Transcript Epothilone D: History and Future Directions in AD

CNDR AD Drug DiscoveryFrom Plaques to Tangles
Kurt R. Brunden, PhD
Director of Drug Discovery
Research Professor
Center for Neurodegenerative Disease Research
University of Pennsylvania
Alzheimer’s Disease
Neuropathology

Amyloid Plaques
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Neurofibrillary tangles
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Extracellular
Compact and diffuse
Comprised of Aβ peptides
Intraneuronal
Comprised of tau protein
Neuron loss
Reactive glial cells
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
Astrocytes
Microglia
Forman et al. 2004
Microglial Activation in AD
Release of Inflammatory Molecules
-Activated microglia are found in the vicinity of senile plaques.
-A number of inflammatory (eicosanoids, cytokines, chemokines)
and reactive (NO, H2O2) molecules are released from activated microglia
that may contribute to AD neuropathology.
IHC from Barton (2006) Nature Rev. Neurosci. 7:254 and schematic from Block (2008) BMC Neuroscience 9(Suppl 2):S8
Thromboxane-Prostanoid (TP) Receptor
Receptor Activation Increases APP & Aβ Expression
Shineman et al. (2008) J. Neurosci. 28:4785-4794.
TP Receptor
Might Other Receptors Affect APP Expression?
 The TP receptor is known to signal
via multiple G-proteins
(predominantly Gαq and Gα12/13).
 Our studies revealed that TP-
mediated APP regulation was via
Gαq.
 Might other Gαq-linked eicosanoid
receptors regulate APP and Aβ levels?
Herbst-Robinson et al. (2015) Scientific Rep. 5:18286
Eicosanoid Biosynthesis
Enzymes and Gαq-linked Receptors
TXB2
3.5
ng/g wet tissue
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ND
AD
Unpaired t test
P value
P value summary
0.0076
**
PGE2
1.75
ng/g wet tissue
1.50
TP
1.25
1.00
0.75
0.50
0.25
EP1/EP3
BLT1
CysLT1
0.00
ND
Unpaired t test
P value
P value summary
AD
0.0117
*
APP mRNA
APP Protein
Herbst-Robinson et al. (2015) Scientific Rep. 5:18286
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Secreted Aβ
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EP3
A40
(fmol/ml/g protein)
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EP1
EP
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APP/GAPDH
(Relative to Veh Ctrl)
transfection Mock
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R.Q. APP/GAPDH
(Relative to Veh Ctrl)
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Eicosanoid Receptors
Multiple Receptors Regulate APP & Aβ
treatment
APP
GAPDH
CysLT1 BLT1
0.15
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0.10
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0.05
<LOD
Eicosanoid Receptors
APP Expression in Rat Hippocampal Neurons
High Agonist Addition
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Low Agonist Addition
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Relative APP/GAPDH
Expression
1.5
1.0
0.5
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LTD4 17PGE2 I+L+17
Herbst-Robinson et al. (2015) Scientific Rep. 5:18286
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0
Control IBOP
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Relative APP/GAPDH
Expression
2.0
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Brain Eicosanoids
Driving Further Plaque Deposition?
TxA2, PGE2, LTD4
SP
SP
Aβ
↑APP
SP
Neuron
5XFAD APP-PS1 Tg Mice
Robust Plaque Deposition
Oakley et al. (2006) J. Neurosci. 26:10129-40
Brain Eicosanoids
Elevated 5-LOX & COX-1 in Aged 5XFAD Tg Mice
5-LOX
GAPDH
COX-1
GAPDH
Brain Eicosanoids
Possible Involvement in 5XFAD Tg Mice
PGE2
6.0
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Non-Tg
PGE2
1.5
1.5
1.5
1.5
6.0
6.0
6.0
1.5
APP
GAPDH
Cortex
1.5
6.0
1.5
1.5
1.5
6.0
1.5
6.0
0.5
*
6.5 Months
TxB2
2
1
Cortex
0.0
1.5 Months
6.5 Months
Herbst-Robinson et al. (2015) Scientific Rep. 5:18286
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TxB2
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Relative APP/GAPDH
3
1.5 Months
6.0
6.0
1.5
APP
GAPDH
Hippo
1.0
0.0
Relative Levels
6.0
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Relative Levels
2.0
Hippocampus
Brain Eicosanoids
COX/5-LOX Inhibition Decreases APP Expression
Cortex
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1.00
0.75
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1.00
0.75
0.50
0.50
COX
1.00
0.75
**
0.50
Vehicle
COX/Licof
0.25
C-term Frag/GAPDH
Vehicle
COX
COX/Licof
1.00
*
0.75
0.50
0.25
0.00
0.00
Vehicle
COX/Lico
Herbst-Robinson et al. (2015) Scientific Rep. 5:18286
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CL
APP
GAPDH
Relative APP/GAPDH
Relative APP/GAPDH
CL
APP
GAPDH
APP
GAPDH
C-term Frag/GAPDH
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APP
GAPDH
Vehicle
COX/Lico
Brain Eicosanoids in AD
Additional Evidence of Involvement
 Genetic knockout of PGE2 receptors (EP1, EP2, EP3
and maybe EP4) reduces Aβ plaque burden in APP Tg
mouse models.
 Genetic knockout or pharmacological inhibition of 5LOX reduces Aβ plaque burden in APP Tg mouse
models.
 NSAIDs generally reduce Aβ plaque burden in APP Tg
mouse models.
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Certain NSAIDS (e.g., ibuprofen) at μM concentration can also
affect γ-secretase cleavage of APP and Aβ42 generation.
 Epidemiological data suggest long-term NSAID use
reduces risk of AD.
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Although NSAID trials in AD have generally been unsuccessful.
Brain Eicosanoids
Dual 5-LOX/COX Inhibitors for AD
 Were NSAID trials truly unsuccessful?
 Most trials were conducted in mid-to-late stage AD patients.
 Many trials utilized COX-2 selective agents, and increasing
evidence suggests COX-1 may be more important in AD.
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In the ADAPT trial, some benefit was observed 2-3 years after study
completion with a non-selective NSAID (naproxen), but not with a COX2 selective drug (celecoxib).
Most non-selective NSAIDs have low brain/plasma exposure ratios
(<0.1) and CSF drug levels or PGs were not assessed.
Inhibition of COX alone will not affect leukotrienes (5-LOX), and
there is evidence of AA shunting to 5-LOX upon COX inhibition.
 We posit that brain-penetrant, dual 5-LOX/COX-1
inhibitors may provide benefit in AD and possibly other
neurodegenerative diseases.

