COX-2, eicosanoids and the resolution of inflammation

Paul Colville-Nash

Department of Experimental Pathology

William Harvey Research Institute

Spector, W.G. & Willoughby, D.A. (1968) Pharmacology of Inflammation.

1500 B.C.Ebers papyrus recommended

dried leaves of myrtle to expel rheumatic pains from womb

In Roman times in Asia,

China, the Americas and Africa, salicylate-containingplants were used

Salicylic acid was synthesized in 1859 by Hermann Kolbe at Marburg University in Germany. His student, von Heyden, adapted the synthesis for industrial production in 1874.

In 1876 the anti-rheumatic effect of salicylic acid was demonstrated in a clinical trial.

Aspirin consumption worldwide

15x1012

tablets per year or 45,000 tons per year(information from Bayer AG)

Proposals for Mode of Action of Aspirin-like Drugs

• Northover and Subramanian (1961) Inhibit kallikrein• Whitehouse (1962) Interfere with oxidative metabolism• Smith MJH (1966) Stabilise capiliary permeability• Collier HOJ (1969) Block a route to mediator receptors,

or block release of an intermediate• McArthur (1971) Displace endogenous anti inflammatory

peptides from plasma proteins• Di Rosa et al (1971) Interfere with migration of leukocytes• Barker (1971) Hyperpolarise nerve membranes• Northover (1971, 1973) Inhibit Ca++ uptake or binding to cellular

membranes• Chang et al (1972) Inhibit leukocyte phagocytosis• Ignarro (1972) Stabilise lysosomal membranes• Sharma (1972) Inhibit generation of lipoperoxides

1960s• Prostaglandins are a family of potent lipid mediators

derived from arachidonic acid• They are made by all cells in the body except the red blood

cells

Roles of Prostaglandins in the 1970’s

• Pyretic (Feldberg & Gupta, 1973; Milton & (Feldberg & Gupta, 1973; Milton & Wendlandt, 1973)Wendlandt, 1973)

• Pro-inflammatory (Willis, 1969)(Willis, 1969)

• Hyperalgesic (Ferreira, 1972)(Ferreira, 1972)

• Inhibit gastric acid secretion (Robert, 1968)(Robert, 1968)

• Contract the uterus (Bergstrom et al .,1968)(Bergstrom et al .,1968)

• Increase renal blood flow (Lonigro et al., 1973)(Lonigro et al., 1973)

1971Discovered that aspirin and similar drugs inhibit the biosynthesis of prostaglandins proposed that this was their mode of action

Inhibition(%)

Indomethacin

Aspirin

Salicylic acid

Log concentration (µg/mL)(Vane, 1971)

100

80

60

40

20

0

0.1 1.0 10 100 1000

4

3

1

2

31 1

33

4

4

1

Clues to COX-2

Flower and Vane (1972) found paracetamol more active

on brain COX than on spleen COX.

“Our results support the idea that a study of

prostaglandins synthetase systems from different systems

will lead to aspirin-like drugs with a greater specificity of

action.”

Clues to COX-2

“Selective inhibition of prostaglandin production in inflammatory exudates and the gastric mucosa.”

Whittle et al. Nature, London (1980)

?COX selectivity to explain the lack of toxicity of

salicylate and BW755 on the stomach.

Discovery of COX-2• Cells may contain two pools of COX, a constitutive COX

enzyme and a different COX which is LPS-inducible and the expression of which is sensitive to glucocorticoid inhibition

Fu, Masferrer, Seibert, Raz and NeedlemanJ Biol Chem, 1990

• In 1991, Dan Simmons published the structure of a protein encoded by an early response gene which was 60% homologous with COX in ram seminal vesicles

Xie et al. Proc. Natl. Acad. Sci. USA88 2692-2696 April 1991

• The three dimensional structure of COX-1 has been published by Garavito et al. and that of COX-2 by Browner et al.

Comparison of NSAID Binding SitesCOX-1 COX-2

M. Browner, et al, 1998Roche Bioscience

Aspirin-like drugs are anti-inflammatory by inhibition of prostaglandin biosynthesis by COX-2 and are

