Presentation on the potential use of Sulforaphane and the NRF2 pathway as a treatment for Duchenne Muscular Dystrophy

1. Isothiocyanates and the NRF2
transcriptional pathway as a
potential therapeutic for Duchenne
Muscular Dystrophy
Jordan Gladman
5/5/2015

2. A disease of multiple dysfunctional biological
pathways
Multiple Pathways disrupted by loss
of Dystrophin
• Inflammation
– Nuclear Factor Kappa B (NF-kB)
– Tumor Necrosis Factor-alpha (TNF-
alpha)
• Intracellular calcium balances
• Fibrosis & Muscle Regeneration
• Free Radical, Redox Dysfunction
& ROS
– Reduction in nNOS/NO
– Increased Levels of SOD-1
• Apoptosis & Necrosis
– p38 MAPK

3. Duchenne Muscular Dystrophy Organ System
Involvement
Skeletal Muscle Involvement Organ System Involvement

4. Nrf-2/Phase II genes and Isothiocyanate activation
Osburn and Kensler (2008) Mutation Research 659: 31-39
Phase II genes regulate the
expression of detoxifying proteins
that protect against oxidative
damage and are anti-inflammatory
• Isothiocyanate is the chemical group –N=C=S
• Naturally abundant in cruciferous vegetables
• Broccoli: Glucoraphanin  SFN (Sulforaphane)
• Wasabi: Sinigrin  AITC (Allyl Isothiocyanate)
• Watercress: Gluconasturtiin  PEITC (Phenethyl isothiocyanate)
• Capable of forming a chemical bond with cellular proteins
Nrf2 is ubiquitously expressed with the
highest concentrations in the kidney,
muscle, lung, heart, liver, and brain

5. Disruption of Nrf2 signaling promotes cell degradation
in skeletal muscle
Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged
skeletal muscle
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1822, Issue 6, 2012, 1038 - 1050

6. Loss of NRF2 results in skeletal myopathy
Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged
skeletal muscle
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1822, Issue 6, 2012, 1038 - 1050
Additional data in exercise induced
muscle damage found that increasing
NRF2 levels lead to increased levels of
ARE target proteins and improved
muscle histology

7. • Glucoraphanin, Sulforaphane’s precursor, is naturally abundant in
broccoli and cauliflower
• Identified as a Cancer Chemopreventive
• Multiple labs are making sulforaphane derivatives
Glucoraphanin (Glucosinolate precursor)
The Isothiocyanate Sulforaphane
Sulforaphane
myrosinase
Sulforaphane

8. Sulforaphane and DMD
• The affects of Sulforaphane
– p38 MAPK
– NF-κB
– Myostatin
• Sulforaphane treatment of MDX Mice
• Potential negative aspects
• Future Steps

9. P38 MAPK myofiber death
• Wissing et al. (2014) Hum Mol Genet.
P38α MAPK underlies muscular
dystrophy and myofiber death through a
Bax-dependent mechanism.
– P38α is over expressed in DMX
– Over-activation of p38α causes
severe muscle-wasting pathology in
mice
• P38 expression inhibits Nrf2 by keeping
it bound to KEAP1
Aurelian (2015) CROSS TALK OF SIGNALING AND APOPTOTIC CASCADES IN THE CNS: TARGET FOR VIRUS MODULATION
Frontiers in Bioscience 10, 2776-2787

10. Expression of p38 MAPK in DMD.
Erin R. Wissing et al. Hum. Mol. Genet. 2014;23:5452-5463

11. P38 MAPK myofiber death
• SFN suppressed activation of
p38 MAPK isoforms by
blocking phosphorylation of
MKK3/6
– Keum et al. (2006) Cancer Res.
Aurelian (2015) CROSS TALK OF SIGNALING AND APOPTOTIC CASCADES IN THE CNS: TARGET FOR VIRUS MODULATION
Frontiers in Bioscience 10, 2776-2787
SB731445 Sulforaphane
P38 activation has been show to increase
Utrophin so it may be incompatible with some
forms of DMD therapy

12. McKinley and Willoughby (2014) Effectiveness of Antioxidant Nutraceuticals in Attenuating Canonical NF-κB Signaling in Human Skeletal Muscle
Resulting From Exercise-Induced Inflammation and Oxidative Stress. Journal of Nutritional Health & Food Engineering
The NF-κB pathway and cross talk in DMD
Survival, Proliferation,
Inflammation, Immune
Regulation
Catabasis, CAT-1004 is SMART
linker conjugate of salicylate and
docosahexaenoic acid (DHA)
designed to enhance the activity
of salicylate and DHA in
modulating the NF-kB pathway.
Guttridge Lab, NBD
delivery improves the
disease phenotype of the
golden retriever model of
Duchenne muscular
dystrophy. Skelet Muscle.
2014.
FN14
TWEAK
The tumor necrosis factor (TNF)-like weak inducer of
apoptosis (TWEAK) has a wasting effect on skeletal
muscle cells both in-vivo and in-vitro

