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Preclinical Drug Development:
Opportunities & Challenges
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
ALS Genetics
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
Its understanding
the molecular mechanismsOxidative Stress
Astrocytosis
Protein Aggregation
ER Stress
De Myelination
Axon Transport
Synaptic
Die Back
RNA Processing
Mitochondrial
Dysfunction
Microglial Activation
Muscle Wasting
Micro hemorrhaging
Leukocyte infiltration
Is There a Problem With
Preclinical Drug Development?
Is hSOD1G93A a Good Preclinical Model
for Drug Development ?
• 2 lines originally characterized
• High and low copy number hSOD1G93A
• Lines deposited in JAX
• TDI purchased the line and established its own colony in 2001
Gurney., Neurol. Science 1997
Kinetics of Disease in ALS and the SOD1 Model
1.0
BL6
SJL
0.9
0.8
0.750
0.7
Surviving
Percent patients surviving
1.000
0.500
0.6
0.5
0.4
0.3
0.250
0.2
0.1
0.000
0.0
30.0
60.0
90.0
120.0
Survival (months)
2108 Patients
Mean age of onset 55.7 (Range 21-85)
1st symptom to vent 30.6 mo +/- 20 mo
hSOD1 Transgenics
G93A: mean survival from 60 days to 164 days
G37R: 375 days
G85R: 243 days
D90A: ~520 days
0.0
70
80
90 100 110 120 130 140 150 160 170 180
DAge
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
Cox-2 Inhibitors Extend Survival in
a Transgenic Model of ALS
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
Drachman et al., Ann Neurol 2002
2 Month PGE2 CSF
Celebrex Clinical Trial In ALS
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
Cudkowicz et al., Ann. Neurol. 2006
Baseline PGE2 CSF
ALS TDI Celecoxib Data:
No Change in Onset or Survival
Celecoxib 300 mg/kg/d in Chow
Survival
100
100
CTRL (88)
DRUG (64)
CTRL (88)
DRUG (64)
75
75
50
50
25
25
0
95
105
115
125
135
145
Onset Age (days)
Onset Age
K-M Product-Limit Survival Fit
Median Time (days)
Prob>ChiSq
CTRL
DRUG Log-Rank Wilcoxon
116
117
0.9707
0.7506
Cox Proportional Hazards
Risk Ratio
Prob>ChiSq
CTRL
DRUG
0.99
1.01
0.9721
105 115 125 135 145 155
Death Age (days)
Death Age
K-M Product-Limit Survival Fit
Median Time (days)
Prob>ChiSq
CTRL
DRUG Log-Rank Wilcoxon
130
130
0.8859
0.9599
Cox Proportional Hazards
Risk Ratio
Prob>ChiSq
CTRL
DRUG
1.02
0.98
0.8897
0
Percent Surviving
Percent with Disease Onset
Disease Onset
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
Slope Change ALSFRS Score
Minocycline Enhances Disease Progression
and Mortality in Phase III Trial
Minocycline
Placebo
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.”
* Gordon et al., Lancet 2007
ALS TDI Data Minocycline:
Exacerbates Disease
Minocycline 200 mg/kg/d in Chow
Survival
Disease Onset
100
100
75
CTRL (24)
DRUG (23)
75
50
50
25
25
CTRL (24)
DRUG (23)
0
90
100
110
120
130
140
Onset Age (days)
Onset Age
K-M Product-Limit Survival Fit
Median Time (days)
Prob>ChiSq
CTRL
DRUG Log-Rank Wilcoxon
125
118
0.0054
0.0332
Cox Proportional Hazards
Risk Ratio
Prob>ChiSq
CTRL
DRUG
2.32
0.43
0.0091
110
120
130
140
0
150
Death Age (days)
Death Age
K-M Product-Limit Survival Fit
Median Time (days)
Prob>ChiSq
CTRL
DRUG Log-Rank Wilcoxon
136
131
0.0325
0.0548
Cox Proportional Hazards
Risk Ratio
Prob>ChiSq
CTRL
DRUG
1.83
0.55
0.0502
Percent Surviving
Percent with Disease Onset
SJL Background
Ceftriaxone Slows Progression and
Improves Survival in hSOD1G93A Mice
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
Rothstein et al., Nature 2005
ALS TDI Data Ceftriaxone:
No Change in Onset or Survival
Ceftriaxone 200 mg/kg/d i.p.
