ARTICLE IN PRESS

Journal of Thermal Biology 31 (2006) 208–219

www.elsevier.com/locate/jtherbio

Review

Cyclooxygenase: Past, present and future. A tribute to John R. Vane

(1927–2004)
Regina M. Botting
The William Harvey Research Institute, The John Vane Science Centre, St. Bartholomew’s and the London School of Medicine and Dentistry,
Queen Mary, University of London, Charterhouse Square, London EC1M 6BQ, UK
Accepted 22 November 2005

Abstract

Study of the prostaglandins led directly to the elucidation of the mode of action of aspirin which inhibits their synthesis. John Vane
elegantly demonstrated in 1971 that non-steroid anti-inflammatory drugs (NSAIDs) blocked cyclooxygenase (COX), the enzyme which
makes prostaglandins. In 1991, Daniel Simmons described the gene which expresses a second cyclooxygenase, COX-2. This discovery
explained the anti-inflammatory actions of NSAIDs, inhibition of COX-2, and their side actions, inhibition of COX-1. Within 8 years
selective COX-2 inhibitors became available for the treatment of inflammation without the disadvantage of gastric toxicity. Recently, a
COX-1 variant protein, named COX-3, sensitive to inhibition with acetaminophen, was characterised, cloned and expressed.
r 2005 Elsevier Ltd. All rights reserved.

Keywords: Prostaglandin; Cyclooxygenase-1; Cyclooxygenase-2; Cyclooxygenase-3; Non-steroid anti-inflammatory drugs; Rofecoxib; Celecoxib;
Lumiracoxib; Valdecoxib; Etoricoxib; Acetaminophen

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
2. Cyclooxygenase-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
3. Cyclooxygenase-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
4. Functions of COX-1 and COX-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
4.1. Cardiovascular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
4.2. Gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
4.3. Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
4.4. Central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
4.5. Reproduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
5. Inhibitors of COX-1 and COX-2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
5.1. The non-steroid anti-inflammatory drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
5.2. Selective COX-2 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
6. Other cyclooxygenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Fax: +44 208 677 9671.

E-mail address: r.m.botting@qmul.ac.uk.

0306-4565/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jtherbio.2005.11.008
 ARTICLE IN PRESS
R.M. Botting / Journal of Thermal Biology 31 (2006) 208–219 209

1. Introduction

Cyclooxygenase (COX) or prostaglandin endoperoxide

synthase (PGHS) is the key enzyme in the synthesis of
prostaglandins from their precursor, arachidonic acid.
Prostaglandins are lipid mediators made by most cells in
the body except for red blood cells and released by almost
any type of chemical or mechanical stimulus. They produce
a remarkably broad spectrum of effects that embraces
practically every biological function as well as being of
prime importance as mediators of pain, fever and swelling
in inflammation. Inhibition of their biosynthesis by the
non-steroid anti-inflammatory drugs (NSAIDs) causes
major changes to the pathophysiological functions of the
organism.
More than 70 years ago, two American gynaecologists,
Kurzrok and Lieb (1930) observed that strips of human
uterus either relaxed or contracted when exposed to human
semen. Some years later, von Euler in Sweden reported the
presence of a substance in seminal fluid that contracted
smooth muscle and lowered blood pressure in experimental
animals. He identified the active material as a lipid-soluble
acid and named it ‘‘prostaglandin’’ as it appeared to
originate from the prostate gland (von Euler, 1936). He
was wrong in this assumption since most of the prosta-
glandins are made in the seminal vesicles. Technical
advances in the 1960s allowed the demonstration that
‘‘prostaglandins’’ were a family of lipid compounds of Fig. 1. The arachidonic acid cascade. The precursor of the prostaglandins,
unique structure. More than 100 related substances derived arachidonic acid, is cleaved from cell membrane phospholipids by
from arachidonic acid and synthesised by COX have now phospholipase A2. It is then converted by COX-1 or COX-2 into unstable
been identified. endoperoxides, PGG2 and PGH2. The endoperoxide, PGH2 is metabolised
Prostaglandin E1 (PGE1) and PGF1a were isolated and by synthases to primary prostaglandins PGD2, PGE2 and PGF2a as well as
TXA2 and PGI2 (prostacyclin). TXA2 is found mostly in platelets and
their structures elucidated in 1962 (Bergström et al., 1962) PGI2 mainly in endothelial cells and in the stomach mucosa. PGE2 is made
and in 1964, PGE2 was synthesised from arachidonic acid in the kidney and at sites of inflammation. TXA2 is metabolised to the
using an enzyme preparation from sheep seminal vesicles inactive TXB2 and PGI2 is converted into the inactive metabolite, 6-keto-
(Bergström et al., 1964). Prostaglandins of the D, E and Fa PGF1a. (Reprinted with permission from ‘‘Principles of Immunopharma-
series are referred to as the ‘‘primary prostaglandins’’ and cology’’, Nijkamp, F.P. and Parnham, M.J. eds. 2005. Birkhäuser Verlag,
Basel, Switzerland.)
