Clinical trial NCT01856868
Conditions: Becker Muscular Dystrophy
Keywords: BMD Becker muscular dystrophy epicatechin clinical trial neuromuscular disease
Sponsor: Craig McDonald, MD
Start date: 2013-05-01; Completion date: 2018-09-01
Enrollment: 7 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
(-)-Epicatechin will be evaluated for the treatment of progressive muscle loss and impaired skeletal muscle function in Becker Muscular Dystrophy (BMD) patients.

Detailed description:
This is a proof-of-concept phase 1/2a pilot and endpoint development study that is designed to provide initial evidence of biological activity of (-)-epicatechin. Primary endpoints include initial assessment of tissue-specific evidence of efficacy from muscle biopsy samples. Secondary endpoints include measures of strength and physical function, and safety and adverse event data. Pilot endpoints include assessment of mRNA and miRNA peripheral blood profiles and validation of non-invasive near-infrared spectroscopy (NIRS) muscle perfusion studies during exercise and a recumbent cycle exercise test that may be employed as endpoints in future clinical trials.

This single center open-label pilot study will enroll 10 adults with genetically-confirmed Becker muscular dystrophy, 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 baseline and at screening, day 1, and weeks 1, 2, 4 and 8.

Arms: arm 1: Purified nutritional extract (-)-epicatechin 100mg/day orally for 8 weeks.
Interventions: intervention 1: (-)-epicatechin
Intervention descriptions: intervention 1: purified nutritional extract (-)-epicatechin 100mg/day orally for 8 weeks.

Protocol documents:
Prot_SAP_000.pdf: 
Use of (-)-Epicatechin in the Treatment of Becker Muscular Dystrophy (Pilot 
Study) 
NCT01856868 
March 8, 2013 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
UCD0113:  An open-label pilot study of purified tea-derived epicatechin to 
improve mitochondrial function, strength and skeletal muscle exercise 
reponse in Becker Muscular Dystrophy  
 
Original:  March 8, 2013 
 
 
 
 
 
 
 
 
STUDY COORDINATING CENTER: 
Neuromuscular Medicine and Rehabilitation 
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): 
Richard T. Abresch, MS 
Lee-Way Jin, MD, PhD 
Frank Sharpe, PhD 
Glenn Jickling, PhD 
Bjorn Oskarsson, MD 
University of California, Davis  
 
DATA MANAGER(S): 
Evan de Bie, BS 
University of California, Davis 
 
PROJECT COORDINATOR: 
TBA 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
UCD0113:  An open-label pilot study of purified tea-derived 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 
TBA 
PROJECT CENTRAL PHARMACIST 
TBA from UC Davis Investigational Drug Pharmacy 
 
INDEPENDENT MEDICAL MONITOR 
TBA (from UC Davis)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
2 
PROTOCOL SIGNATURE PAGE 
 
UCD0113:  An open-label pilot study of purified tea-derived epicatechin to improve 
mitochondrial function, strength and skeletal muscle exercise reponse in Becker Muscular 
Dystrophy  
 
Original:  March 8, 2013 
 
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
3 
TABLE OF CONTENTS 
1 
STUDY SUMMARY ................................................................................................................................... 7 
1.1 
BACKGROUND ...................................................................................................................... 7 
1.2 
STUDY PURPOSE ................................................................................................................. 9 
1.3 
STUDY DESIGN..................................................................................................................... 9 
1.4 
SPECIFIC AIMS ..................................................................................................................... 9 
2 
BACKGROUND AND SIGNIFICANCE .................................................................................................. 11 
2.1 
BECKER MUSCULAR DYSTROPHY ........................................................................................ 11 
2.2 
JUSTIFICATION OF CURRENT APPROACH ............................................................................. 11 
3 
EPICATECHIN OVERVIEW.................................................................................................................... 12 
3.1 
PHARMACOKINETICS  / PHARMACODYNAMICS ....................................................................... 12 
4 
PRELIMINARY STUDIES ....................................................................................................................... 14 
4.1 
PRECLINICAL DATA ............................................................................................................. 14 
4.1.1 
(-)-Epicatechin Increases Mitchondrial Respiration and Biogenesis in Cultured Human 
Skeletal Muscle Cells (unpublished data) .............................................................................. 14 
4.1.2 
(-)-Epicatechin Induces Mitochondrial Biogenesis Pathway in Human Coronary Artery 
Endothelial Cells (unpublished data) ...................................................................................... 16 
4.1.3 
(-)-Epicatechin Induces Expression of Mitochondrial Biogenesis Factor PGC-1α in Aged Mice 
(unpublished data) .................................................................................................................. 16 
4.1.4 
(-)-Epicatechin Induces Expression of Mitochondrial Biogenesis Factor PGC-1α and 
Mitochondrial Proteins in δ-sarcoglycan KO Mice (unpublished data) .................................. 17 
4.1.5 
(-)-Epicatechin Increases In Vivo Mitchondrial Biogenesis and Muscle Function in Aged 1 
Year Old Mice ......................................................................................................................... 17 
4.1.6 
(-)-Epicatechin Increases Mitchondrial Biogenesis and Improves Myocardial Function in Rats
 
19 
4.1.7 
(-)-Epicatechin Modulates the Synthesis of Muscle Growth and Differentiation Proteins in 
Cultured Cells and Mice (Aged and MD)(unpublished data) .................................................. 19 
4.1.8 
(-)-Epicatechin Stimulates the Expression of MD-Relevant Muscle Structural Proteins in Aged 
and MD Mice (unpublished data) ........................................................................................... 21 
4.1.9 
(-)-Epicatechin Reduces Fibrosis in  δ-Sarcoglycan KO Mice (unpublished data) .. 22 
4.1.10 
(-)-Epicatechin Reduces Plasma Marker of Muscle Damage and Improves Muscle Strength in 
MDX Mouse Model of MD (unpublished data) ....................................................................... 23 
4.1.11 
(-)-Epicatechin Blocks Calcium-Induced Pore Formation in Cardiac Mitochondria (unpublished 
data) 
24 
5 
HUMAN STUDIES .................................................................................................................................. 25 
5.1 
POC CLINICAL STUDY #1:  MITOCHONDRIAL BIOGENESIS AND MUSCLE IMPROVEMENT  IN PATIENTS WITH 
HEART FAILURE AND TYPE 2 DIABETES PATIENTS(UCSD) .............................................................. 25 
5.1.1 
Markers of Mitochondrial Biogenesis, Structure and Function ................................ 25 
5.1.2 
Markers of Tissue Oxidative Stress ......................................................................... 26 
5.2 
POC CLINICAL STUDY #2:  AGE-RELATED CHANGES IN MUSCLE REGULATORY PROTEINS  AND EFFECT OF 
(-)-EPICATECHIN ON MUSCLE FUNCTION AND PROTEINS  IN HEALTHY VOLUNTEERS (UCSD) ............ 29 
5.3 
CLINICAL STUDY #3:  PHARMACOKINETICS OF (-)-EPICATECHIN IN HEALTHY VOLUNTEERS (UCSF)
 
