Ottavio Arancio, MD, PhD, Columbia University

Novel PDE5 Inhibitors as a
Therapeutic Tool Against
Alzheimer’s Disease
Ottavio Arancio, M.D., Ph.D.
Columbia University
Synaptic alterations are highly correlated with
the severity of clinical dementia
glutamate
NMDA
AMPA
The earliest amnesic symptoms in AD are likely
to be due to subtle changes in the way in which
cell communicate at synaptic level
HYPOTHESIS
Can we prevent
β-amyloid-induced
impairment
of memory formation
using drugs acting
at the downsream level
of β-amyloid production?
Aβ
NOS
AC
NO
Ca2+
cAMP
Uch-L1
PKA
Proteasome
Calpains
CREB
GC
cGMP
PKG
Histones
gene
transcription
Synaptic plasticity
Memory
Aβ modulation of NO/cGMP/PKG/CREB pathway
Aβ
NOS
L-citrulline
L-Arg
NO
GTP
NO-donors
The Journal of Neuroscience, July 20, 2005 • 25(29):6887– 6897 • 6887
sGC
sGC
Amyloid- Peptide Inhibits Activation of
the Nitric Oxide/cGMP/cAMPResponsive Element-Binding Protein
5’-cGMP
cGMP-analogs
cGMP Synaptic
Pathway during
Hippocampal
PDEV
Plasticity
Neurobiology of Disease
PDE5 inhibitors
PKG
cGK
The Journal of Neuroscience, June 24, 2009 • 29(25):8075– 8086 • 8075
PKG agonists
Development/Plasticity/Repair
Phosphodiesterase 5 Inhibition
Improves Synaptic Function,
Memory, and Amyloid- Load in an
Alzheimer’s Disease Mouse Model
synaptic plasticity
CREB
memory
Puzzo et al, J. Neurosci. 2005
Can we improve synaptic and cognitive
abnormalities at early stages of amyloid
deposition?
Recovery of LTP impairment
in 3 month-old APP/PS1 mice
by Sildenafil (Viagra)
400
APP/PS1, vehicle
APP/PS1, sildenafil
APP/PS1, no tetanus
APP/PS1, sildenafil, no tetanus
fEPSP slope (% of baseline)
350
300
250
200
150
100
50
0
0
20
40
60
80
100
120
140
Time (min)
Puzzo et al, J. Neurosci. 2009
Sildenafil improves contextual conditioning
in 3 month-old APP/PS1 mice
WT, vehicle
WT/WT
WT/WT,
Sildenafil
WT, sildenafil
APP/PS1
APP/PS1, vehicle
APP/PS1,
APP/PS1, Sildenafil
sildenafil
50
% freezing
40
30
20
10
0
Baseline
24 h
Puzzo et al, J. Neurosci. 2009
Sildenafil improves spatial working memory
in 3 month-old APP/PS1 mice
WT, vehicle
WT, sildenafil
APP/PS1, vehicle
APP/PS1, sildenafil
7
6
Errors
5
4
3
2
1
A1
A2
A3
A4
R
Trial
Puzzo et al, J. Neurosci. 2009
Can we improve synaptic and cognitive
abnormalities when a substantial plaque
load already exists?