they are synthesised in a stepwise manner by a complex
system of microsomal enzymes beginning with COX. Thus,
the precursor, arachidonic acid, is first cyclised to form the
endoperoxide derivative, prostaglandin G2 and then the enzyme, prostacyclin synthase. PGI2 has a double-ring
reduced by peroxidase to form prostaglandin H2. These structure, closed by an oxygen bridge between carbons 6
two endoperoxides, which are chemically unstable, are then and 9 (Fig. 1). It is hydrolysed non-enzymatically to a
isomerised enzymatically or non-enzymatically into differ- much less active, stable compound, 6-keto-PGF1a.
ent products, such as PGD2, PGE2 or PGF2a. The presence of the different prostaglandin synthases
The endoperoxide, PGH2, is also metabolised into two varies from tissue to tissue. For example, lung and spleen
unstable and highly biologically active compounds with tissues are able to synthesise the whole range of products
structures that differ from those of the primary prosta- but other tissues cannot; hence, platelets synthesise mainly
glandins. One of these is thromboxane A2 (TXA2), formed TXA2, whereas the blood vessel walls primarily produce
by an enzyme, thromboxane synthase, first isolated from PGI2 (Bunting et al., 1983). PGE2 is an important mediator
platelets (Needleman et al., 1976). TXA2 has a very short in the kidney and in inflammation, whereas PGD2 is made
chemical half-life of about 30 s; it breaks down non- in mast cells and in the brain.
enzymatically into the stable and relatively inactive
thromboxane B2 (TXB2). 2. Cyclooxygenase-1
The other route of metabolism of PGH2 is to prostacy-
clin or prostaglandin I2 (PGI2), yet another unstable In 1976, COX-1 with a molecular mass of 71 kDa was
compound with a half-life of around 3 min, formed by isolated from sheep seminal vesicles (Hemler and Lands,
 ARTICLE IN PRESS
210 R.M. Botting / Journal of Thermal Biology 31 (2006) 208–219
1976) and it was cloned by three separate groups in 1988
(DeWitt and Smith, 1988; Merlie et al., 1988; Yokoyama
et al., 1988). This enzyme exhibits both COX and
hydroperoxidase activities and is derived from an mRNA
of 2.8 kb. Garavito and his colleagues (Picot et al., 1994)
determined the three-dimensional structure of COX-1,
which consists of three independent folding units: an
epidermal growth factor-like domain, a membrane-binding
section and an enzymatic domain (Fig. 2). The sites for
COX and peroxidase activity are adjacent but spatially
distinct. The enzyme integrates into only a single leaflet of
the membrane lipid bilayer and thus the position of the
COX channel allows arachidonic acid to gain access to the
active site from the interior of the bilayer. The COX active
site is a long, hydrophobic channel and Garavito et al.
(Malkowski et al., 2000) provide evidence to suggest that
some of the NSAIDs such as flurbiprofen inhibit COX-1 by
excluding arachidonate from the upper portion of the
channel. Binding sites for arachidonic acid and NSAID,
tyrosine 385 and serine 530, are positioned at the apex of
the long active site whereas arginine 120 lies close to the
opening of the COX channel. There may be other sub-sites
for binding of the precursor in this narrow channel.
COX-1 is the constitutive isoform of COX which has
clear physiological functions. It performs a ‘‘housekeep-
ing’’ function to synthesise prostaglandins which regulate
normal cell activity (Fig. 3). Its activation leads, for
instance, to the production of prostacyclin which, when
released by the endothelium, is anti-thrombogenic (Mon-
cada et al., 1976) and when released by the gastric mucosa,
it is cytoprotective (Whittle et al., 1978). It is also COX-1 in
platelets that leads to TXA2 production, causing aggrega-
tion of the platelets to prevent inappropriate bleeding
(Funk et al., 1991). The concentration of the enzyme
Fig. 2. COX-1 and COX-2 catalytic channels. The hatched areas in COX-1
are those that are more accessible in COX-2 due to amino acid
substitutions. Tyr 385 and Arg 120 function as binding sites for
arachidonic acid and NSAIDs, while Ser 530 is acetylated irreversibly
by aspirin. SP ¼ side pocket; ES ¼ extra space. (Reprinted with permis-
sion from ‘‘Principles of Immunopharmacology’’, Nijkamp, F.P. and
Parnham, M.J. eds. 2005. Birkhäuser Verlag, Basel, Switzerland.)
Fig. 3. The side effects of NSAIDs are caused by inhibition of the
constitutive enzyme, COX-1, which synthesises prostaglandins that serve
essential physiological functions such as causing appropriate platelet
aggregation, protection of the gastric mucosa, inhibition of thrombogen-
esis and maintenance of renal function. The therapeutic effects of NSAIDs
are due to inhibition of COX-2, an enzyme induced by inflammatory
factors released by bacteria, the vascular endothelium or other cells
involved in the inflammatory response. (Reprinted with permission from
‘‘Principles of Immunopharmacology’’, Nijkamp, F.P. and Parnham, M.J.
eds. 2005. Birkhäuser Verlag, Basel, Switzerland.)
largely remains stable, but small (2- to 4-fold) increases in
expression can occur in response to stimulation with
hormones or growth factors (DeWitt, 1991; Wu et al.,
1991).
