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