30 
5.3.1 
Safety and Tolerability ............................................................................................. 31 
5.3.2 
Pharmacokinetics ..................................................................................................... 31 
5.3.3 
Conclusions ............................................................................................................. 31 
6 
RESEARCH DESIGN AND METHODS ................................................................................................. 32 
6.1 
SELECTION AND WITHDRAWAL OF PARTICIPANTS ................................................................. 32 
6.1.1 
Criteria for Enrollment .............................................................................................. 32 
6.1.2 
Participant Screening Schedule ............................................................................... 32 
6.1.3 
Randomization ......................................................................................................... 33 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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6.2 
WITHDRAWAL OF PARTICIPANTS .......................................................................................... 33 
6.2.1 
Follow-Up of Withdrawn Participants ....................................................................... 33 
6.3 
TREATMENT OF PARTICIPANTS ............................................................................................ 33 
6.3.1 
Epicatechin Administration ...................................................................................... 33 
6.3.2 
Criteria for Dose Reductions .................................................................................... 34 
6.3.3 
Study Drug Holiday as Result of Surgical Procedure .............................................. 34 
6.3.4 
Concomitant Therapy............................................................................................... 34 
6.3.5 
Patient Care Outside UC Davis ............................................................................... 34 
7 
PHARMACY ............................................................................................................................................ 34 
7.1 
DOSING INFORMATION AND SAFETY STUDIES ....................................................................... 34 
7.1.1 
Preclinical Rodent Safety Studies ............................................................................ 34 
7.1.2 
Human Safety Studies ............................................................................................. 35 
7.2 
STUDY DRUG FORMULATION AND PROCUREMENT ................................................................ 35 
7.2.1 
(-)-Epicatechin as a Natural Test Substance (Cardero Therapeutics) .................... 35 
7.2.2 
(-)-Epicatechin as supplies as a natural test product ............................................... 36 
7.3 
TREATMENT CYCLES DRUG DISPENSATION ......................................................................... 36 
7.4 
INVESTIGATIONAL PRODUCT ACCOUNTING PROCEDURES ..................................................... 36 
7.5 
MAINTENANCE OF RANDOMIZATION CODES AND EMERGENCY UNBLINDING ........................... 36 
8 
ASSESSMENT OF EFFICACY............................................................................................................... 36 
8.1 
LABORATORY EFFICACY PARAMETERS ................................................................................ 36 
8.2 
FUNCTIONAL EFFICACY PARAMETERS  ................................................................................. 38 
9 
ASSESSMENT OF SAFETY .................................................................................................................. 39 
9.1 
SAFETY PARAMETERS ........................................................................................................ 39 
9.2 
PROCEDURES FOR REPORTING ADVERSE EVENTS ............................................................... 40 
9.3 
DATA AND SAFETY MONITORING BOARD / MEDICAL MONITOR .............................................. 40 
9.4 
DURATION OF PARTICIPATION / FOLLOW-UP ........................................................................ 40 
9.5 
TRIAL STOPPING RULES OR DISCONTINUATION CRITERIA ..................................................... 40 
10 
HUMAN PARTICIPANTS .................................................................................................................... 41 
10.1 
CHARACTERISTICS OF THE STUDY POPULATION ................................................................... 41 
10.2 
PARTICIPATION OF CHILDREN, WOMEN AND MINORITY POPULATIONS ................................... 41 
10.3 
SOURCES OF RESEARCH MATERIAL FROM LIVING PARTICIPANTS .......................................... 41 
10.4 
RECRUITMENT OF PARTICIPANTS ........................................................................................ 41 
10.4.1 
Advertising ............................................................................................................... 41 
10.4.2 
Screening ................................................................................................................. 41 
10.5 
INFORMED CONSENT/ASSENT AND ETHICAL CONSIDERATIONS ............................................. 41 
10.6 
RETENTION OF PARTICIPANTS ............................................................................................. 42 
10.7 
POTENTIAL RISKS ............................................................................................................... 42 
10.7.1 
Risks of Epicatechin ................................................................................................. 42 
10.7.2 
Risks of Blood Tests ................................................................................................ 42 
10.7.3 
Risks of Muscle Biopsy ............................................................................................ 43 
10.7.4 
Risks of QMT ........................................................................................................... 43 
10.7.5 
Risks of PFT ............................................................................................................ 43 
10.7.6 
Risks of EKG ............................................................................................................ 43 
10.7.7 
Risks of Functional Evaluation and Muscle Strength Testing.................................. 43 
10.7.8 
Risks of DEXA ......................................................................................................... 43 
10.7.9 
Risks of Multi-Frequency Bioimpedence Assessment (MFBIA) .............................. 43 
10.7.10 Risks of Upper Extremity Range of Motion Evaluation ............................................ 43 
10.8 
PROCEDURES FOR MINIMIZING RISKS .................................................................................. 43 
10.9 
JUSTIFICATION OF RISKS TO PARTICIPANTS ......................................................................... 43 
10.10 BENEFITS ........................................................................................................................... 44 
10.11 FINANCIAL CONSIDERATIONS .............................................................................................. 44 
11 
STATISTICS ........................................................................................................................................ 44 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
5 
12 
DATA COLLECTION .......................................................................................................................... 44 
12.1 
DATA MANAGEMENT SYSTEM .............................................................................................. 44 
12.2 
DATA QUALITY CONTROL AND QUALITY ASSURANCE ............................................................ 44 
12.3 
SECURITY AND BACKUPS .................................................................................................... 44 
12.4 
DATA MONITORING ............................................................................................................. 45 
13 
REFERENCES .................................................................................................................................... 45 
 
 
 
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
6 
TABLES 
 
Table 1: Effects of (-)-epicatechin on progressive muscle loss parameters................................................... 12 
Table 2: Selected human oral bioavailability studies of (-)-epicatechin ......................................................... 14 
Table 3: Study procedures ............................................................................................................................. 33 
Table 4: Dose reduction schedule. ................................................................................................................. 34 
Table 5: Contents of the 25mg (-)-epicatechin capsule.................................................................................. 36 
 
FIGURES 
 
Figure 1: Chemical structures of flavonoid families and (-)-Epicatechin .......................................................... 7 
Figure 2: Major (-)-epicatechin metabolites in plasma. .................................................................................. 13 
Figure 3: (-)-Epicatechin metabolite profile in plasma .................................................................................... 13 
Figure 4: (-)-Epicatechin enhances mitochondrial respiration ........................................................................ 15 
Figure 5: (-)-Epicatechin increases mitochondrial number and cristae density in human skeletal muscle cells 
(unpublished data) ................................................................................................................................... 15 
Figure 6: (-)-Epicatechin induces estrogen-related receptor alpha translocation (activation) into the nucleus 
of HCAEC ................................................................................................................................................ 16 
Figure 7: (-)-Epicatechin increases PGC1-alpha in aged mice. ..................................................................... 17 
Figure 8: (-)-Epicatechin increases PGC1-alpha and mitochondrial proteins in gamma-sarcoglycan knock 
out mice. .................................................................................................................................................. 17 
Figure 9: (-)-Epicatechin increases mitochondrial proteins in 1-year old mice. ............................................. 18 
Figure 10: (-)-Epicatechin increases mitochondrial volume density and mitochondrial membrane surface 
area in 1-year old mice. ........................................................................................................................... 18 
Figure 11: Muscle differentiation in time course in C2C12 cells +/- (-)-epicatechin. ...................................... 20 
Figure 12: Muscle regulatory protein expression in 6 and 26 month old mice +/- (-)-Epicatechin ................. 20 
Figure 13: (-)-Epicatechin modulates muscle regulatory proteins in diaphragm muscle in MDX mice. ......... 21 
Figure 14: (-)-Epicatechin increases DAPC proteins in middle aged mice. ................................................... 22 
Figure 15: (-)-Epicatechin increases alpha-sarcoglycan protein expression in gamma-sarcoglycan knock out 
mouse muscle. ........................................................................................................................................ 22 
Figure 16: (-)-Epicatechin reduces fibrosis in the gamma-sarcolgycan knock out mouse. ............................ 23 
Figure 17: (-)-Epicatechin reduces plasma CK activity (muscle injury biomarker) in MDX mouse model of 
muscular dystrophy. ................................................................................................................................ 23 
Figure 18: (-)-Epicatechin improves skeletal muscle strength in the MDX mouse model of muscular 
dystrophy. ................................................................................................................................................ 24 
Figure 19: (-)-Epicatechin blocks calcium-induced pore formation in cardiac mitochondria in a dose-
dependent manner. ................................................................................................................................. 24 
Figure 20: (-)-Epicatechin-rich cocoa increases active PGC-1a and decreases acetylated (inactive) PGC1a 
in HF/DM2 patients. ................................................................................................................................. 25 
Figure 21: (-)-Epicatechin-rich cocoa increases mitochondrial complex V protein and cristae abundence in 
HF/DM2 patients. .................................................................................................................................... 26 
Figure 22: (-)-Epicatechin-rich cocoa increases PGC1a and FOX01 association to chromatin from 
quadriceps of HF/DM2 patients. .............................................................................................................. 26 
Figure 23: (-)-Epicatechin-rich cocoa increases antioxidant proteins and total tissue thiols (reduced 
glutathione) in HF/DM2 patients. ............................................................................................................. 27 
Figure 24: (-)-Epicatechin-rich cocoa increases quadriceps muscle nNOS expression in HF/DM2 patients.
 ................................................................................................................................................................. 27 
Figure 25: Muscle growth/differentiation protein expression +/- (-)-epicatechin-rich cocoa in HF/DM2 
patients. ................................................................................................................................................... 28 
Figure 26: Muscle structural proteins induced by  (-)-epicatechin-rich cocoa in HF/DM2 patients. ............... 28 
Figure 27: Changes in sarcomere ultrastructure in representative HF/DM2 patient before and after 
treatment with (-)-epicatechin-rich cocoa. ............................................................................................... 29 
Figure 28: Age-related muscle protein changes in human skeletal muscle samples. ................................... 30 
Figure 29: Changes in muscle strength and follistatin/myostatin ratio following 7 day of (-)-epicatechin 
treatment. ................................................................................................................................................ 30 
Figure 30: Vital signs following (-)-epicatechin administration in healthy subjects. ....................................... 31 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin 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.   
 
