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The Biological Basis for the Cardiovascular Consequences of COX-2 Inhibition
Tilo Grosser
Institute for Translational Medicine and TherapeuticsUniversity of Pennsylvania
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Membrane phospholipids
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Differential Inhibition of COX isoforms
Grosser et al., JCI, 2006;116(1):4-15
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Selectivity for COX-2 is a continuous, rather than a discrete variable
FitzGerald and Patrono NEJM 2002
COX-1
CO
X-2
COX-1 selectivity
COX-2 selectivity
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A large fraction of human prostacyclin biosynthesis is COX-2 dependent
1PGI-M = 2,3-dinor-6-keto-PGF1 ; † P<0.01 vs Placebo; *P<0.05 vs Placebo.
Catella-Lawson et al.JPET. 1999;289:735.
Indomethacin50 mg tid
Placebo Rofecoxib50 mg qd
†
†
0
40
80
120
160
n=12 n=12 n=10
PG
I-M
1 ±
SE
(pg/
mg
Cre
atin
in)
Placebo Celecoxib 400 mg
Ibuprofen 800 mg
0
40
80
120
160
†
*
n=7 n=7 n=7
McAdam et al. PNAS. 1999;96:272
Placebo Celecoxib 200 mg
Rofecoxib25 mg
n=50 n=50 n=500
40
80
120
160
†
†
Fries, Grosser, FitzGerald, Gastroenterology 2006; 130:55
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pg/mg creatinine pg/mg creatinine
2,3-Dinor-6-Keto-PGF12,3-Dinor-TXB2
Controls
Non-cardiac chestpain
Unstable angina
Myocardial infarction
Fitzgerald, D.J., NEJM 1986
Urinary Thromboxane and Prostacyclin metabolites are increased in acute coronary syndromes
[pg/ml] [pg/ml]
3000
2000
1000
0N = 4 6 16 14
3000
2000
1000
0N = 4 6 16 14
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TxA2
COX-1
Thrombosis
Platelet activation and aggregation
COX-1 COX-2
PGI2
Thromboxane A2 amplifies platelet activation and recruits additional platelets to the site of clot formation
??
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Cheng et al., Science 296:593, 2002
Prostacyclin modulates the bioactivity of Thromboxane
* p<0.05 vs. wild type
Common carotid artery injury
flexible wire
7.5
5.0
2.5
0.0IP TP IPTP
Urin
ary
2,3
dino
r T
xB2
(Fol
d ov
er b
asal
)
*
*
wild typeknock-out
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COX-2 disruption and thrombosis
(12.5 25
0
100
200
300 WTCOX-2 KO
COX-2Y385F
Collagen(g)
* *Pla
tele
t co
un
ts (
x103
/ l)
**
Platelet depletion
(14)
(12)(10)
* *
0
20
40
60
80
100
TxA
2 a
go
nis
t-in
du
ced
th
rom
bo
sis
(p
erce
nt
mo
rtal
ity)
WT COX-2 KO COX-2Y385F
Mortality
Cheng Y, et al. J Clin Invest. 2006;116:1391-1399.
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Prostacyclin modulates thrombosis dose dependently
+/+
P<0.01
P<0.05
Co
mp
lete
occ
lusi
on
tim
e (m
in)
+/- -/-
IP
0
20
40
60
80
100
120
COX-1 KD
vehicle DFU
P<0.05P<0.01
P<0.05
vehicle DFU
wildtype
Cheng Y, et al. J Clin Invest. 2006;116:1391-1399.
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TxA2
ADP
Thrombin
Thrombosis
COX-2 derived prostacyclin acts as a constraint on all thrombotic stimuli
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COX-2 inhibition augments thrombosis
• COX-2 disruption or inhibition augments the thrombotic response to various stimuli in vivo by depression of prostacyclin biosynthesis.
• All coxibs depress prostacyclin biosynthesis = class effect
• Suppression of COX-2 dependent prostacyclin does not cause spontaneous thrombosis, but augments the response to thrombogenic stimuli
• Hazard from coxibs particularly in those otherwise predisposed to thrombosis ?
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Are there differences in hazard between distinct compounds?
- Selectivity for COX-2
- COX-2 unrelated effects (“off-target effects”)?
