Ottavio Arancio, M.D., Ph.D.Columbia University

Novel PDE5 Inhibitors as a Therapeutic Tool Against

Alzheimer’s Disease

AMPANMDA

glutamate

The earliest amnesic symptoms in AD are likely to be due to subtle changes in the way in which

cell communicate at synaptic level

Synaptic alterations are highly correlated with the severity of clinical dementia

HYPOTHESIS

Can we prevent β-amyloid-induced

impairment of memory formation using drugs acting

at the downsream level of β-amyloid production?

Aβ

Histones

cAMP

PKA

AC

Proteasome

Uch-L1 Ca2+

Calpains

GC

cGMP

PKG

NO

NOS

gene transcription Synaptic plasticity

Memory

CREB

Aβ modulation of NO/cGMP/PKG/CREB pathway

NOGTP

cGMP

L-Arg L-citrulline

NOS

5’-cGMPPDEV

cGK PKG

CREB

sGC sGC

Aβ

NO-donors

cGMP-analogs

Puzzo et al, J. Neurosci. 2005

PDE5 inhibitors

PKG agonists

The Journal of Neuroscience, July 20, 2005 • 25(29):6887– 6897 • 6887

Neurobiology of Disease

Amyloid- Peptide Inhibits Activation of the Nitric Oxide/cGMP/cAMP-Responsive Element-Binding Protein Pathway during Hippocampal Synaptic Plasticity

The Journal of Neuroscience, June 24, 2009 • 29(25):8075– 8086 • 8075

Development/Plasticity/Repair

Phosphodiesterase 5 Inhibition Improves Synaptic Function,Memory, and Amyloid- Load in an Alzheimer’s Disease Mouse Modelsynaptic plasticity

memory

Can we improve synaptic and cognitive abnormalities at early stages of amyloid

deposition?

Recovery of LTP impairmentin 3 month-old APP/PS1 mice

by Sildenafil (Viagra)

Time (min)

0 20 40 60 80 100 120 140

fEPS

P sl

ope

(% o

f bas

elin

e)

0

50

100

150

200

250

300

350

400 APP/PS1, vehicleAPP/PS1, sildenafilAPP/PS1, no tetanusAPP/PS1, sildenafil, no tetanus

Puzzo et al, J. Neurosci. 2009

Sildenafil improves contextual conditioningin 3 month-old APP/PS1 mice

Puzzo et al, J. Neurosci. 2009

% fr

eezin

g

0

10

20

30

40

50

WT/WTWT/WT, SildenafilAPP/PS1APP/PS1, Sildenafil

Baseline 24 h

WT, vehicleWT, sildenafil APP/PS1, vehicleAPP/PS1, sildenafil

Sildenafil improves spatial working memoryin 3 month-old APP/PS1 mice

1

2

3

4

5

6

7

WT, vehicleWT, sildenafilAPP/PS1, vehicleAPP/PS1, sildenafil

Erro

rs

TrialA1 A2 A3 A4 R

Puzzo et al, J. Neurosci. 2009

Can we improve synaptic and cognitive abnormalities when a substantial plaque

load already exists?

sildenafil3 weeks 8 weeks

test

Prolonged Beneficial Effect by Sildenafil onSynaptic Dysfunction and

Memory Loss

Time (min)

0 20 40 60 80 100 120 140

fEPS

P sl

ope

(% o

f bas

elin

e)

0

50

100

150

200

250

300

350

400

450

500APP/PS1, vehicleAPP/PS1, sildenafilAPP/PS1, no tetanusAPP/PS1, sildenafil, no tetanus

APP/PS1, vehicle,tetanusAPP/PS1, sildenafil, tetanusAPP/PS1, vehicle,no tetanusAPP/PS1, sildenafil, no tetanus

