrial membrane potential. Importantly for safety 
reasons, neither endogenous nor resting oxidative phosphorylation in muscle cells is affected.  
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
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Figure 4: (-)-Epicatechin enhances mitochondrial respiration 
 
 
Treated cells were then examined using electron microscopy (Fig 5).   The electron micrographs 
illustrate two effects. One is the marked increase in mitochondrial number. Even more unusual is 
the increased number of cristae per mitochondrion, suggestive of the potential for increased ATP 
synthesis per mitochondrion. To our knowledge, cristae density within a mitochondrion has not 
previously been demonstrated to be acutely modulatable before.  Mitochondrial biogenesis can 
also be blunted by the use of eNOS inhibitors, supporting a role for the NO system in the 
mechanism of action for (-)-epicatechin’s effects.   
Figure 5: (-)-Epicatechin increases mitochondrial number and cristae density in human skeletal 
muscle cells (unpublished data)  
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
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4.1.2 (-)-Epicatechin Induces Mitochondrial Biogenesis Pathway in Human Coronary 
Artery Endothelial Cells (unpublished data) 
Recent unpublished experiments established that (-)-epicatechin initiates mitochondrial replication via the 
activation of two co-factors that participate in the classical transcription pathway for mitochondrial 
biogenesis: The Estrogen Related Receptor (ERR) pathway and the activation of PGC1α.   The ERR 
pathway comprises an orphan nuclear receptor complex consisting of 3 subunits, α, β and γ. When 
exposed to the unknown ligand, they trimerize in the cytoplasm and form a complex with PGC1α, and 
subsequently localize to the nucleus, where they initiate the transcription pathway for mitochondrial 
biogenesis [51]. Nuclear localization of the α subunit is accepted as an indicator of ERR activation. Human 
coronary artery endothelial cells (HCAEC) exposed to (-)-epicatechin demonstrated localization of ERRα to 
the nucleus within 30 minutes (Fig 6).  
Figure 6: (-)-Epicatechin induces estrogen-related receptor alpha translocation (activation) into 
the nucleus of HCAEC 
 
 
                 4.1.3 (-)-Epicatechin Induces Expression of Mitochondrial Biogenesis Factor PGC-1α in 
Aged Mice (unpublished data) 
The peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC-1α) is a 
transcriptional coactivator of nuclear receptors and other transcriptional factors that can enhance 
multiple aspects of cellular energy metabolism, including mitochondrial biogenesis and 
angiogenesis [52, 53]. Expression of PGC-1α in cultured mammalian cells or specific tissues of 
transgenic mice increases number and mass of mitochondria together with a strong enhancement 
of cellular respiratory capacity[17], suggesting that PGC-1α would be expected to enhance 
mitochondrial biogenesis and promote muscle repair and regeneration. The effects of (-)-
epicatechin on PGC-1α levels were examined in young (6 mo), and old (26 mo) mice.   Senile 
mice treated with epicatechin for two weeks exhibited increased expression of PGC-1α across all 
tissues evaluated (Fig 7A). These animals also demonstrated correlative mitochondrial biogenesis 
(not shown). When PGC-1α level of expression was compared in the senile mice to young mice, 
they were markedly diminished, and stimulated by epicatechin treatment in as little as two weeks 
(Fig 7B).  
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
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Figure 7: (-)-Epicatechin increases PGC1-alpha in aged mice. 
 
 
4.1.4 (-)-Epicatechin Induces Expression of Mitochondrial Biogenesis Factor PGC-1α and 
Mitochondrial Proteins in δ-sarcoglycan KO Mice (unpublished data) 
The effects of (-)-epicatechin on PGC-1α levels were examined in the δ-sarcoglycan KO mouse 
model of MD.   3 month old WT and δ-sarcoglycan mice were treated with (-)-epicatechin or water 
for 1 month. In the δ-sarcoglycan KO mice, the loss of PGC1α is striking, as is its stimulation with 
1 month of epicatechin treatment (Fig 8A). The change in PGC1α correlated with loss and regain 
of mitochondrial density (Fig 8B).   The KO mice treated with water exhibited a significant 
depletion of mitochondrial proteins – enzymes and structural proteins, during a phase where they 
developed fibrotic cardiomyopathy. Epicatechin restored mitochondrial protein levels to normal 
levels, suggesting that (-)-epicatechin induced PGC-1a-mediated mitochondrial biogenesis. 
Figure 8: (-)-Epicatechin increases PGC1-alpha and mitochondrial proteins in gamma-
sarcoglycan knock out mice.  
 
 
4.1.5 (-)-Epicatechin Increases In Vivo Mitchondrial Biogenesis and Muscle Function in 
Aged 1 Year Old Mice  
To test for the in vivo effects of (-)-epicatechin on muscle performance and indicators of mitochondrial 
structure (porin, mitofilin) and biogenesis (Tfam), studies were conducted in mice to compare the effects of 
(-)-epicatechin +/- daily exercise with vehicle +/- daily exercise[54].  Aged one year old male mice were 
subjected to two weeks of (-)-epicatechin treatment (1 mg/kg BID, dissolved in water) by oral gavage. 
Significant increases in treadmill performance (≈50%) and enhanced in situ muscle fatigue resistance 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
17
(≈30%) were observed with (-)-epicatechin. Components of oxidative phosphorylation complexes, mitofilin, 
porin, nNOS, and Tfam as well as mitochondrial volume and cristae abundance were significantly higher 
with (-)-epicatechin treatment for hindlimb and cardiac muscles than exercise alone (Figures 9 and 10). In 
addition, there were significant increases in skeletal muscle capillarity. The combination of (-)-epicatechin 
and exercise resulted in further increases in oxidative phosphorylation complexes proteins, mitofilin, porin, 
and capillarity than (-)-epicatechin alone. These findings indicate that (-)-epicatechin alone or in 
combination with exercise induces an integrated response that includes structural and metabolic changes in 
skeletal and cardiac muscles resulting in greater endurance capacity.  
Figure 9: (-)-Epicatechin increases mitochondrial proteins in 1-year old mice. 
 
Effect of (-)-epicatechin and exercise on A) mitochondrial oxidative phosphorylation complexes and B) mitochondrial membrane 
proteins.  Ctrl = water only, Epi=1 mg/kg BID.  Exercise = 30 minutes of treadmill exercise 5 times per week. 
Figure 10: (-)-Epicatechin increases mitochondrial volume density and mitochondrial membrane 
surface area in 1-year old mice. 
 
 
Electron microscopic examination of plantaris muscle to calculate A) mitochondrial volume density (% of cytoplasm occupied by 
mitochondria) and B) mitochondrial membrane surface area for cristae and inner membrane, normalized to outer membrane area.   
 
These results are consistent with a recent study by Hutteman et al which described the ability of (-)-
epicatechin to maintain exercise-induced improved capillarity and mitochondrial capacity in mice after 
discontinuation of exercise training, in part by induction of mitochondrial complex proteins [55]. 
In a follow up study the capacity of (-)-epicatechin treatment to stimulate myocardial angiogenesis was 
examined in the same animals.  Results indicate that exercise training or (-)-epicatechin significantly 
stimulated myocardial angiogenesis by 30-35% above control levels (as judged by biochemical and 
histological measures) whereas the use of both lead to further significant increases (to ~50%). Exercise 
training effects were associated with significant increases in protein levels and/or activation (i.e. 
phosphorylation) of canonical angiogenesis pathway associated events [vascular endothelial growth factor 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
18
(VEGF), eNOS, NO and cGMP]. In most cases, (-)-epicatechin generated comparable degrees of 
stimulation of these pathways. The use of combined treatment led from incremental to additive outcomes in 
these signaling pathway endpoints.  
4.1.6 (-)-Epicatechin Increases Mitchondrial Biogenesis and Improves Myocardial 
Function in Rats  
Results from a series of studies in a rat myocardial infarction model suggests that (-)-epicatechin may 
facilitate preservation of myocardial function via preservation of mitochondrial structure and function.  In the 
first study [56] (-)-epicatechin (1 mg/kg/day) or water (control) pretreatment was administered daily via oral 
gavage to male rats for 2 or 10 days. Ischemia was induced via a 45-min coronary occlusion. Reperfusion 
was allowed until 48 h or 3 wk while treatment continued. With 2 days of treatment, no reductions in 
myocardial infarct (MI) size occurred. After 10 days, a significant ~50% reduction in MI size occurred in the 
(-)-epicatechin-treated animals. (-)-Epicatechin rats demonstrated no significant changes in hemodynamics. 
Tissue oxidative stress was reduced significantly with (-)-epicatechin treatment. Matrix metalloproteinase-9 
activity demonstrated limited increases in the infarct region with (-)-epicatechin. By 3 wk, a significant 32% 
reduction in MI size was observed with treatment, accompanied with sustained hemodynamics and 
preserved chamber morphometry. 
 
A subsequent study mirroring the one noted above was performed but using a more severe modality of 
myocardial injury which over time can trigger the development of heart failure (permanent coronary 
occlusion) [57]. Results from this study also indicated significant reductions in MI size by (-)-epicatechin (1 
mg/kg/day) early after occlusion (48 h) that were sustained over time (3 weeks). Treatment did not alter 
hemodynamics. These effects were accompanied by the significant long-term (3 week) preservation of 
myocardial structure and function. 
 
