1. Preclinical Drug Development: Opportunities & Challenges

2. Amyotrophic Lateral Sclerosis/ Motor Neuron Disease (ALS/MND) Degeneration of both upper and lower motor neurons Median Survival: 3-5 yrs Average Age onset: 50 10% cases genetic Genes for 70% of cases known 90% cases sporadic Clinical presentation identical

3. ALS Genetics

4. Genetics in the Context of Drug Development The identification of a genetic mutation does NOT always lead to an effective treatment for patients SOD1 (ALS) in 1993: No effective treatment CF (Cystic Fibrosis) in 1989: First treatments 2011 No directly linked genetic mutations: Multiple Sclerosis: 12 FDA approved drugs

5. Astrocytosis Microglial Activation Synaptic Die Back De Myelination Micro hemorrhaging Leukocyte infiltration Muscle Wasting Oxidative Stress Axon Transport Mitochondrial Dysfunction ER Stress Protein Aggregation RNA Processing Its understanding the molecular mechanisms

6. Is There a Problem With Preclinical Drug Development?

7. Is hSOD1 G93A a Good Preclinical Model for Drug Development ? Gurney., Neurol. Science 1997 2 lines originally characterized High and low copy number hSOD1 G93A Lines deposited in JAX TDI purchased the line and established its own colony in 2001

8. Kinetics of Disease in ALS and the SOD1 Model Percent patients surviving 0.250 0.500 0.750 1.000 0.000 0.030.060.090.0120.0 Survival (months) 2108 Patients Mean age of onset 55.7 (Range 21-85) 1 st symptom to vent 30.6 mo +/- 20 mo ALS-TDI Colonies SJL/BL6J n = 499 mean survival = 131 days BL6J n =46 mean survival = 164 days JAX Colony (Greg Cox; PC) SJL/BL6J n = 67, mean survival = 129 days BL6J n =140, mean survival = 161 days hSOD1 Transgenics G93A: mean survival from 60 days to 164 days G37R: 375 days G85R: 243 days D90A: ~520 days

9. Cox-2 Inhibitors Extend Survival in a Transgenic Model of ALS Drachman et al., Ann Neurol 2002 Conclusions Celebrex inhibited prostaglandin E2 Celebrex delayed disease onset Celebrex increased lifespan by 25% Observations to keep in mind Number of animals per group is 9 Limited info on study design gender and littermate

10. Celebrex Clinical Trial In ALS Cudkowicz et al., Ann. Neurol. 2006 Baseline PGE2 CSF 2 Month PGE2 CSF Summary 99 Placebo patients 201 Celebrex 800 mg/daily 12 months No impact on ALSFRS, FVC, or motor function No impact on prostaglandin levels in CSF Safe and well tolerated

11. ALS TDI Celecoxib Data: No Change in Onset or Survival

12. Minocyline Delays Onset and Extends Survival in hSOD1G93A Mice Conclusions Minocycline delays disease onset Minocycline extends survival Minocycline inhibits cytochrome c release from mitochondria Observations to keep in mind Number of animals per group is 10 Limited info on study design gender and littermate * Zhu et al., Nature 2002

13. Minocycline Enhances Disease Progression and Mortality in Phase III Trial * Gordon et al., Lancet 2007 Slope Change ALSFRS Score MinocyclinePlacebo Summary ALSFRS Score Decreased 9.4 units in placebo Decreased 11.7 units in Minocycline Failure endpoint was higher in Minocycline Group 32 in placebo 41 in Minocycline “ Four published reports have shown that minocycline delays disease progression in the ALS transgenic mouse.” “Our results suggest either that the current approach to translational neuroscience is unsatisfactory or the transgenic mouse model is a poor representation of ALS.”

14. ALS TDI Data Minocycline: Exacerbates Disease

15. Ceftriaxone Slows Progression and Improves Survival in hSOD1 G93A Mice Rothstein et al., Nature 2005 Conclusions Ceftriaxone decreases motor neuron loss and muscle weakness Ceftriaxone increases lifespan Ceftriaxone increased GLT1 expression in spinal cord Observations to keep in mind Median Survival of controls Limited info on study design gender and littermate

16. ALS TDI Data Ceftriaxone: No Change in Onset or Survival

17. Lithium Delays Disease Onset and Improves Survival in the G93A Model of ALS Fornai et al., PNAS 2008 Control Lithium Summary Clinical Study 16 patients on Rilutek plus Lithium 28 Rilutek alone Treatment improved respiratory function Treatment improved motor function Treatment impacted survival Many caveats to the study design Summary Pre Clinical Data Increased lifespan by 36% delayed progression by 300% Improved Mn survival Decreased astrocytosis Decreased protein aggregation Increases autophagic vacuoles

18. ALS TDI Data Lithium: No Change in Onset or Survival Disease Onset Survival 40 mg/kg/day

19. Questions: Are there unrecognized variables that need to be controlled in the study design? How do we quantify the contribution of each variable? The search for a positive control: What drugs actually work in this animal model?

