Steroid Approaches: NF-kappaB, Delta 9-11 and other Potential Therapies for DMD

Steroid Approaches: NF-kappaB, Delta 9-11 and other Potential Therapies for DMD Kanneboyina Nagaraju, DVM, PhD Associate Director, Research Center for Genetic Medicine Children s National Medical Center Washington DC

Disclosure Member, Board of Directors, ReveraGen Biopharma (Formerly Validus Biopharma)

Muscle inflammation

Gene expression profiling of skeletal muscle from 3 distinct stages of cellular and clinical pathophysiology Fetus: 13-24 weeks of gestation DMD (n=2); Control (n=2) Infant: 8-10 months of age DMD (n=4); Control (n=4) Children: 5-10 years of age DMD (n=10); Control (n=5) Chen et al., 2005

NF-kB target genes are up-regulated early in disease in DMD Crash of energy production Persistence of regeneration NF-kB pathway Chen et al., 2005

NF-kB activation in DMD muscle Normal control DMD Chen et al., 2005

NF-κB controls several inflammatory genes in Skeletal Muscle

NF-kB inhibitors tested in mdx mouse model of dystrophy Cytokines Viruses Endogenous Immune Response Bacterial NBD Peptide Negative dominant IKKα and IKKβ via AAV Curcumin P65-shRNA via AAV Other NF-κB inhibitors L-arginine I-glutamine N-Acetylcysteine Genistein (Soy Isoflavone) Flavocoxid Green Tea Extract IRFI-042 Pyrollidine dithiocarbamate Ursodeoxycholic acid Proteosomal degradation

NF-kB inhibition in mdx mouse model Intervention Histology Function Comments Reference AAV-p65-shRNA NBD NBD No Immune response Heart/diaphragm Systemic delivery Yang et al., 2012 Delfin et al., 2011 Peterson et al., 2011 Reay et al., 2011 AAV-IKKa-dn or - IKKbdn No Immune response Tang et al., 2010 Pyrollidine dithiocarbamate UDCA Curcumin Curcumin Flavocoxid IRFI-042 Genistein EGCG L-Arginine L-Glutamine NAC No No No No No No Other effects Chemical chaperone Worsens function Anti-oxidant lipid peroxidation Anti-oxidant Anti-oxidant NO Anti-oxidant Anti-oxidant Carlson et al., 2005/Graham et al., 2010 Messina et al., 2006 Siegel et al., 2011;2008 Pan et al., 2007 Durham et al., 2006 Messina et al., 2009 Messina et al., 2006 Messina et al., 2006 Evans et al., 2010 Hnia et al., 2010 Mok et al., 2008 Whitehead et al., 2008

Glucocorticoid treatment for DMD 47 clinical studies- 6 double blind studies Beneficial effects Improve muscle strength and function Prolong walking for several years Improve breathing and heart function Lower the risk of scoliosis and enhance the quality of life Positive effects prominent if started early (daily dose) Side effects Excessive weight gain Cushingoid face (moon-faced) Bone fractures Behavioral changes How does GCs work, and can they be made to work better?

Long term (6M) effects of prednisone (1mg/kg) in mdx mice 5.5 5.0 *** Untreated Prednisone KGF/KG 4.5 4.0 * 3.5 3.5 0.0 0 50 100 180 Prednisone treatment (days)

Effect of prednisone on fibrosis and heart function in mdx mice UNTX PREDNISONE LV EF % 65 60 55 50 45 40 35 30 25 20 15 10 5 0 UNTX *** PRED LV SF % 35 30 25 20 15 10 5 0 UNTX *** PRED

General mechanisms of action of glucocorticoids Membrane stabilization Beneficial effects Metabolic side effects Rhen and Cidlowski 2005

The 11-beta hydroxy and 11-keto substituents are key to hormonal activity 11 11 11 11 11 9 9 The delta 9,11 double bond is the key to the enhanced safety profile of VBP Compounds

Side effects: GRE transcription

Beneficial effects via NF-κB inhibition Myoblasts 100 Prednisolone VBP2 VBP5 VBP6 90 VBP7 VBP15 % NF-kB Activity 80 70 60 50 40 30-10.0-9.5-9.0-8.5-8.0-7. 5-7.0-6.5-6.0-5.5-5.0 Log [Drug] M

Effect of VBP15 on membrane stabilization FM1-43 Fluorescence Intensity 600 500 400 300 200 100 DMSO VBP Prednisone FM1-43 Fluorescence Intensity 250 200 150 100 50 DMSO VBP Prednisone 0 0 Time elapsed (min) Time elapsed (sec)

MDX Mouse preclinical trial (4M) 1. Untreated 2. VBP15: 5mg/kg /day 15mg/kg/day 45 mg/kg/day 3. Prednisolone: 5 mg/kg/day

Effect of VBP-15 on muscle inflammation in mdx mice

ffect of VBP-15 on behavioral activity in mdx mice

Effect of VBP-15 on in vitro force contractions and serum CK levels in mdx mice

VBP 15 profile No GRE inducbon. Doesn t acbvate glucocorbcoid response element containing promoters. No GR binding. No downregulabon of glucocorbcoid receptor in MDX and wild type Inhibits NF- kb signaling in myotubes and myoblasts In myoblasts VBP15= 22nM, prednisolone = 99 nm In myotubes VBP15 = 28nM, prednisolone = 140 nm Membrane stabilizer as measured in C2C12 myoblast laser injury model superior to pred 4 month MDX trial: 5, 15, and 45 mg/kg/day vs 5 m/kg/day prednisolone. 15 mg/kg dose best % change in specific force (in vitro force contracbon). 15% improvement with VBP15 at 15mg/kg and 7% with pred. Similar results with another analog 22% reducbon in cathepsin B acbvity vs 27% pred (anb- inflammatory) No change in body wt, spleen weight and muscle weight, significant losses in all three with pred

THERAPEUTICS FOR RARE & NEGLECTED DISEASES (TRND) http://trnd.nih.gov/

Acknowledgements Raju Lab Andrea Pons, PhD Arpana Sali, MS Jack VanderMeulen, PhD Vanessa Jhanke, PhD Heather Alger, PhD Qing Yu, PhD Will Coley, BS Beryl Ampong, PhD Sree Rayavarapu, DVM Sarah Jordan, BS Aditi phadke, MS Andreas Baudy, PhD Chris Spurney MD ReveraGen Biopharma John McCall, PhD Erica Reeves, PhD Ed Connor, MD Beth Burnside, PhD Jesse Damsker, PhD Blythe Dillingham, BS CNMC Eric Hoffman, PhD Jyoti Jaiswal, PhD Terry Partridge, PhD Department of Defense Foundation to Eradicate Duchenne Muscular Dystrophy Association-VP Cure Duchenne National Institutes of Health