COX-2, eicosanoids and the resolution of inflammation

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COX-2, eicosanoids and the resolution of
inflammation
Paul Colville-Nash Department of Experimental
Pathology William Harvey Research Institute
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Spector, W.G. Willoughby, D.A. (1968)
Pharmacology of Inflammation.
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1500 B.C. Ebers papyrus recommended dried leaves
of myrtle to expel rheumatic pains from womb
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• In Roman times in Asia,
• China, the Americas and Africa,
  salicylate-containingplants were used

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• 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.

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• Aspirin consumption worldwide
• 15x1012 tablets per year or 45,000 tons per year
• (information from Bayer AG)

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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

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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 1970s

• 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)

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1971

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

Indomethacin
Inhibition ()
100
1
1
3
4
80
1
Aspirin
60
3
3
Salicylic acid
40
3
2
1
4
4
20
0
0.1
1.0
10
100
1000
Log concentration (µg/mL)
(Vane, 1971)
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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.
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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.
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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

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

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Comparison of NSAID Binding Sites
COX-2
COX-1
M. Browner, et al, 1998Roche Bioscience
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• Aspirin-like drugs are anti-inflammatory by
  inhibition of prostaglandin biosynthesis by COX-2
  and are ulcerogenic through inhibition of COX-1

PhysiologicalStimulus
InflammatoryStimulus
Macrophages/Other Cells
COX-1 constitutive
COX-2 Induced
PGI2 endothelium stomach mucosa
PGE2 Kidney
TXA2 platelets
PGs
Proteases
Other Inflammatory Mediators
Inflammation
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Summary of COX Selectivities in WHRI Blood /
A549 Assay
100
COX-1 selective
COX-2 selective
10
1
(IC50 ratio (COX-2/COX-1)
0.1
0.01
0.001
NS398
celecoxib
etodolac
sulindac
salicylate
Tolmetin
L745337
diclofenac
nimesulide
meloxicam
Naproxen
Aspirin
Ibuprofen
ketoprofen
Flurbiprofen
Diflunisal
Indomethacin
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NSAIDs and Joint Destruction

• 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.

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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
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Willoughby, D.A. (1975). Annals of Rheumatic
Disease, 34.
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• 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
  15deoxyD12-14PGJ2, these prostanoid's levels
  reduced on treatment with COX inhibitors
• Replacement of these prostanoids during COX
  inhibitor treatment reverses exacerbation of
  inflammation at 48hours

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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 Marys
School of Medicine and Dentistry Charterhouse
Square London, EC1M 6BQ. United Kingdom Nature
Medicine, 5, p698, 1999.
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Leukocyte NF-?B activity
EMSA
6h
48h
l
A
A
l
Time (h)
e
e
0
0
l
l
e
e
5
5
R
R
R
R
p
p
3
6
24
48
c
S
S
N
N
c
NF-?B
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)
1.5
200
6
0
1
(


)
s
l
l
m
l

1.0
e
(

c
e

t
y
100
a
r
d
o
t
u
0.5
a
x
m
E
m
a
l
f
0.0
0
n
24h
con
PDTC
I
24h
con
PDTC
)
1.5
200
6
0
1
(

)

l
s
m
l

l
(
e
1.0

c
e

t
y
a
100
r
d
o
u
t
0.5
x
a
E
m
m
a
l
f
0.0
0
n
I
24h
con
MG132
24h
con
MG132
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Possible role for NF-kB 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
Marys School of Medicine and Dentistry Charterhou
se Square London, EC1M 6BQ. United
Kingdom Nature Medicine, 7(12), p1291, December
2001.
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Apoptosis overrides survival signals through a
caspase-mediated dominant-negative NF-?B loop.
Levkau et al. Nat. Cell Biol. 1227 (1999) Ravi
et al. Cancer Res. 58882 (1998) Kang et al. J.
Biol. Chem. 27624638 (2001)
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Endogenous 15deoxy?12-14PGJ2 regulates leukocyte
apoptosis in vivo




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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)
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• 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 Marys 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

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Alternative approaches
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Arachidonic acid release and metabolism