3. Cyclooxygenase-2
There were various clues in the literature suggesting that
there may be a second COX enzyme. As early as 1972,
Smith and Lands (1972) and Flower and Vane (1972)
speculated on the existence of isoenzymes. Others (Lysz
and Needleman, 1982; Lysz et al., 1988) also suggested two
distinct forms of COX.
Rosen et al. (1989) were studying the regulation of COX
in cultures of epithelial cells from sheep trachea and found
an increase in activity of COX during prolonged cell
culture. The increase in activity was not accounted for by a
coordinate increase in 70 kDa COX protein, and a
corresponding increase in the mRNA of 2.8 kb. However,
they found an increase in a second mRNA of 4.0 kb and
suggested that this was derived from a distinct COX-
related gene which encoded for a protein with COX
activity.
Needleman and his group (Raz et al., 1989; Fu et al.,
1990; Masferrer et al., 1990) reported that bacterial
lipopolysaccharide (LPS) increased the synthesis of pros-
taglandins in human monocytes in vitro and in mouse
peritoneal macrophages in vivo. This increase, but not the
basal level of enzyme, was inhibited by dexamethasone and
associated with de novo synthesis of new COX protein.
This further gave rise to the concept of ‘‘constitutive’’ and
‘‘inducible’’ forms of COX.
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R.M. Botting / Journal of Thermal Biology 31 (2006) 208–219
The breakthrough came from biologists outside the field
of prostaglandins. Simmons and his colleagues were
studying immediate early genes and discovered a gene
encoding a second form of COX, induced by v-src, serum
or phorbol esters in chicken embryo fibroblasts (Simmons
et al., 1989; Xie et al., 1991). It was encoded by a 4.1 kb
mRNA, similar in size to that reported by Rosen et al.
(1989). They cloned the gene, deduced the structure of the
enzyme protein and found it homologous to COX but to
no other known protein. Some months later, Herschmann
and his colleagues (Kujubu et al., 1991) found a similar
gene in the mouse, as did Simmons et al. (1991). Others
confirmed a distinct isoform of COX, COX-2 (O’Banion
et al., 1991; Sirois and Richards, 1992).
Both COX-1 and COX-2 have a molecular weight of
71 kDa and the amino acid sequence of COX-2 shows a
60% homology with the sequence of the non-inducible
enzyme. The mRNA for the inducible enzyme approx-
imates 4.5 kb and that of the constitutive enzyme, 2.8 kb.
The inhibition by the glucocorticoids of the expression of
COX-2 is an additional aspect of the anti-inflammatory
action of the corticosteroids. The levels of COX-2,
normally very low in cells, are tightly controlled by a
number of factors including cytokines, intracellular mes-
sengers and by the availability of substrate. Thus, increased
synthesis of prostaglandins is achieved in inflammation
due to up-regulation of COX-2 by inflammatory stimuli
(Fig. 3).
The three-dimensional structure of COX-2 has also been
published (Luong et al., 1996). It closely resembles the
structure of COX-1, except that the COX-2 active site is
slightly larger and can accommodate bigger structures than
those which are able to fit into the active site of COX-1. A
secondary internal side pocket of COX-2 contributes
significantly to the larger volume of the active site in this
enzyme (Fig. 2), although the central channel is also wider
by approximately 17%.
4. Functions of COX-1 and COX-2
4.1. Cardiovascular system
Blood platelets contain only COX-1, which converts
arachidonic acid to the potent aggregatory and vasocon-
strictor eicosanoid TXA2, the major COX product formed
by platelets. TXA2 has a half-life at body pH and
temperature of 30 s, degrading to inactive TXB2 (Needle-
man et al., 1976). Prostacyclin generated in the vessel wall
may be the physiological antagonist of this system in
platelets. Normal vascular endothelial and smooth muscle
cells express COX-1 which produces mainly prostacyclin.
According to this concept, prostacyclin and TXA2 repre-
sent biologically opposite poles of a mechanism for
regulating platelet–vessel wall interaction and the forma-
tion of haemostatic plugs and intra-arterial thrombi
(Moncada and Vane, 1982). By stimulating IP receptors,
prostacyclin potently inhibits aggregation of platelets and
211
relaxes vascular smooth muscle resulting in vasodilatation.
Both effects are brought about through activation of a Gs
protein, stimulation of adenylate cyclase and accumulation
of cyclic AMP (Tateson et al., 1977; Oliva and Nicosia,
1987). Prostacyclin inhibits platelet aggregation (platelet–
platelet interaction) at much lower concentrations than
those needed to inhibit adhesion (platelet–collagen inter-
action) (Higgs et al., 1978). Thus, prostacyclin may permit
platelets to stick to and interact with damaged vascular
tissue, so allowing platelets to participate in the repair of a
vessel wall while at the same time preventing or limiting
thrombus formation.