An additional study was implemented to test the potential of (-)-epicatechin to exert cardioprotection during 
I/R via modulation of mitochondrial function [56]. Ischemia was induced in rats via a 45 min occlusion, 
followed by reperfusion for 1 h, 48 h, or 3 weeks (wk). (-)-Epicatechin (10 mg/kg) was administered IV 15 
min prior to reperfusion for the single dose group and again 12 h later for the double dose group. Controls 
received water. A single dose of (-)-epicatechin significantly reduced infarct size by 27% and 28% at 48h 
and 3 wk, respectively, compared to controls. Double dosing further decreased infarct size at 48 h by 80%, 
which was sustained at 3 wk (52% reduction). In order to assess if (-)-epicatechin-induced cardioprotection 
was mediated by protection of mitochondrial function, mitochondria were isolated from the left ventricle of 
sham, I/R, and I/R + (-)-epicatechin animals 1 h after ischemia. I/R animals had a significant decrease in 
mitochondrial O2 consumption, significant increase in mitochondrial Ca2+ levels, and decreased ATP and 
NADH pools. (-)-Epicatechin protected against these changes and had levels similar to sham animals. 
Taken together, results suggest that (-)-epicatechin preserves myocardial bioenergetics which likely 
underlies the cardioprotection observed. 
4.1.7 (-)-Epicatechin Modulates the Synthesis of Muscle Growth and Differentiation 
Proteins in Cultured Cells and Mice (Aged and MD)(unpublished data) 
The ability of (-)-epicatechin to effect markers of muscle growth and differentiation were assessed in 
cultured C2C12 myoblasts, 6 and 26 month old mice and mdx mice (MD model).   
Muscle Regulatory Protein Expression in C2C12 Cells  C2C12 cells were cultured +/- (-)-epicatechin in the 
presence of 1% horse serum, which induces differentiation (myoblast to myotube transition). Figure 11 
depicts the three day time course of differentiation (by Western blots).  (-)-Epicatechin treatment 
accelerated and increased protein levels of the muscle growth factor follistatin and those of differentiation 
(myogenin, MyoD), while decreasing levels of the inhibitor myostatin, suggesting that (-)-epicatechin 
upregulates muscle differentiation.   
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
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Figure 11: Muscle differentiation in time course in C2C12 cells +/- (-)-epicatechin. 
 
 
4.1.8  Muscle Regulatory Protein Expression in 6 and 26 month old Mice 
The expression of muscle growth, differentiation and senescence proteins in quadriceps SkM biopsies from  
6 and 26 month old mice, either untreated (control) or treated with 1 mg/kg (-)-epicatechin for 15 days, was 
quantified by Westerns (Figure 12).   There were clear age-related changes in protein expression, with 
notable increases in SkM myostatin and decreases in follistatin protein levels. Modest decreases were 
observed in MEF2, MyoD and myogenin with larger differences noted for Myf5. As expected, large 
increases were observed in senescence-associated β-galactosidase: (SA-β-Gal), a cell senescence 
marker, in older animals.  As in the C2C12 cells, (-)-epicatechin treatment decreased myostatin while 
increasing the levels of differentiation-promoting factors, with three of the factors (MyoD, MEF2 and 
myogenin) reaching levels in the old mice that were similar or greater than those seen in young control 
mice.  
 
Figure 12: Muscle regulatory protein expression in 6 and 26 month old mice +/- (-)-Epicatechin 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
20
                 4.1.8.1 Muscle Regulatory Protein Expression in mdx Mouse Model of MD 
Figure 13 depicts representative images obtained from Western blots performed on diaphragm muscle 
samples from mdx mice treated for month with (-)-epicatechin starting at 12-16 weeks of age. Muscle 
samples were probed to evaluate changes in protein levels for the muscle growth modulators, myostatin 
and follistatin. As can be observed in the upper panels, in mdx mice there is a significant upregulation of 
myostatin that did not improve with treatment. In contrast, follistatin levels in the diaphragm were 
significantly reduced in water treated mdx mice and recovered with (-)-epicatechin. Follistatin is a trophic 
muscle hormone well known to promote muscle regeneration and well as inhibit fibrosis and inflammation. 
Changes in recognized regulators of muscle differentiation were evaluated (Myf5, MEF2A, MyoD and 
myogenin). As can be observed, with the exception of MyoD where (-)-epicatechin treatment did not fully 
recover protein levels, all other modulators were restored to wild type (WT) levels with treatment. Similar 
results were obtained in an analysis of the mdx gastrocnemius muscle (data not shown). 
Figure 13: (-)-Epicatechin modulates muscle regulatory proteins in diaphragm muscle in MDX 
mice. 
 
4.1.9 (-)-Epicatechin Stimulates the Expression of MD-Relevant Muscle Structural 
Proteins in Aged and MD Mice (unpublished data) 
The effects of (-)-epicatechin in animals experiencing progressive muscle loss was examined in aged 
normal mice (model of sarcopenia) and δ-sarcoglycan KO mice (model of limb-girdle muscular dystrophy).  
In examining muscle loss associated with age, 26 month old mice, within 6 months of the end of their 
natural lifespan, were treated with (-)-epicatechin (1mg/kg) for just two weeks. All mice demonstrated 
increases in dystrophin, the sarcoglycans, and desferlin when compared by Western blot to age-matched 
controls (Fig 14).  
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
21
Figure 14: (-)-Epicatechin increases DAPC proteins in middle aged mice. 
 
The effect of epicatechin’s stimulation of the dystrophin protein complex was also examined in the δ-
sarcoglycan KO mouse. As shown in Fig.15, such mice also manifest a marked loss of α sarcoglycan 
protein expression, a loss which was quickly reversed by two weeks of epicatechin treatment, 1 mg/kg/twice 
a day, potentially representing compensatory sarcoglycan expression.    
Figure 15: (-)-Epicatechin increases alpha-sarcoglycan protein expression in gamma-
sarcoglycan knock out mouse muscle. 
 
4.1.10 (-)-Epicatechin Reduces Fibrosis in δ-Sarcoglycan KO Mice (unpublished data) 
Treated animals from the experiment described above also showed a dramatic decrease in expression of 
collagen III, a marker of fibrosis (Fig 16, left panel).  The reduction in collagen III translated into reduction of 
myocardial fibrosis as judged by quantitative histology of heart valve tissue sections (Fig 5.13, right panel).  
These data suggest the potential of (-)-epicatechin to reduce cardiomyopathy often associated with MD. 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
22
Figure 16: (-)-Epicatechin reduces fibrosis in the gamma-sarcolgycan knock out mouse.  
 
4.1.11 (-)-Epicatechin Reduces Plasma Marker of Muscle Damage and Improves Muscle 
Strength in MDX Mouse Model of MD (unpublished data) 
In order to confirm ongoing muscle injury in the mdx mouse model and assess the effect of (-)-epicatechin 
on plasma creatine kinase (CK), mice were treated with water or (-)-epicatechin (1mg/kg, bid) by oral 
gavage for 4 weeks; normal mice (WT) were treated with water.  Excess activity of CK in plasma is 
recognized as a marker of skeletal and/or cardiac muscle damage.  As shown in Fig 17, the levels of CK 
activity rise in water treated mdx mice and they are significantly reduced with epicatechin treatment to 
levels similar of those obtained in wild type animals.   
Figure 17: (-)-Epicatechin reduces plasma CK activity (muscle injury biomarker) in MDX mouse 
model of muscular dystrophy. 
 
 
In a separate study to evaluate the effect of (-)-epicatechin on muscle strength, mdx mice were treated with 
(-)-epicatechin (1mg/kg, qd) by oral gavage for 4 weeks; normal mice (control) were treated with water. 
Muscle strength testing was performed at baseline and at 4 weeks, consisting of measuring hang time of 
each mouse from a wire grid (30), with percent change from baseline calculated for each mouse.   Figure 
18 depicts (mean±SD) percent change in hang time for control and mdx mice.  Control and mdx mice 
receiving water showed modest 18% and 14% increases, respectively, while (-)-epicatechin treatment 
resulted in a 55% increase in hang time.   Although this was a small pilot study, the results are consistent 
with the ability of (-)-epicatechin to improve muscle function in animals lacking dystrophin, providing initial 
proof-of-concept MD efficacy data.    
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
23
Figure 18: (-)-Epicatechin improves skeletal muscle strength in the MDX mouse model of 
muscular dystrophy. 
 
4.1.12 (-)-Epicatechin Blocks Calcium-Induced Pore Formation in Cardiac Mitochondria 
(unpublished data) 
Cardiac myocyte mitochondria were isolated from rat hearts and used to assess (-)-epicatechin’s ability to 
block calcium influx and prevent damage.  Mitochondrial swelling (measured by changes in light 
transmission) was induced by exposure to 33 µM calcium chloride alone or calcium chloride plus increasing 
doses of (-)-epicatechin (10-8-10-4M)(Figure 19). (-)-Epicatechin blocked the opening of the mitochondrial 
PTP in a dose-dependent manner, suggesting that (-)-epicatechin could be effective at preserving 
mitochondrial function in calcium-overloaded MD muscle cells and thus reducing mitochondrial-related 
muscle necrosis in MD patients.  
Figure 19: (-)-Epicatechin blocks calcium-induced pore formation in cardiac mitochondria in a 
dose-dependent manner. 
 