13. The NF-κB pathway is regulated by Nrf2 and
Sulforaphane
• Nrf2 overexpression is capable of inhibiting the NF-κB
– Cuadrado A J et al. (2014) Biol Chem. Transcription factors NRF2 and NF-κB
are coordinated effectors of the Rho family, GTP-binding protein RAC1 during
inflammation.
• NFκB could directly repress Nrf2 transcription.
– Liu GH et al. (2008) Biochim Biophys Acta. NF-kappaB/p65 antagonizes Nrf2-
ARE pathway by depriving CBP from Nrf2 and facilitating recruitment of
HDAC3 to MafK.
• SFN treatment was found to be able to inhibit IKK/IκB
phosphorylation and p65 NFκB subunit nuclear translocation
– Xu C et al. (2005) Oncogene. Suppression of NF-kappaB and NF-kappaB-
regulated gene expression by sulforaphane and PEITC through IkappaBalpha,
IKK pathway in human prostate cancer PC-3 cells.
• SFN prevented NF-κB binding to its consensus DNA sequence
– Elke Heiss et al. (2001) J Biol Chem. Nuclear factor kappa B is a molecular
target for sulforaphane-mediated anti-inflammatory mechanisms.

14. Sulforaphane
Sulforaphane

15. Sulforaphane represses Myostatin mRNA levels
• Sulforaphane acts as a histone
deacetylase (HDAC) inhibitors
• SFN treatment significantly
represses MSTN expression, in
porcine myoblast cells
• Protein levels were not
examined
Burks and Cohn (2011) Role of TGF-β signaling in inherited and acquired
myopathies. Skeletal Muscle 1:19
Fan et al. (2012) Sulforaphane causes a major epigenetic repression
of myostatin in porcine satellite cells. Epigenetics 7:12
• Myostatin is a secreted TGF beta
family member that inhibits muscle
differentiation
• Animals lacking myostatin or animals
treated with substances that block the
activity of myostatin have significantly
larger muscles
• Beneficial in MDX mouse model

16. Sulforaphane alleviates muscular
dystrophy in mdx mice
Chengcao Sun , Cuili Yang , Ruilin Xue , Shujun Li , Ting Zhang , Lei
Pan , Xuejiao Ma , Liang Wang , Dejia Li (2015) Journal of
Applied Physiology. Vol. 118 no. 2, 224-237
• Examined the effects of Sulforaphane in mdx mice
– Weight of various tissues
– Grip strength and run endurance
– Plasma Creatine Phosphokinase and Lactate Dehydrogenase Levels
– Muscle integrity and histology
– Nrf2 genes
– Inflammation

17. Sulforaphane (SFN) increased muscle weight and improved myocardial hypertrophy in mdx
mice.
Chengcao Sun et al. J Appl Physiol 2015;118:224-237
©2015 by American Physiological Society
• C57BL/10ScSn-Dmdmdx/NJU
• 2 mg per kg body weight per day in DMSO/Corn oil by oral gavage
• Data not normalized to body weight

18. Increase of the forelimb grip and running distance in SFN-treated mdx mice after 8 wks of
treatment.
Chengcao Sun et al. J Appl Physiol 2015;118:224-237
©2015 by American Physiological Society
• Data not normalized to body weight

19. SFN alleviated the oxidative stress of mdx mice.
Chengcao Sun et al. J Appl Physiol 2015;118:224-237
©2015 by American Physiological Society
Assessing Muscle
Damage:

20. SFN activated (Nrf2)/(ARE) signalling pathway.
Chengcao Sun et al. J Appl Physiol 2015;118:224-237
©2015 by American Physiological Society
Assessing Muscle
oxidative stress:

21. Reduction in the levels of Evans blue staining in skeletal muscles from SFN-treated mdx
mice.
Chengcao Sun et al. J Appl Physiol 2015;118:224-237
©2015 by American Physiological Society
EBD -Marker of
muscle integrity

22. Reduction in central nucleation and fiber size variability in SFN-treated mdx muscle (TA)
fibers.
Chengcao Sun et al. J Appl Physiol 2015;118:224-237
©2015 by American Physiological Society

23. Chengcao Sun et al. J Appl Physiol 2015;118:224-237
©2015 by American Physiological Society
SFN ameliorates inflammation (CD45) in mdx muscle fibers
CD45 was used to
detect all lymphocytes

24. Potential negative aspects
• Activation of NRF2 may promote the development of de
novo cancerous tumors as well as the development of
atherosclerosis by raising plasma cholesterol levels and
cholesterol content in the liver
• Sulforaphane in the literature
– Mechanisms of action are based on cancer cell lines and often
not separated from activation of NRF2
– Studies are descriptive rather than mechanistic in the literature
– uM levels used however there is evidence this is obtainable in
vivo by oral administration.
• Sulforaphane may be a therapeutic option for DMD but it
may be difficult to commercialize
– Many Supplements available
– Use of a sulforaphane derivative may be a better choice but no
therapeutic data is available

25. Collaboration is possible
• Evgen Pharma
– Sulforadex® , a novel, synthetic and stabilized version
of the naturally occurring compound sulforaphane
– Pipeline: SFX-01: Prostate cancer and Multiple
sclerosis
– SFX-02 and SFX-03 in development
• Multiple labs are making sulforaphane derivatives
• Other Isothiocyanates may provide equal or
better results

26. Next Steps
• Develop a collaboration with Evgen Pharma or
a laboratory that produces a sulforaphane
derivative or possibly other Isothiocyanates
• Additional rigorous studies need to be done in
animal models and patient cell lines
• Toxicology and optimal dosing should be
investigated