Immunocytochemical studies suggest that expression of
COX-2 in normal blood vessels is very low (Crofford et al.,
1994; Schonbeck et al., 1999). However, shear stress
induced by laminar but not by turbulent flow upregulates
COX-2 in vascular endothelial cell cultures (Topper et al.,
1996). In fact, studies in human volunteers given selective
COX-2 inhibitors show, by estimating the prostacyclin
metabolite (2,3-dinor-6-keto-PGF1a) in urine, that most of
the prostacyclin produced by the endothelium derives from
induced COX-2 (Catella–Lawson et al., 1999; McAdam
et al., 1999). However, high levels of COX-2 can be
detected in inflamed tissues, angiogenic microvessels and
atherosclerotic plaques (Sano et al., 1992).
4.2. Gastrointestinal tract
In most species, including humans, the bulk of the
gastroprotective prostaglandins is synthesised by COX-1
although small amounts of COX-2 can also be found
expressed constitutively in the normal rat stomach (Karg-
man et al., 1996). The so-called ‘‘cytoprotective’’ action of
the prostaglandins in preventing gastric erosions and
ulceration is mainly brought about by endogenously
produced prostacyclin and PGE2. Their synthesis can be
demonstrated to occur in every part of the gastrointestinal
tract with the highest concentrations being produced in
gastric smooth muscle and gastric mucosa (Whittle and
Salmon, 1983). This ‘‘cytoprotective’’ action in preventing
gastric damage is complex but depends on the reduction in
secretion of gastric acid (Robert et al., 1967), increase in
gastric mucosal blood flow through vasodilatation of
mucosal blood vessels (Whittle et al., 1978) and increased
secretion of viscous mucus (Johansson and Kollberg, 1979)
and of duodenal bicarbonate thus effectively neutralising
stomach acid production (Allen and Garner, 1980).
Surprisingly, transgenic mice with a deleted COX-1 gene
did not spontaneously develop stomach ulcers (Langen-
bach et al., 1995). The reason for this unexpected finding
became clear when rats were treated with a combination of
a selective COX-1 and a selective COX-2 inhibitor and
rapidly developed gastric damage (Wallace et al., 2000).
Thus, inactivation of both COX-1 and COX-2 results in
the development of gastrointestinal damage but not
inhibition of activity of either enzyme alone (Gretzer
et al., 2001). This result was confirmed in transgenic
 ARTICLE IN PRESS
212 R.M. Botting / Journal of Thermal Biology 31 (2006) 208–219
COX-1 gene-deficient mice, in which selective inhibition of
COX-2 caused small bowel ulcers (Sigthorsson et al., 2002).
Thus, the lack of spontaneous intestinal damage in COX-1
knockout mice may be explained by the upregulation of
COX-2 protein when COX-1 is absent (Tanaka et al.,
2002).
Although very little COX-2 is found in healthy intestinal
tissues, COX-2 is highly expressed in human and animal
colon cancer cells as well as in human colorectal
adenocarcinomas (Thun et al., 1991; Luk, 1996). A clinical
trial of celecoxib in patients with familial adenomatous
polyposis showed a 30% reduction in intestinal polyps
(Steinbach et al., 2000) and this indication for the drug has
been allowed by the FDA. Human gastric and breast
tumours also express higher levels of COX-2 protein than
surrounding normal tissues (Ristimäki et al., 1997; Parrett
et al., 1997). These tumours may also be susceptible to
treatment with selective COX-2 inhibitors.
4.3. Kidney
The normal kidney cortex produces mainly PGE2 and
prostacyclin as well as small amounts of TXA2 (Farman et
al., 1987) while the renal medulla produces mostly PGE2
for which it has a synthetic capacity approximately 20
times that of the cortex (Zusman and Keiser, 1977). Renal
PGE2 is mostly synthesised by COX-1, however mesangial
cells in the macula densa contain constitutive COX-2 which
synthesises prostacyclin that may directly stimulate renin
secretion (Harris et al., 1994; Harris, 1996).
Production of prostaglandins is not essential for the
maintenance of normal kidney function. However, vasodi-
lator prostaglandins are important in patients with kidneys
compromised by disease, for example congestive heart
failure, liver cirrhosis or renal insufficiency and in animal
models of disease states. Inhibition of COX by NSAIDs
may result in renal ischaemia when prostaglandin synthesis
is reduced.