Therapeutic Activities  Recent studies by scientists at the University of California, San Diego (UCSD) 
have suggested that (-)-epicatechin could reverse progressive muscle loss associated with aging and 
muscular dystrophy through stimulation of muscle-promoting proteins and mitochondrial biogenesis, and 
reduction in tissue oxidative stress, inflammation and fibrosis [4-7].  A detailed description of published and 
unpublished preclinical and clinical data supporting the evaluation of (-)-epicatechin in BMD is presented in 
Sections 5 and 6. 
 
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.  


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
8 
 
Cocoa flavonoids have also been intensively studied as potential therapies due to their antioxidant and 
cardiovascular protective activities (reviewed in [8, 9]), and a recent meta-analysis of randomized controlled 
studies of flavonoid-rich cocoa (FRC) determined that FRC consumption significantly improves several 
cardiovascular risks, including blood pressure, insulin resistance, lipid profiles, and flow-mediated vascular 
dilation (FMD)[10].   (-)-Epicatechin was shown to be the FRC component primarily responsible for positive 
effects on vascular endothelium [11], mediated in part by stimulation of the nitric oxide pathway, which 
plays a pivotal role in maintaining vascular endothelial health (reviewed in [12-14]).   
 
Safety and Pharmacokinetics  Published reports indicate that (-)-epicatechin and flavanol-rich extracts 
have thus far been well tolerated in animals and humans, with no significant adverse events have been 
reported in acute or chronic studies (reviewed in [8, 9]).  Human pharmacokinetic (PK) studies in the 
literature indicate that (-)-epicatechin is orally bioavailable, with well defined absorption (plasma Tmax ~ 2 
hours) and clearance (plasma elimination half-life  of ~2 hours) rates for (-)-epicatechin and its major 
metabolites [15-22].   The majority of (-)-epicatechin and metabolites are cleared from plasma by 8 hours.  
Recently a human PK study was completed at the University of San Francisco (UCSF) evaluating (-)-
epicatechin provided by Cardero Therapeutics;  this study is described in detail in Section 6.3. 
 
Preclinical Studies - University of California, San Diego (UCSD)  Collaborators at UCSD examined the 
muscle-promoting effects of (-)-epicatechin in human skeletal muscle cells in vitro and in mice and rats.   In 
human cells treated with (-)-epicatechin in culture, treated cells had a statistically significant increase in 
mitochondrial cristae membrane area relative to controls, suggesting (-)-epicatechin-treated cells have a 
greater capacity of ATP generation (unpublished data).   In mice, significant increases in muscle 
performance, myocardial angiogenesis and indicators of mitochondrial structure and biogenesis were 
observed in (-)-epicatechin-treated animals [4].   A comparison of recognized regulators (markers) of 
skeletal muscle (SkM) growth (myostatin, follistatin), differentiation (myogenin, MyoD, MEF2A, Myf5) and 
senescence (senescence-associated β-galactosidase ,SA-β-Gal) in young and old mice revealed that aging 
is associated with decreases in muscle-promoting capacity (manuscript submitted for publication).  Two 
weeks of (-)-epicatechin (1 mg/kg bid) significantly improved the profile of muscle markers in old mice.  
Recent (-)-epicatechin studies in the mdx and δ-sarcoglycan KO mouse models of muscular dystrophy (MD) 
have shown positive structural and functional effects on damaged muscle, supporting the evaluation of (-)-
epicatechin as a myopathy therapy for BMD patients. 
 
Completed Clinical Studies Three clinical studies have been completed by collaborators of Cardero 
Therapeutics.   Two proof of concept clinical studies were conducted at UCSD to examine the effects of (-)-
epicatechin on skeletal muscle (SkM) structure and function and mitochondrial biogenesis, while a 
pharmacokinetic (PK) study in healthy volunteers was conducted at UCSF.   These studies are described in 
detail in Sections 6.1-6.3. 
 
The first study was performed in five heart failure, type 2 diabetes mellitus patients using (-)-epicatechin-
enriched cocoa to assess the effects on skeletal muscle mitochondria structure and indicators of 
mitochondrial biogenesis [7]. Apparent major losses in normal mitochondria and muscle (sarcomere) 
structure were observed before treatment.  Epicatechin-enriched cocoa increased protein and/or activity of 
mediators of biogenesis and cristae abundance, and in a follow-up study, reduced markers of tissue 
oxidative stress.  Increases in several muscle structural proteins, including dystrophin, were noted, as well 
as improvements in sarcomere organization. 
 