Epi = (-)-epicatechin.   Epi-4 – Epi-8  = (-)-epicatechin concentrations  of 10-4 – 10-8M. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
24
5.HUMAN STUDIES 
5.1 Clinical Study #1:  Mitochondrial Biogenesis and Muscle Improvement in Patients with Heart 
Failure and Type 2 Diabetes Patients (UCSD) 
A proof of concept (POC) study was performed at UCSD in five heart failure, type 2 diabetes mellitus 
patients using (-)-epicatechin rich cocoa (mixture of a beverage and chocolate) in order to assess the 
effects on skeletal muscle mitochondria structure, indicators of mitochondrial biogenesis and SkM proteins 
and structure [58]. Published reports indicate that in patients suffering from heart failure or diabetes there is 
a significant compromise in mitochondrial structure and muscle bioenergetics [59-62]. Thus, altered 
mitochondrial bioenergetics may explain (at least in part) the impaired exercise capacity that is present in 
these patient populations. Changes were assessed in protein and/or activity levels of oxidative 
phosphorylation proteins, porin, mitofilin, nNOS, nitric oxide, cGMP, SIRT1, PGC-1α, Tfam, and 
mitochondria volume and cristae abundance by electron microscopy in skeletal muscle biopsies. Apparent 
major losses in normal mitochondria structure were observed before treatment.  
5.1.1 Markers of Mitochondrial Biogenesis, Structure and Function 
Fig 20 demonstrates that (-)-epicatechin-rich cocoa increased active PGC-1α with correlative decreases in 
acetylated (inactive) PGC1α, consistent with induction of mitochondrial biogenesis. This upregulation in 
mitochondrial biogenesis correlated with increased mitochondrial protein expression and cristae abundance 
(Figure 21).  
Figure 20: (-)-Epicatechin-rich cocoa increases active PGC-1a and decreases acetylated 
(inactive) PGC1a in HF/DM2 patients. 
 
 
 
 
b = before 
epicatechin 
a = after 
epicatechin 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
25
Figure 21: (-)-Epicatechin-rich cocoa increases mitochondrial complex V protein and cristae 
25bundance in HF/DM2 patients. 
 
The same patients demonstrated a marked increase in PGC-1α localized to the nuclear chromatin (22). 
There was a concurrent co-localization of Fox01, a cofactor with PGC-1α for inducing transcription of 
endogenous anti-oxidant pathways (and an important regulator of muscle energy metabolism) [63]. One of 
the characteristics of muscles in sarcopenia is evidence of marked oxidative injury. These anti-oxidant 
enzymes (e.g., superoxide dismutase, catalase, see Fig 23) would be predicted to have a beneficial effect 
on the injured muscle of muscular dystrophy.  
Figure 22: (-)-Epicatechin-rich cocoa increases PGC1a and FOX01 association to chromatin 
from quadriceps of HF/DM2 patients. 
 
5.1.2 Markers of Tissue Oxidative Stress  
Using the same group of patients but incorporating into the study a group of control subjects of similar ages 
(n=4) markers of tissue oxidative stress were compared before and after treatment with (-)-epicatechin-rich 
chocolate (Fig 23).  
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
26
Figure 23: (-)-Epicatechin-rich cocoa increases antioxidant proteins and total tissue thiols 
(reduced glutathione) in HF/DM2 patients. 
 
SOD2 and catalase expression were restored to levels exceeding age-matched controls and approaching 
levels normally associated with the young.  A significant ~60% decrease in SkM tissue thiol levels were 
observed before treatment and these were fully restored to levels similar to controls by (-)-epicatechin-rich 
chocolate. Protein nitrosylation levels (determined using a slot blot) can be used as a general indicator of 
tissue oxidative stress levels. SkM samples of control and HF/DM2 patients were examined (data not 
shown). Prior to treatment, patient protein nitrosylation levels were significantly higher by ~2.5 fold than 
controls (=100%) and were restored to ~1.5 fold by treatment with (-)-epicatechin-rich chocolate. Altogether 
these results indicate that the use of (-)-epicatechin-rich chocolate has a potential to restore tissue 
mitochondria structure and oxidative stress levels back towards control.  
5.1.3 nNOS Expression 
Patient muscle biopsies were assessed for nNOS expression, as myopathy, particularly BMD, is often 
associated with both depletion of neuronal NO synthase (nNOS) and its dislocalization from the 
sarcolemma.  Loss of nNOS, in turn, is linked with impaired mitochondrial biogenesis, increased muscle 
atrophy and autophagy. Depletion and dislocalization of nNOS has a negative impact on muscle function in 
muscular dystrophy, causing increased muscle fatigability and functional ischemia during exercise[10, 64]. 
In this clinical trial, HF/DM2 patients treated with (-)-epicatechin-rich chocolate showed a statistically 
significant increase in quadriceps muscle nNOS expression compared to pre-treatment baseline (Fig 24).  
Figure 24: (-)-Epicatechin-rich cocoa increases quadriceps muscle nNOS expression in HF/DM2 
patients. 
 
Muscle Protein Expression and Sarcomere Organization Patient SkM biopsies were evaluated for levels of 
muscle differentiation and structural proteins, and for sarcomere organization (unpublished data).  Similar to 
the results of studies in C2C12 cells and mice, treatment with (-)-epicatechin increased the expression of 
proteins involved in muscle differentiation (follistatin, myogenin, MyoD), while levels of the inhibitor 
myostatin decreased (Figure 25). 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
27
Figure 25: Muscle growth/differentiation protein expression +/- (-)-epicatechin-rich cocoa in 
HF/DM2 patients. 
 
Biopsies from the HF/DM2 patients were analyzed by Western blot to assess the effect (-)-epicatechin-rich 
cocoa on the dystrophin-associated protein complex (DAPC), a key structural component in muscle cells.  
As shown in Fig 26, prior to treatment there was a striking deficiency of dystrophin, which recovered to 
control levels with treatment. Significant increases with treatment were also noted in all the sarcoglycans, 
beta-dystroglycan and utrophin.  
Figure 26: Muscle structural proteins induced by (-)-epicatechin-rich cocoa in HF/DM2 patients. 
 
HF/DM2 patients before (b) and after (a) 30 day treatment with epicatechin-rich cocoa.  Muscle control = healthy volunteers.  SG: 
sarcoglycans, DG: dystroglycan     
 
As the perturbation of dystrophin levels can greatly compromise sarcomere organization and thus, 
contractile function, SkM samples were evaluated using electron microscopy. Figure 27 shows a 
representative sample from one patient obtained before vs after treatment. As observed, at baseline 
sarcomere “organization” was very poor (no clear definition of Z and other bands). With treatment, 
sarcomere organization markedly recovered. Results were “quantified” using blinded, naïve graders and a 
highly significant improvement (P<0.0001) was observed in sarcomere organization (1.7±0.5 before vs. 
3.1±0.5 after out of a maximum scale of 4) after scoring 100 EM images (10 before, 10 after/patient).  
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
28
Figure 27: Changes in sarcomere ultrastructure in representative HF/DM2 patient before and 
after treatment with (-)-epicatechin-rich cocoa.  
 
5.2 Clinical Study #2:  Age-Related Changes in Muscle Regulatory Proteins and Effect of (-)-
Epicatechin on Muscle Function and Proteins in Healthy Volunteers (UCSD) 
A two part study was conducted to evaluate age-related muscle changes and the effects of (-)-epicatechin 
on those changes (manuscript submitted for publication, 2012).   In the first part, tissue bank SkM samples 
were obtained from young (28.5±7 years, n=6) and old (62±2 years, n=6) subjects and analyzed for the 
amounts of muscle regulatory proteins.   Levels of SkM growth (myostatin, follistatin), differentiation 
(myogenin, MyoD, MEF2A, Myf5) and senescence (senescence-associated β-galactosidase: SA-β-Gal) 
proteins in each group are shown in Figure 28.   Muscle levels of myostatin (differentiation inhibitor) and 
SA-β-Gal (senescence) were increased in the older group, while those of follistatin, MyoD and myogenin 
decreased, suggesting an age-related loss of muscle growth and differentiation potential.   These data 
defined a profile of age-related changes in muscle regulatory proteins and informed the selection of muscle 
protein outcome measures to be used in the second part of the study:  (-)-epicatechin treatment of middle 
aged subjects.  
 
In part 2, a small pilot treatment study was performed in human subjects (n=6, average age 41±5 years) to 
assess the effects of (-)-epicatechin on muscle strength and blood levels of myostatin and follistatin.  
Subjects were treated for 7 days with 25 mg of (-)-epicatechin in capsules BID.  Muscle strength was 
assessed by hand grip dynamometry (thrice with each hand, alternating hands between trials and resting 
for 10 seconds in order to prevent fatigue, maximum strength attained was used for analysis). Myostatin 
and follistatin was measured in blood samples taken before and after the 7 days of treatment.  Treatment 
resulted in a bilateral increase in hand strength of ~7% which was accompanied by a significant increase in 
the ratio follistatin/myostatin (Figure 29).  This coupling of functional and molecular changes suggests that 
(-)-epicatechin has potential as a therapy to reduce progressive muscle loss.  
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
29
Figure 28: Age-related muscle protein changes in human skeletal muscle samples. 
 
 
Figure 29: Changes in muscle strength and follistatin/myostatin ratio following 7 day of (-)-
epicatechin treatment. 
 
5.3 Clinical Study #3:  Pharmacokinetics of (-)-Epicatechin in Healthy Volunteers (UCSF) 
An open label study in was carried out by Dr. Christopher Barnett at the University of California, San 
Francisco (UCSF) to assess the safety and pharmacokinetics of a single dose of purified (-)-epicatechin in 
healthy volunteer subjects (n=9). (-)-Epicatechin doses of 50, 100 or 200 mg were administered in 40 ml of 
water. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
30
5.3.1 Safety and Tolerability 
No adverse effects were reported or observed in any of the 9 subjects. Subjects specifically denied 
symptoms of light headedness, dizziness, fatigue, chest discomfort or changes in breathing during the 
observation period. Data from vital sign monitoring is shown in Figure 30.   
 
