Debio-025 as a candidate to reduce muscular dystrophy pathology

Doug Millay Lab of Jeff Molkentin Cincinnati Children’s Hospital Medical Center Muscular Dystrophy Pathology and Treatment

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Dystrophin (or other DGC) Wt MD Mutation Hallmarks of MD Pathology -Influx of Inflammatory Cells (white arrow) -Central Nuclei (regeneration, black arrow) -Fiber Size Variability (arrowhead) Therapy -Fibrosis

? Degeneration

Inflammation Pharmacologic Agents Regeneration Stem Cell Therapy The Dystrophin-Glycoprotein Complex (DGC)

Lama2-/- laminin Severe phenotype Typically die within 2 months of age Scgd-/- Resembles human disease closely Reduced survival

αDG sarcoglycan complex extracellular β DG

CAV 3 TRPCs syn intracellular NOS db dystrophin actin

Function: Connects the cytoskeleton to the extracellular matrix, stabilizing the mdx sarcolemma The DGC, Ca2+, and Degeneration

2+ Ca Ca2+ Ca2+ 2+ Ca Ca2+

Contraction DGCX DGCX Ca2+ Entry via Sarcolemmal Lesions What are the pathways by which And/or Channel Activity elevated intracellular Ca2+ causes damage?

Ca2+ Membrane Repair Proteins

Apoptotic Calpain ROS Mitochondria Signaling

Necrosis Fiber Death Mitochondrial Calcium Overload

Adapted from Dr. Christopher Baines Muscular Dystrophy Mice Have Swollen Mitochondria

Wt Absorbance, Swelling 0.6 Scgd-/- 0.5 0.4 * 0.3 0.2

Absorbance 0.1 6 6 0 Wt Scgd-/-

Millay DP et al. Nat. Med. (2008) The Mitochondrial Pore

¾ D (gene=Ppif) is a critical component of the mitochondrial pore.

¾ Ca2+ and oxidative damage can lead to pathological opening and cell death, HOWEVER Ppif-/- mitochondria are resistant.

OMM

IMM

CypD Ca2+ ROS Loss of cyclophilin D Rescues Muscle Weights

Wt Scgd-/- Scgd-/- Ppif-/- Ppif-/-

8 6 wks * 12 8 Mo. # # 10 6 * 8 4 # 6 # 4 * 2 2 * MW/TL (mg/mm) MW/TL

6 7 6 3 6 7 6 3 (mg/mm) MW/TL 0 0 9 10 10 3 9 10 10 3 Gastroc. Quad. Gastroc. Quad.

Millay DP et al. Nat. Med. (2008) Genetic Ablation of cyclophilin D Reduces Pathology and 25 #

Increases Function in Scgd-/- Muscle ) 2 20 * 15 (N/cm -/- 6 6 6 6 Wt Scgd Scgd-/- Ppif-/- Specific force 10 Wt Scgd-/- Scgd-/- Diaph. Ppif-/- Ppif-/- Gastroc. Quadriceps

Millay DP et al. Nat. Med. (2008) Mechanism of Protection

Wt Scgd-/- Scgd-/- Ppif-/-

5,000X

10,000X

Millay DP et al. Nat. Med. (2008) Mechanism of Protection

Wt Scgd-/- Scgd-/- Ppif-/-

30,000X

Millay DP et al. Nat. Med. (2008) Analysis of Cardiac Defects

Wt

40 # 30 * Scgd-/- 20 FS (%)

10 11 7 11 0

Wt Scgd-/- Ppif-/- Scgd-/- Scgd-/- Ppif-/-

Millay DP et al. Nat. Med. (2008) Increased Lifespan and Body Weight in Lama2-/- Mice Lacking cyclophilin D Wt Lama2-/- Lama2-/- Ppif-/-