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Differential recovery from steady-state inhibition of platelet COX-1 by low-dose Aspirin and Naproxen
Capone et al., Circulation. 2004; 109(12):1468-71
P<.01
Naproxen (N = 9)
Aspirin (N = 8)
P<.01P=.074
1 3 12 24
0
25
50
75
100
% T
hro
mb
oxa
ne
inh
ibit
ion
hours after last dose
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Opposing roles of COX-2 and COX-1 products
in blood pressure regulation and atherogenesis
80
90
100
110
120
130
140
150
** **
Sy
sto
lic
BP
(m
mH
g)
WT
COX-2 KOCOX-2Y385F
WT/Celecoxib
##
COX-1 KD/Celecoxib
Cheng et al., J Clin Invest 116; 1391, 2006
* p<0.05 vs WT; ** p<0.01 vs WT; ## p<0.01 vs WT/Celecoxib
Kobayashi et al, JCI 114: 784-94, 2004
+/+
0
5
10
15P<0.05
P<0.01
% le
sio
n a
rea
TP -/- IP -/-
APOE -/-APOE -/-
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Heterogeneity of response to traditional NSAIDs
Garcia Rodriguez et al 2005
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• Opposing roles of COX-1 and -2 products in thrombosis, blood pressure regulation and atherogenesis
• Non-isoform selective NSAIDs (e.g. naproxen) may afford prolonged platelet inhibition throughout the dosing intervals in some individuals
• COX-2 critical in maintaining renal medullary blood flow under conditions of increased vasoconstrictor tone; Hypertension on NSAIDs relates to inhibition of COX-2
• Predisposition to atherosclerosis and hypertension attenuated by coincident inhibition of COX-1
• Differences in the degree of COX-2 selectivity are likely to affect cardiovascular risk
COX-2 selective NSAIDs vs isoform non-selective NSAIDs
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Are there differences in hazard between distinct compounds?
- Selectivity for COX-2
- COX-2 unrelated effects (“off-target effects”)?
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0.001 0.01 0.1 1 10
Plasma concentration (M)Celecoxib
0.001 0.01 0.1 1 10
Plasma concentration (M)Rofecoxib
COX-1
COX-2
COX-1
COX-2
Selection of the drug concentration in hASMC
hASMC
In vivo
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Veh Cel Rof Veh Cel Rof Veh Cel Rof Veh Cel RofBas
-2 hrs 0 hrs 2 hrs 8 hrs 24 hrs
interleukin 1
All regulated Genes
(FDR <0.1)
relative expression level
low
high
High-thruput screen for off-target genomic effects
0 1 3 4Differentially regulated Genes (FDR <0.1)
3
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Expression profiles of genes differentially regulated by rofecoxib and celecoxib
Affymetrix qRT-PCR Affymetrix qRT-PCR
0 2 8 24 hrs
2
3
4
*
2059
26_a
t (l
og 2
exp
ress
ion)
IL27RA
0 2 8 24 hr
0.0
2.5
5.0
RofecoxibControl
Celecoxib
IL27
R m
RN
A x
107/
18s
rRN
A
0 2 8 24 hrs
2.5
3.0
3.5
4.0
*
2049
46_s
_at
(log
2 e
xpre
ssio
n)
TOP3A
0 hr 2 hr 8 hr 24 hr
12
16
TOP3
A m
RN
A x
107/
18s
rRN
A
PTGIS
0 2 8 24 hrs
13
14
15 *
2081
31_s
_at
(log
2 e
xpre
ssio
n)
0 hr 2 hr 8 hr 24 hr
0
400
800
*
PTG
IS m
RN
A x
107/
18s
rRN
A
0 2 8 24 hrs
7.5
10.0
12.5
*
2187
29_a
t (lo
g 2
expr
essi
on)
LXN
0 hr 2 hr 8 hr 24 hr
10
20
LTX
mR
NA
x10
7/18
s rR
NA
*
0 2 8 24 hrs
2
3
4
*
2184
79_s
_at (
log
2 ex
pres
sion
)
XPO4
0 hr 2 hr 8 hr 24 hr2.5