At time +0.5 hours there was a trend towards reduced mean systolic blood pressure with a concomitant 
decrease in heart rate that is consistent with subjects being relaxed, and not with a blood pressure lowering 
from (-)-epicatechin. In one subject blood pressure at time +0.5 hours decreased markedly, but the value 
was obtained immediately upon waking, the subject denied symptoms and the heart rate was also reduced 
from baseline, findings that are consistent with relaxation and not (-)-epicatechin induced vasodilatation.  
Figure 30: Vital signs following (-)-epicatechin administration in healthy subjects. 
 
5.3.2 Pharmacokinetics  
Pharmacokinetic parameters for each dose administered are summarized presented in detail in the 
investigator’s brochure. The (-)-epicatechin concentration and metabolite concentration rose rapidly 
following oral administration consistent with rapid absorption and first pass metabolism as has been 
previously described [34, 37-44, 46]. Complete plasma concentration profiles of (-)-epicatechin and its 
metabolites after oral administration can be found in the complete PK study report in the investigator’s 
brochure. The shape of the natural log transformed plasma concentration curves was consistent with first 
order pharmacokinetics. 
 
The ratio of the AUC for (-)-epicatechin and each metabolite to administered dose of (-)- epicatechin is 
given in Table 6.4.  The AUC/dose ratio remains nearly constant for (-)-epicatechin but increases with 
increasing dose of each of the metabolites suggesting either dose dependent excretion of these 
metabolites, or perhaps saturation of some competing pathway for epicatechin metabolism. Investigator 
brochure Table 6.5 shows that AUCmetabolite/AUCepicatechin  is high for each of the metabolites suggesting that 
one or more of the metabolites may be an important mediator of biological effects. 
5.3.3 Conclusions 
 (-)-Epicatechin was well tolerated over the 50-200 mg dose range, with rapid absorption and first pass 
metabolism.  Non-metabolized (-)-epicatechin represented <5% of the total (-)-epicatechin compounds by 
30 minutes.   (-)-Epicatechin and its metabolites were rapidly cleared from the body with a plasma 
elimination half-life of approximately 2.5 hrs for the 100 and 200 mg doses.  Plasma concentration of (-)-
epicatechin was generally proportional to the administered dose. The 3 dominant groups of metabolites in 
our study were glucuronidated, sulfated and methyl sulfated, consistent with a recently published  
evaluation of (-)-epicatechin pharmacokinetics and metabolism [37]. A novel finding in this study was that 
the AUCMetabolite/AUCepicatechin ratio increased with increasing doses suggesting dose dependency of (-)-
epicatechin metabolism.   
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
31
5.4 Clinical Study #4: Epicatechin in Becker phenotype dystrophinopathy patients. 
As a proof of concept in adults with dystrophinopathies, we conducted a single-center open-label proof-of-
concept pilot study of oral epicatechin 50mg twice daily in ambulatory adults with genetically-confirmed 
Becker muscular dystrophy from August 2013 to June 2014. 
Study participants:  This was a single-center proof-of-concept pilot study and due to the limited availability 
of both genetically-confirmed BMD patients and a limited drug supply, we employed a convenience sample 
for this study.  The main criterion for success of the study was presence of one or more biologic or strength 
and performance outcome measures that yield a response magnitude that allows for sufficient power in a 
Phase II B study with a sample size of 30 individuals.  A maximum of ten participants were to be enrolled in 
our study.  Participants were males between the ages of 18 and 60 years with a genetically-confirmed 
diagnosis of BMD, average to low daily physical activity and the ability to ambulate at least 75 meters 
without the use of assistive devices.   
Study drug:  Participants received epicatechin by mouth 50mg twice per day (100mg per day total dose) 
for 8 weeks.   
Study procedures and evaluation:  Participants had a total of 7 study visits each at baseline and at 
screening, day 1, and weeks 1, 2, 4 and 8.  A comprehensive safety review was conducted at each visit, 
and a medical monitor reviewed suspected study-related adverse events throughout the duration of the 
study.  Evaluations of efficacy are shown in the table below. 
 
Table 2:  BMD epicatechin pilot study efficacy evaluations. 
1) assessment of peripheral blood biomarker profiles;  
2) assessment of baseline and post-treatment muscle biopsy by histology, Western blot and electron microscopy, and;  
3) assessment of strength by isokinetic dynamometry and quantitative grip testing [7, 65], 4) exercise performance and metabolic 
testing during a standardized graded recumbent cycle test[66, 67],  
5) muscle perfusion of the vastus lateralis by NIRS during the exercise test [68], and  
6) the 6-minute walk test.   
                5.4.1 Results 
Recruitment and baseline data: Seven participants with 
sufficient genetic diagnostics passed screening.  One 
participant who failed screening was not sufficiently 
ambulatory to perform the required functional evaluations.  
One participant withdrew consent during screening.  The 
remaining participants all began treatment.  Baseline 
characteristics of the final study cohort are shown in Table 
3.  One participant was unable to participate in the full set of 
strength testing measures at baseline due to the advanced 
level of his disease.  Tissue for electron microscopy was 
limited to 3 patients due to poor tissue quality and an error in 
tissue fixation for one sample. 
               5.4.2 Biomarker outcomes at 8 weeks 
All enrolled participants completed 8 weeks of follow-up on study medication.  One participant sustained a 
fall at home (unrelated to study medications) that resulted in a minor knee injury and temporary limitation of 
mobility just prior to his 8-week evaluation and was unable to undergo week 8 exercise testing or biopsy.   
Biomarkers of mitochondrial biogenesis from muscle biopsy tissue: Notable changes in muscle tissue 
as measured by Western blot included significant increases in transcription factors LKB1, PGC1a, AMPK, 
and mitofilin associated with mitochondrial biogenesis and function (Figure 1).  We were able to collect 
bicep muscle tissue of sufficient quality to directly image mitochondria for 3 participants.  Those participants 
showed increases in both numbers of mitochondria and cristae abundance supportive of our overall 
concept (Figure 2).  One participant demonstrated approximately double the number of muscle 
mitochondria per unit area after 8 weeks of treatment (Figure 2, first panel).  
 
Table 3:  Participant characteristics at baseline. 
Measure 
N Mean SD 
Min 
Max 
Age at screening (Yrs) 
7 
46.6 
9.5 
31.5 
60 
Height (cm) 
7 179.2 
4.3 172.5 185.5 
Weight (kg) 
7 
83.2 
18.3 53.3 104.5 
Forced Vital Capacity (%) 7 
93.4 
12.5 
77 
116 
6MWD (m) 
7 
372 
87.2 
245 
502 
Time to Stand (s) 
5 
5.5 
2.3 
2.9 
8.1 
Time to Climb 4 Stairs (s) 7 
7.4 
7.4 
4.4 
12.3 
Time to Run 10m (s) 
7 
7.9 
7.9 
5.1 
12.8 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
32
Figure 1:  Increased muscle / mitochondrial transcription factors. 
 
 
 
Figure 2:  Increases in mitochondrial count and cristae abundance in 4 participants with viable muscle biopsies. 
        
         
 
Biomarkers of regeneration from muscle biopsy tissue: Western blot results from muscle biopsy also 
indicated significant alterations in expression of tissue follistatin and myostatin (Figure 3), which are major 
regulators of muscle growth and repair, suggesting a shift toward proliferation of mitochondria and muscle 
tissue overall.  
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
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Figure 3:  Pro-repair regulation of muscle growth and regeneration-related proteins. 
 
 
Dystrophin-related proteins from muscle biopsy tissue: Increases were seen in muscle tissue Western 
blot for key structural proteins dystrophin, utrophin and dysferlin (Figure 4), with non-significant increasing 
trends in other related skeletal muscle proteins suggesting a downstream pattern of growth and repair that 
is consistent with the previously mentioned increases in regulatory proteins. 
 
Figure 4:  Increased dystrophin-associated proteins in muscle by Western blot. 
 
 
Biomarkers of muscle regeneration and mitochondrial growth from plasma: Changes in muscle tissue 
follistatin and myostatin were replicated in circulating plasma as well, with increased follistatin and 
decreased myostatin expression.  The striking change in the ratio between plasma levels of growth 
potentiating follistatin and growth inhibiting myostatin suggests a likely biomarker of initiation of a tissue 
repair state (Figure 5) that will be useful in future clinical trials.  Mass spectrometry yielded statistically 
significant increases (p<0.05) of between 8-fold and 140-fold in mitochondrial proteins acyl-coenzyme A 
thioesterase 2, acyl-CoA synthetase family 3, mitochondrial chaperone BCS1, paraplegin, 28-s ribosomal 
protein S31 (mitochondrial) and threonine synthase-like 1, all of which are indicators of upregulated 
mitochondrial production. 
 
 
 
 
 
 
 
 
 
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
34
Figure 5: Follistatin and myostatin changes in circulating plasma act as a biomarker of muscle growth and repair. 
 
 
Biomarkers of heart failure and NO reserve from plasma: In addition to indicators of improved 
energetics and muscle repair, we demonstrate significant increases in plasma nitrates, including nitric oxide 
(NO), a key regulator of vascular tone that promotes tissue oxygenation during exercise (Figure 6).  Nitric 
oxide synthetase (NOS), which is responsible for NO production, co-localizes with dystrophin and is known 
to be reduced in individuals with dystrophinopathies.  We also noted significant reductions in plasma B-
natriuretic peptide (BNP) which is expressed during heart failure and by skeletal muscle satellite cells in 
response to ischemic stress (Figure 6) [69]. These results again suggest an overall pattern of growth and 
repair and normalization of function.   
 
