Drug discovery for the treatment of Alzheimer’s disease · Drug discovery for the treatment of Alzheimer’s disease - Moving away from symptomatic therapies ... BSI (45 mpk) 2-Photon

Drug discovery for the treatment ofAlzheimer’s disease -Moving away from symptomatic therapies

Menelas N. PangalosExecutive Vice President & Head of

Discovery Research

AD drug discovery – March 2009

2

Today’s talk

Introduction

PAI-1 inhibitors

Summary

secretase inhibitors



3

Today’s talk

Introduction

PAI-1 inhibitors

Summary




4

Alzheimer’s disease statistics

A person develop dementiaevery three minutes

AD currently affects more than24 million people world wide

Expected to reach 80 million by 2040

Accounts for >60% of all dementias

Prevalence- 1% between the ages of 60-64

- 50% in people aged 85 and over

1 in 8 baby boomers will get AD

Through course of the diseasepatients will require full timecare

Direct and indirect costs in theUS are estimated in excess of$150B/yr

Could reach $1 trillion by 2050

NormalNormal AlzheimerAlzheimer’’ss

NormalNormal ModerateModerate AdvancedAdvanced


5

Wyeth’s AD pipeline uses all three of ourtechnology platforms

Vaccines

ACC-001†

ACC-002†

Proteins

Bapineuzumab†

AAB-002†

AAB-003†

† Alliance with Elan

Small Molecules

GSI-953

PAZ-417

SAM-531

SAM-760

NSA-789

HTC-867


6

Today’s talk

Introduction

PAI-1 inhibitors

Summary




7

Inhibition of the amyloid pathway –Prevention of amyloid plaque formation

NeuropathologyLeading to Cognitive

Dysfunction

AAmyloid Protein

Beta amyloiddeposits or plaques

Beta Secretase Gamma Secretase

Beta amyloidpeptide

A

AA

ABeta amyloidaggregates

Gamma Secretase orBACE Inhibitors


8

Gamma secretase is a multi-transmembraneprotein complex

From Marjaux et al (2004) Neuron 42:189-192


9

Cell-based assays demonstrate GSI-953 is apotent inhibitor of A production

GSI-953

WAY-952

-10 -8 -6 -4

-10

10

30

50

70

90

110

Compound (log[M])

Pe

rce

nt

Ve

hic

le

0

20

40

60

80

100

DAPT

A 40 A 42

CMPDEC50 (nM)

A 40 A 42

DAPT41.7± 9.5

27.7± 4.8

GSI-95314.8± 1.2

12.4± 0.8

WAY-952 >10,000 >18,000

LY4115750.32

± 0.010.36

± 0.01

LY45013927.5± 1.2

26.0± 1.0

Mayer SC et al. J. Med. Chem. 2008, 51(23), 7348-7351


10

GSI-953: Increases levels of CTF fragments

WAY-Change

EC50 (nM)

210953CTF A Tot

6.5 7.9

% C

on

tro

l

WAY-210953 [nM]

Radiolabeled CTF AssayLumen TD Cytoplasm

APP

CTF

A

+

APPs

CTF

0

100

200

300

400

500

600

0 10 20 30 40 50

Lumen TD Cytoplasm

APP

CTF

A

+

APPs

CTF CTF

0

100

200

300

400

500

600

0 10 20 30 40 50

0

100

200

300

400

500

600

0 10 20 30 40 50


11

-Secretase inhibition and potential alteredNotch processing

A

+

A

APP

NotchNotch

CTFCTF

AICDAICD

NotchNotch

NICDNICD

A

Lumen TD Cytoplasm

F “ “

+

Notch cleaved by -secretase

NICD translocates tonucleus

NICD is a transcriptionalfactor regulatingdevelopment and cellulardifferentiation (eg,gastroendothelium)


12

GSI-953 selectively inhibits APP processingover Notch

CompoundEC50 (nM) Ratio

Notch/ANotch A 42

DAPT14.9± 0.9

27.7± 4.8

0.5

GSI-953208.5± 27.3

12.4± 0.8

16.8

WAY-952 Inactive >18,000 N.D.