In the second study, healthy adult subjects (average age of 41 years) were treated for 7 days with 25 mg of 
(-)-epicatechin in capsules BID in order to assess treatment effects on muscle strength and circulating 
markers of SkM growth (follistatin, myostatin).   A pre-study examination of tissue bank muscle samples 
from young and old subjects had determined that aging is associated with a decrease in follistatin (promoter 
of muscle growth) and an increase in myostatin (inhibitor of muscle growth), in addition to changes in other 
markers of muscle growth and differentiation.  (-)-Epicatechin treatment significantly improved hand-grip 
strength and increased the ratio of follistatin to myostatin in the plasma. Taken together, these POC clinical 
results suggest that (-)-epicatechin may be a useful therapy for the progressive loss of muscle function 
associated with Becker Muscular Dystropy.  
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
9 
In the PK study, (-)-epicatechin was well tolerated over the 50-200 mg dose range, with rapid absorption 
and first pass metabolism.  (-)-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. 
1.2 
Study Purpose 
This is a proof-of-concept phase 1/2a pilot and endpoint development study that is designed to provide 
initial evidence of biological activity of (-)-epicatechin.  Primary endpoints include initial assessment of 
tissue-specific evidence of efficacy from muscle biopsy samples.  Secondary endpoints include measures 
of strength and physical function, and safety and adverse event data.  Pilot endpoints include assessment 
of mRNA and miRNA peripheral blood profiles and validation of non-invasive near-infrared spectroscopy 
(NIRS) muscle perfusion studies during exercise and a recumbent cycle exercise test that may be 
employed as endpoints in future clinical trials. 
1.3 
Study Design 
This single center open-label pilot study will enroll 10 adults with genetically-confirmed Becker muscular 
dystrophy, 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 baseline and at screening, day 1, and weeks 1, 2, 4 and 8.  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.   
Evaluations of efficacy will include: 
• 
Assessment of peripheral blood creatine phosphokinase, follistatin, (-)-epicatechin 
pharmacokinetics, blood lactate during exercise, and baseline and post-treatment mRNA and 
miRNA biomarker profiles. 
• 
Assessment of baseline and post-treatment muscle biopsy by histology, Western blot, immunostain 
and electron microscopy. 
• 
Assessment of strength by isokinetic dynamometry and quantitative grip testing, exercise 
performance during a standardized recumbent cycle test, muscle perfusion of the quadriceps by 
NIRS during the exercise test, a 6-minute walk test, and body composition by dual-energy x-ray 
absorptiometry (DEXA). 
 
Evaluations of safety will include comprehensive review of 
• 
vital signs, height and weight 
• 
medical history 
• 
physical and neurological exam 
• 
previous and concomitant medication history 
• 
electrocardiogram 
• 
laboratory safety panels for hematology and coagulation, standard blood chemistries (lipid, hepatic 
and metabolic profiles) 
This is a greater than minimal risk study with prospect for direct benefit.  A medical monitor will review 
safety-related evaluations throughout the duration of the study. 
1.4 
Specific Aims 
Aim 1. (Efficacy Primary Endpoint): Evaluation of (-)-epicatechin on blood and muscle tissue 
markers of mitochondrial biogenesis and muscle regeneration in adults with Becker muscular 
dystrophy.  Treatment with 8 weeks of (-)- epicatechin 100mg daily will induce mitochondrial biogenesis, 
muscle regeneration, and improved histological appearance in sarcomere morphology. 
Hypothesis: (-)- Epicatechin will show evidence of mitochondrial biogenesis by Western blot assays 
of biceps brachii  muscle biopsies through increases in ETC1, ETC5 and SOD2 proteins. 
Hypothesis:  (-)- Epicatechin will show evidence of induction of muscle regeneration through 
increases in PGC1a, decreases in acetylated (inactive) PGC1a,  and and increases in follistatin, 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
10
myogenin, and MyoD compared to pre-treatment baseline as determined by Western blot assays of 
biceps brachii  muscle biopsies.    
Hypothesis: Administration of (-)- epicatechin will be associated with increases in plasma 
concentrations of follistatin, a known hormonal regulator of muscle regeneration and a potential 
pharmacodynamic biomarker.  
Hypothesis: Administration of (-)- epicatechin will be associated with electron microscopy evidence 
of increases in mitochondrial number and cristae density within the mitochondria compared to pre-
treatment baseline.  
Hypothesis: Administration of (-)- epicatechin will be associated with increased nNOS expression in 
skeletal muscle compared to pre-treatment baseline and this increased expression will be 
associated with improved muscle perfusion during exercise. 
Aim 2 (Efficacy Secondary Endpoints):  Evaluation of the effects of (-)-epicatechin on exercise 
capacity in adults with Becker muscular dystrophy.  Individuals with Becker muscular dystrophy who 
receive 100mg/day of (-)-epicatechin will demonstrate improved exercise performance and strength that is 
associated with increases in mitochondrial biogenesis and muscle regeneration. 
Hypothesis:  Using the 6 minute walk test, and a standardized recumbent cycle exercise test with 
measurement of lactic acid levels, VO2 max, Work max, anaerobic threshold, and NIRS-defined 
skeletal muscle perfusion (measures of oxyhemoglobin, deoxyhemoglobin, and tissue saturation 
index) we will detect pre-/post-treatment differences in exercise capacity and endurance that will be 
clinically significant (≥ 5-10%) and reflect increases in mitochondrial biogenesis. 
Hypothesis:  Changes in grip strength (≥ 5%), elbow flexion/extension isometric strength (≥ 5%), 
knee extension isometric muscle strength (≥ 1 pound = 4.448 Newtons), pulmonary function 
derived maximal static airway pressures (MIP and MEP ≥ 10 cm H2O), and regional lean tissue 
mass by DEXA (≥ 2%) will be clinically significant and correlate with biomarker evidence for muscle 
regeneration and histological improvement in sarcomere morphology. 
Hypothesis:  Pilot data from exercise performance and strength evaluations in adults with Becker 
muscular dystrophy will determine the magnitude of response over time that will enable accurate 
power calculations for future clinical trials.   
Aim 3 (Safety Secondary Endpoints):  Evaluation of safety and pharmacokinetic profiles of (-)-
epicatechin in adults with Becker muscular dystrophy.  Assessments of safety will include a standard 
clinical safety panel including hematologic, hepatologic, renal and metabolic profiles.  Pharmacokinetic 
studies will include repeat assessments of trough, 2 hour-post (peak) and 4-hour post dose (-)-epicatechin 
levels. 
Hypothesis (safety):  (-)-epicatechin given at 100mg/d orally will display an acceptable safety profile 
in BMD patients. 
Hypothesis (pharmacokinetics): (-)-epicatechin given at 100mg/d orally in BMD patients will 
produce pharmacokinetic profiles similar to those seen in previously described studies in the 
literature. 
Aim 4 (Pilot Biomarker Endpoints): Pilot evaluation of disease- and epicatechin-specific circulating 
mRNA and miRNA blood 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. 
Hypothesis: Using gene array assays directed toward fibrosis- and inflammatory-oriented 
pathways, we expect treatment with (-)-epicatechin to induce detectable pre-/post-treatment 
differences in mRNA and miRNA expression that may be utilized as biomarkers of treatment effect 
for future clinical trials. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
11
2 
BACKGROUND AND SIGNIFICANCE 
2.1 
Becker Muscular Dystrophy 
Muscular dystrophies are a group of diverse genetic diseases featuring progressive muscle weakness, 
degradation of muscle fibers, and loss of function [23].  BMD is an inherited disease primarily seen in 
males, with an incidence of 3-6 per 100,000 live births [24].  Onset of symptoms can occur over a wide age 
range, with most patients diagnosed between 5 and 15 years of age [25].    BMD is characterized by 
progressive muscle loss (sarcopenia), with loss of strength, muscle injury, degeneration, atrophy, and 
eventually fibrosis [25]. Most patients can no longer walk by age 25-30, and lung (e.g., breathing problems, 
infections) and heart (e.g., cardiomyopathy) complications can significantly reduce lifespan [25].  
 
Many muscular dystrophies, including BMD and the more common Duchenne Muscular Dystrophy (DMD), 
result from mutations in the gene encoding dystrophin, a key subsarcolemmal protein in the dystrophin-
associated protein complex (DAPC) at the muscle cell membrane [26, 27].   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 [28, 29]. 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  [30].   BMD is attributed to quantitative or qualitative 
decrements in dystrophin expression rather than complete or near-complete loss of dystrophin as in DMD.   
 