Figure 6: Changes in NO and BNP expression in plasma show reduced ischemic stress. 
   
 
 
               5.4.3 Strength and function and safety outcomes at 8 weeks. 
Metabolic indicators during graded exercise cycle testing: Overall, duration of exercise and maximal 
attainable workload did not increase on the graded cycle exercise test.  We did observe significant 
improvements in metabolic exercise performance that are consistent with and similar in magnitude to those 
seen in response to short-duration (8-10 weeks) exercise training regimens.  During the graded cycle 
exercise test, participants demonstrated significant decreases from baseline at 8 weeks in VO2, blood 
lactate and heart rate for a given level of workload in watts (Figure 7) indicating a reduced energy cost of 
exercise, and improved muscle energy utilization and respiratory efficiency.  These effects are highly 
consistent with our biological marker, and further support our concept that supplementation with epicatechin 
provides an exercise training-like effect for muscle in people with dystrophinopathies in a manner consistent 
with moderate exercise but without the damaging mechanical stress.  Participants demonstrated significant 
changes in tissue oxygen saturation index (TSI) by near infrared spectroscopy (NIRS) in the vastus lateralis 
during the six-minute walk test (p<0.001).   These changes suggest an improved degree of vasodilation 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
35
during exercise that is consistent with the overall increase in expression of plasma NO observed in the 
biomarker studies. 
Figure 7: Metabolic exercise test parameters show efficiency gains during exercise. 
 
Clinical measures of strength and function:  Despite metabolic testing evidence demonstrating reduced 
work of exercise via V02, lactate and heart rate reductions at a given level of work, we failed to show 
significant groupwise improvements in commonly-used clinical measures of timed motor performance, 
strength and pulmonary function that we included in our evaluation set.  However, our participant who 
demonstrated a doubling of mitochondrial volume as noted above also increased his 6MWT distance from 
350m to 494m while other participants showed mild increases or stability over 8 weeks.   We feel that the 
lack of response in clinical measures is due mostly to the short duration of the study and the small sample 
size.  It has been demonstrated in randomized clinical drug trials in patients with more rapidly progressing 
Duchenne muscular dystrophy diagnoses that evidence of groupwise differences between treated and non-
treated participants do not become evident until between 24 and 48 weeks of treatment [70].  Given the 
current evidence there is a clear indication of the need for long-term studies of epicatechins in 
dystrophinopathy patients.   
5.4.4 Safety outcomes at 8 weeks 
No serious adverse events occurred during the treatment phase of the study.  Mild to moderate health 
events were consistent with community living (e.g. cold, headache) and were not present in a pattern that 
suggested that they were related to epicatechin therapy.  One participant experienced a biopsy wound 
infection that resolved with oral antibiotics.  Moderate short-duration bruising from muscle biopsies was 
present in all participants and was consistent with standard clinical care, and resolved without incident. 
 
6 
     RESEARCH DESIGN AND METHODS 
6.1 Selection and Withdrawal of Participants  
       6.1.1 Criteria for Enrollment 
Up to ten participants will be studied in this initial pilot project.    
Participant Inclusion Criteria 
• 
Prior participation in UCD0113 BMD epicatechin pilot study 
• 
Male 
• 
Age 18 years to 70 years 
• 
Average to low daily physical activity 
• 
Ability to ambulate for 75 meters without assistive devices 
• 
Diagnosis of BMD confirmed by at least one the following:  


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
36
o 
Dystrophin immunofluorescence and/or immunoblot showing partial dystrophin deficiency, 
and clinical picture consistent with typical BMD, or 
o 
Gene deletions test positive (missing one or more exons) of the dystrophin gene, where 
reading frame can be predicted as ‘in-frame’, and clinical picture consistent with typical 
BMD, or 
o 
Complete dystrophin gene sequencing showing an alteration (point mutation, duplication, 
or other mutation resulting in a stop codon mutation) that can be definitely associated with 
BMD, with a typical clinical picture of BMD, or 
o 
Positive family history of BMD confirmed by one of the criteria listed above in a sibling or 
maternal uncle, and clinical picture typical of BMD. 
• 
Nutritional, herbal and antioxidant supplements taken with the intent of maintaining or improving 
skeletal muscle strength or functional mobility have been discontinued at least 2 weeks prior to 
screening (daily multivitamin use is acceptable). 
• 
Hematology profile within normal range 
• 
Baseline laboratory safety chemistry profile within normal range 
• 
No plan to change exercise regimen during study participation 
Participant Exclusion Criteria 
 
• 
Currently enrolled in another treatment clinical trial. 
• 
History of significant concomitant illness or significant impairment of renal or hepatic function. 
• 
Use of regular daily aspirin or other medication with antiplatelet effects within 3 weeks of first dose 
of study medication. 
• 
Regular participation in vigorous exercise. 
• 
Symptomatic heart failure with cardiac ejection fraction <25% 
 
6.1.2 
Participant Screening Schedule 
The entry procedures will be conducted according to the schedule in Table 3.  After potential participants 
have completed the screening visits and the site has verified they meet all study inclusion criteria the 
participant will be entered into the treatment phase of the study. 
 
 
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
37
Table 3: Study procedures 
 
 
6.1.3   Randomization  
This is an open-label pilot study, and will not utilize randomization in the study design. 
     6.2 Withdrawal of Participants 
Participants are free to withdraw from the study at any time. Study investigators may elect to withdraw a 
participant from the study for reasons including an adverse event and inability to comply with study 
procedures.  Reasons for withdrawal of all participants will be recorded for review by the Study Medical 
Monitor. 
6.2.1 Follow-Up of Withdrawn Participants 
Those participants who are withdrawn from study medication, but are still willing to finish study participation 
per protocol, will be followed with all specified testing for the remainder of the 48-week study period.  If 
participant agrees to follow-up, adverse event and medical event follow-up, along with basic safety data, will 
also be collected at scheduled study visits or via phone every 2 weeks for the remainder of the study period 
(through week 48). 
6.3 Treatment of Participants 
6.3.1 Epicatechin administration  
Participants will receive epicatechin by mouth 50mg twice per day (100mg per day total dose).  Study 
medication will be supplied as a clear 25mg (#3) gelatin capsule without any inert fillers.  Participants will 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
38
take two capsules in the morning at approximately 7:30AM at least 15 minutes before the morning meal and 
two with the evening at approximately 7:30PM at least 1 hour after the evening meal as absorption is limited 
by some milk products. 
For details on participant enrollment, please refer to the study procedures chart (Table 3) and pharmacy 
sections.  When medications have been received, they should be dispensed to participants from their 
assigned investigational drug supply along with the medication instructions handout for parents and 
physicians.  
6.3.2 Criteria for Dose Reductions 
Dose reductions will occur due to adverse drug events and participants will not have their doses re-
escalated during the course of the study.  Criteria for dose reduction will include: 
• 
Recurrent non-manageable headache 
• 
Increase in baseline safety evaluations >1.5 ULN or 100% change from baseline, excepting CK, 
AST, ALT 
• 
Abnormal coagulation studies (PT/PTT) 
• 
Other Grade 4 adverse event 
If participants require a dose reduction based on the criteria listed above follow the guidelines in Table 4 
when reducing a participant’s study medication dose. 
Table 4: Dose reduction schedule. 
Dose Reduction 1 
Reduce participant’s dose by 50mg/day (25mg AM, 25mg PM dose).  
Dose Reduction 2 
Withdraw participant from study medication 
The Common Terminology Criteria for Adverse Events (CTCAE) published by the Cancer Therapy 
Evaluation Program will be used to grade adverse events for this trial. 
6.3.3 Study Drug Holiday as Result of Surgical Procedure 
If a participant experiences an SAE that necessitates an unanticipated surgical procedure, the study drug 
should be withdrawn and a “drug holiday” begins. The day the study drug is withdrawn (first day not taking 
study drug) is Day One of the drug holiday.  A drug holiday can last up to 2 weeks (14 calendar days).   
Study drug should be withdrawn 2-5 days prior to the scheduled surgery date. The study drug should be 
restarted 2 days post surgery unless PI has medical rational for other regimen for restarting study drug. 
6.3.4 Concomitant Therapy 
No new medications may be taken, except over the counter cold remedies, daily multivitamin, and Zantac, 
during the study period without the agreement of the Study Chair. The exception is any case where such 
medications are used to prevent injury or disability due to unforeseen adverse events. 
6.3.5 Patient Care Outside UC Davis 
Participants participating in UC Davis studies may not receive protocol therapy at non-UC Davis institutions.  
However, the following are acceptable: 
• 
For interim blood work done at a non-UC Davis site, a copy of the laboratory slip or other 
documentation MUST be forwarded to UC Davis for inclusion in the medical record and case report 
forms. 
These guidelines do NOT override any federal, international or sponsoring agency requirements. 
Records from any non-UC Davis institutions must be available for audit. 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
39
7 
PHARMACY 
7.1 Dosing Information and Safety Studies 
The safety of this supplement has not been proven in the BMD population. We propose to use a dose at 
approximately 1.1-1.4 mg/kg/day spit between the morning and evening doses. The maximum dose in our 
population will not exceed 100 mg/day. 
7.1.1 Preclinical Rodent Safety Studies 
Studies in rodents indicate that (-)-epicatechin can be delivered safely at doses far in excess of the 
anticipated 1mg/kg dose in human studies.   In a study with green tea extracts containing (-)-epicatechin 
given orally to rats daily for 6 months, the no-observable-adverse-effect level (NOAEL) corresponded to 85 
mg (-)-epicatechin/kg [71].   In a developmental toxicity study in pregnant rats using a different tea extract, 
the no-observable-adverse-effect level (NOAEL) corresponded to 100 mg (-)-epicatechin/kg (the highest 
dose tested)[72].   The intraperitoneal (-)-epicatechin median lethal dose (LD50) reported for mice is 1000 
mg/kg as stated in the MSDS provided by suppliers such as Sigma-Aldrich. Clarke and Clarke proposed 
that any substance with an intraperitoneal LD50 of above 1000 mg/kg may be regarded as safe [73]. 
7.1.2 Human Safety Studies 
Clinical studies with purified (-)-epicatechin (1-2 mg/kg) in the dose range of Cardero’s proposed studies did 
not reported any adverse events[42, 48].   An extensive literature of human studies using high-flavanol 
cocoa or chocolate human studies exists, indicating that flavanols could be administered safely at doses up 
to 1008 mg flavanols per day for 15 days and 444 mg flavanols per day for 6 weeks (reviewed in [74, 75]).   
A safety study of a green tea extract containing ~110 mg (-)-epicatechin per dose showed that once a day 
dosing for 4 weeks yielded the same safety profile as placebo, with no significant differences in hemotologic 
or clinical chemistry [76].    
 