4.4. Central nervous system
Neurones throughout the brain express COX-1, but it is
most abundant in the forebrain (Yamagata et al., 1993;
Breder et al., 1995) where it may be involved in sensory
processing and control of the autonomic nervous system. It
is the most prolific isoenzyme in the mouse brain since
deletion of the COX-1 gene reduces PGE2 levels by 70%
(Ayoub et al., 2004). However, ‘‘basal’’ levels of COX-2
mRNA and COX-2 protein are expressed in the forebrain
without stimulation by pro-inflammatory stimuli (Yama-
gata et al., 1993; Breder et al., 1995; Cao et al., 1995), but
their expression can be increased in neuronal, glial and
endothelial cells by treatment with LPS, IL-1 or TNF
(Breder and Saper, 1996; Cao et al., 1996, 1998). The so-
called constitutive COX-2 is particularly high in neonate
brains where it is probably induced by intense nerve
activity in the developing brain. Electrical seizures increase
COX-2 mRNA in neurones of the hippocampus (March-
eselli and Bazan, 1996) and acute stress raises levels in the
cerebral cortex (Yamagata et al., 1993). Constitutive
expression of COX-2 mRNA which may be involved with
nociceptive processing is found in rat spinal cord (Beiche
et al., 1996; Yaksh and Svensson, 2001).
There is good evidence that COX-2 induced in endothe-
lial cells of hypothalamic blood vessels (Cao et al., 1995,
1996, 1998; Matsumura et al., 1998) by circulating LPS or
IL-1 (Ek et al., 2001) is involved in fever generation. Fever
is produced by PGE2 which enters the organum vasculo-
sum laminae terminalis (OVLT) and activates the thermo-
regulatory centre (Blatteis and Sehic, 1997). The febrigenic
response to LPS or IL-1 is abolished in COX-2 knockout
mice (Li et al., 1999, 2001; Steiner et al., 2005) suggesting
that the PGE2 which causes fever originates from the COX-
2 gene. Microsomal prostaglandin E synthase-1, expressed
in endothelial cells, also plays an important role in the
development of fever. Mice deficient in this enzyme showed
no fever or PGE2 synthesis in the central nervous system in
response to injection of LPS. However, these mice
responded with a fever to an intracerebral injection of
PGE2 (Engblom et al., 2003).
4.5. Reproduction
Both COX-1 and COX-2 are expressed in the pregnant
uterus, fetal membranes and umbilical cord. COX-2
mRNA increases in the human amnion and placenta
immediately before and after the start of labour (Gibb and
Sun, 1996) and preterm labour can be delayed by
administration of selective COX-2 inhibitors (Sawdy
et al., 1997). Female COX-2 knockout mice are mostly
infertile, producing very few offspring due to a reduction in
ovulation (Lim et al., 1997). In addition, the kidneys of
these mice do not develop fully after birth which reduces
their life span to between 8 and 16 weeks (Dinchuk et al.,
1995; Morham et al., 1995). Kidney maturation also ceased
prematurely when selective COX-2 inhibitors were fed
chronically to normal mice during pregnancy (Kömhoff
et al., 2000). However, mice of a mixed strain did not
exhibit this severe kidney pathology (Laulederkind et al.,
2002) and lived a full life span.
The mouse uterine epithelium is a major source of
prostaglandins during labour and female COX-1 knockout
mice experience parturition failure (Reese et al., 2000)
reversed by administration of prostaglandins. Prostaglan-
dins synthesised by COX-1 are also essential for the
survival of fetuses during parturition, since the majority of
offspring born to homozygous COX-1 knockout mice do
not survive (Langenbach et al., 1995). This high mortality
of the pups may be due to the failure of the ductus
arteriosus to close after birth. Patency of the ductus
arteriosus as well as its closure is dependent on prosta-
glandins made by COX-1 and COX-2 as offspring with a
double COX-1 and -2 knockout die shortly after birth with
an open ductus (Reese et al., 2000; Loftin et al., 2001).
 ARTICLE IN PRESS
R.M. Botting / Journal of Thermal Biology 31 (2006) 208–219
Thus, the patency of the ductus arteriosus in wild type
animals depends on the vasodilator action of PGE2 acting
on EP4 receptors in the ductus (Segi et al., 1998). However,
the compensatory dilator action of nitric oxide has been
suggested as the mechanism which maintains a patent
ductus in COX-1 and -2 null mice (Baragatti et al., 2003;
Coceani et al., 2005). The use of indomethacin as a
tocolytic has the disadvantage of closing the ductus in
utero (Heymann et al., 1976).
5. Inhibitors of COX-1 and COX-2
5.1. The non-steroid anti-inflammatory drugs
The NSAIDs were so named to distinguish them from
the anti-inflammatory steroids also used for the treatment
of inflammation. The oldest of these is aspirin or
acetylsalicylic acid, a derivative of salicylate originally
obtained from plant sources. Its main therapeutic actions
are analgesic, antipyretic and anti-inflammatory and its
major side actions are gastrotoxic, anti-thrombotic and
delay of the birth process. Aspirin was synthesised by Felix
Hoffman at the Bayer Company in Germany in 1897, but
its mechanism of action remained unknown until 1971.