LY411575 0.3± 0.1

0.36± 0.0

0.9

LY45013962.9

± 19.526.0± 1.0

2.4

RBP-Jk (SuH)

HES SEAP-Reporter

mDENotch

-Secretase

SP

NICD

NICD


13

P8-11

APP processing in rat cortical brain slicesbiolistically transfected with APP


14

-Secretase inhibitors block A formation

in brain slices

Vehicle GSI-1

- APP

- CTF

- A


15

GSI-953 reduces brain A levels in a time anddose-dependent manner in Tg2576 mice

0

20

40

60

80

0 0.25 0.5 1 2 4 6 10 24

Ab 40

Ab42

A R

ed

ucti

on

(%

)

****

Time (hours)

A 40 A 42

*p < 0.5***p < 0.001

A R

ed

ucti

on

(%

)

0

10

20

30

40

50

0 1.0 2.5 5.0

GSI-953 (mg/kg)

***

***

******

A 40 A 42

*p < 0.5 **p < 0.01***p < 0.001


16

Contextual fear conditioning (CFC)

Testing (Day 2)

24h 300s

Hippocampal-dependentconditioning in mice

Learning involves a shockassociated with a specificenvironment (context)

Memory is expressed bycontext-dependent freezing inthe absence of the shock

1.5 mAmp 1.5 mAmp

120s 2s 120s 2s 30s

Training (Day1)


17

GSI-953 reverses hippocampal contextualmemory deficits in Tg2576 animals

*sig.diff. from WT vehicle; ^sig.diff. from APP Tg Vehicle

0

10

20

30

40

50

0 2.5 5 10 30

Wild-Type Tg2576

WAY-952GSI-953 (mg/kg)PBS

301052.50

^

** *


18

-Secretase Inhibitors exhibit an A “rebound”effect in plasma but not in brain

GSI (30mg/kg, po)

*p<0.05, **p< 0.001)

% A

Levels

-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

50%

60%

70%

0 2 4 6 8 10

A 40 BrainA 42 BrainA 40 Plasma

**

**

**

*

**

**

****

*

*

*

**

Time (h)

Reduced A

Increased A


19

GSI-953 reduces plasma A levels in human:target engagement and biomarker strategy

0 1 2 3 4-50

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

0

3

10

30

60

100

150

200

300

450

600

Dose (mg)

Time (hours)

Pla

sm

a A

40

(% c

han

ge f

rom

baselin

e)


20

Today’s talk

Introduction

PAI-1 inhibitors

Summary




21



Dysfunction

AAmyloid Protein



Beta amyloidpeptide

A

AA


Gamma Secretase orBACE Inhibitors


22

Develop a reproducibleassay

Run screen - approximately1,000,000 compoundstested

Take identified chemical hitsand confirm their activity in asecond assay usinghuman hands

Use X-ray structure to guidechemical drug design

Evolution of a BACE drug discovery program


23

Chemists work with biologists to modify the initialchemical hit to fit “perfectly” on the biologicaltarget and have drug-like properties

Chemical LeadChemical Hit

Next step – optimizing “lead” molecules toimprove effectiveness


24

Polar/Charged Hydrophobic

S1S3 S2’

S1’

FLAP

Para position providesbest opportunity toincrease potency viaS3 pocket:

N

HN

O

NH2

NH

Confirming and understanding an earlychemical ‘hit’ for our BACE program

S1 – S3 formslarge hydrophobicbinding pocket


25

BACE1 IC50

3700 nM

Start with lead compound

Drug discovery – optimizing a chemical ‘hit’

N

HN

O

NH2

NH

S1


26

600 nM--unstable

N

HN

O

NH2

NH



Examine each pocket

S1

BACE1 IC50

3700 nM


27

N

HN

O

NH2

NHS3

800 nM

X = H, O

Good stability


Examine each pocket

Combine results

S1

X


BACE1 IC50

3700 nM


28

S1’

290 nMX = H, O


Examine each pocket

Continue optimization


NH

OH

N

HN

O

NHS3

S1

X

BACE1 IC50

800 nM


29

S2’60 nM

X = H, O


Examine each pocket

Optimized tool compound


Cl

S1’

NH

OH

N

HN

O

NHS3

S1

X

BACE1 IC50

3700 nM


30

S1

S2’

S1’

S3

FLAP

Development of more potent BACE inhibitors

A few thousandcompounds synthesizedto explore chemistryaround initial hits

Potency improvedmore than a thousandfold

Stability and brainpenetration maintainedand improved

Polar/Charged Hydrophobic


31

Guanidine extracted 8 h takedown, n=10/group

Brain A 40 Brain A 42

Our BACE inhibitors do not lower brain A

acutely in wild type mice

vehic

le 3 10

30

100

0

20

40

60

80

100

120

BSI (mg/kg)

A40 (

% v

eh

icle

co

ntr

ol)

vehic

le 3 10

30

100

0

20

40

60

80

100

120

BSI (mg/kg)A

42 (

% v

eh

icle

co

ntr

ol)


32

Brain A 42 Brain A 40

Veh 3 10 30 1000

50

100

150

***

A40 (

% o

f veh

icle

co

ntr

ol)