LTP

1

2

3

4

5

6

7WT, vehicleWT, sildenafilAPP/PS1, vehicleAPP/PS1, sildenafil

Erro

rs

Trials

A1 A2 A3 A4 R

RAWM

Puzzo et al, J. Neurosci. 2009

% fr

eezi

ng

0

5

10

15

20

25

30 APP/PS1 VehicleAPP/PS1 SildenafilWT/WT VehicleWT/WT Sildenafil

Fear Cond

WTvehicle

No tetanus Tetanus

APP/PS1vehicle

1

4

2

3

1

2

3

4

5 6

WTSildenafil

APP/PS1Sildenafil

5

6

7

87 8

No tetanus Tetanus

WTveh

APP/PS1veh

WTSild

APP/PS1Sild

IF(%

ofco

ntro

l)

020406080

100120140160180200 Immediate

No tetanus Tetanus

WTveh

APP/PS1veh

WTSild

APP/PS1Sild

IF(%

ofco

ntro

l)

020406080

100120140160180200 Immediate

020406080

100120140160180200

WTSild

APP/PS1Sild

IF(%

ofco

ntro

l)

WTveh

APP/PS1veh

Prolonged

020406080

100120140160180200

WTSild

APP/PS1Sild

IF(%

ofco

ntro

l)

WTveh

APP/PS1veh

Prolonged

Sildenafil re-establishes normal levelsof CREB phosphorylation

in hippocampal slices from APP/PS1 mice

Sildenafil decreases Aβ levels in APP/PS1 mice

Is there any PDE5 in human hippocampus?

Database of human brain Gene Logic’s ASCENTA System

0/24

20/24

3/24

0/28

23/28

3/28

Rel

ativ

e ex

pres

sion

( ×10

-6)

0

40

80

120

160

200

heart whole brain hippocampus cerebrum

primer-1primer-2primer-3

0

1

2

3

4

5

6

7

8

heart whole brain hippocampus cerebrum

primer-1primer-2primer-3

Expr

essi

on(f

old

incr

ease

)

Expression levels of PDE5 mRNA in heart, whole brain, hippocampus and cerebrum of

humans (Quantitative RT-PCR)

A

B

Agents increasing cGMP levels by enhancing CREB phosphorylation

produce a beneficial effecton synaptic transmission and

cognition

PDE Superfamily

Our aim is to move the PDE5 inhibitory project forward to the stage where it

not only provides new biological insights but also, when appropriate, can serve as the basis for future development of new

therapeutic strategies

Currently used PDE5 inhibitors

O

NN

HNN

O

SN

N

OO

Sildenafil f Vardenafil

O

N

HNNH

N

O

SN

N

OO

NBAN

O

S OOR2

GD

ER3

R4

OR1

HN

N N

N

O

H2N

O

O PO

OH

OHOc-GMP c-GMP-based

PDE5 inhibitors

Fig 7

HN

N

OO

NO

O

Tadalafil

BBBPerm.

IC50against PDE5

Half life Select. ratio for

PDE1

Select. ratio for

PDE6

SILDENAFIL + 6 nM 3-4 hrs 180 12

VARDENAFIL ? 0.17 nM 4-5 hrs >1000 3.5

TADALAFIL - 5 nM 17-18 hrs >1000 1000

None of the commercially available PDE5 inhibitors possesses the selectivity required

for chronic administration to an elderly population with comorbid conditions

We have launched a computer-aided med/chem program to identify

molecules that counteract synaptic and memory dysfunction by inhibiting

PDE5

Our goals are to obtain novel drugs with. high specificity and potency. good PK, bioavailability and CNS

penetration. safety. novel compositions of matter with an

unobstructed intellectual property path to development

Given that many PDE5 inhibitors have been developed in the past decades, we

avoided wasting resources to develop an entirely new scaffold with high potency

and excellent selectivity:

XHN

RO

R2

Ia (x=C or N)