ulcerogenic through inhibition of COX-1

COX-1constitutive

PhysiologicalStimulus

PGI2

endothelium

stomach mucosa

COX-2Induced

Macrophages/Other Cells

PGs

Inflammation

InflammatoryStimulus

Proteases Other Inflammatory

Mediators

PGE2

KidneyTXA2

platelets

Summary of COX Selectivities in WHRI Blood / A549 Assay

COX-1 selectiveCOX-2 selectiveL7

4533

7ni

mes

ulid

eN

S398

mel

oxic

amce

leco

xib

etod

olac

sulin

dac

salic

ylat

edi

clof

enac

Ibup

rofe

n

Difl

unis

al

Asp

irin

Flur

bipr

ofen

(IC

50 r

atio

(C

OX

-2/C

OX

-1) 100

1

0.1

0.01

0.001

10

keto

prof

enIn

dom

etha

cin

Tolm

etin

Nap

roxe

n

Rhe um a to id Arthritis - Pa tho lo g ic a l C ha ng e s

C a rtila g e De struc tio n

No rm a l Syno vium Infla m e d syno vium

C linic a l Dise a se

NSAIDs clinically efficacious: reduce joint pain and swelling

But

NSAIDs may promote joint damage: • Newman and Ling, Lancet, 1985; • De Brito et al, Br J Rheum, 1986• Brandt, Am J Med, 1987; • Bottomley et al, Br J Pharm, 1988; • Pettipher et al, Ann Rheum Dis, 1989; • Bulstra et al, Clin Orthop, 1992.

NSAIDs and Joint Destruction:

A role in the resolution of inflammation?

Cyclooxygenase (COX)

A key enzyme in prostaglandin synthesis

Two isoforms: COX-1, constitutive, maintenance of physiological processes COX-2, inducible, major target for new generation NSAIDs

with reduced side effects e.g. less injurious to normal gastric mucosa

BUT

COX-2 associated with wound healing phase of gastric ulcersCOX-2 selective inhibitors delay healing of gastric ulcers in rodents

Willoughby, D.A. (1975). Annals of Rheumatic Disease, 34.

H o u rs

2 6 1 2 2 4 4 8

0

1 0 0

5 0

7 5

2 5

0 0

0 .5 0 .5

1 .0 1 .0

1 .5 1 .5

2 .0 2 .0

Infl

amm

ator

y ex

udat

e vo

lum

e /m

l

Total Inflamm

atory cell number / 10

6

CO

X-2 protein expression / arbitrary unitsT im e c o u rse o f c h a n g e s in in f la m m a to ry e x u d a te v o lu m e ,in f la m m a to ry c e ll n u m b e r a n d C O X -2 p ro te in e x p re s s io n .

Con

trol

Con

trol0 .1 1 .0 1 0 .0

N S -3 9 8m g /k g

0 .1 1 .0 1 0 .0

N S -3 9 8m g /k g

****

**

***

0 .3 1 .0 3 .0

In d o m e th a c inm g /k g

0 .3 1 .0 3 .0

In d o m e th a c inm g /k g

**

**

***

*** **

*

***

0

0 .1

0 .2

0 .3

0 .4

0 .5

Exu

date

vol

ume

/ml

0

2

4

6

8

Tota

l inf

lam

mat

ory

cell

num

ber

/106

E ffe c ts o f C O X in h ib ito rs o n in f la m m a to ry ex u d a te v o lu m e a n d in f la m m a to ry c e ll n u m b e r a t 2 h o u rs p o s t- in d u c tio n o f ca rrag ee n an p le u risy

Con

trol

Con

trol

0

0 .2

0 .4

1 .2

1 .0

0 .8

0 .6

Exu

date

vol

ume

/ml

0

2 0

4 0

6 0

8 0

Tota

l inf

lam

mat

ory

cell

num

ber

/106

E ffe c ts o f C O X in h ib ito rs o n in f la m m a to ry ex u d a te v o lu m e a n d in f la m m a to ry c e ll n u m b e r a t 4 8 h o u rs p o s t- in d u c tio n o f c a rra g e e n a n p le u risy

0 .1 1 .0 1 0 .0

N S -3 9 8m g /k g

0 .1 1 .0 1 0 .0

N S -3 9 8m g /k g

**

*

0 .3 1 .0 3 .0

In d o m e th a c inm g /k g

0 .3 1 .0 3 .0

In d o m e th a c inm g /k g

***

2 6 1 2 2 4 4 8H o u rs

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

[PG

E] (pg/m

l exudate) 2

CO

X a

ctiv

ity

2 6 1 2 2 4 4 8

[PG

D]

pg/m

l exu

date

2[15deoxy

PG

J] pg/m

l exudate

12-142

T im e a fte r in d u c tio n /h o u rs

T im e c o u rse o f c h a n g e s in e x u d a te co n c e n tra tio n s o f P G D a n d 1 5 d e o x y P G J d u rin g a c a rra g e e n a n - in d u c e d p le u risy.

2

21 2 -1 4

[PG

D]

pg/m

l exu

date

2

[15d

eoxy

PGJ

] pg

/ml e

xuda

te12

-14

2*** **

*

***

***

*** **

*

***

***

***

***

***

***

E ffe c ts o f C O X in h ib ito rs o n e x u d a te c o n c e n tra tio n s o f P G D a n d 1 5 d e o x y P G J 4 8 h o u rs p o s t- in d u c tio n o f c a rra g e en a n p le u risy.