Veh 3 10 30 1000

50

100

150

***

*

A4

2 (

% o

f v

eh

icle

co

ntr

ol)

BSI (mg/kg) BSI (mg/kg)

Our BACE inhibitors do lower brain A acutely

in PGP KO mice


33

0

10

20

30

40

50 Wild-TypeTg2576

Mem

ory

Defi

cit

s (

% F

reeze)

(mg/kg; po)

100

*0 10 30 10050

*sig. diff. from WT vehicle; ^sig. diff. from Tg2576 vehicle

0

10

20

30

40

50 Wild-TypeTg2576

Mem

ory

Defi

cit

s (

% F

reeze)

(mg/kg; po)

0 100

*0

*0 1010 3030 1001005050

^

*

^

**

^

^

Oral dosing of a BACE inhibitor reversescognitive deficits in an AD mouse model


34

8 weeks 10 weeks 12 weeks 14 weeks

16 weeks 18 weeks 20 weeks 22 weeks

We can see Amyloid deposits increase overa period of a few weeks


35

Control

BSI (45 mpk)

2-Photon microscopy in live animals

… And Our BACE inhibitors can reduce theamyloid load in the brains of these AD mice

WAY-264116 (mg/kg)

0 22 45

*

* n=3 Animals, P=0.017

0

100

200

300

400

500

600

700

800

900

Pla

qu

e C

ross-S

ecti

on

al A

rea

( μ

m 2 )

BSI (mpk)


36

PSAPP+ vehicle

PSAPP+ BACE-1

Vehicle BACE Inh-1 Wild type0

2

4

6

8

10

A lo

ad

(%

)

***

***

***

Hippocampus

0

2

4

6

8

10

n=19

n=20

n=20

A lo

ad

(%

)***

***

*** Cortex

Vehicle BACE Inh-1 Wild type

Plaque load is significantly reduced bychronic administration of a BACE Inhibitor


37

Today’s talk

Introduction

PAI-1 inhibitors

Summary




38



Dysfunction

AAmyloid Protein



Beta amyloidpeptide

A

AA


PAI-1 Inhibitors


39

A activates and is cleaved by the tPAplasmin cascade

tPA

+ mRNA

Plasminogen

Proteolyticcleavage

tPA affinity for Plgn

tPA

Plasmin

A

aggregationA + A A (n)


40

A pathology increases PAI-1 levels andinhibits tPA and decreases plasmin formation

tPA

+ mRNA

Plasminogen

Proteolyticcleavage


tPA

Plasmin

A

aggregationA + A A (n)

PAl-1


41

Decreased plasmin activity results in reduced A catabolism and further PAI-1 expression

tPA

+ mRNA

Plasminogen

Proteolyticcleavage


tPA

Plasmin

A + A A (n)

PAl-1

A

aggregation


42

A is a plasmin substrate plasmin and cancleave it into numerous fragments

Data from Steve Estus lab; Tucker et al. J. Neurosci. 2000, 20:3937

HPLC Peptide Cleavage AnalysisA 40

1

Abs

10 200 30

A 40

1 23 4

5 7 7

10 200 30

1

+ Plasmin


43

tPA and plasminogen are required for Aclearance in vivo

Data from Sidney Strickland lab; Melchor et al. J. Neurosci. 2003, 23:8867

WT tPA-/- plg-/- WT tPA-/- plg-/- WT tPA-/- plg-/-

A Immunoreactivity Microglial Activation Neurodegeneration


44

0.01 0.1 1 100

20

40

60

80

100

120

PAI-1 Inhibitor (uM)

PA

I-1 A

cti

vit

y (%

)

In Vitro PAI-1 Inhibitor assay: tPA activitycleaves chromogenic substrate

PAI-1 IC50 (nM)Inhibitor Avg. ± SEM

PAI-749 288 ± 41PAZ-417 655 ± 29PAI-039 1731 ± 203

PAI tPA SP

PAI tPAIS

Jacobsen et al. PNAS. (2008) 105: 8754-8759


45

PAI-1 inhibitors activate A and A oligomerdegradation in vitro

tetramertrimer

dimer

monomer


46

Inhibitors of PAI-1 restore deficits of tPAactivity in hippocampus of Tg2576 mice

PAZ-417

Tg

AP

P

WT

tPA

IHC (Protein)

*

Veh PAZ Veh PAZ

WT TgAPP

tPA

A

cti

vit

y (

%)

0

5

10

15

20

25

30

35 ** *p<0.04**p<0.004

Vehicle

Zymograph (tPA Activity)

PAI-1


47

PAZ-417 reduces plasma levels of A 40 inTg2576 mice

MED=10 mg/kg (6 hr)