In addition to formal clinical studies, cocoa, chocolate and tea have been used globally on a daily basis for 
centuries, suggesting that these compounds pose no significant safety risk.   Tea is considered a Generally 
Regarded As Safe (GRAS) compound by the FDA (21 CFR 182.20).    
7.2 Study Drug Formulation and Procurement 
Epicatechin for this study will be obtained from Cardero Therapeutics, Inc. and will be supplied as 25mg 
clinical trial grade (#3) clear gelcaps in bulk containers.  Distribution of medication will be supervised by the 
UC Davis Investigational Drug Service Pharmacy. 
7.2.1 (-)-Epicatechin as a Natural Test Substance (Cardero Therapeutics) 
(-)-Epicatechin is a naturally-occurring product found in chocolate and tea.  It is a member of the flavonoid 
family. The (-)-epicatechin is purchased commercially and is obtained from tea extracts:  
 
Manufacturer 
 
Sigma-Aldrich 
Product # 
 
E1753 
Country of Origin: 
India 
 
It has >90% purity by HPLC, and has been purified in a GMP facility as follows: it is dissolved in ethanol, 
treated with charcoal, filtered to remove insolubles, the solvent is exchanged to purified water, and then the 
solvent is removed by lyophilization.    
 
The final test substance is tested in a GMP analytical lab using appropriate qualified HPLC methods for 
impurities, including chirality testing.   Release specifications require > 90% purity, <5% of the enantiomer, 
and 5% of catechin.  It is also tested for other characteristics typical in GMP materials (identity by 1H NMR, 
IR; water content by Karl Fischer titration;  ethanol content by GC;  and the general USP tests of residue on 
ignition and heavy metals).   Based on test results, a Certificate of Analysis is generated (see Appendix) 
and a %-content by weight is calculated.    
 
Chemical name:    
(-)-Epicatechin or (2R, 3R)-2-(3,4-dihydroxyphenyl)chroman-3,5,7-triol 
Molecular formula: 
C15H14O6 
CAS #:  
 
490-46-0 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
40
Molecular weight: 
290.27 grams/mole 
Appearance: 
 
off-white to light brown solid 
Solubility: 
 
>5 mg/ml in water at 20oC 
pKa: 
 
 
neutral 
Taste:  
 
moderately bitter, resembling aspirin in degree of bitterness 
 
(-)-Epicatechin is a single enantiomer containing two defined chiral centers.   The structure of (-)-
epicatechin is depicted in Figure 1.   The absolute stereochemistry of (-)-epicatechin is as shown because it 
is either isolated from natural sources and further purified, or it is synthesized and compared to the natural 
isomer. 
    
Stability  (-)-Epicatechin has been demonstrated to be stable in the lyophilized state for at least six months 
under refrigeration.   A stability study is ongoing and retest dates will be generated based on results. 
7.2.2 (-)-Epicatechin as supplies as a natural test product 
The intended route of administration is oral.  (-)-Epicatechin isolated from tea will be supplied by Cardero 
Therapeutics in gelatin capsules, each containing 25 mg (-)-epicatechin combined with excipients.  The test 
product is used as is, and swallowed with some water.  Table 5 describes the (-)-epicatechin capsule 
formulation.  
Table 5: Contents of the 25mg (-)-epicatechin capsule. 
Component 
Role 
Amount 
Epicatechin 
Natural Ingredient 
  25.00mg 
Microcrystalline cellulose@ 
(Avicel PH 102) 
Diluent 
127.00mg 
Crospovidone@ (Kollidon CL) 
Disintegrant 
    4.80mg 
Citric acid monohydrate@ 
Acidifying agent 
    1.60mg 
Colloidal Silicon dioxide@ 
Glidant 
    0.80mg 
Magnesium Stearate@ 
Lubricant 
    0.80mg 
Total 
160.00mg 
 
Gelatin Capsule  The gelatin has been formulated into hard gelatin capsule shells for use in human 
pharmaceuticals.  The gelatin itself is limed bone gelatin, and has been obtained from Rousselot SAS 
(production site: Chemin Moulins Premiers, France – 84800 Isle-Sur-La-Sorgue).  The applicant has 
obtained Certificate of Suitability No. R1-CEP 2000-029-Rev 03, dated 22 July 2011, from 
Rousselot.  According to this certificate, the substance GELATIN meets the criteria described in the current 
version of the monograph Products with risk of transmitting agents of animal spongiform encephalopathies 
(no. 1483 of the European Pharmacopoiea). 
 
Sterility and pyrogen testing  Sterility and pyrogen testing has not been conducted, as test product is 
supplied as lyophilized powder for oral use only. All products to be included in (-)-epicatechin formulation 
(capsules) will be USP grade (vitamin C, EDTA, cellulose, lactose).   Sterile water will be used to prepare 
liquid doses for immediate and single use. 
 
Stability   A stability study of the test product in capsules is ongoing.   Retained samples of clinical trial 
supplies will be tested to confirm potency. 
7.3 Treatment Cycles Drug Dispensation 
Medications will be dispensed for the entire period of time between study visits, and will be clearly marked 
as investigational drugs per UC Davis Investigational Drug Service Pharmacy procedures.  The site will 
dispense study medication/placebo to the participant and will keep a medication log to document all study 
medication dispensed to participants. The participant will be instructed to take the study medication per 
protocol dosing schedule, every day during the eight-week treatment period. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
41
7.4 Investigational Product Accounting Procedures 
At each study visit, participants will be asked to return used medication containers from the previous 
month(s), as well as any unused medications.  The participating centers will measure any unused 
medication and the site coordinator will keep records of the participant’s medication use. 
7.5 Maintenance of Randomization Codes and Emergency Unblinding 
This study is open-label and no emergency unblinding procedures are necessary. 
8 
ASSESSMENT OF EFFICACY 
8.1 Laboratory Efficacy Parameters 
• 
Peripheral Blood Tests:  Peripheral venous blood will be collected at points specified in the study 
visit chart to evaluate blood biomarkers as noted in the specific aims. 
8.2 Functional Efficacy Parameters  
• 
Anthropometric Measurements (5 minutes) 
Standing height will be measured to the nearest 0.1cm in all participants.  Weight will be measured 
to the nearest 0.1kg.  Waist and hip circumference will be measured to the nearest 0.1cm and 
waist-hip ratio will be calculated. 
• 
Body Composition Assessment by Dual energy X-ray absorptiometry (DEXA) (15 minutes) 
DEXA scans are primarily used to evaluate bone mineral density.  It can also be used to measure 
total body composition and fat content with a high degree of accuracy.  For this study it will be used 
to measure total and regional body composition. DEXA uses X-rays to assess bone mineral density 
or measure total body composition.  However, the radiation dose is approximately 1/10th that of a 
standard chest X-ray. Bone density will be evaluated based on whole-body and subcranial total 
bone mass and areal bone mass (adjusted mass for bone size).   
• 
Functional Evaluation (1 minute) 
The functional classification used by CINRG utilizes a scale modified from the upper extremity 
scale reported by Brooke et al [77] and the lower extremity scales used by Vignos et al [78]. The 
functional grades consist of six levels of function for the upper extremities and eleven levels for the 
lower extremities.  Specific methods are outlined in the CQMS User’s Manual.   
• 
Upper Extremity Range of Motion Testing (60 minutes) 
The Kinect camera is a product for Xbox designed by Microsoft that captures motion 
(http://msdn.microsoft.com/en-us/library/jj131033.aspx). The Kinect camera will be used to 
measure reachable workspace and fatigue of the participant using the protocol developed and 
published by Kurillo, Han et al. [79-84].  
While being monitored by the Kinect, the participant will: 
o 
perform a series of standardized range of motion and functional upper extremity tasks 
o 
perform a series of standardized range of motion and functional upper extremity tasks while 
holding a weight 
 