5.0
7.5
10.0
XPO4
mRN
A x1
07/18
s rRN
A
0 2 8 24 hrs
5
10
15
*
2024
81_a
t (l
og 2
exp
ress
ion)
DHRS3
0 hr 2 hr 8 hr 24 hr
100
200
300
DH
R3
mR
NA
x10
7/18
s rR
NA
*
0 2 8 24 hrs
2.50
2.75
3.00
3.25
IL-1
*
2130
54_a
t (l
og 2
exp
ress
ion)
KIAA0841
0 hr 2 hr 8 hr 24 hr
1
2
3
4
5
6
7
KIA
A08
41 m
RN
A x
107/
18s
rRN
A
IL-1
0 2 8 24 hrs
10.0
12.5
15.0
*20
1010
_s_a
t (lo
g 2
expr
essi
on)
TXNIP
0 hr 2 hr 8 hr 24 hr
1000
2000
TXNI
P m
RNA
x107
/18s
rRNA *
0 2 8 24 hrs
1.75
2.25
2.75
*
2130
54_a
t (lo
g 2
expr
essi
on)
PIK3C2B
0 hr 2 hr 8 hr 24 hr
0.0
2.5
5.0
PIK
3C2B
mR
NA
x10
7/18
s rR
NA
GALNT12
0 2 8 24 hrs
7
8
9
10
IL-1
2188
85_s
_at (
log
2 ex
pres
sion
) *
0 hr 2 hr 8 hr 24 hr
5
10
15
20
25
IL-1
GA
LNT1
2 m
RN
A x
107/
18s
rRN
A
*
0 2 8 24 hrs
3
4
5
6*
2071
43_a
t (l
og 2
exp
ress
ion)
CDK6
0 hr 2 hr 8 hr 24 hr
100
200
CD
K6
mR
NA
x10
7/18
s rR
NA
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Placebo Celecoxib Rofecoxib 200 mg 25 mg
post dose / pre dose ratio
low(decrease)
high (increase)
compounds
known: 92unknown: 137
median post / pre dose ration = 50 healthy subjects
Metabolomic plasma profiles of celecoxib, rofecoxib are more similar than any drug to placebo
Metabolon Inc.Robert Mohney Felice de Jong
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Can individuals at risk for CV complications be identified early during treatment?
Towards an individualization of NSAID therapy
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Interindividual variability in the pharmacological response to COX-2 inhibition
0.2
0.5
1
2
5
10
20
Co
x-2
sele
ctiv
ity
(CO
X-2
in
hib
itio
n /
CO
X-1
in
hib
itio
n)
Fries, Grosser, FitzGerald, Gastroenterology, 2006
Placebo
n=50
celecoxib (200 mg)
n=50
rofecoxib(25 mg)
n=50
Attained COX-2 selectivityAttained COX-2 selectivity
100
200
300
400500600700
Se
rum
Tx
B2
0 4 SS 0 SS 4 hours
COX-1 activity ex vivo
10
20
30
4050
Se
rum
PG
E2
0 4 SS 0 SS 4
Single dose steady statehours
Single dose steady state
COX-2 activity ex vivo
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0 4 8 12 24 36
0
1000
2000
3000
Ce
lec
ox
ib [
ng
/ml]
0 4 8 12 24 36
0
1000
2000
3000
Time after last dose (hrs)
Ce
lec
ox
ib [
ng
/ml]
Time after last dose (hrs)
CYP2C9*3 +/+
CYP2C9*3 +/-
Genetic contribution to interindividual variability (i)
Fries, Grosser, FitzGerald, Gastroenterology, 2006
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Cardiovascular complications from inhibition of COX-2 relate most plausibly to Inhibition of COX-2 derived prostacyclin formation.
Prostacyclin acts as a constraint on all thrombotic stimuli.
The cardiovascular hazard pertains to all coxibs and likely to traditional NSAIDs with high COX-2 selectivity.
Hazard would be expected to relate to - baseline cardiovascular risk- attained drug selectivity in vivo- dose and duration of exposure- interindividual differences in drug response.
High interindividual variability in the pharmacological response to coxibs.Genetic variability in PK and PD contributes to variance.
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Garret A. FitzGeraldSusanne Fries Yan ChengYing YuDairong WangEmanuela Ricciotti