Dual 5-LOX/COX inhibitors are being developed for peripheral
inflammation (e.g., licofelone). Evidence of improved safety
relative to NSAIDS, but not brain-penetrant.
Brain Eicosanoids
Brain-Penetrant, Dual 5-LOX/COX Inhibitors
Scaffold 2
10
7.5
2.5
PGD2
LTB4
0.0
-4
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2
PGD2
LTB4
2
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-6.5
-3
-6.0
-3
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5PG
CNDR-51756 [log M]
-4
10
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PGD2
LTB4
-6
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-3.5
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-4.0
PGD2 (ng/g)
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LTC4 (pg/g)
6
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3h after 50 mg/kg
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CNDR-51785 (log [M])
LTB4 (ng/mL)
PGD2 (ng/mL)
Ibuprofen [log M]
-5.5
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PGD2 (ng/mL)
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Tau Loss-of-Function
Altered Microtubules & Axonal Transport
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Tau is an abundant microtubule (MT)-associated protein found predominantly
in axons which promotes MT polymerization and stabilization.
Tau hyperphosphorylation, as observed in AD and FTLD, reduces the binding
of tau to MTs and can promote aggregation.
There is evidence of MT deficits in AD and Tg mouse models with tau
pathology.
Brunden et al. (2009) Nature Rev. Drug Disc., 8:783-93
MT-Stabilizing Agents
Potential Neurodegenerative Therapeutics
 Oncology drugs such as Taxol (paclitaxel) and
Taxotere (docetaxel) kill cancer cells by affecting
mitotic spindle MT dynamics during cell division.
 Could low doses of a drug like Taxol help stabilize
MTs in the AD brain without causing the side-effects
seen in cancer treatment?
 Can brain-penetrant microtubule-stabilizing drugs be
identified?
Epothilone D
Unique PK/PD Properties
15
Brain epoD (ng/g)
1000
ng/ml or g
100
10
1
Plasma
Brain
10
5
0.1
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25
Time (hours)
Day Post-Dosing
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0.75
g/
kg
m
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cl
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kg
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CNDR66 (EpoD)
1.00
m
O
OH
1.25
6
OH
g/
kg
O
m
O
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**
3
N
Relative Ac-Tubulin
1.50
S
6
Brunden et al. (2010) J. Neurosci., 30:13861-66 & Brunden et al. (2011) Pharmacol. Res., 63:431-51
10
EpoD in PS19 (Tau P301S) Tg Mice
Intervention Study Design
9M PS-19 (males)
n≥9
Vehicle Control
Weekly i.p. Injections
9M PS-19 (males)
n≥9
0.3 mg/kg EpoD
Weekly i.p. Injections
3 Months Dosing
9M PS-19 (males)
n≥9
1.0 mg/kg EpoD
Weekly i.p. Injections
9M Non-Tg (males)
n≥9
Vehicle Control
Weekly i.p. Injections
Zhang et al. (2012) J. Neurosci., 32:3601-3611
Efficacy Testing
1. Axonal Dystrophy
2. MT Density
3. FAT
4. Behavioral Testing
5. CNS Pathology
Safety Testing
1. Behavioral Observations
2. Body/Organ Weights
3. CBCs
EpoD Interventional Study
Optic Nerve Axonal Dystrophy & MT Density
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*
3.5
*
4
MT/0.035 m2
3.0
2.5
2.0
1.5
1.0
3
2
1
0.5
m
1
Ep
oD
19
PS
Ep
oD
19
g/
kg
kg
0.
3
m
g/
Ve
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PS
PS
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W
m
PS
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Ep
oD
1
0.
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Ep
oD
19
PS
g/
kg
g/
kg
m
Ve
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PS
T
W
Zhang et al. (2012) J. Neurosci., 32:3601-3611
Ve
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0.0
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Dystrophic Axons/128 m Area
**
EpoD Interventional Study
Improved Neuronal Survival
Wild-Type Vehicle
*
Normalized NeuN Area
*
0.8
0.6
0.4
0.2
oD
m
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1.
19
PS
PS
19
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m
g/
kg
Ep
oD
Ep
g/
kg
19
PS
W
Ve
h
Ve
h
0.0
T
PS19 Vehicle
1.0
EpoD also prevented synapse loss
and improved FAT
Zhang et al. (2012) J. Neurosci., 32:3601-3611
EpoD Interventional Study
Reduction of Tau Pathology
WT Vehicle
WT Vehicle
WT Vehicle
WT Vehicle
PS19 Vehicle
PS19 Vehicle
PS19 Vehicle
PS19 Vehicle
AT8 IHC
MC1 IHC
D.
2
Zhang et al. (2012) J. Neurosci., 32:3601-3611
g/
kg
m
g/
kg
Ep
oD
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g/
kg
m
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Ep
oD
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g/
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Ep
oD
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0.0
g/
kg
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m
g/
kg
1
Ep
oD
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1.0
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oD
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E.
Relative Insoluble Tau
4
MC1 Score
4
EpoD 1mg/kg
*
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AT8 Score
C.
EpoD 1mg/kg
EpoD 1mg/kg
Ep
oD
EpoD 1mg/kg
MT-Stabilizer Clinical Trials
EpoD & TPI-287
 BMS Phase Ib Study of BMS-241027 in AD patients