2

21 2 -1 4

E ffec ts o f rep lacem en t o f P G D an d 1 5 deo xy P G J o n ex ace rba tio n o f in flam m atio n b y N S -39 8 trea tm en t

a t 4 8h o u rs p o st-in d u ctio n o f c arrag eenan p leu risy.

2 212 -1 4

Summary

•Initial peak in COX-2 protein expression at 2hours, associated with maximal PGE2 synthetic activity ex vivo and raised PGE2 levels in exudates

•Second greater peak in COX-2 protein expression at 48hours during resolution associated with minimal PGE2 synthetic activity ex vivo and minimal levels of PGE2 in exudates

•Non-selective and selective COX-2 inhibitors exacerbate inflammation if given during resolution phase of carrageenan pleurisy

•Second peak of COX-2 protein expression associated with raised levels of PGD2 and 15deoxy12-14PGJ2, these prostanoid's levels reduced on treatment with COX inhibitors

•Replacement of these prostanoids during COX inhibitor treatment reverses exacerbation of inflammation at 48hours

Inducible cyclooxygenase (COX-2) mayhave anti-inflammatory properties

Derek Gilroy, Paul Colville-Nash, Dean Willis, Joanne Chivers,Mark Paul-Clark and Derek Willoughby

Department of Experimental PathologyWilliam Harvey Research Institute

Barts and The London, Queen Mary’s School of Medicine and DentistryCharterhouse SquareLondon, EC1M 6BQ.

United Kingdom

Nature Medicine, 5, p698, 1999.

NF-B

3 6

6h

24 48

48h

S SN NRel

A

Rel

A

cRel

cRel

p50

p50

Time (h)EMSA

Leukocyte NF-B activity

Infl

am

mat

ory

ce

lls (

10

)6

0.0

0.5

1.0

1.5

Exu

dat

e (

ml)

24h24h concon PDTCPDTC0

100

200

******

0.0

0.5

1.0

1.5

Exu

dat

e (m

l)

24h24h concon MG132MG132 Infl

amm

ato

ry c

ells

(10

)6

0

100

200

******

******

Possible role for NF-B in theresolution of inflammation

Toby Lawrence, Derek W Gilroy, Paul Colville-Nashand Derek A Willoughby

Department of Experimental PathologyWilliam Harvey Research Institute

Barts and The London, Queen Mary’s School of Medicine and DentistryCharterhouse SquareLondon, EC1M 6BQ.

United Kingdom

Nature Medicine, 7(12), p1291, December 2001.

Apoptosis overrides survival signals through a caspase-mediated dominant-negative NF-B loop. Levkau et al. Nat. Cell Biol. 1:227 (1999)Ravi et al. Cancer Res. 58:882 (1998) Kang et al. J. Biol. Chem. 276:24638 (2001)

RHD

NLS TA1 TA2

RelA (p65) Caspase 3

Endogenous 15deoxy12-14PGJ2 regulates leukocyte apoptosis in vivo

0

25

50

75

0

5

10

15

****

**

Indom ethacinNS39815d PGJ12-14

2

-- -- -- -- -- -- ----

-- ++ ++++++ ++

++

Tota

l leu

kocy

tes

(10

)6

Ap

op

toti

c c

ells

(%

)

****

**

Proinflammatory cytokines (e.g.IL-1, TNF, IL-6)

Matrix metalloproteases (e.g. MMP-1, MMP-2)

Inflammatory cell adhesion molecules (e.g. ICAM-1)

Inflammatory enzyme systems (e.g. iNOS)

15deoxy12-14PGJ2

X X

X

X

X

X

X

NSAID

COX-1, COX-2, and COX-3 and the future treatment of chronic inflammatory disease

Derek A Willoughby, Adrian R Moore and Paul Colville-Nash

Department of Experimental Pathology, William Harvey Research InstituteBarts and The London, Queen Mary’s School of Medicine and Dentistry

Charterhouse Square, London, EC1M 6BQ. United Kingdom

Lancet, 355 (9204), p646, 2000.

•Treatment during disease flare with NSAIDs beneficial

•Treatment during periods of disease remission perhaps less desirable

•Options: a) Stop NSAID treatment during remission - difficultb) Replace endogenous mechanisms - not available yetc) Block reactivated pro-inflammatory systems

Alternative approaches

Arachidonic acid release and metabolism

Arachidonic acid

O2H

OH

COOHO

O

PGG2

COOHO

O

PGH2

Cyclooxygenase

COOH

OH

O

OH

PGE2

COOH

OH

OH

OH

PGF2

COOH

OH

O

O

TxA2

OH

COOH

OH

O

PGI2

COOH

OH

OH

O

PGD2

COOH

O

15deoxy12-14PGJ2

dehy

drat

ion

hPGD2S

mPGE 2S/cP

GE 2S

PG

I 2 syn

thas

e

PGF2 synthase

TxA2 synthase

iPLA2 sPLA2cPLA2

5-, 12- & 15 lipoxygenaseCytochrome p450