While a dystrophin mutation is the primary defect, the progressive loss of muscle function in BMD is due to 
a secondary cascade of chronic destructive events, including calcium influx and overload, mitochondrial 
swelling and apoptosis, disruption of nNOS/NO leading to muscle ischemia during exercise, inflammation, 
increased levels of ROS (oxidative stress) and protease activation, that eventually exhaust muscle repair 
mechanisms and result in muscle degeneration [26-31].  Mitochondrial damage appears to play a central 
role in this muscle damage cascade as evidenced by studies in sarcopenia.  SkM from older subjects 
demonstrate increases in mitochondrial ETC abnormalities (accumulation of cytochrome c oxidase negative 
and succinate dehydrogenase hyper-positive fibers), increases in multiple markers of oxidative stress, 
accumulation of mtDNA mutations, and transcriptome changes indicative of mitochondrial dysfunction [32-
34]. Studies in mitochondrial DNA mutator mice (mtDNA polymerase proof-reading mutation) demonstrated 
that mutations in mtDNA leads to skeletal muscle apoptosis and sarcopenia  [35].  Myocyte apoptosis, the 
end result of severe mitochondrial dysfunction, is mediated in part by the opening of the mitochondrial 
permeability transition pore (mPTP), the rupture of the outer mitochondrial membrane and release of 
mitochondrial apoptotic signals [36].  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 this secondary damage 
cascade and/or improve skeletal muscle function.    
 
Published studies in mouse models of MD support targeting the secondary damage cascade to preserve 
mitochondria and improve muscle function.   Muscle cell calcium overload can lead to calcium-induced 
mitochondrial permeability transition (MPT), resulting in swelling, loss of function, rupture and necrotic cell 
death [37, 38]. Genetic or pharmacologic inhibition of cyclophilin D, the enzyme that regulates calcium-
dependent MPT, in mouse models of MD (mdx, scgd-/-) significantly reduced mitochondrial swelling, 
improved myofiber organization and reduced fibrosis and necrosis [39].   In a separate study, stimulating 
mitochondrial metabolism by the provision of an endurance mimetic compound AICAR, which activates 
AMP-activated protein kinase (AMPK) activity and promotes mitochondrial biogenesis and metabolism, led 
to markedly improved muscle function and overall activity in mdx mice and restored structural integrity of 
muscle fibers [40]. 
2.2 
Justification of Current Approach 
 (-)-Epicatechin has been shown to improve muscle structure and function by positively influencing several 
aspects of the secondary damage cascade leading to progressive muscle loss in MD.   These animal and 
human data are outlined in Table 1. (-)-Epicatechin is an attractive therapeutic candidate due to 
combination of its muscle-restoring and promoting activities and excellent safety profile. 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
12
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 PGC1α 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 δ-sarcoglycan KO mouse model of MD 
 
3 
EPICATECHIN OVERVIEW 
3.1 
Pharmacokinetics  / Pharmacodynamics 
(-)-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 [41, 42], allowing 
negligible amounts of native (-)-epicatechin in the mesenteric circulation.  
 
In the liver, further glucuronidation, methylation, and sulfation can take place [41, 43, 44]. Several studies 
have determined the presence of these conjugates in the plasma and urine of rodents and humans [15, 19, 
44-47], as well as in rat bile [43] and brain [48]. In plasma, (-)-epicatechin is present almost exclusively as 
conjugated metabolites [19, 47] .    
 
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 [11, 18-21, 45, 47-50]. 
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 [47].  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 [47].   
 
Relative amounts of (-)-epicatechin metabolites identified in plasma in two separate studies are shown in 
Figure 3.   
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
13
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 [19].  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 [19]. Unmodified (-)-
epicatechin represented <0.5%.      B.  Profile of (-)-epicatechin metabolites in plasma after ingestion of 100g of dark chocolate ((79 
mg (-)-epicatechin)) [47].  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 [17, 19, 21].  
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 [51], 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 [17]. 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
14
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  [52] 
(-)-Epicatechin 
200 
3570 
17.85 
Ottaviani, J.I., et al., 
2011 [18] 
 (-)-epicatechin added to 
cocoa vehicle 
125 (mean) 
~900 
~7.2 
Ottaviani, J.I., et al., 
2012 [19] 
Cocoa drink 
135 (mean) 
1245 
9.3 
Actis-Goretta, L., et 
al., 2012 [47] 
Dark Chocolate 
79 
873 
11.1 
Donovan, J.L., et al., 
2006 [53] 
Cocoa drink 
55 
630 
11.5 
Schroeter, H., et al., 
2006 [11] 
Cocoa flavanols: 
 917  mg  
 
174 
 
~1950 
 
~11.2 
Baba, S., et al., 2000 
[15] 
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 
[52], while administration of an ~75 mg dose (1 mg/kg body weight) resulted in significant improvement in 
vascular function (FMD) [11].   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 [54]. 
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
15
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
16
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 [55]. 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 [56, 57]. 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 [58], 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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
17
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 [4].  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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
18
(≈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 [59]. 
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
19
(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 [60] (-)-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) [61]. 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 [60]. 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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
20
Figure 11: Muscle differentiation in time course in C2C12 cells +/- (-)-epicatechin. 
 
 
4.1.1.1 
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
21
4.1.1.2 
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. 
 
4.1.8 
 (-)-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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
22
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. 
 
4.1.9 
 (-)-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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
23
Figure 16: (-)-Epicatechin reduces fibrosis in the gamma-sarcolgycan knock out mouse.  
 
4.1.10  (-)-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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
24
Figure 18: (-)-Epicatechin improves skeletal muscle strength in the MDX mouse model of 
muscular dystrophy. 
 
4.1.11  (-)-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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
25
5 
HUMAN STUDIES 
5.1 
POC 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 [7]. Published reports indicate that in patients suffering from heart failure or diabetes there is a 
significant compromise in mitochondrial structure and muscle bioenergetics [62-64]. 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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
26
Figure 21: (-)-Epicatechin-rich cocoa increases mitochondrial complex V protein and cristae 
abundence 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) [65]. 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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
27
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.  
5.1.1.1 
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 [28, 66]. 
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
28
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
29
Figure 27: Changes in sarcomere ultrastructure in representative HF/DM2 patient before and 
after treatment with (-)-epicatechin-rich cocoa.  
 
5.2 
POC 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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
30
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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
31
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 [11, 18-21, 45, 47-50]. 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 [47]. 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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
32
6 
RESEARCH DESIGN AND METHODS 
6.1 
Selection and Withdrawal of Participants  
6.1.1 
Criteria for Enrollment 
Ten participants will be studied in this initial pilot project.    
Participant Inclusion Criteria 
• 
Male 
• 
Age 18 years to 60 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:  
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 
Potential participants for the study will be evaluated using a two visit screening method that will allow 
investigators to evaluate the participant’s ability to provide a reproducible (15%) (primary endpoint) test scores 
to be enrolled.  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 randomized into the study. 
 
 
 
 
 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
33
Table 3: Study procedures 
Event 
Screening1 
(Week -2 to 
Day 1) 
Baseline 
(Day 1) 
Week  
1 
Week 
2 
Week 
4 
Week 
8 
Early 
with-
drawal 
Informed consent 
X 
 
 
 
 
 
 
Medical history 
X 
X 
 
X 
X 
X 
X 
Vital signs 
X 
X 
 
X 
X 
X 
X 
Physical examination 
X 
X 
 
X 
X 
X 
X 
Blood & urine  
collection (safety, efficacy, 
PK ) 
X 
X 
 
X 
X 
X 
X 
ECG 
X 
X 
 
X 
X 
X 
 
Other medications  
and dietary  
supplements 
X 
X 
 
 
X 
X 
X 
Adverse events 
 
X 
 
X 
X 
X 
X 
Functional assessments   
strength, 6MWT,  
recumbent graded 
exercise test, NIRS, PFTs 
 
X 
 
 
X 
X 
X 
Blood tests: baseline and 
efficacy 
 
X 
 
 
X 
X 
X 
Muscle Biopsy Tests: 
baseline and efficacy 
 
X 
 
 
 
X 
 
Dispense Epicatechin 
 
X 
 
X 
 
 
 
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 (method) testing for the remainder of the 8-week study period (8 months 
from participant’s baseline visit).  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 8). 
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 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 and randomization, 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.  