4

3 # 2 # * # 1 * * MW/TL (mg/mm) MW/TL 10 7 6 10 7 6 10 7 6 0

Millay DP et al. Nat. Med. (2008) movie Ca2+ Ca2+ 2+ 2+ 2+ Ca2+ Ca Ca Ca Ca2+ DGC Calcium-Induced X Ca2+ Wt -/- Degeneration through the Mitochondrial Pore

Mitochondrial Pore

CypD

Inhibitors of cyclophilin D (CyclosporineA) (Debio-025) Cyclophilin D Inhibitors

¾ Cyclosporine A ¾ Inhibitor of mitochondrial pore, but also calcineurin (regeneration). Is also an immuno-suppressant. ¾ Effective in reducing mitochondrial abnormalities in patients lacking collagen VI (Merlini L et. al. Proc Natl Acad Sci U S A. 2008 Apr 1;105(13):5225-9)

¾ Debio-025 (Db25) ¾ Cyclosporine analog and cyclophilin inhibitor (10X more potent than cyclosporine A). No effect on calcineurin.

¾Reduced response to in cells from patients with collagen VI deficiency (Angelin A et. al. Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):991-6)

¾ Already proved to be safe in humans for treatment of Hepatitis C virus (Currently in Stage II Clinical Trials). Debio-025 Reduces Disease Progression in mdx Mice

12 0.10 * 10 0.08 Wt-Veh * # Wt-Db25 8 0.06 # mdx-Veh 6 # 0.04 mdx-Db25 *

absorbance 4 0.02 # Δ 7 7 9 9 (mg/mm) MW/TL 2 * 0 7 7 7 9 9 PEG 0 7 7 9 9 7 9 9 Gastroc. Quad. TA

Millay DP et al. Nat. Med. (2008) Debio-025 Reduces Disease Progression in mdx Mice vehicle Debio-025

Wt Wt

mdx mdx

Millay DP et al. Nat. Med. (2008) Debio-025 Reduces Disease Progression in mdx Mice

Wt-Veh Wt-Db25

mdx-Veh mdx-Db25 Millay DP et al. Nat. Med. (2008) Conclusions

¾ Inhibition of cyclophilin D may serve as a novel approach for treating MDs.

¾ Debio-025 may be a drug that can be used in humans to reduce MD disease progression and prolong life. Future Directions (Transition of Debio-025 into Clinic)

Gene Replacement (Cure) Gene Therapy Stem Cell Therapy

RNA Manipulations (Subset of Patients)

Pharmacologic Agents

Combinatorial Therapy Future Directions (Transition of Debio-025 into Clinic)

Dystrophin Deficient Golden Retriever October 2008—Puppies Born

Mid-November 2008—Analysis of CK levels and quadriceps biopsy

Late November 2008—Separation of affected puppies into control and treatment groups based on disease severity

December 2008—Begin treatment at 2 months of age

Collaborators: Monthly Tests: -Blood Draws (CK levels, toxicity) -Muscle Biopsy (assessment of pathology) - Research Center of the University of São Paulo, -Video Recording (functional analysis) São Paulo Brazil

Echocardiography at 6 and 10 months of age -DEBIOPHARM GROUP (Debio-025) (cardiac function)

-Parent Project Muscular Dystrophy October 2009—End treatment at 10 months of age (8 months of Debio-025 treatment) Jeff Molkentin Scott Blair Acknowledgements Jop VanBerlo John Elrod Joerg Heineke Chris Baines Jennifer Kwong Allen York Xu Wu Majorie Maillet Hiro Nakayama Mannix Auger-Messier Matt Benard Michelle Sargent Sanjeewa Goonasekera Jeff Lynch Qinghang Liu Izhak Kehat Xu Wu Robbins Lab (EM) Erin Wissing Functional Analysis Jeffrey Robbins H. Lee Sweeney- UPenn Angela Lorts Hanna Osinska Elisabeth Barton- UPenn Jennifer Schwanekamp Jason Karch Funding NIH Jain Foundation PPMD