The Biological Basis for the Cardiovascular Consequences of COX-2 Inhibition

Tilo Grosser

Institute for Translational Medicine and TherapeuticsUniversity of Pennsylvania

Membrane phospholipids

Differential Inhibition of COX isoforms

Grosser et al., JCI, 2006;116(1):4-15

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

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

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

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

??

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

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.

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.

TxA2

ADP

Thrombin

Thrombosis

COX-2 derived prostacyclin acts as a constraint on all thrombotic stimuli

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 ?

Are there differences in hazard between distinct compounds?

- Selectivity for COX-2

- COX-2 unrelated effects (“off-target effects”)?

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

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 -/-

Heterogeneity of response to traditional NSAIDs

Garcia Rodriguez et al 2005

• 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

Are there differences in hazard between distinct compounds?

- Selectivity for COX-2

- COX-2 unrelated effects (“off-target effects”)?

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

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

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

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

Can individuals at risk for CV complications be identified early during treatment?

Towards an individualization of NSAID therapy

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

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

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.

Garret A. FitzGeraldSusanne Fries Yan ChengYing YuDairong WangEmanuela Ricciotti