Prostaglandins and glucocorticoids are potent mediators of inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) exert their effects by inhibition of prostaglandin production. The pharmacological target of NSAIDs is cyclooxygenase (COX, also known as PGH synthase), which catalyses the first committed step in arachidonic-acid metabolism. Two isoforms of the membrane protein COX are known: COX-1, which is constitutively expressed in most tissues, is responsible for the physiological production of prostaglandins; and COX-2, which is induced by cytokines, mitogens and endotoxins in inflammatory cells, is responsible for the elevated production of prostaglandins during inflammation. The structure of ovine COX-1 complexed with several NSAIDs has been determined. Here we report the structures of unliganded murine COX-2 and complexes with flurbiprofen, indomethacin and SC-558, a selective COX-2 inhibitor, determined at 3.0 to 2.5 A resolution. These structures explain the structural basis for the selective inhibition of COX-2, and demonstrate some of the conformational changes associated with time-dependent inhibition.

Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of Inlammator dimseas, but sg t side effects such as g oi l erosion and renal damage lmit their use. NSAIDs Inhibit the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to p (PGs) and thromboxane. Two forms of COX have been identlfled-COX-1, which is constitutively expressed in most tissues and organs, and the Inducible enzyme, COX-2, whih has been localized primarily to ilmmatory cells and tsues. In an animal model of acute inflammation (Injection ofcarrageenan into the footpad), edema was produced that was associated with marked accumulation of COX-2 mRNA and thromboxane. A selective inhibitor of COX-2 (SC-58125) inhibited edema at the inmatory site and was analgesic but had no effect on PG production In the stomach and did not cause gastric toxicity. These data suggest that selective inhibition of COX-2 may produce superior an mmatory drugs with substantial safety advantages over existing NSAIDs. bone density, fluid and electrolyte imbalance, and "Cushinglike" symptoms. These untoward effects limit glucocorticoid use in chronic inflammatory disorders such as rheumatoid arthritis (13).Studies were designed to evaluate the role ofCOX-2 in vivo at the site of inflammation. We report that in a model of inflammation useful in the characterization of NSAIDs, the carrageenan-injected rat paw, COX-2 was expressed locally in response to the proinflammatory stimulus and that the induction of COX-2 mRNA coincided with the synthesis of proinflammatory PGs and the development of edema and hyperalgesia. COX-1 mRNA was detectable in the normal rat paw, but its expression did not change following the onset of the inflammatory reaction. Furthermore, a selective inhibitor of COX-2 {SC-58125: 1-[(4-methylsulfonyl)phenyll-3-trifluoromethyl-5-(4-fluorophenyl)pyrazole} blocked edema and hyperalgesia in vivo following an inflammatory insult, without causing gastric mucosal damage.

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammaion in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by hih levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-direc antiinfla tory drugs.Nonsteroidal antiinflammatory drugs (NSAIDs) are used to treat acute and chronic inflammatory disorders such as rheumatoid arthritis. The antiinflammatory mechanism of NSAIDs is due to a reduction ofprostaglandin (PG) synthesis by the direct inhibition of cyclooxygenase (COX; prostaglandin-endoperoxide synthase, EC 1.14.99.1) (1). Unfortunately, inhibition of PG production in organs such as stomach and kidney can result in gastric lesions, nephrotoxicity, and increased bleeding.COX exists in two forms. COX-1 is in most tissues and is involved in the physiological production of PGs. COX-2 is cytokine-inducible and is expressed in inflammatory cells (2-9). The identification of constitutive and inducible COX enzymes led to the hypothesis that COX-2 is primarily responsible for PGs produced in inflammation and COX-1 for PGs involved in normal homeostasis (4-6, 10, 11).The rat air pouch is a convenient model to study acute inflammation (12). It is formed by the subcutaneous injection of air over several days and is composed of a lining of cells that consists primarily of macrophages and fibroblasts. Injection of carrageenan into the fully formed air pouch produces an inflammatory granulomatous reaction characterized by a marked production of biochemical mediators in the fluid exudate, including PGs and leukotrienes, as well as a significant influx of polymorphonuclear leukocytes (PMNs) and macrophages (13). Using molecular and pharmacological reagents, we studied the role of COX-2 in this model of inflammation by specific examination of the induction of COX-2 mRNA and protein as well as the production of PGs in the pouch exudate. The results indicate that induction of COX-2 is responsible for the production of PGs at the site of inflammation, whereas the normal synthesis of PGs in the stomach appears to depend on constitutive...