Survival
100
100
CTRL (38)
DRUG (38)
CTRL (38)
DRUG (38)
75
75
50
50
25
25
0
80
90
100
110
120
Onset Age (days)
Onset Age
K-M Product-Limit Survival Fit
Median Time (days)
Prob>ChiSq
CTRL
DRUG Log-Rank Wilcoxon
101
102
0.2467
0.3848
Cox Proportional Hazards
Risk Ratio
Prob>ChiSq
CTRL
DRUG
1.30
0.77
0.2752
105
115
125
135
145
Death Age (days)
Death Age
K-M Product-Limit Survival Fit
Median Time (days)
Prob>ChiSq
CTRL
DRUG Log-Rank Wilcoxon
126
126
0.9697
0.8640
Cox Proportional Hazards
Risk Ratio
Prob>ChiSq
CTRL
DRUG
0.99
1.01
0.9711
0
Percent Surviving
Percent with Disease Onset
Disease Onset
Lithium Delays Disease Onset and Improves
Survival in the G93A Model of ALS
Control
Summary Pre Clinical Data
• Increased lifespan by 36%
• delayed progression by 300%
• Improved Mn survival
• Decreased astrocytosis
• Decreased protein aggregation
• Increases autophagic vacuoles
Fornai et al., PNAS 2008
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
ALS TDI Data Lithium:
No Change in Onset or Survival
Disease Onset
40 mg/kg/day
Survival
Product-Limit Survival Fit Age at Death
Survival Plot Time to event:
DAge
1.0
Average Neurological Score (R+L, M+F)
CTRL
DRUG
0.9
0.000
0.8
0.500
0.7
1.500
CTRL
2.000
DRUG
Surviving
NSAve (0-4)
1.000
0.6
0.5
0.4
2.500
0.3
3.000
0.2
3.500
0.1
0.0
4.000
50
60
70
80
90
100
110
120
130
140
110
150
120
Age (days)
K-M Product-Limit Survival Fit
Sex
Both
Female
Male
Treatment
Median Time
CTRL
DRUG
CTRL
DRUG
CTRL
DRUG
2Age
117
114
121
114
113
115
130
140
150
DAge
Test
Log-Rank Wilcoxon
Prob>ChiSq
0.6345
0.5785
0.1876
0.1814
0.7586
0.6293
K-M Product-Limit Survival Fit
Sex
Both
Female
Male
Treatment
Median Time
CTRL
DRUG
CTRL
DRUG
CTRL
DRUG
DAge
127
124
129
124
126
124
Test
Log-Rank Wilcoxon
Prob>ChiSq
0.9304
0.9397
0.6587
0.7357
0.7726
0.7483
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?
Variables Contributing to Noise in the Model
Variable #1: Censoring
Variable #2: Low Copy
Animals
Variable #3: Gender
Variable #4: Litter
Controlling Noise Variables and
Optimization of Study Design
Optimized Study Design
Effect of increasing 'n' on Noise for Each Study Design Variable
•
•
Noise: Frequency of Apparent Effects
60.00%
•
•
50.00%
40.00%
•
30.00%
•
20.00%
•
10.00%
0.00%
n=4
N=10
N=16
N=20
n=24
n=30
n=40
n=50
•
-10.00%
Number Per Group
UU+Censored & Low Copy
UU+Censored
UU
MB
•
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
Historical Issue with Pre-clinical
Animal Model Development
Celebrex Slows
Disease & Improves
Survival in SOD1
Mouse Model, 2002
Minocycline Slows
Disease & Improves
Survival in SOD1
Mouse Model, 2004
First Mutation
Associated with
ALS Identified:
SOD1 Gene, 1993
1993
Lithium Slows
Disease & Improves
Survival in SOD1
Mouse Model, 2008
1995
1997
1999
2001
Transgenic mouse
model expressing
human mutant
SOD1, 1997
2003
2005
2007
Celebrex ALS
Clinical Trial Fails
in Phase III, 2006
Optimized
Experimental Design
for Preclinical Drug
Screening in the ALS
Mouse Model, 2007
2009
To make the Model
10 Years
To Validate the Model
2013
Lithium ALS Clinical
Trial Fails in Phase
III, 2010
Minocycline ALS
Clinical Trial Fails
in Phase III, 2007
4 Years
2011
Flaws in Preclinical Execution Lead to
Failures in Clinical Development
• 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
Perrin, Nature 2014
TDP43 Mutations in ALS
TDP43 Mutations in ALS: déjà vu?