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
34
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: 
• 
Peak (-)-epicatechin PK level >4umol/ml 
• 
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 (See Appendix F). 
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. 
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 [67].   In a developmental toxicity study in pregnant rats using a different tea extract, 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
35
the no-observable-adverse-effect level (NOAEL) corresponded to 100 mg (-)-epicatechin/kg (the highest 
dose tested)[68].   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 [69]. 
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 [11, 52].   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 [8, 9]).   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 [16].    
 
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 
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. 
    


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
36
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. 
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: 
• 
Creatine phosphokinase (CPK) 


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UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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• 
Follistatin 
• 
mRNA and miRNA biomarker profiles 
• 
Pharmacokinetics: epicatechin metabolites [serum tested at trough, 2 hours (Tmax), 4 hours 
after dose at baseline, week 2, week 4 and week 8]. 
• 
Blood lactate testing during exercise:  Blood lactate levels will be collected at baseline and 2-
minute intervals by finger-stick or ear-stick during 6-minute cycle testing. 
• 
Muscle Biopsy (Biceps Brachii) 
Open biopsies of the mid-biceps will be conducted at baseline and after 8 weeks of study 
medication dosing.  The muscle biopsy samples will be collected by open biopsy or according to 
standard hospital procedures. The minimum amount of muscle tissue required is a small piece of 
muscle of at least 0.5 x 1.0 x 1.0 cm. If possible, the tissue collected will be of sufficient size to 
provide 4 (four) pieces of tissue (each 0.5x0.5x0.5cm). Muscle for histological evaluation will be 
sent fresh to hospital pathology.  Muscle tissue for evaluation at outside labs will be immediately 
frozen in liquid nitrogen-cooled 2-methylbutane and stored at -80°C or -70°C till shipment.  Biopsy 
at baseline will be collected from the lateral aspect of the midpoint of the biceps muscle without the 
use of depolarizing agents or Bovie catheterization prior to tissue extration. Biopsy at 8 weeks will 
be collected from medial aspect of the midpoint of the biceps muscle of the same arm, and must be 
separated by a margin of at least 1cm to avoid scar tissue from the earlier biopsy. 
 
Histology samples will be evaluated (Lee-Way Jin lab, UC Davis) to examine: 
• 
Fiber size variation 
• 
Tissue necrosis 
• 
Degree of central nucleation 
• 
Presence of basophilic/regenerating fibers 
• 
Fiber type (myosin IHC stains) 
• 
NADH-TR and succinic dehydrogenase (mitochondria) 
 
Muscle biopsy samples will be examined by Western blot (Robert Henry lab, UC San Diego) to 
evaluate: 
 
Initial Phase (done immediately upon biopsy) 
• 
Muscle regeneration: follistatin, MyoD, myogenin, dystrophin, sarcoglycans, dysferlin 
• 
Oxidation/oxidative stress:  SOD1, SOD2, catalase, GSK, nNOS, eNOS 
• 
Mitochondrial function: ETC-I, ETC-V, mitofilin, porin 
• 
Transcriptional regulation: PGC1-alpha 
• 
Collagen deposition: Collagen I, collagen III, fibronectin 
 
Delayed Phase (completed on stored material at a later date if warranted by initial data) 
• 
Glucose transport: Glut1, Glut 4 (and ratio) 
• 
Apoptosis: cytochrome-c, caspase 
• 
Dystrophin-associated complex (DAP) (Steve Moore lab, University of Iowa): sarcoglycans 
(alpha, beta, gamma, delta, epsilon), laminin-2 (alpha, beta, gamma), alpha dystrobrevin-2, 
syntrophin 
• 
Matrix metalloproteinase-9 
 
Muscle biopsy samples will be examined by electron microscopy (Robert Henry lab, UC San Diego) 
to evaluate: 
 
Initial Phase (done immediately upon biopsy) 
• 
Sarcomeric structure 
• 
Mitochondrial number and localization 
 
Delayed Phase (completed on stored material at a later date if warranted by initial data) 
• 
Myofiber volume 
• 
Mitochondrial volume per myofiber volume 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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• 
Mitochondrial christae density 
8.2 
Functional Efficacy Parameters  
• 
Six-Minute Walk Test 
Participants will complete the Duchenne muscular dystrophy six-minute walk test (6MWT) as described 
by McDonald et al [70].  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.  During the 6MWT, participants will undergo respiratory gas exchange testing using a 
Cosmed “backpack” portable metabolic system and muscle perfusion testing using a non-invasive 
portable near-infrared spectroscopy (NIRS) system. 
• 
Graded exercise test using a recumbent cycle ergometer 
Each participant will perform a graded exercise test on an electronically braked recumbent cycle 
ergometer as previously described  [71, 72].  Modifications for Becker muscular dystrophy related 
proximal weakness will be made as described by others [72, 73]. 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 [74].  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. [75].  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. 
• 
Quantitative Muscle Testing 
Isometric and isokinetic QMT of elbow and knee flexors and 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. 
• 
Range of Motion Testing 
Passive ranges of motion for knee extension, ankle dorsiflexion, elbow extension and wrist extension 
will be measured to the nearest 5 degrees using goniometry techniques as described by Pandya et al  
[76] and Fowler [77].  Knee and elbow extensions ranges from 20 to -150 degrees.  Ankle dorsiflexion 
range is from 20 to -80 degrees, with 0 degrees considered full passive range of motion. Wrist 
extension with fingers extended range is from 100 to -90 degrees, with 90 degrees considered full 
range of motion. 
• 
Pulmonary Function Tests 
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 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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Timed Function Testing has frequently been used to assess functional abilities in three important areas: 
rising from the floor, 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 [78]. 
• 
Functional Evaluation 
The functional classification used by CINRG utilizes a scale modified from the upper extremity scale 
reported by Brooke et al [79] and the lower extremity scales used by Vignos et al [80]. 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.   
• 
Anthropometric Measurements 
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).   
• 
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, 
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.   
• 
Upper Extremity Range of Motion Testing (20 minutes) 
Upper Extremity Range of Motion Testing (20 minutes).  The subject will be asked to perform the 
following range of motion activities while wearing the Cal-FIT physical activity motion analysis system 
and recorded by the stereo-camera optical measurement system: 
1. move their elbow through a complete range of motion from maximally flexed to maximal extension 
2. move the forearm through a complete range of motion from maximal pronation to maximal 
supination 
3. move the wrist through a complete range of motion from maximal extension (dorsiflexion) to 
maximal flexion (palmar flexion) and from radial deviation through ulnar deviation 
4. move the shoulder through a complete range of motion from maximal extension to maximal 
flexion,  maximal abduction to maximal adduction, and from maximal internal rotation to maximal 
external rotation. 
While being monitored by a motion sensing system (KINECT): 
1. perform a series of standardized range of motion and functional upper extremity tasks 
2. perform a series of standardized range of motion and functional upper extremity tasks while holding 
a weight 
 
9 
ASSESSMENT OF SAFETY 
9.1 
Safety Parameters 
Safety parameters for this study will include: 
• 
Review of Systems 
• 
Review of Adverse Events 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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• 
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 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 eight to ten weeks from the date of 
enrollment (eight weeks from start of study drug administration).   
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:  