20. Variables Contributing to Noise in the Model Variable #1: Censoring Variable #2: Low Copy Animals Variable #3: Gender Variable #4: Litter

21. Controlling Noise Variables and Optimization of Study Design Optimized Study Design 48 total mice. Tx group 12m+12f, control group 12m+12f. Same gender litter matching Observers should be blind to treatment Single, uniform endpoint criterion Confirmation of transgene copy number prior to study enrollment Tracking and censoring from final analysis all non-ALS deaths Statistical analysis, a log rank method is typically necessary for any survival analysis; since we have shown the SOD1G93A model has multiple variables, the Cox Proportional Hazards model is most appropriate We recommend no multi-arm studies

22. Historical Issue with Pre-clinical Animal Model Development Optimized Experimental Design for Preclinical Drug Screening in the ALS Mouse Model, 2007 10 Years To Validate the Model 19931995199719992001200320052007200920112013 First Mutation Associated with ALS Identified: SOD1 Gene, 1993 Transgenic mouse model expressing human mutant SOD1, 1997 4 Years To make the Model Celebrex Slows Disease & Improves Survival in SOD1 Mouse Model, 2002 Minocycline Slows Disease & Improves Survival in SOD1 Mouse Model, 2004 Lithium Slows Disease & Improves Survival in SOD1 Mouse Model, 2008 Celebrex ALS Clinical Trial Fails in Phase III, 2006 Minocycline ALS Clinical Trial Fails in Phase III, 2007 Lithium ALS Clinical Trial Fails in Phase III, 2010

23. Flaws in Preclinical Execution Lead to Failures in Clinical Development Perrin, Nature 2014 ALS TDI has failed to replicate more than a dozen published studies ALS TDI published optimized experimental design for preclinical testing (Scott, 2008) These failed studies wasted hundreds of millions of dollars in clinical trial efforts Most importantly it squandered patient opportunity

24. TDP43 Mutations in ALS 19931997200120052009201320172021 Mutations in theTDP43 Gene in ALS Patients, 2006 Transgenic mouse model expressing human mutant TDP43, 2010 TDP43 Mutations in ALS: déjà vu? 4 Years Does it have to take 10 Years to Validate the Model ? To make the Model First Mutation Associated with ALS Identified: SOD1 Gene, 1993 Transgenic mouse model expressing human mutant SOD1, 1997 10 Years To Validate the Model

25. Original Data: Survival data for publically available data of prpTDP43 colonies: (A)TDP43 colony at Washington University, St. Louis, MO (B)TDP43 colony maintained for distribution at the Jackson Laboratories, Bar Harbor, ME Generation of a Validated Preclinical Model of TDP43 Transgenic Mouse Challenges of Original Mouse Models of TDP43 The kinetics of survival for the colony are too broad for drug screening It would require 400 animals per group to detect a 5% drug effect on survival with 95% confidence A B

26. Generation of a new TDP43 Colony at ALS TDI by Congenic Back Crossing Initial Results fro TDI: Kinetics of disease progression within a gender are now very tight. A power analysis of 1000 animals optimized a study design for PD outcomes with power to detect 5% changes

27. Characterizing the Phenotype is Critical prpTDP43 animals have minimal nmj loss They do not die from progressive neurodegeneration prpTDP43 animal succumb from an acute bowel obstruction

28. How Do You Leverage a Validated Preclinical Model?

29. Molecular Profiling Neurodegenerative Models Animals Genotyped Animals Genotyped Animals re genotyped for copy number and assigned to a study Animals re genotyped for copy number and assigned to a study d30d45d50 Treatment Initiation Treatment Initiation d70-80 Disease onset Tail Paralysis Disease onset Tail Paralysis d80-120 Disease progression Paralysis hind limbs to forelimbs Disease progression Paralysis hind limbs to forelimbs D120-150 End stage disease 5 non transgenics, 5 wt animals SOD1 G93A Loa (Dynein heavy chain mutant) prpTDP43 A315T (2012) Groups harvested at 10 day intervals starting at day 30 Tissues extracted and flash frozen on dry ice Brain, spinal cord, skeletal muscle, brown fat, white fat, sciatic nerve, blood Laser captured motor neurons and surrounding tissue, NMJs Profiled on Affymetrix MOE430vII gene chips and Affymetrix Ex1.0 exon arrays Luminex protein microarrays 5 non transgenics, 5 wt animals SOD1 G93A Loa (Dynein heavy chain mutant) prpTDP43 A315T (2012) Groups harvested at 10 day intervals starting at day 30 Tissues extracted and flash frozen on dry ice Brain, spinal cord, skeletal muscle, brown fat, white fat, sciatic nerve, blood Laser captured motor neurons and surrounding tissue, NMJs Profiled on Affymetrix MOE430vII gene chips and Affymetrix Ex1.0 exon arrays Luminex protein microarrays

