Upload
zared
View
32
Download
0
Embed Size (px)
DESCRIPTION
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 - PowerPoint PPT Presentation
Citation preview
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