• 
A6MCT for Arms and Legs (15 minutes) 
Participants will perform an assisted 6-minute cycling test to assess endurance as described by 
Jansen et all [85].  The exercise test will begin by positioning the participant on a motorized 
assisted mobility trainer (KPT Cycla, Kinetec, France) that can be used with a participant’s personal 
(electric) wheelchair or a regular chair with a backrest.  The goal of the test is to cycle as fast as 
possible and keep it up for 6-minutes.  The KPT Cycla is set in passive mode 1 with no-load speed 
of 7 RPM. Participant are seated either in their wheelchair or a chair with back support in front of 
the KPT Cycla, with the hips and knees are positioned at 90 degrees of flexion for one leg while the 
other will be submaximally extended.  The arm pedal axis of the KPT cycla will be adjusted a few 
centimeters (max of 5 cm) below shoulder level. The distance from the chair to the bicycle is 
determined by allowing participants to move their legs and arms over submaximal range of motion, 
producing a feeling of stretch.  Verbal encouragement is given every 15 seconds.  Participants are 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
42
also told of the time completed and time left every minute.  Rest is allowed if fatigued but 
participants are encouraged to continue cycling as soon as possible.  The outcome of the test is the 
number of revolutions achieved in 6-minutes.  Revolutions per minute (cumulative) and rest periods 
are also recorded.  
• 
Graded exercise test using a recumbent cycle ergometer (45 minutes) 
Each participant will perform a graded exercise test on an electronically braked recumbent cycle 
ergometer as previously described  [66, 67].  Modifications for Becker muscular dystrophy related 
proximal weakness will be made as described by others [67, 85]. The exercise test will began by 
participants pedaling at a rate of 60 revolutions/min with no load for 1 min (warm up). After the 
warm-up period, the work rate (WR) on the cycle ergometer will start at 10 W and increase either 
10 W/min or every other minute for Becker muscular dystrophy participants until volitional 
exhaustion. Level of exertion will be monitored by heart rate and the Borg visual analogue scale 
[86].  Increments will be adjusted so that the duration of the test will be kept between 12 and 15 
min. Twelve-lead ECG, blood pressure, O2 consumption (V˙O2), CO2 production (V˙CO2), and 
ventilation (Vmax, SensorMedics/VIASYS Healthcare) will be continuously measured. After 
volitional exhaustion, participants will be immediately placed in the supine position, where a 
postexercise ECG will be performed within 15–30 s. Exercise tests will be considered to be 
maximal if the peak heart rate (HR) is 85% of that predicted for age (220 – age) and/or the peak 
respiratory exchange ratio (RER; V˙CO2/V˙O2) is ≥ 1.15.   Muscle perfusion testing of a quadriceps 
muscle will be performed during cycle exercise using a non-invasive portable near-infrared 
spectroscopy (NIRS) system as described by Allart et al. [68].  We will use a non-invasive three 
channel, portable continuous-wave NIRS device (PORTAMON, Artinis Medical Systems, Zetten, 
The Netherlands).  Participants will undergo blood lactate testing at baseline and at 2-minute 
intervals as specified in the “Peripheral Blood Tests” section. 
• 
Six-Minute Walk Test (10 minutes) 
Participants will complete the Duchenne muscular dystrophy six-minute walk test (6MWT) as 
described by McDonald et al [87].  Methods will be adapted for the adult population by removing the 
element of constant verbal encouragement.  Measurements recorded will include 25-meter split 
times and total distance traveled.   
• 
Quantitative Muscle Testing (20 minutes) 
Isometric elbow flexors and knee extensors will be conducted using the BIODEX ergometer. The 
highest value of three consecutive maximal strength testing efforts will be recorded. Specific 
methods for testing can be found in the BIODEX manual of operations.  Quantitative hand grip 
strength will be assessed using the CiTEC handheld ergometer, again recording the highest value 
of three consecutive tests for each hand.  Methods for testing can be found in the CiTEC manual of 
operations. 
• 
Pulmonary Function Tests (30 minutes) 
Standard PFTs will be done using the Renaissance II spirometer. Spirometry has been used 
extensively as a measure of respiratory function. PFTs include maximal inspiratory pressure (MIP), 
maximal expiratory pressure (MEP), forced vital capacity (FVC), forced expiratory volume 1 (FEV1), 
peak expiratory flow (PEF), maximum ventilatory volume (MVV) and peak cough expiratory flow 
(PCEF).  
• 
Timed Function Testing (5 minutes) 
Timed Function Testing has frequently been used to assess functional abilities in three important 
areas: climbing four standard stairs and walking/running 10 meters. Each of these skills is timed to 
provide a quantitative measure of function. Specific methods are outlined in Florence et al [88]. 
 
• 
Multifrequency Bioimpedence Assessment (MFBIA) 
Participants will undergo multifrequency bioimpedence assessment (MFBIA) testing using a Xitron 
model 4000 MFBIA system to painlessly and non-invasively assess the amount of total body water, 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
43
intracellular water, extracellular water and fat-free mass.  One electrode is placed to bisect the 
ulnar head, and the other electrode is placed just behind the middle finger.  On the ankle, one 
electrode is placed to bisect the medial malleolus, and the other electrode is placed just behind the 
middle toe.  Impedence is measured between upper and lower extremity electrodes.   
 
• 
Echocardiogram. 
 
Participants will undergo echocardiogram with collection of left ventricular (LV) circumferential and 
longitudinal strain, LV shortening fraction, LV ejection fraction, end systolic volume, and end 
diastolic volume as described by [89].  
 
9 
ASSESSMENT OF SAFETY 
9.1 Safety Parameters 
Safety parameters for this study will include: 
• 
Review of Systems 
• 
Review of Adverse Events 
• 
Laboratory Assessments: Concurrent with peripheral blood draws listed in the efficacy section and 
according to the study visit procedure chart, participants will provide blood samples for standard 
clinical safety testing.  Safety panels will include: 
CBC/Diff 
PT/PTT 
CPK 
Total cholesterol  
Albumin 
Total Bilirubin 
Conjugated Bilirubin 
GGT 
ALT/AST 
Alkaline Phosphatase 
Sodium 
Potassium 
CO2 
Chloride 
Calcium 
BUN 
Serum Creatinine 
Serum Glucose 
Serum Total Protein 
Fasting lipid profile 
BNP 
• 
EKG 
• 
Review of Medical History 
• 
Review of Medical and Surgical Events 
• 
Physical and Neurological Exam 
• 
Review of Medication History  
• 
Review of Current Medications and Therapies 
• 
Review of any Adverse Events 
• 
Collection of Vital Signs 
• 
Collection of Height & Weight 
9.2 Procedures for Reporting Adverse Events 
Adverse events must be recorded in the study source documentation and eCRFs. All events occurring after 
the informed consent is signed must be reported regardless of whether or not they are believed to be 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
44
related to the study drugs or procedures. During study participation, adverse events should be followed by 
the study site principal investigator until resolved. All adverse events are graded according to the CTCAE.  
9.3 Data and Safety Monitoring Board / Medical Monitor 
This study will not employ a Data and Safety Monitoring Board (DSMB) but adverse events will be reviewed 
by an appointed Medical Monitor who is otherwise not affiliated with the study.   
In the event of a SAE, the Medical Monitor will be notified within 24 hours of notice of the event to the 
Coordinating Center, and details of the event will be provided for review at that time.  Additional follow-up 
information will be provided to the Medical Monitor within 15 days of the initial event.  Summaries of all 
other reported adverse events will be reviewed by the Medical Monitor on a quarterly basis. 
9.4 Duration of Participation / Follow-Up 
Participants will participate in the study for a period of approximately 48-50 weeks from the date of 
enrollment (including screening). 
9.5 Trial Stopping Rules or Discontinuation Criteria 
Participants may withdraw from the study at any time without prejudice.  Study investigators may withdraw 
participants at any time for reasons such as:  
• 
Inability to comply with study protocol 
• 
SAEs 
• 
Non-tolerable or non-manageable adverse events (AE) 
• 
Severe or sustained unexplained laboratory abnormalities  
• 
Recommendation of suspension of the study by the Medical Monitor 
 
10 HUMAN PARTICIPANTS 
a. Characteristics of the Study Population 
Ambulatory male adults with a confirmed diagnosis of BMD, 18 years to 70 years, will be recruited. 
b. Participation of Children, Women and Minority Populations 
BMD is an X-linked recessive disease affecting only males. However, female carriers of the disease can be 
symptomatic due to skewed X-inactivation. We have opted to study the most commonly affected population, 
males, to ensure patient homogeneity. 
c. Sources of Research Material from Living Participants 
Study data and blood samples collected will only be for research use or monitoring of study safety. 
d. Recruitment of Participants 
Participants will be recruited through the clinics of participating investigators, advertising and medical record 
screening in participating clinics. This trial will also be listed and updated on www.clinicaltrials.gov. 
i. Advertising 
Any local advertising through newsletters or muscular dystrophy-associated organizations’ mailings will be 
submitted to the local institutional review board (IRB) that provides overview of this project. 
ii. Screening 
Patients for this study will be identified from the prior study cohort enrolled in the UCD0113 proof of concept 
BMD epicatechin study.  Personnel who review existing patient medical records must be designated by the 
study site’s principal investigator and must be an employee of that institution.  A list of those personnel must 
be supplied to the institution’s IRB. 
e. Informed Consent/Assent and Ethical Considerations 
Informed consent/assent must be documented for each participant.  The date and time of the 
consent/assent must be prior to the initiation of any study-related tests or procedures, including diagnostics 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
45
that might be required to confirm a participant’s study eligibility.  The consent/assent form should 
supplement, not replace dialogue between the study principal investigator and the patient. 
f. 
Retention of Participants 
Care of each participant will be supervised by the participating site’s principal investigator and study 
coordinator, who will schedule all visits and assessments. 
If a participant is non-compliant, efforts will be made by site staff to ensure participant compliance, including 
participant/family teaching and increased frequency of phone contact to reiterate necessary information. If 
compliance does not improve, the investigator may decide to withdraw the participant from the trial. Any 
withdrawn participant will complete a follow-up visit along with an overview of systems and any adverse 
events that were noted during their study participation. Any ongoing adverse events will be monitored for 30 
days after the participant’s follow-up/withdrawal visit. 
g. Potential Risks 
i. Risks of Epicatechin 
Animal and human safety studies suggest that (-)-epicatechin at doses of 1-2 mg/kg should be safe and 
well tolerated.  In a study with green tea extracts containing (-)-epicatechin given orally to rats daily for 6 
months, the no-observable-adverse-effect level (NOAEL) corresponded to 85 mg (-)-epicatechin/kg [71].  
For the human dosing plan of 1 mg/kg, this provides a safety margin relative to the rat NOAEL of at least 
85X.   The dosing plan is also supported by published human studies, in which (-)-epicatechin doses of 1 
and 2 mg/kg were administered with no adverse events were reported [42].  Potential risks based on the 
biological activities of (-)-epicatechin include: 
 
