Methods and Findings in Experimental and Clinical Pharmacology
Vol. 22, No. 5, 2000, pp. 291-298
ISSN 0379-0355
Copyright 2000 Prous Science, S.A.
CCC: 0379-0355/2000
http://www.prous.com

Cyclooxygenase Isoenzymes and Newer Therapeutic Potential for Selective COX-2 Inhibitors

S.K. Kulkarni, N.K Jain and A. Singh¹

Instituto de Farmacoepidemiología, Universidad de Valladolid, Valladolid, Spain

SUMMARY

Cyclooxygenase (COX), also known as prostaglandin G/H synthase, is a membrane-bound enzyme responsible for the oxidation of arachidonic acid to prostaglandins that was first identified over 20 years ago. In the past decade, however, more progress has been made in understanding the role of cyclooxygenase enzymes in various pathophysiological conditions. Two cyclooxygenase isoforms have been identified and are referred to as COX-1 and COX-2. COX-1 enzyme is constitutively expressed and regulates a number of housekeeping functions such as vascular hemostasis and gastroprotection, whereas COX-2 is inducible (i.e., sites of inflammation) by number of mediators such as growth factors, cytokines and endotoxins. Nonsteroidal antiinflammatory drugs (NSAIDs) produce their therapeutic effects through inhibition of COX, the enzyme that makes prostaglandins. Nonselective inhibition of COX isoenzyme leads to not only beneficial therapeutic effects but also a number of detrimental effects. Beneficial effects are due to inhibition of COX-2 and detrimental effects are due to inhibition of physiological COX-1. The present review discusses the biology as well as the role of these COX isoenzymes in various pathophysiological conditions. © 2000 Prous Science. All rights reserved.

Key words: Cyclooxygenase enzyme - Nonsteroidal antiinflammatory drugs - Selective COX-2 inhibitors - Newer therapeutic potentials

INTRODUCTION

Cyclooxygenase (COX), the enzyme that catalyses the synthesis of cyclic endoperoxides from arachidonic acid to form prostaglandins (PG), was isolated in 1976 and cloned in 1988 (1).This membrane-bound hemo- and glycoprotein has a molecular weight of 71 kDa, and is found in the greatest amounts in the endoplasmic reticulum of prostanoid forming cells (2). It exhibits cyclooxygenase activity, which cyclizes arachidonic acid, and adds the 15-hydroxy group to form prostaglandin G₂. The hydroperoxy group of prostaglandin G₂ is reduced to the hydroxy group of prostaglandin H₂ by a peroxidase that uses a wide variety of compounds to provide the requisite pair of electrons. Both cyclooxygenase and hydroperoxidase activities are contained in the same dimeric protein molecule.

The 1990s has seen a new dawn in inflammatory research, with biological studies demonstrating increased COX activity in a variety of cells after exposure to endotoxin, pro-inflammatory cytokines, growth factors, hormones and tumor promoters. This activity requires new protein synthesis and is amenable to inhibition by corticosteroids. This observation gave rise to the concept that there might be a constitutive COX activity, referred to as COX-1 and an inducible one subsequently referred to as COX-2 (3). The two isoforms of COX are almost identical in structure but have important differences in substrate and inhibitor selectivity and in their intracellular locations (4, 5). Protective prostaglandins, which preserve the integrity of the stomach lining and maintain normal renal function in a compromised kidney, are synthesized by COX-1. In addition COX-1 is also present in the platelets and leads to thromboxane A₂ production, causing aggregation of the platelets to prevent inappropriate bleeding (6). The existence of the inducible isoform, COX-2, was first suspected when Needleman and his group showed (7) that it could be induced by inflammatory stimuli and by cytokines in migratory and other cells, suggesting that the antiinflammatory actions of NSAIDs are due to the inhibition of COX-2, whereas the unwanted side effects such as damage to the stomach lining and toxic effects on the kidney are due to inhibition of the constitutive enzyme COX-1 (8).