Multi-center, randomized, double-blind placebo-controlled study of
BMS-241027 at 0.003-0.03 mg/kg i.v. once weekly for 9 weeks
(equiv. to mouse doses of 0.04-0.4 mg/kg).
Outcomes were safety, CSF tau and NFL, cognitive performance and
MRI.
Study concluded with no demonstration of a biomarker change (no
direct biomarker of MT stabilization). No reported safety issues.
 UCSF/Cortice Bioscience Phase Ib Study of TPI-287 in
PSP/CBD Tauopathy Patients



TPI-287 at 2, 6.3 or 20 mg/m2 (0.05, 0.17 or 0.54 mg/kg) i.v. once
every 3 weeks for 9 weeks.
Outcomes are safety and tolerability, including MTD
Secondary outcomes are blood and CSF drug levels, plasma PK, CSF
tau/p-tau and NFL and brain MRI
Dictyostatin Interventional Study
Improved Brain Measures
 Our prior work revealed that dictyostatin, like EpoD, was brain-
penetrant and showed a much greater brain than plasma half-life.
 Study design similar to the EpoD interventional study.
 Efficacy results were generally similar to what was observed with
EpoD, including increased MTs, reduced axonal dystrophy and tau
pathology, and a strong trend toward increased neuronal survival.
 Unlike EpoD, PS19 mice showed GI side-effects that resulted in the
death of some study mice.
Brain-Penetrant MT-Stabilizers
Non-Natural Products
 Natural products like EpoD and dictyostatin are difficult to
synthesize and generally require i.v. dosing.
 There could be value in identifying small molecule MTstabilizing agents that are simpler to synthesize, orally
bioavailable, and free of Pgp interactions.
 We are presently pursuing triazolopyrimidine and
phenylpyrimidine small molecules that increase markers of
stable MTs and are brain-penetrant (Lou et al., J. Med.
Chem. 57:6116-27, 2014 and Kovalevich et al, JPET
357:432-450, 2016).
 Jane Kovalevich will discuss these efforts in the next
presentation.
CNDR AD Drug Discovery
Summary
 Our objective is to pursue therapeutic strategies
that are complementary, but not directly
competitive, to existing large pharma programs.
 Preference is for druggable targets where
modulation might also provide benefit in other
neurodegenerative conditions


CNS eicosanoids have been implicated in PD, ALS and MS.
Evidence of MT dysfunction in PD, ALS and TBI
 We also work in partnership with pharma to
advance their internal programs.
Acknowledgements
CNDR Drug Discovery
(Biology/Pharmacology)
Alex Crowe
Michael James
Jane Kovalevich
Amy Lam
Li Liu
Maggy Nitla
Mandy Yao
Mansi Khanna
Bin Zhang
NIA, CART, Marian S. Ware &
Woods Foundations,
BrightFocus, MJFF, other
philanthropic donations
AstraZeneca, Biogen, BMS,
GSK, J & J, Sanofi, Pfizer
Drug Discovery
Director
Kurt Brunden
CNDR Directors
Virginia Lee
John Trojanowski
Past CNDR DD Contributors
Jenna Carroll
Bryant Gay
Anne-Marie Hogan
Edward Hyde
Francesco Piscitelli
Justin Potusak
Katie Herbst-Robinson
James Soper
Shimpei Sugiyama
Laurel Vana
CNDR Drug Discovery
(Chemistry)
Amos B. Smith, III
Carlo Ballatore
Anne-Sophie Cornec
Heeon Han
Pierrick Lassalas