Transcript Document

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-containing
plants 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
12
15x10 tablets per year or 45,000 tons per year
(information from Bayer AG)
Proposals for Mode of Action of
Aspirin-like Drugs
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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 &
Wendlandt, 1973)
• Pro-inflammatory (Willis, 1969)
• Hyperalgesic (Ferreira, 1972)
• Inhibit gastric acid secretion (Robert, 1968)
• Contract the uterus (Bergstrom et al .,1968)
• Increase renal blood flow (Lonigro et al., 1973)
1971
Discovered that aspirin and similar drugs inhibit the
biosynthesis of prostaglandins proposed that this was their
mode of action
Inhibition
(%)
100
Indomethacin
1
1
3
4
80
1
Aspirin
60
3
3
40
20
3
2
4
1
Salicylic acid
4
0
0.1
1.0
10
Log concentration (µg/mL)
100
1000
(Vane, 1971)
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 LPSinducible and the expression of which is sensitive to
glucocorticoid inhibition
Fu, Masferrer, Seibert, Raz and Needleman
J 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. USA
88 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 Sites
COX-1
M. Browner, et al, 1998
Roche Bioscience
COX-2
Aspirin-like drugs are anti-inflammatory by inhibition of
prostaglandin biosynthesis by COX-2 and are
ulcerogenic through inhibition of COX-1
Physiological
Stimulus
Inflammatory
Stimulus
Macrophages/Other Cells
COX-1
constitutive
COX-2
Induced
TXA2
PGI2
PGE2
platelets
endothelium
Kidney
Proteases
PGs
stomach mucosa
Inflammation
Other
Inflammatory
Mediators
(IC50 ratio (COX-2/COX-1)
Summary of COX Selectivities in
WHRI Blood / A549 Assay
100
10
1
0.1
0.01
0.001
COX-2 selective
COX-1 selective
Rheumatoid Arthritis - Pathologic al Changes
Normal Synovium
Inflamed synovium
Cartilage Destruction
Clinic al Disease
NSAIDs and Joint Destruction:
NSAIDs clinically efficacious: reduce joint pain and swelling
But
NSAIDs may promote joint damage:
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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.
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 ulcers
COX-2 selective inhibitors delay healing of gastric ulcers in rodents
Willoughby, D.A. (1975). Annals of Rheumatic Disease, 34.
100
1.5
75
1.0
50
0.5
25
2.0
1.5
1.0
0.5
6
0
0
2
6
12
24
Hours
48
0
COX-2 protein expression / arbitrary units
2.0
Total Inflammatory cell number / 10
Inflammatory exudate volume /ml
Time course of changes in inflammatory exudate volume,
inflammatory cell number and COX-2 protein expression.
***
0.1
0
0.3 1.0 3.0
0.1 1.0 10.0
Indomethacin
mg/kg
NS-398
mg/kg
4
**
***
0.2
*
***
***
0.3
6
**
***
**
**
*
8
2
0
Control
0.4
Control
Exudate volume /ml
0.5
Total inflammatory cell number /106
Effects of COX inhibitors on inflammatory exudate volume and inflammatory
cell number at 2hours post-induction of carrageenan pleurisy
0.3 1.0 3.0
Indomethacin
mg/kg
0.1 1.0 10.0
NS-398
mg/kg
0.6
0.4
0.2
0
0.3 1.0 3.0
0.1 1.0 10.0
Indomethacin
mg/kg
NS-398
mg/kg
80
60
40
20
0
Control
**
0.8
Control
Exudate volume /ml
1.0
Total inflammatory cell number /106
1.2
*
***
Effects of COX inhibitors on inflammatory exudate volume and inflammatory
cell number at 48hours post-induction of carrageenan pleurisy
0.3 1.0 3.0
Indomethacin
mg/kg
0.1 1.0 10.0
NS-398
mg/kg
4000
COX activity
2000
1000
0
2
6
12
24
Hours
48
[PGE2] (pg/ml exudate)
3000
Time course of changes in exudate concentrations of PGD2 and
15deoxy12-14PGJ2 during a carrageenan-induced pleurisy.
[PG D2] pg/ml exudate
[15deoxy12-14PGJ2] pg/ml exudate
2
6
12
24
Time after induction /hours
48
***
***
***
***
***
***
[15deoxy12-14PGJ2] pg/ml exudate
***
***
***
***
***
***
[PG D2] pg/ml exudate
Effects of COX inhibitors on exudate concentrations of PGD2 and
12-14
15deoxy PGJ2 48hours post-induction of carrageenan pleurisy.
Effects of replacement of PGD2 and 15deoxy12-14PGJ2
on exacerbation of inflammation by NS-398 treatment
at 48hours post-induction of carrageenan pleurisy.
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
15deoxy12-14PGJ2, 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) may
have anti-inflammatory properties
Derek Gilroy, Paul Colville-Nash, Dean Willis, Joanne Chivers,
Mark Paul-Clark and Derek Willoughby
Department of Experimental Pathology
William Harvey Research Institute
Barts and The London, Queen Mary’s School of Medicine and Dentistry
Charterhouse Square
London, EC1M 6BQ.
United Kingdom
Nature Medicine, 5, p698, 1999.
Leukocyte NF-B activity
EMSA
6h
48h
Time (h)
3 6 24 48
NF-B
S N
S N
200
6
Inflammatory cells (10 )
***
1.0
0.5
0.0
24h
con
PDTC
6
1.5
Exudate (ml)
200
Inflammatory cells (10 )
Exudate (ml)
1.5
***
1.0
0.5
0.0
24h
con
MG132
***
100
0
24h
con
PDTC
***
100
0
24h
con
MG132
***
***
Possible role for NF-B in the
resolution of inflammation
Toby Lawrence, Derek W Gilroy, Paul Colville-Nash
and Derek A Willoughby
Department of Experimental Pathology
William Harvey Research Institute
Barts and The London, Queen Mary’s School of Medicine and Dentistry
Charterhouse Square
London, EC1M 6BQ.
United Kingdom
Nature Medicine, 7(12), p1291, December 2001.
RelA (p65)
Caspase 3
RHD
NLS
TA1
TA2
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)
75
50
**
**
*
*
25
0
Indomethacin
NS398
15d12-14PGJ2
-- -+ -+ -+ -+
- - + - +
Apoptotic cells (%)
Total leukocytes (106)
Endogenous 15deoxy12-14PGJ2 regulates
leukocyte apoptosis in vivo
15
**
**
*
*
10
5
0
-- -+ -+ -+ -+
- - + - +
NSAID
15deoxy12-14PGJ2
Proinflammatory cytokines
(e.g.IL-1b, TNFa, IL-6)
Matrix metalloproteases
(e.g. MMP-1, MMP-2)
Inflammatory cell adhesion molecules
(e.g. ICAM-1)
Inflammatory enzyme systems
(e.g. iNOS)
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 Institute
Barts 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 - difficult
b) Replace endogenous mechanisms - not available yet
c) Block reactivated pro-inflammatory systems
Alternative approaches
Arachidonic acid release and metabolism
iPLA2
cPLA2
sPLA2
Arachidonic acid
Cytochrome p450
5-, 12- & 15 lipoxygenase
PGG2
O
COOH
O
O2H
OH
PGH2
PGD2
hPGD2S
Cyclooxygenase
O
COOH
O
TxA2
COOH
OH
dehydration
OH
O
COOH
OH
PGI2
COOH
OH
O
PGE2
15deoxy12-14PGJ2
COOH
O
PGI2 synthase
O
PGF2a
OH
COOH
COOH
O
OH
OH
O
OH
OH
OH