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H 2 , the precursor of PGs and thromboxane. These lipid mediators play important roles in inf lammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inf lammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inf lammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC 50 ‫؍‬ 0.009 M; COX-2 IC 50 ‫؍‬ 6.3 M). SC-560 inhibited COX-1-derived platelet thromboxane B 2 , gastric PGE 2 , and dermal PGE 2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inf lammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inf lammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal f luid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inf lammatory pain that is mediated at least in part by COX-2.

Abstract

In this study, all dosages of celecoxib were efficacious in the treatment of rheumatoid arthritis and did not affect COX-1 activity in the GI tract mucosa as evidenced by less frequent incidence of endoscopic ulcers compared with naproxen.

Postnatal development and adult function of the central nervous system are dependent on the capacity of neurons to effect long-term changes of specific properties in response to neural activity. This neuronal response has been demonstrated to be tightly correlated with the expression of a set of regulatory genes which include transcription factors as well as molecules that can directly modify cellular signaling. It is hypothesized that these proteins play a role in activitydependent responses. Previously, we described the expression and regulation in brain of an inducible form of prostaglandin synthase/cyclooxygenase, termed COX-2. COX-2 is a ratelimiting enzyme in prostanoid synthesis and its expression is rapidly regulated in developing and adult forebrain by physiological synaptic activity. Here we demonstrate that COX-2 immunoreactivity is selectively expressed in a subpopulation of excitatory neurons in neo-and allocortices, hippocampus, and amygdala and is compartmentalized to dendritic arborizations. Moreover, COX-2 immunoreactivity is present in dendritic spines, which are specialized structures involved in synaptic signaling. The developmental profile of COX-2 expression in dendrites follows well known histogenetic gradients and coincides with the critical period for activitydependent synaptic remodeling. These results suggest that COX-2, and its diffusible prostanoid products, may play a role in postsynaptic signaling of excitatory neurons in cortex and associated structures.Neural activity results in specific structural and functional modifications of the cerebral cortex. This activity-dependent process is essential for achieving the appropriate synaptic relationships during development and for normal function of the mature cortex (1). Recent studies are beginning to identify molecular mechanisms underlying activity-dependent changes (2). There is abundant correlative evidence linking neural activity and transcription factor (TF) expression (3). Members of the Fos, Jun, and zinc finger TF families are naturally expressed at high levels in specific populations of cortical neurons and this expression is tightly regulated by synaptic activity (4, 5). Furthermore, these TFs are also rapidly and transiently induced in different paradigms of synaptic plasticity consistent with the notion that they regulate the expression of specific effector genes that underlie long-term plasticity (3).In addition to TFs, the initial genomic response to neural activity includes proteins that can directly modify cellular function. Among them is an inducible form of the enzyme prostaglandin synthase/cyclooxygenase, termed COX-2 (6-9). Cyclooxygenase is the first enzyme in the prostaglandin/ prostacyclin/thromboxane pathway and converts arachidonic acid to prostaglandin G2/prostaglandin H2. There are presently two known forms of cyclooxygenase: a constitutively expressed form termed COX-1 (10) and the inducible formThe publication costs of this article were defrayed in part by page charge payment. This article must the...

Prostaglandins formed by the cyclooxygenase (COX) enzymes are important mediators of inflammation in arthritis. The contribution of the inducible COX-2 enzyme to inflammation in rat adjuvant arthritis was evaluated by characterization of COX-2 expression in normal and arthritic paws and by pharmacological inhibition of COX-2 activity. The injection of adjuvant induced a marked edema of the hind footpads with coincident local production of PGE 2 . PG production was associated with upregulation of COX-2 mRNA and protein in the affected paws. In contrast, the level of COX-1 mRNA was unaffected by adjuvant injection. TNF-␣ and IL-6 mRNAs were also increased in the inflamed paws as was IL-6 protein in the serum. Therapeutic administration of a selective COX-2 inhibitor, SC-58125, rapidly reversed paw edema and reduced the level of PGE 2 in paw tissue to baseline. Interestingly, treatment with the COX-2 inhibitor also reduced the expression of COX-2 mRNA and protein in the paw. Serum IL-6 and paw IL-6 mRNA levels were also reduced to near normal levels by SC-58125. Furthermore, inhibition of COX-2 resulted in a reduction of the inflammatory cell infiltrate and decreased inflammation of the synovium. Notably, the antiinflammatory effects of SC-58125 were indistinguishable from the effects observed for indomethacin. These results suggest that COX-2 plays a prominent role in the inflammation associated with adjuvant arthritis and that COX-2 derived PGs upregulate COX-2 and IL-6 expression at inflammatory sites. ( J. Clin. Invest. 1996. 97:2672-2679.)