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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• 
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 
10.1 
Characteristics of the Study Population 
Ambulatory male adults with a confirmed diagnosis of BMD, 18 years to 60 years, will be recruited. 
Local Participant Population Available 
The incidence of BMD is equal across racial and ethnic groups.  It is assumed that the participant population 
of the study site should closely mirror the racial and ethnic distribution of the US population. 
Projected Number of Participants Needed for this Study 
  
American 
Indian or 
Alaskan 
Native 
Asian or 
Pacific 
Islander 
Black, 
not of 
Hispanic 
Origin 
Hispanic 
White, 
not of 
Hispanic 
Origin 
Other 
Total 
MALE 
0 
1 
1 
2 
6 
0 
10 
10.2 
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. 
10.3 
Sources of Research Material from Living Participants 
Study data, blood and tissue samples collected will only be for research use or monitoring of study safety. 
10.4 
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. 
10.4.1 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. An advertisement 
is included in Appendix C. 
10.4.2 Screening 
Patients for this study will be identified through advertising, self-referral, referrals from other physicians and 
review of patients’ clinic medical records currently under the care of the study site’s principal investigator or 
co-investigators.  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.  A telephone script (See Appendix B) will be used for the initial contact of 
potential study participants. If the potential participant is to be contacted by mail, the mailing letter template 
(Appendix B) should be used. 
10.5 
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 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. 


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10.6 
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. 
10.7 
Potential Risks 
10.7.1 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 [67].  
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 [11].  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 [81].  With cocoa based studies, 
blood pressure reducing effects are only reported in humans that have high blood pressure [81]. 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 [82]. 
The consumption of cocoa products has been reported to be associated with increased likelihood of 
migraine development [83, 84]. 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 [85]. 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)[68].  No information is available on levels of (-)-epicatechin in breast milk.Risks of  
10.7.2 Risks of Blood Tests 
The risks of blood drawing include soreness or bruising at the site of the needle. A local numbing cream (EMLA) 
will 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. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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10.7.3 Risks of Muscle Biopsy 
After the muscle biopsy patient may feel pain at the place of the biopsy. Patients usually find the pain easy 
to tolerate and that they rarely need to take a painkiller although Tylenol or ibuprofen is appropriate as 
needed for the next 24 hours. The muscle biopsy may leave a small scar and it is possible that the strength 
of that muscle might be slightly reduced in the short term. 
10.7.4 Risks of QMT 
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.   
10.7.5 Risks of PFT 
These tests may cause dizziness and lightheadedness during and shortly after the test. 
10.7.6 Risks of EKG 
The EKG has no known risks. 
10.7.7 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.   
10.7.8 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). 
10.7.9 Risks of Multi-Frequency Bioimpedence Assessment (MFBIA) 
There are no known risks of MFBIA testing.  
10.7.10 Risks of Upper Extremity Range of Motion Evaluation 
There are no known risks of upper extremity range of motion evaluation. 
10.8 
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.   
10.9 
 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  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
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10.10 
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 
10.11 
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 generally 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 
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.  Baseline and screening measures will be used to calculate test-
retest reliability using the intraclass correlation coefficient.  Level of statistical significance is set at <0.05.  
Simple comparative analyses involving chi squares ttests and correlations will be used to assess and 
understand simple relationships, including defining first order interactions. Where possible we will perform 
longitudinal analyses using mixed models (using xtreg or xtmixed in STATA 12) to evaluate raw scale and 
percent change over time in study outcomes measures.  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.  If feasible with the small number of participants in the study and because there is 
little available data for individuals with Becker muscular dystrophy, this 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 study personnel. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
45
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.   
 
13 REFERENCES 
 
1. 
Crozier, A., I.B. Jaganath, and M.N. Clifford, Dietary phenolics: chemistry, bioavailability and effects 
on health. Nat Prod Rep, 2009. 26(8): p. 1001-43. 
2. 
Landolino, A.B. and D.R. Cook, Phenylpropanoid metabolism in plants: biochem- istry, functional 
biology, and metabolic engineering. In: Fraga, C.G. (Ed.), Plant Phenolics and Human Health: 
Biochemistry, Nutrition, and Pharmacology. Wiley, Hoboken, 2010: p. 489-563. 
3. 
Steinberg, F.M., M.M. Bearden, and C.L. Keen, Cocoa and chocolate flavonoids: implications for 
cardiovascular health. J Am Diet Assoc, 2003. 103(2): p. 215-23. 
4. 
Nogueira, L., et al., (-)-Epicatechin enhances fatigue resistance and oxidative capacity in mouse 
muscle. J Physiol, 2011. 589(Pt 18): p. 4615-31. 
5. 
Ramirez-Sanchez, I., et al., (-)-epicatechin activation of endothelial cell endothelial nitric oxide 
synthase, nitric oxide, and related signaling pathways. Hypertension, 2010. 55(6): p. 1398-405. 
6. 
Ramirez-Sanchez, I., et al., (-)-Epicatechin induces calcium and translocation independent eNOS 
activation in arterial endothelial cells. Am J Physiol Cell Physiol, 2011. 300(4): p. C880-7. 
7. 
Taub, P.R., et al., Alterations in skeletal muscle indicators of mitochondrial structure and biogenesis 
in patients with type 2 diabetes and heart failure: effects of epicatechin rich cocoa. Clin Transl Sci, 
2012. 5(1): p. 43-7. 
8. 
Katz, D.L., K. Doughty, and A. Ali, Cocoa and chocolate in human health and disease. Antioxid 
Redox Signal, 2011. 15(10): p. 2779-811. 
9. 
Corti, R., et al., Cocoa and cardiovascular health. Circulation, 2009. 119(10): p. 1433-41. 
10. 
Shrime, M.G., et al., Flavonoid-rich cocoa consumption affects multiple cardiovascular risk factors 
in a meta-analysis of short-term studies. J Nutr, 2011. 141(11): p. 1982-8. 
11. 
Schroeter, H., et al., (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular 
function in humans. Proc Natl Acad Sci U S A, 2006. 103(4): p. 1024-9. 
12. 
Kolluru, G.K., J.H. Siamwala, and S. Chatterjee, eNOS phosphorylation in health and disease. 
Biochimie, 2010. 92(9): p. 1186-98. 
13. 
Moncada, S. and E.A. Higgs, The discovery of nitric oxide and its role in vascular biology. Br J 
Pharmacol, 2006. 147 Suppl 1: p. S193-201. 
14. 
Sessa, W.C., Regulation of endothelial derived nitric oxide in health and disease. Mem Inst 
Oswaldo Cruz, 2005. 100 Suppl 1: p. 15-8. 
15. 
Baba, S., et al., Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human 
volunteers. Free Radical Research, 2000. 33(5): p. 635-641. 
16. 
Chow, H.H., et al., Pharmacokinetics and safety of green tea polyphenols after multiple-dose 
administration of epigallocatechin gallate and polyphenon E in healthy individuals. Clin Cancer Res, 
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17. 
Lee, M.J., et al., Pharmacokinetics of tea catechins after ingestion of green tea and (-)-
epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. 
Cancer Epidemiol Biomarkers Prev, 2002. 11(10 Pt 1): p. 1025-32. 
18. 
Ottaviani, J.I., et al., The stereochemical configuration of flavanols influences the level and 
metabolism of flavanols in humans and their biological activity in vivo. Free Radic Biol Med, 2011. 
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19. 
Ottaviani, J.I., et al., Structurally related (-)-epicatechin metabolites in humans: assessment using 
de novo chemically synthesized authentic standards. Free Radic Biol Med, 2012. 52(8): p. 1403-12. 
20. 
Rein, D., et al., Epicatechin in human plasma: in vivo determination and effect of chocolate 
consumption on plasma oxidation status. J Nutr, 2000. 130(8S Suppl): p. 2109S-14S. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
46
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Richelle, M., et al., Plasma kinetics in man of epicatechin from black chocolate. Eur J Clin Nutr, 
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Wang JF, S.D., Holt RR, Ensunsa JL, Fraga CG, Schmitz HH, Keen CL., A dose-response effect 
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Donovan, J.L., et al., Catechin Is Present as Metabolites in Human Plasma after Consumption of 
Red Wine. The Journal of Nutrition, 1999. 129(9): p. 1662-1668. 


