Preclinical Drug Development: Opportunities &
Challenges
Slide 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
Slide 3
ALS Genetics
Slide 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
Slide 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
Slide 6
Is There a Problem With Preclinical Drug Development?
Slide 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
Slide 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
Slide 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
Slide 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
Slide 11
ALS TDI Celecoxib Data: No Change in Onset or Survival
Slide 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
Slide 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.
Slide 14
ALS TDI Data Minocycline: Exacerbates Disease
Slide 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
Slide 16
ALS TDI Data Ceftriaxone: No Change in Onset or Survival
Slide 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
Slide 18
ALS TDI Data Lithium: No Change in Onset or Survival Disease
Onset Survival 40 mg/kg/day
Slide 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?
Slide 20
Variables Contributing to Noise in the Model Variable #1:
Censoring Variable #2: Low Copy Animals Variable #3: Gender
Variable #4: Litter
Slide 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
Slide 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
Slide 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
Slide 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:
dj 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
Slide 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
Slide 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
Slide 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
Slide 28
How Do You Leverage a Validated Preclinical Model?
Slide 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
Slide 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
Slide 31
Activation of Costimulatory Pathway 0 1 d50d60d80d90d100d110
Additional therapeutics targeting aspects of the costimulatory
pathway Anti CD40L
Slide 32
In Vivo Experiments: Blocking Ab to CD40Lg Pharmacokinetic (Pk)
Analysis in mSOD1 mice Determine 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
Slide 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
Slide 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
Slide 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
Slide 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
Slide 37
ALS TDI Drug Studies ALS TDI has run more drugs in SOD1 mice
than the entire academic community combined % Survival %
progression Published % Survival
Slide 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
Slide 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