CYCLOOXYGENASE ISOENZYMES

COX isoforms are bifunctional hemoproteins that catalyze both the bioxygenation of arachidonic acid to form PGG₂ and the peroxidative reduction of PGG₂ to form PGH₂. For a given COX isoform there is approximately 90% (81-98%) identity between species. The tissue distribution of COX-1 and COX-2 differs notably between species and can be heterogenous within the same tissue. For example, COX-2 is the major isoform in rat and mouse brain, whereas similar levels of both isoforms have been detected in human brain. Both isoforms were detected in the stomach with COX-1 being the most abundant in mouse and rat, whereas COX-2 was found to be expressed to a similar extent as COX-1 in human tissue. In the rat stomach, COX-2 was found in the surface mucous cell while COX-1 was found in mucous neck cells (Table 1) (9, 10).

In addition, COX isoenzymes also partition at the cellular level since COX-1 function primarily in the endoplasmic reticulum, whereas COX-2 activity is located both in the endoplasmic reticulum and in the nuclear envelope (11). This compartmentalization suggests that COX isoenzymes may represent two temporally and spatially separated prostanoid bisynthetic systems with COX-2 producing prostanoids for intracellular differential or replicative events (2, 12). The compartmentalization of COX isoenzymes is an attempt to explain their respective role at the cellular level when gene distribution techniques may allow an understanding of their biological relevance.

Cyclooxygenase-1 (COX-1)

Picot et al. (13) reported the three dimensional structure of COX-1, providing a new therapeutic understanding for the actions of COX inhibitors. This bifunctional enzyme is composed of three independent folding units--an epidermal growth factor-like domain, a membrane-binding motive and an enzymatic domain. The sites for cyclooxygenase and peroxidase are adjacent but spatially distinct. The COX active site is a long, hydrophobic channel. Aspirin-like drugs, such as flurbiprofen, inhibit COX-1 by excluding arachidonate from the upper portion of the channel. Tyrosine 385 and serine 530 are at the apex of the long active site. Aspirin irreversibly inhibits COX-1 by acetylation of the serine 530, thereby excluding access for arachidonic acid (14).

Cyclooxygenase-2 (COX-2)

The COX-2 enzyme is dimeric and each monomer consists of a catalytic domain and a membrane-binding domain, connected by the N-terminal EGF domain. The membrane-binding domain forms a channel, which leads to the active site (15).

The roentgenogram crystal structure of COX-2 closely resembles COX-1 and the binding sites for arachidonic acid for these enzymes are also very similar. The active site of COX-2 is slightly larger and can accommodate bigger structures than those which are able to reach the active site of COX-1. Selectivity for COX-2 inhibitors can be conferred by replacing the His513 and Ile523 of COX-1 with Arg and Val, respectively. This replacement removes the constriction at the mouth of the secondary side channel and allows the more bulky selective COX-2 inhibitors (4).

FUNCTIONS OF COX-1 AND COX-2

COX-1 performs a housekeeping function to synthesize PGs which regulate normal cell activity. Normally little or no COX-2 is found in resting cells but its expression can be increased dramatically after exposure of cells to lipopolysaccharides, phorbol esters, cytokines or growth factors.

Gastrointestinal tract

In most species, including humans, the bulk of the cytoprotective PGs are synthesized by COX-1. These prostanoids reduce gastric acid secretion, exert direct vasodilator action on the vessels of the gastric mucosa and stimulate secretion of mucous and bicarbonate which forms a protective barrier (15). COX-2 can also be expressed in the normal rat stomach. But recently it is reported that COX-2 is highly expressed in human and animal colon cancer cells as well as in human colorectal adenocarcinomas (15, 16). Mizuno et al. (17) reported that COX-2 mRNA and protein are involved in the repair process of ulcer healing and are specific antagonists of these delays healing in mice. These results have recently been extended by Schmassman et al. (18) who showed marked inhibition of gastric ulcer healing in the rat by a COX-2 inhibitor (L-745337). Moreover, COX-2 expression in healthy stomach is low and expression at the site of ulceration is considerably higher and it may be expected that the COX-2 enzyme plays a crucial role in the maintenance of gastrointestinal mucosal integrity, particularly when the mucosa is ulcerated or inflamed (19, 20).