*p<0.02

0 3 10 30 1000

5

10

15

20

25

30

35

* * *

PAZ-417 (mg/ml)

% P

las

ma

A L

ow

eri

ng


48

PAZ-417 reduces plasma and brain levels ofA in Tg2576 mice

20 mg/kg, po 6 hrs post-treatment

*p<0.01

31% 20% 15%

Plasma A 40 Brain A 40 Brain A 42

* * *

%

A L

ev

els

0

20

40

60

80

100

120

Veh PAZ Veh PAZ Veh PAZ


49

Tg

-30 -20 -10 0 10 20 30 40 50 60 70 80 9075

100

125

150

175

200

225

250

275

300

325Tg Vehicle

Tg PAZ-417

*

Time (min)

% B

as

eli

ne

fE

PS

P S

lop

e

< WT level< deficit

PAZ-417 restores LTP deficits in the dentategyrus of TgAPP mice

-30 -20 -10 0 10 20 30 40 50 60 70 80 9075

100

125

150

175

200

225

250

275

300

325

WT PAZ-417

WT Vehicle

Time (min)

% B

as

eli

ne

fE

PS

P S

lop

e

WT

PAZ-417 Vehicle


50

PAZ-417 reverses hippocampal contextualmemory deficits

*

^ ^

Vehicle 10 30 100

Significant difference from WT (*) or vehicle-treated APP Tg (^)

0

10

20

30

40

50M

em

ory

(%

Fre

eze

)

PAZ-417 (mg/kg, 1x po)

WT

Tg2576


51

Today’s talk

Introduction

PAI-1 inhibitors

Summary

-secretase inhibitors


52

Wyeth CNS pipeline

Phase 0 (9) Phase 1 (4/2) Phase 2 (3) Phase 3 (3)

ACC-001 (AD)

SAM-531 (AD/Schz)

Vabicaserin (SCA-136) (Schz/MDD)

Lecozotan (HOLD)

Bapineuzumab(Alzheimer'sDisease)

AAB-002 (AD) GSI-953 (AD)

NSA-789 (AD/Schz)

Pristiq™(Depression)

NRI-470 (Pain)

Pristiq™ (Neuropathic Pain)

Pristiq™(Fibromyalgia)

Registration/Approval (1)

ACC-002 (AD)

PAZ-417 (AD)

ILS-920 (Stroke)

MMS-255 (ND)SBI-087 (MS)

HTC-867 (AD/Schz)

SAX-187 (HOLD)

AAB-003 (AD)

VRA-175 (Pain)

SAM-760 (AD) Pristiq(VMS)


53

Acknowledgements and many thanks….

Questions?

Menelas N PangalosVice President

Neuroscience Discovery Research


55

WAY-210953: Lowers A levels withoutalteration of expression or secretion

Radiolabeled Cellular Assay

% C

on

tro

l

WAY-210953 [nM]

Lumen TD Cytoplasm

APP

0

20

40

60

80

100

0 100 200 300

CTF+

APPs

0

20

40

60

80

100

0 100 200 300

0

20

40

60

80

100

0 100 200 300

+

APPs

CTF

CTF

AA

Analyte Change

APP no

APPs no

A EC50 ~35 nM


56

GSI-953 reduces A in Tg2576 mouse model(time-course effects, 100 mpk, po)

Plasma

A R

ed

ucti

on

(%

)

0

20

40

60

80

100

120

0 2 4 6

Time (hours)

*** ****** A 40

***p < 0.001

CSF

A R

ed

ucti

on

(%

)

0

20

40

60

80

100

120

0 2 4 6

**

******

*** *** *** A 40 A 42

**p < 0.01***p < 0.001

Brain

A R

ed

ucti

on

(%

)

0

20

40

60

80

0 2 4 6

*** ***

****** *** *** A 40

A 42***p < 0.001


57

GSI-953 binds to -secretase: There is a clearrelationship of binding to inhibitory activity

GSI-953

WAY-210952

L-685458

LY450139

Compound ( M)

0.001 0.01 0.1 1 10

Pe

rce

nt

Re

sid

ua

l C

PM

10

20

30

40

50

60

70

80

90

100

110

A 42 EC50 (nM)B

ind

ing

IC

50 (

nM

)

10510410310210110-110-2

104

103

102

101

10-1

10-2

r2 = 0.99

˜

˜

˜˜

˜˜

˜

˜ ˜


58

tetramertrimer

dimer

monomer

Dose dependent cleavage of A and Aoligomers by a PAI-1 inhibitor

Documents

Drug discovery for the treatment of Alzheimer’s disease · Drug discovery for the treatment of Alzheimer’s disease - Moving away from symptomatic therapies ... BSI (45 mpk) 2-Photon