Before 1971, inhibition of several enzymes by aspirin had
been observed and there were different hypotheses about
its mode of action. Against this background of knowledge,
Piper and Vane took over the investigation of the mode of
action of aspirin. They employed the technique of
continuous bioassay using the cascade bioassay system
(Vane, 1964) developed by Vane in the mid-1960s for use
with blood or an artificial salt solution (Fig. 4). Piper and
Vane found that the anaphylactic release of prostaglandins
from isolated perfused guinea pig lungs was blocked by
aspirin and similar drugs (Palmer and Piper, 1970). Vane
postulated that the various stimuli which released pros-
taglandins were in fact ‘‘turning on’’ the synthesis of these
Fig. 4. Diagram of the blood-bathed organ technique. Blood or salt
solution superfuses a series of isolated organs, the movements of which, in
response to drugs, are recorded.
213
mediators and that aspirin might well be blocking their
synthesis. Using the supernatant of a homogenate from
guinea pig lung as a source of prostaglandin synthase,
Vane found a dose-dependent inhibition of prostaglandin
formation by aspirin, salicylate and indomethacin but not
by morphine (Vane, 1971). Two other reports from the
same laboratory supported and extended his finding. Smith
and Willis found that aspirin prevented the release of
prostaglandins from aggregating human platelets (Smith
and Willis, 1971) and Ferreira, Moncada and Vane
demonstrated that aspirin-like drugs blocked prostaglandin
release from the perfused, isolated spleen of the dog
(Ferreira et al., 1971).
Following the elucidation of the mechanism of action of
aspirin, many more NSAIDs were developed by pharma-
ceutical companies. These include naproxen, ibuprofen,
piroxicam, diclofenac and many others. The inhibition of
prostaglandin synthesis explains all the actions of the
NSAIDs. They prevent the pathological over-production
of prostaglandins by COX-2 which contribute to the
inflammatory process (therapeutic effects) and prevent
the physiological formation of prostanoids by COX-1 (side
effects). For example, the ulcerogenic activity of aspirin
arises from the inhibition of prostaglandin production in
the stomach which has an important cytoprotective
function in the gastric mucosa. The inhibition by aspirin
is due to the irreversible acetylation of serine 530 in the
COX-1 active site or the serine 516 in the active site of
COX-2 (Van der Ouderaa et al., 1980). In contrast to this
irreversible action of aspirin, other NSAIDs such as
indomethacin or ibuprofen produce reversible COX
inhibition by competing with the substrate, arachidonic
acid, for binding to the active site of COX-1 or COX-2
(Vane et al., 1990). This inhibition is responsible for the
analgesic, antipyretic and anti-inflammatory actions of the
NSAIDs as well as their gastrotoxicity, anti-thrombotic
action (Fig. 3) and delay of the birth process. The anti-
thrombotic action is reversed when the NSAID is
eliminated but the anti-thrombotic action of aspirin is
irreversible until new platelets are formed after 8–10 days
(Vane et al., 1998).
5.2. Selective COX-2 inhibitors
In 1998, an estimated 5.8 billion dollars was spent world
wide on anti-inflammatory drugs and 1.8 billion dollars of
this accounted for sales of NSAIDs in the USA. However,
all NSAIDs at anti-inflammatory doses cause side effects,
the most serious being irritation, bleeding and ulcers of the
gastrointestinal tract. Indeed, several epidemiological
studies have estimated the degree of gastric damage caused
by different NSAIDs (Henry et al., 1996) since 34–46% of
patients on NSAID therapy will have some form of
gastrointestinal gastric event (Coles et al., 1983). It has
been estimated that in the USA alone, some 100,000
patients are hospitalised each year because of perforations,
ulcers or bleeding (PUBs) in the stomach (Fries, 1999) and
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214 R.M. Botting / Journal of Thermal Biology 31 (2006) 208–219
10,000–20,000 of these patients die in intensive care.
Gastric side effects range from mild dyspepsia to PUBs
and even ibuprofen, which is recognised as a mild gastric
irritant, causes problems in a significant proportion of
patients. For many years, there was a clear need for anti-
inflammatory drugs which did not damage the stomach.
The discovery of the inducible COX-2 highlighted the
differences in pharmacology of the two COX enzymes
(Mitchell et al., 1993). Aspirin, indomethacin and ibupro-
fen are much less active against COX-2 than against COX-
1 (Meade et al., 1993). Indeed, the strongest inhibitors of
COX-1 such as aspirin, indomethacin and piroxicam are
the NSAIDs which cause the most damage to the stomach
(Lanza, 1989). The spectrum of activities of some 10
standard NSAIDs against the two enzymes ranges from a
high selectivity towards COX-1 (166-fold for aspirin;
Mitchell et al., 1993; Akarasereenont et al., 1994) through
to equiactivity on both (0.5-fold for diclofenac; Warner et
al., 1999).
The range of activities of NSAIDs against COX-1
compared with COX-2 explains the variations in the side
effects of NSAIDs at their anti-inflammatory doses. Drugs
which have a high potency against COX-2 and a low COX-
2/COX-1 activity ratio will have potent anti-inflammatory
activity with few side effects on the stomach and kidney.