30. Activation of Co-Stimulatory Pathway 0 1 d50d60d80d90d100d110 Longitudinal gene expression changes from the SOD1 mouse model Co-Stimulatory pathway is an immune modulatory pathway Activated in spinal cord, skeletal muscle, sciatic nerve Spinal CordMuscleSciatic Nerve Drugable pathway present in 3 diseased tissues in the SOD1 pre-clinical model

31. Activation of Costimulatory Pathway 0 1 d50d60d80d90d100d110 Additional therapeutics targeting aspects of the costimulatory pathway Anti CD40L

32. In Vivo Experiments: Blocking Ab to CD40Lg Pharmacokinetic (Pk) Analysis in mSOD1 mice Determine ½ life of the drug in mice Determine biodistribution, tolerability Dose Ranging Efficacy Studies A1) Female 1 mg/kg A2) Male 1.34 mg/kg B1) Female 2 mg/kg B2) Male 2.67 mg/kg C1) Female 4 mg/kg C2) Male 5.35 mg/kg Biomarker Drug Response Dose dependent marker Amenable to clinical development Shorten and facilitate phase II trial Pk analysis

33.  CD40L Ab is Efficacious in SOD1 G93A Mice Females: 5.22 mg/kg loading dose 1 mg/kg weekly IP Males: 6.75 mg/kg loading dose 1.34 mg/kg weekly IP Day 50 start A. Time required to attain peak body weight. Time to peak was not significantly changed B. Time from peak body weight until death. BW maintenance was significantly improved C. Time to disease onset (Ns =2). Disease onset was significantly delayed by D. Survival was significantly prolonged Females: 5.22 mg/kg loading dose 1 mg/kg weekly IP Males: 6.75 mg/kg loading dose 1.34 mg/kg weekly IP Day 50 start A. Time required to attain peak body weight. Time to peak was not significantly changed B. Time from peak body weight until death. BW maintenance was significantly improved C. Time to disease onset (Ns =2). Disease onset was significantly delayed by D. Survival was significantly prolonged pVal= 0.286pVal= 0.046 pVal= 0.001 pVal= 0.003 Lincecum, 2010

34. Meta Analysis of Anti CD40L Treatment 30 female MR1 treated mice 30 litter matched controls 500 historical female controls Median Survival Female control: 127 days MR1 treated 139 days pValue: 0.0002 SIM LIMS historical female data Monte Carlo analysis 18 non treated females Random assign treatment/control Frequency of detecting a false positive

35. CD68+ Cell Counts By Treatment CD68+ Cell Counts S100b+ Distal Nerve (5 fields/count) All age matched females, 18 ug/week, I.p. 50 day start, sacrificed at 103 days Biological replicates; double blind analysis All age matched females, 18 ug/week, I.p. 50 day start, sacrificed at 103 days Biological replicates; double blind analysis No treatment: 00846 treatment - Tx + Tx Cnt  CD40L Treatment Reduces Axonal Recruitment of Macrophages

36. GFAP, Dapi 100 day Lumbar Spinal Cord 53 days treatment 1mg/kg Anti-CD40L Control Anti CD40L Treatment Decreases Astrocytosis and Improves Motor Neuron Survival Nissel Staining 100 day lumbar spinal cord 53 days treatment 1mg/kg Anti-CD40L Control

37. ALS TDI Drug Studies ALS TDI has run more drugs in SOD1 mice than the entire academic community combined %  Survival %  progression Published % Survival

38. Summary Characterize the phenotype well Some aspects of the disease will be represented other pathophysiologies may not be part of the model Perform a robust power analysis on kinetics of the phenotype of interest to optimize experimental design Remember its just a model of the disease

39. Acknowldgements Sean Scott James Heywood Al Gill Bashar Al Nakhala John Lincecum Monica Wang Ricky Sanchez Isa Carrion Fernando Vieira Januce Kranz Jeff Cole Jeyanthi Ramasubbu Alan Bostrom Ken Thompson Theo Hatzipetros Carlos Maya Andy Moreno Matt Ferola Josh Kidd