Hypotension   Given the reported effects of (-)-epicatechin on blood vessels, it is reasonable to assume 
that a potential risk may be associated with vasodilation. Of note is the fact that so far, no blood pressure 
reducing effects by (-)-epicatechin have been reported in normal subjects [90].  With cocoa based studies, 
blood pressure reducing effects are only reported in humans that have high blood pressure [90]. There is 
the possibility that patients undergoing pharmacologic treatment for high blood pressure if given (-)-
epicatechin may develop hypotension through additive or synergistic effects. 
 
Migraines The facilitation of migraines has been associated with the action of vasoactive substances [91]. 
The consumption of cocoa products has been reported to be associated with increased likelihood of 
migraine development [92, 93]. Thus, it is reasonable to surmise that (-)-epicatechin may increase the 
chances for migraine development in susceptible individuals.  
 
Bleeding Anti-clotting like effects have been described for multiple members of the flavonoid family 
including (-)-epicatechin [94]. There is no report as to the effects that (-)-epicatechin per se may have on 
phenomena such as platelet aggregation or clotting times. There is a limited number of reports on the 
effects of cocoa on these endpoints. In general, they report effects on platelet aggregate 
As with any compound, hypersensitivity reactions may occur, although no such reactions have been seen 
to date. 
 
Contraindications and Warnings No contraindications are known.  However, due to the possibility that (-)-
epicatechin may interact with drugs with known antiplatelet effects to potentiate their activity, participants 
who are currently on long-term therapy with any such agents will be excluded from the study. 
 
Overdose Neither the effects of overdose of (-)-epicatechin nor an antidote to overdose are known.  The 
intraperitoneal (-)-epicatechin median lethal dose (LD50) reported for mice is 1000 mg/kg as stated in the 
MSDS provided by suppliers such as Sigma-Aldrich.     
 
Pregnancy and Lactation  In a developmental toxicity study in pregnant rats using a green tea extract, the 
no-observable-adverse-effect level (NOAEL) corresponded to 100 mg (-)-epicatechin/kg (the highest dose 
tested)[72].  No information is available on levels of (-)-epicatechin in breast milk.Risks of  


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
46
ii. Risks of Blood Tests 
The risks of blood drawing include soreness or bruising at the site of the needle. A local numbing cream 
(EMLA) may be applied to the area at the participant’s request. There are no side effects associated with the 
use of this cream. Rarely, a more serious injury, such as hematoma (bleeding under skin) or infection may 
develop. 
iii. Risks of QMT Strength Testing 
QMT will be done at each visit to measure strength in the participant’s muscles. At this time, there are no 
known risks associated with functional evaluation or muscle strength testing methods used in this protocol. 
However, the participant may experience mild muscle soreness the day after muscle testing.   
iv. Risks of PFT 
These tests may cause dizziness and lightheadedness during and shortly after the test. 
v. Risks of EKG 
The EKG has no known risks. 
vi. Risks of Functional Evaluation and Muscle Strength Testing 
At this time, there are no known risks associated with functional evaluation or muscle strength testing 
methods used in this protocol. However, the participant may experience mild muscle soreness the day after 
muscle testing.   
vii. Risks of DEXA 
DEXA scans involve exposure to a very small amount of radiation (<1.0mrem).  DEXA is contraindicated in 
pregnancy (not applicable in this project). 
viii. Risks of Multi-Frequency Bioimpedence Assessment (MFBIA) 
There are no known risks of MFBIA testing.  
ix. Risks of Upper Extremity Range of Motion Evaluation 
There are no known risks of upper extremity range of motion evaluation. 
h. Procedures for Minimizing Risks 
Confidentiality of medical information will be maintained throughout the study.  Participants will be assigned 
identification numbers that will be used on all case report forms. No personally identifiable information will 
be released beyond the Study Coordinating Center without the participant’s prior written consent.  Data 
entered into electronic case report forms will be handled by the RedCap online CRF system and managed 
in compliance with FDA privacy and data retention standards for electronic clinical research data collection. 
Safety data recorded during the conduct of this study will be transmitted directly to the Medical Monitor 
through the RedCap system.  Data will be collected at each study visit and over the phone (family reported 
AEs) between study visits.  Both AEs and SAEs will be reviewed by the Medical Monitor.  AEs will be 
reviewed on a quarterly basis by the Medical Monitor.   
i. 
 Justification of Risks to Participants 
Due to the low toxicity profiles of (-)-epicatechin, risks to subjects associated with participation in this study 
are less than or similar to standard clinical interventions in patients with BMD.   


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
47
j. 
Benefits 
The intended benefits outweigh the risks of using the study medication and study procedures in BMD 
patients. The participants may experience an increase in muscle strength or a delay in strength decline. 
The participants will receive additional care during study participation including muscle strength training, 
contracture measurements and spirometry during each study visit. Medical and adverse event history will 
be closely monitored. The additional medical monitoring allows for increased interaction with medical staff 
above expected routine clinical care. The increased monitoring of safety labs will also be performed.   
It is possible the participants will not experience any direct benefit as a result of their study participation. 
However, the data collected during this trial may provide information that will benefit the scientific 
community as well as other individuals with BMD 
k. Financial Considerations 
No financial compensation will be given to participants or their families for participation in this trial other 
than minor assistance with transportation expenses (parking, etc.).  
11  STATISTICS 
Presented herein is the overall analysis plan. The main criterion for success of the study will be presence of 
one or more biologic or strength and performance outcome measures (Aims 1 and 2) that yield a response 
magnitude that allows for sufficient power in a Phase II B study with a sample size of 30 individuals. 
Analysis, employing both statistical and graphical presentations of data, will proceed from descriptions and 
simple comparisons to multiple variable models. This will help to ensure proper understanding of the data at 
each level before proceeding to the next. Descriptive analysis of means and standard deviations or 
frequencies and proportions will characterize study participants overall as well as evaluate assumptions of 
normality and homoscedasticity. Any significant departures from these assumptions especially for the 
measurement variables will lead to normalizing or variance stabilizing transformations or, in the unlikely 
event these are not successful, to conversion to ranks.  Level of statistical significance is set at 
<0.05.  Simple comparative analyses will be used to assess and understand simple relationships, including 
defining first order interactions.  We will compare continuous variables across groups using the Student’s t-
test and analysis of variance.  We will compare differences between groups using Chi Square and Fisher’s 
Exact tests.  We will compare simple relationships between variable using parametric or nonparametric 
correlations as appropriate.  Power calculations will be based on simple between-group comparisons of 
means for treated / non-treated individuals at specified time periods.  However, we will also explore 
complex relationships between variables and differences between groups using a combination of linear 
regression and longitudinal multivariate mixed model approaches (using xtreg or xtmixed in 
Stata12).  These procedures will allow us to account for fixed and random effects as well as correlation 
between observations when we introduce repeated measures on the same individuals.  Pilot data will be 
used to determine estimates of minimally important clinical difference (MCID) for each measure (defined as 
1/3 S.D. for the population).  Safety data will be compiled as tables of frequency and severity of adverse 
events by body system. 
 
12 DATA COLLECTION 
 
12.1 Data Management System 
A web-based clinical data entry system, RedCap is being used for electronic case report form (eCRF) data 
collection. The data management system complies with all federal regulations pursuant to 12 CFR Part 11.  
12.2 Data Quality Control and Quality Assurance 
The RedCap System will provide validity checks on participant and visit identifier fields, as they are entered, 
to ensure that the visit is unique and appropriately timed according to protocol criteria. A secure web-based 
‘smart’ eCRF system will detect some inaccuracies in data entry immediately to alert site study staff prior to 
data submission.   
    12.3 Security and Backups 
To ensure patient confidentiality, no patient identifiers are entered into this system. Sequential study 
numbers are assigned to all participants. All computers are password protected and accessible only to 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0115B: Epicatechin Extension in Becker Muscular Dystrophy 
 
48
study personnel. All data entered into the RedCap System are copied by the UC Davis CTSC Data 
Coordinating Center to a secure back-up server at another site several times per day.  
12.4 Data Monitoring  
Over the course of the study, the Study Chair, Project Manager, biostatisticians and Medical Monitor will 
require access to the entire study dataset.  This may be for the purposes of monitoring a specific site’s data, 
performing quality control, performing periodic data analysis or for safety or efficacy monitoring.  The above 
named individuals will have read-only access to the study data via the RedCap web interface.   
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