NXR1

O

OR2

R3N

S XR1

R2

R3

R

O ON

XR1

R2

R3

MeO2C

O

Ib Ic T1056

Fig. 15

N

R3R1NHO

N

R3R1NH

SR2

O

N

R3R1O

R2O

N

NH

R2

R4

R3

R5

R1

IIeR2

N

N

R3N

R2IIa IIb IIc IId

I

II

we identified quinoline derivatives as the top candidates for the design and synthesis of novel PDE5 inhibitors to be optimized

against AD

IC50 against PDE5 = 0.05 nMselect. ratios > 7800 vs PDE1-4

160 vs PDE6)

However, … Unknown,selectivity for the remaining PDEs, in vivoefficacy in an AD model or other diseases, PK including BBB penetration, toxicity,

and solubility.In addition only a few substituents on the quinoline ring were investigated and just

one compound was dominant.

YF012403PCT/US,2008 appl.61/140,315

YF012403 was easily prepared in six steps

Fig. 13

PDE5A1 Activity

-3 -2 -1 0 1 2 30

102030405060708090

100110

Substrate=100nM cGMP

IC50=0.27nM

YF012403 (log[nM])

% A

ctiv

yty

In vitro activity of YF012403 against PDE1-11

Concentration-Time curve of YF012403 in mouse brain tissue and plasma

PK parameters of YF012403 in mouse brain tissue and plasma

YF012403 ameliorates the LTP deficit in Aβ42-treated slices

fEP

SP

Slo

pe (%

of b

asel

ine)

0

50

100

150

200

250

300

vehicle n=8YF012403 (50nM) n=6 Aβ (200nM)-YF012403 (50nM) n=7Aβ (200nM) n=6

P<0.05P<0.05

YF012403 ameliorates the contextual fear memory deficit

in Aβ42-infused mice.

Baseline 24h

% F

reez

ing

0

10

20

30

40

Vehicle (n=12)YF012403 3mg/kg (n=10)YF012403 3mg/kg + Aβ200nM (n=13)YF012403 10mg/kg (n=10)YF012403 10mg/kg + Aβ200nM (n=11)Aβ200nM (n=15)

% fr

eezi

ng

P<0.05

YF012403 binding structure with human PDE5 and PDE6 protein (in purple) determined by molecular docking

Experimental Plan

We will focus our research design on modifications of YF012403 to optimize its

druggability

The primary benzylic alcohol at the 3-position (C3) is very likely to be oxidized by

microsomes generating benzaldehyde and consequently causing first-pass metabolism

problems and severe side effects due to subsequent conjugate addition to proteins

A cyclopropyl group at the 8-position (C8) may not be stable in vivo by undergoing ring

opening, and thus representing an electrophilic liability

CONCLUSIONS

-PDE5 inhibition rescues synapticand memory dysfunctions byamyloid-β elevation.

-The quinoline scaffold was selectedfor high potency and selectivity ofknown compounds with this scaffold.

Based on the results of a biological screening against synaptic

dysfunction, only changes resulting in improvement compared to YF012403

activity against PDE5 are being advanced further.

This work was supported by: NIH (NS49442, AG027468), ADDF.

Acknowledgments

F. AzizM. Fa’Y. FengI. FrancisB. GongG.

Hashimoto

M. SakuraiA. StaniszewskiF. TrincheseH. ZhangF. MichelassiS. Varhade

O. Arancio

E. Leznik B. LeeI. OrozcoL. PriviteraD. Puzzo

Columbia U.K. DuffU. of

MinnesotaK. Hsiao-

Ashe

Columbia UniversityD.W. LandryShi Xian Deng

UKYC.G. Zhan

U. CataniaA. Palmeri

Columbia UniversityM. Shelanski

Based on SAR analysiswe propose new scaffolds

meeting the following criteria:

2) Readily synthesized from readily available starting materials

3) Sufficient sites that can be modified to generate a relatively large number of compounds for screening

4) Novel composition of matter with no impediment for development

1) A fused ring system with an H-bond acceptor or donor