Garcia Rodriguez and Jick (1994) have published a
comparison of epidemiological data on the side effects of
NSAIDs. Piroxicam and indomethacin in anti-inflamma-
tory doses showed high gastrointestinal toxicity. These
drugs have a much higher potency against COX-1 than
against COX-2 (Vane and Botting, 1995). Thus, when
epidemiological results are compared with COX-2/COX-1
ratios, there is a parallel relationship between gastrointest-
inal side effects and COX-2/COX-1 ratios.
Nimesulide, etodolac and meloxicam were identified in
the 1980s as potent anti-inflammatory drugs with low
ulcerogenic activity in the rat stomach. In some instances,
this was also shown to parallel low activity against
prostaglandin synthesis in the rat stomach. After the
characterisation of the COX-2 gene, these three drugs were
each found preferentially to inhibit COX-2 rather than
COX-1, with a variation in their COX-2/COX-1 ratios of
between 0.1 and 0.01 depending on the test system used.
Ratios obtained by the human whole blood assay, which
measures inhibition of COX-1 in platelets and COX-2 in
mononuclear cells stimulated with LPS, are now generally
accepted as the best reflection of the inhibitory activity of
the drugs in humans (Patrignani et al., 1994).
Selective inhibitors of COX-2 were introduced in 1999.
The first NSAIDs to be introduced as selective COX-2
inhibitors were celecoxib (Celebrex) and rofecoxib (Vioxx).
In place of the carboxyl group of the non-steroid anti-
inflammatory acids, the structure of celecoxib contains a
sulfonamide group and that of rofecoxib contains a
methylsulfone. The sulphur-containing phenyl rings of
these drugs bind into the side pocket of the COX catalytic
channel of COX-2 but interact weakly with the active site
of COX-1 (Kurumbail et al., 1996). Thus, they are potent
inhibitors of COX-2 and weak inhibitors of COX-1. When
the selectivity is estimated by the human whole blood
assay, celecoxib has a selectivity of 7.6 and rofecoxib a
selectivity of 35 in favour of COX-2 (Riendeau et al., 2001).
Second-generation selective COX-2 inhibitors have now
been developed with higher selectivities for COX-2. The
successor to celecoxib, valdecoxib (Bextra) has a selectivity
ratio of 30 and etoricoxib (Arcoxia), the successor to
rofecoxib, has a selectivity of 106 (Riendeau et al., 2001).
Parecoxib, an injectable pro-drug of valdecoxib, is also
available for the treatment of acute pain (Cheer and Goa,
2001) and lumiracoxib is currently in development (Ding
and Jones, 2002) and has undergone clinical trials
(Schnitzer et al., 2004). Lumiracoxib differs in structure
from other coxibs. It is a phenyl acetic acid derivative
instead of a sulfonamide or sulfone, and compared to non-
selective NSAIDs causes no more cardiovascular side
effects and less gastrointestinal complications (Schnitzer
et al., 2004).
The Celecoxib Long-Term Arthritis Safety Study
(CLASS) in 8000 patients for 6 months demonstrated a
lower incidence of ulcer complications in patients receiving
celecoxib than those on ibuprofen or diclofenac. However,
in patients taking aspirin as well as celecoxib, the incidence
of ulcers was no better than in the comparator group
(Silverstein et al., 2000). Moreover, when the trial was
continued for 12 months, there was no difference in the
number of gastric adverse events between the celebrex and
comparator NSAIDs treated patients (FDA Celecoxib
Hearing July 2, 2001).
The ‘‘Vioxx’’ Gastrointestinal Outcomes Research (VIG-
OR) Trial (Bombardier et al., 2000) comparing rofecoxib
with naproxen in 8000 patients for 9 months reported fewer
serious gastrointestinal adverse events with rofecoxib.
Patients who were taking aspirin were excluded from the
trial. However, the incidence of myocardial infarction was
higher among patients in the rofecoxib group than among
those in the naproxen group (0.4% versus 0.1%). The
higher incidence of heart attacks may be a general risk
factor for all selective COX-2 inhibitors. Recent trials
investigating the efficacy of celecoxib (Solomon et al., 2005)
and rofecoxib (Bresalier et al., 2005) in preventing color-
ectal adenomas demonstrated a higher incidence of
cardiovascular events, including myocardial infarction, in
the drug treated group compared to the placebo. It has
been suggested that selective COX-2 inhibitors prevent the
synthesis of the anti-thrombotic prostaglandin, prostacy-
clin, by endothelial cells while leaving unopposed the action
of the pro-thrombotic thromboxane in platelets (McAdam
et al., 1999). Recent epidemiological studies show that all
NSAIDs in anti-inflammatory doses increase the risk of
myocardial infarction (Hippisley-Cox and Coupland,
2005). This may be difficult to explain since the majority
of NSAIDs have a greater selectivity for COX-1 than for
COX-2, which is the basis for their gastrotoxicity and for
their anti-thrombotic action.