Mutations in
theTDP43 Gene in
ALS Patients, 2006
Transgenic mouse
model expressing
human mutant
TDP43, 2010
First Mutation
Associated with
ALS Identified:
SOD1 Gene, 1993
1993
1997
Transgenic mouse
model expressing
human mutant
SOD1, 1997
2001
2005
2009
4 Years
To make the Model
2013
2017
2021
10 Years
To Validate the Model
Does it have to take 10 Years to
Validate the Model ?
Generation of a Validated Preclinical
Model of TDP43 Transgenic Mouse
Challenges of Original
Mouse Models of
TDP43
A
• 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
B
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 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
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
How Do You Leverage a
Validated Preclinical Model?
Molecular Profiling Neurodegenerative Models
Animals re genotyped
for copy number and
assigned to a study
d30
Animals
Genotyped
d45
d50
Treatment
Initiation
Disease onset
Tail Paralysis
d70-80
End stage disease
d80-120
D120-150
Disease progression
Paralysis hind limbs to
forelimbs
• 5 non transgenics, 5 wt animals
• SOD1G93A
• Loa (Dynein heavy chain mutant)
• prpTDP43A315T (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
Activation of Co-Stimulatory Pathway
Spinal Cord
Muscle
• 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
Drugable pathway present in 3 diseased tissues
in the SOD1 pre-clinical model
Sciatic Nerve
-1
0
1
d50 d60 d80 d90 d100 d110
Activation of Costimulatory Pathway
Anti CD40L
• Additional therapeutics targeting aspects of the costimulatory pathway
-1
0
1
d50 d60 d80 d90 d100 d110
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
aCD40L Ab is Efficacious in SOD1G93A 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
Proportion at Peak
1.00
A
0.75
0.50
0.25
0.00
CTRL
DRUG
30
40
50
60
pVal= 0.286
Proportion Onset or Survival
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
B
70
80
15
Time (days)
25
35
45
55
65
pVal= 0.046
1.00
C
D
0.75
0.50
0.25
0.00
95
105 115 125 135 145
pVal= 0.001
110
Age (days)
120
130
140
150
pVal= 0.003
Lincecum, 2010
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
aCD40L Treatment Reduces Axonal Recruitment
of Macrophages CD68+ Cell Counts
No treatment:
CD68+ Cell Counts
S100b+ Distal Nerve (5 fields/count)
By Treatment
- Tx
+ Tx Cnt
All age matched females, 18 ug/week, I.p.
50 day start, sacrificed at 103 days
Biological replicates; double blind analysis
00846 treatment
Anti CD40L Treatment Decreases Astrocytosis and
Improves Motor Neuron Survival
Control
Control
Anti-CD40L
Anti-CD40L
• GFAP, Dapi
• 100 day Lumbar Spinal Cord
• 53 days treatment 1mg/kg
• Nissel Staining
• 100 day lumbar spinal cord
• 53 days treatment 1mg/kg
ALS TDI Drug Studies
ALS TDI has run more drugs in SOD1 mice than the entire academic community combined
% d Survival
% d progression
Published % Survival
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
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