sildenafil
3 weeks
test
8 weeks
Prolonged Beneficial Effect by Sildenafil on
Synaptic Dysfunction and
Memory Loss
LTP
500
APP/PS1,vehicle
vehicle,tetanus
APP/PS1,
APP/PS1,sildenafil
sildenafil, tetanus
APP/PS1,
APP/PS1,
tetanus tetanus
APP/PS1,no
vehicle,no
APP/PS1,
nono
tetanus
APP/PS1,sildenafil,
sildenafil,
tetanus
450
fEPSP slope (% of baseline)
400
350
300
250
200
150
100
50
0
0
20
40
60
80
100
120
140
Time (min)
RAWM
Fear Cond
30
25
WT, vehicle
WT, sildenafil
APP/PS1, vehicle
APP/PS1, sildenafil
7
APP/PS1 Vehicle
APP/PS1 Sildenafil
WT/WT Vehicle
WT/WT Sildenafil
6
Errors
% freezing
5
20
4
15
3
10
2
5
1
A1
0
A2
A3
A4
R
Trials
Puzzo et al, J. Neurosci. 2009
Sildenafil re-establishes normal levels
of CREB phosphorylation
in hippocampal slices
from APP/PS1 mice
No tetanus
Tetanus
1
WT
vehicle
1
2
2
IF (% of control)
No tetanus
3
APP/PS1
vehicle
3
4
6
6
7
APP/PS1
Sildenafil
7
8
8
IF (% of control)
5
Immediate
WT APP/PS1 WT APP/PS1
veh
veh Sild Sild
4
5
WT
Sildenafil
200
180
160
140
120
100
80
60
40
20
0
Tetanus
200
180
160
140
120
100
80
60
40
20
0
Prolonged
WT APP/PS1 WT APP/PS1
veh
veh Sild Sild
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
A
Relative expression (×10-6)
Expression levels of PDE5 mRNA in heart,
whole brain, hippocampus and cerebrum of
humans (Quantitative RT-PCR)
200
primer-1
primer-2
primer-3
160
120
80
40
0
heart
B
whole brain
hippocampus
cerebrum
Expression(fold increase)
8
primer-1
7
6
primer-2
primer-3
5
4
3
2
1
0
heart
whole brain
hippocampus
cerebrum
Agents increasing cGMP levels by
enhancing CREB phosphorylation
produce a beneficial effect
on 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
Fig 7
O
O
R1
O
O
N
HN
H2N
N
N
OH
O
c-GMP
O
O
O P O
OH
O
R3
E
N
A
D
B G
N
R4
O
c-GMP-based
PDE5 inhibitors
O
HN
NH
N
N
N
O
N
H
N
O
O S O
N
S O
R2
O
N
HN
N
Sildenafil f
O S O
N
N
N
O
N
Tadalafil
Vardenafil
BBB
Perm.
IC50
against
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:
Fig. 15
H
we
identified
quinoline derivatives as
the
O
N
X
O
O O
O
R
X
R
R and Rsynthesis
R
S
X
top candidates
for
the
design
N
N
R
R
X
N
I of novel PDE5 inhibitors
MeO
C
R
to
be
optimized
O
R
R
R
against AD
R
T1056
1
1
1
3
3
3
2
2
2
2
2
Ia (x=C or N)
N
N
II
R1
O
NH
R2
IIa
Ic
Ib
R3 R1
O
S
O
R2
R3
NH
IIb
R2
N
N
R1
N
R1
R3
R2
O
N
IIc
R2
R3
R3
N
IId
R4 R5
IIe
IC50 against PDE5 = 0.05 nM
select. ratios > 7800 vs PDE1-4
160 vs PDE6)
NH
However, … Unknown,
selectivity for the remaining PDEs, in vivo
efficacy 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.
YF012403
PCT/US,2008 appl.61/140,315
YF012403 was easily prepared
in six steps
Fig. 13
PDE5A1 Activity
% Activyty
Substrate=100nM cGMP
110
100
90
80
70
60
50
40
30
20
10
0
IC50=0.27nM
-3
-2
-1
0
1
YF012403 (log[nM])
2
3
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
fEPSP Slope (% of baseline)
YF012403 ameliorates
the LTP deficit in Aβ42-treated slices
300
250
vehicle n=8
YF012403 (50nM) n=6
Aβ (200nM)-YF012403 (50nM) n=7
Aβ (200nM) n=6
200
P<0.05
P<0.05
150
100
50
0
YF012403 ameliorates
the contextual fear memory deficit
in Aβ42-infused mice.
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)
%%freezing
Freezing
30
20
P<0.05
10
0
Baseline
24h
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 synaptic
and memory dysfunctions by
amyloid-β elevation.
-The quinoline scaffold was selected
for high potency and selectivity of
known 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.
Acknowledgments
O. Arancio
F. Aziz
M. Sakurai
M. Fa’
A. Staniszewski
E. Leznik
Y. Feng
F. Trinchese
B. Lee
I. Francis
H. Zhang
I. Orozco
B. Gong
L. Privitera F. Michelassi
G.
S. Varhade
D. Puzzo
Hashimot
o
Columbia
University
Columbia University
D.W. Landry
M. Shelanski
Shi Xian Deng
UKY
C.G. Zhan
Columbia U.
U. Catania
K. Duff
A. Palmeri
U. of
Minnesota
K. HsiaoAshe
This work was supported by:
NIH (NS49442, AG027468), ADDF.
Based on SAR analysis
we propose new scaffolds
meeting the following
criteria:
1) A fused ring system with an H-bond acceptor
or donor
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