              Principal Investigator/Program Director:  McDonald, CM  
UCD0113: Epicatechin in Becker Muscular Dystrophy 
 
47
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Ellinger, S., et al., Epicatechin ingested via cocoa products reduces blood pressure in humans: a 
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transcription coactivators. Cell Metab, 2005. 1(6): p. 361-70. 
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ICF_001.pdf: 
Use of (-)-Epicatechin in the Treatment of Becker Muscular Dystrophy 
(Pilot Study) 
NCT01856868 
3/24/2014 


Permission to Take Part in a Human Research Study 
Page 1 of 8 
 
Version Date: 3/19/2013 
Revised: 3/24/2014 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: February 22, 2013 
 
Title of research study: UCD0113:  An open-label pilot study of purified tea-derived epicatechin 
to improve mitochondrial function, strength and skeletal muscle exercise response 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 a research study because you are an adult diagnosed with Becker 
muscular dystrophy.  
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.  
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 916-734-0384 
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 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
454352
03/24/2014


Permission to Take Part in a Human Research Study 
Page 2 of 8 
 
Version Date: 3/19/2013 
Revised: 3/24/2014 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: February 22, 2013 
 
If you call the UCDMC Hospital Operator, tell the Operator you are participating in a research study 
and you wish to talk to Dr. Craig McDonald. 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/IRBAdmin.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: 
• 
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.  (-)-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. 
How long will the research last? 
We expect that you will be in this research study for 8 weeks for a total of 6 visits.  
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   
(Week -2 
to Day 1) 
Baseline 
(Day 1) 
Week 
2 
Week 
4 
Week 
8 
Early 
Withdrawal 
Informed consent 
X 
  
  
  
  
  
Medical history 
X 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
X 
X 
Physical examination 
X 
X 
X 
X 
X 
X 
Blood & urine collection and tests 
X 
X 
X 
X 
X 
X 
ECG 
X 
X 
X 
X 
X 
  
Other medications and dietary 
supplements 
X 
X 
  
X 
X 
X 
Adverse events 
  
X 
X 
X 
X 
X 
Functional assessments 
  
X 
  
X 
X 
X 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
454352
03/24/2014


Permission to Take Part in a Human Research Study 
Page 3 of 8 
 
Version Date: 3/19/2013 
Revised: 3/24/2014 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: February 22, 2013 
 
Muscle Biopsy 
  
X 
  
  
X 
  
Dispense Epicatechin 
  
X 
X 
  
  
  
 
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 and medications (10 mins) (At Each Visit) 
We will ask you questions about medical conditions and medications that you are taking. 
Vital signs and physical exam (30 mins) (At Each Visit) 
We will measure your weight, height, blood pressure and breathing rate.  
Blood & urine collection and tests (30 mins) (At Each Visit) 
We will collect approximately 13 ml (about 2.6 teaspoons) of your blood and a urine sample for 
safety assessments. 
Electrocardiogram (ECG) (15 mins) (At Each Visit) 
A test to determine how well your heart is functioning 
Adverse events (10 mins) (At Each Visit Following Screening) 
We will ask you if anything has happened since the last visit that hurt or was out of the ordinary 
with regard to your health. 
Functional assessments (3 hours) (At Each Visit Following Screening) 
You will be asked to perform several exercise tests including the following measures: 
• 
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 
• 
Strength test—we will measure the strength of your elbows and knees with a BIODEX 
ergometer 
• 
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. 
• 
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. 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
454352
03/24/2014


Permission to Take Part in a Human Research Study 
Page 4 of 8 
 
Version Date: 3/19/2013 
Revised: 3/24/2014 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: February 22, 2013 
 
Impedence is measured between upper and lower extremity electrodes. You will not feel 
anything during the test. 
• 
 Muscle Biopsy (60 mins) (At Baseline and Week 8) 
The muscle biopsy samples will be collected by open biopsy according to standard hospital 
procedures. The minimum amount of muscle tissue required is a small piece of muscle of at least 
0.5 x 1.0 x 1.0 cm. Biopsy at baseline will be collected from the halfway point of the biceps muscle. 
A second biopsy at 8 weeks will be collected from the same muscle at a place about 1 inch away 
from the first biopsy. The biopsies will be done with local anesthesia (numbing) like you would have 
for routine dental procedures. The doctor will close the incision with some small stitches or tape 
called “steri-strips.” The biopsy will have a small scar about an inch long. 
Study Medication-Epicatechin 
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. 
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. 
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. 
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 leave the research, contact the investigator so that the investigator 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 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
454352
03/24/2014


Permission to Take Part in a Human Research Study 
Page 5 of 8 
 
Version Date: 3/19/2013 
Revised: 3/24/2014 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: February 22, 2013 
 
that patients undergoing drug treatment for high blood pressure if given (-)-epicatechin may 
develop hypotension.  
Migraines. 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 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) will 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. 
Risks of Muscle Biopsy  
After the muscle biopsy you may feel pain at the place of the biopsy. Patients usually find the pain 
easy to tolerate and that they rarely need to take a painkiller although Tylenol or ibuprofen is 
appropriate as needed for the next 24 hours. The muscle biopsy may leave a small scar and it is 
possible that the strength of that muscle might be slightly reduced in the short term. Additional 
risks of the muscle biopsy include: bleeding, wound separation, and/or possible infection. 
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) 
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. 
Will being in this study help me in any way? 
We cannot promise any benefits to you or others from your taking part in this research. However, 
possible benefits include: 
• 
You may experience an increase in muscle strength or a delay in strength decline.  
 
 
 
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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Version Date: 3/19/2013 
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Document Revision Date: February 22, 2013 
 
• 
You will receive additional care during study participation including muscle strength training, 
contracture measurements and spirometry during each study visit.  
• 
Your medical and adverse event history will be closely monitored 
• 
You will experience additional medical monitoring that will allow for increased interaction 
with medical staff above expected routine clinical care (i.e. the increased monitoring of 
safety labs) 
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. 
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 medical records may be looked at by 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? 
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 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 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. 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
454352
03/24/2014


Permission to Take Part in a Human Research Study 
Page 7 of 8 
 
Version Date: 3/19/2013 
Revised: 3/24/2014 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: February 22, 2013 
 
Are there other research opportunities? 
If you are interested in being contacted for future research, please provide your phone number 
and/or email. This is completely optional. 
 
(initials) Yes, I am willing to be contacted for future research opportunities. My phone  
number and/or email is:  
 
 
 
 
 
 
 
 
 
. 
 
 
 
APPROVED by the Institutional Review Board 
at the University of California, Davis 
Protocol 
Approved 
 
 
454352
03/24/2014


Permission to Take Part in a Human Research Study 
Page 8 of 8 
 
Version Date: 3/19/2013 
Revised: 3/24/2014 
For IRB Use  
 
 
 
 
 
 
 
Document Revision Date: February 22, 2013 
 
Signature Block for Capable Adult 
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 
 
 
 
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 
 
 
454352
03/24/2014