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R.M. Botting / Journal of Thermal Biology 31 (2006) 208–219
In 2004, Merck Frosst withdrew rofecoxib from the
market and is currently involved in litigation with victims
of cardiovascular adverse events who were treated with
rofecoxib. Selective COX-2 inhibitors are now required to
carry a warning of the risk of cardiovascular side effects.
Valdecoxib causes severe toxic skin rashes as well as
possible myocardial infarctions and the FDA has recom-
mended that its sales should be suspended (Scrip, 2005).
Valdecoxib and parecoxib are associated with an increased
incidence of cardiac events if used for pain after coronary
artery bypass grafting (Nussmeier et al., 2005).
6. Other cyclooxygenases
Acetaminophen is generally considered an NSAID but
its mechanism of action has not been fully resolved. It is a
weak inhibitor of isolated COX-1 and COX-2 but
demonstrates antipyretic and analgesic properties when
taken internally. It has no anti-inflammatory actions. In
1972, Flower and Vane postulated the existence of a COX
in dog brain more sensitive to inhibition with acetamino-
phen than the COX in rabbit spleen. More recently,
Simmons and his colleagues characterised and cloned a
COX enzyme in dog brain which, unlike COX-1 and COX-
2, was sensitive to inhibition with acetaminophen (Chan-
drasekharan et al., 2002). This was a splice variant of
COX-1 termed by the authors, COX-3, which consists of a
COX-1 mRNA that retains intron-1. In dogs, intron-1 is 90
nucleotides in length and represents an in frame insertion
into the portion of the COX-1 open reading frame
encoding the N-terminal hydrophobic signal peptide. This
variant produces enzyme protein containing the encoded
intron-1 sequence when expressed in insect cells. The
activity of the protein is preferentially inhibited by
analgesic antipyretic drugs such as acetaminophen and
may explain the therapeutic actions of this class of
compounds. However, although intron-1 is of similar size
in all species, it is out of frame in humans and rodents.
Thus, it would require additional mechanisms such as the
use of alternative splice sites, ribosomal frameshifting or
RNA editing to make a functional protein (Chandrase-
kharan et al., 2002; Dinchuk et al., 2003; Simmons, 2003;
Simmons et al., 2004). However, COX-1 variant protein
has been identified in both mouse and human tissues
(Chandrasekharan et al., 2002; Dou et al., 2004; Qin et al.,
2005), although the candidate protein identified by Qin
et al. (2005) is not selectively inhibited by acetaminophen.
Since high levels of peroxides (such as those that exist in
inflamed tissues) abolish the action of acetaminophen, an
alternative mechanism has been proposed to explain its
action. This hypothesis proposes that the inhibitory
activity of acetaminophen on COX depends on the level
of peroxides in the target cells, (Ouellet and Percival, 2001;
Boutaud et al., 2002).
An inducible COX-2 enzyme sensitive to inhibition with
low concentrations of acetaminophen was described in
1999 (Simmons et al., 1999). Cultured J774.2 mouse
215
macrophages when incubated with diclofenac for 48 h
expressed a COX-2-like activity which was more sensitive
to inhibition by acetaminophen than LPS-induced COX-2.
In addition, LPS-induced COX-2 was a membrane-bound
enzyme whereas COX-2 induced with diclofenac existed in
the cytoplasm of the cells. If such a COX-2 variant is
identified in vivo, it may explain some therapeutic actions
of acetaminophen which cannot be attributed to inhibition
of a variant of COX-1.
7. Conclusions
The prostaglandin field has been one of the most exciting
areas of research in recent times. More than 70 years after
the marketing of aspirin, John Vane established its
mechanism of action. Just 20 years later, with the increase
in research into the properties of COX, a second
isoenzyme, COX-2, was revealed by Daniel Simmons in
1991. In the eight years that followed, the eagerly awaited
selective inhibitors of COX-2 reached the market, which
alleviated the symptoms of inflammatory disease without
damaging the stomach. The discovery of COX-2 inhibitors
generated great enthusiasm and competition to design
more effective drugs. However, increasing selectivity for
COX-2 also increased toxicity, since the anti-thrombotic
prostacyclin is formed by COX-2 and inhibiting its
synthesis precipitated heart attacks. The problem of this
side action has not yet been resolved. Is it possible to
obtain the benefit of low gastrotoxicity and avoid the
danger of a heart attack? Perhaps lumiracoxib has
provided the solution (see above). It now appears that by
taking any NSAID, patients risk experiencing a heart
attack.
A third COX, COX-3, has also been postulated, based
on the sensitivity of a COX-1 variant from dog brain to
acetaminophen. However, when the COX-3 mRNA of
mouse or human is translated into an enzyme protein, the
intron-1 insertion is out of frame. Thus, the mechanism by
which conversion of mRNA occurs into active enzyme
protein in mouse and human requires further investigation.
When this question is resolved, the mechanism of action of
a drug even older than aspirin will be clarified.
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