Kidney

Maintenance of kidney function is dependent on prostaglandins, therefore the risk of various disorders is indicated when PG synthesis is reduced by NSAIDs. COX-1 is the predominant isoform in the vasculature, glomerulus and collecting duct. COX-2 metabolites are involved in the macula densa, regulation of renin-angiotensin system and/or glomerular hemodynamics (21). Renal blood flow and glomerular filtration rate become progressively dependent upon PG synthesis under conditions of volume depletion or reduced renal perfusion pressure (10).

Central nervous system

COX-1 is distributed in neurons throughout the brain but it is most abundant in the forebrain, where PGs may be involved in complex integrative functions such as control of the autonomic nervous system, and in sensory processing (22-24). Intense nerve stimulation leading to seizures expresses COX-2 mRNA in discrete neurons of the hippocampus, whereas acute stress raises levels in the cerebral cortex (22). COX-2 mRNA is also constitutively expressed in the spinal cord of normal rats and likely to be involved with processing of nociceptive stimuli (25). Endogenous fever producing PGE₂ is thought to originate from COX-2, induced by LPS or IL-1 in endothelial cell lining on the cerebral blood vessels (26). The role of COX-2 in various neurodegenerative disorders has been also reported (27).

Lungs

Airway hyperreactivity, a feature of allergic asthma, is associated with inflammation of the airways. Increased level of COX-2 mRNA and of enzyme protein, with no change in COX-1 levels, has been detected in the airway smooth muscle cells treated with proinflammatory cytokines (28). COX-2 is probably upregulated in the inflamed lungs of the asthmatics resulting in increased production of bronchoconstrictor PGs which exert an exaggerated effect on the bronchiolar smooth muscle that has become hyperreactive to constrictor agents. Up-regulation of COX-2 with simultaneous down regulation of COX-1 by LPS has been reported (29). Cyclooxygenase product of PGE₂ is the weak bronchodilator but PGF₂a and TXA₂ are the potent bronchoconstrictor prostanoids.

Gestation and parturition

Prostaglandins are essential for maintenance of healthy pregnancy and induction of labor. PGs synthesized by COX-1 are apparently essential for the survival of fetuses during parturition. Expression of COX-1 is much greater than that of COX-2 in fetal hearts, kidney, lungs and brains as well as in the decidual lining of the uterus (30, 31). Both COX-1 and COX-2 are expressed in the uterine epithelium at different times in early pregnancy and may be important for implantation of the ovum and in the angiogenesis needed for establishment of the placenta. Prostaglandins originating from COX-2 may play a role in birth process since COX-2 mRNA in the amnion and placenta increases markedly immediately before and after the start of labor (30).

BIOLOGICAL TESTS TO DETERMINE NSAID COX SELECTIVITY

It is well established that selective COX inhibition by NSAIDs is a therapeutically desirable goal and has led to the development of various in vitro and in vivo tests to assess the comparative COX selectivity (Table 2). These systems may differ in the source of enzyme, the purity of enzymatic activity, the partition of NSAID within the system, the time needed for drug binding equilibrium (pre-incubation), the source of arachidonate as substrate and, if arachidonate is added, its concentration, the incubation time, the nature of the metabolite to be studied in similar systems, the drug protein binding in the medium and the biotransformation of the drug in the system.

Most tests assess the IC₅₀ value (the concentration of the substance that, under controlled conditions, inhibits 50% of the activity of COX enzyme). The IC₅₀ value does not predict the actual amount of enzyme inhibition in vivo, nor does it directly translate into a measure of clinical efficacy. The utility of the IC₅₀ is to compare, under in vitro circumstances the potency of inhibition, not the actual clinical effect (32). Table 3 lists the COX-1 and ­2 inhibitory activity of various NSAIDs.

COX-2 SELECTIVE INHIBITION: NEWER THERAPEUTIC TARGETS

The potential improvement in the therapeutic ratio of NSAIDs which inhibit inducible COX-2 at the inflamed site but have no effect on constitutive COX-1, is likely to change the use of the classical NSAIDs. Beside their therapeutic indication, these selective COX-2 inhibitors (Table 4) might have potential use in various diseases such as colorectal cancer and neurodegenerative disease of the Alzheimer type.

COX-2 inhibitors in colon cancer

Various laboratory studies suggested that NSAIDs reduce the risk of colon cancer and that inhibition of colon carcinogenesis is mediated through modulation of prostaglandin by COX isoenzyme. Over expression of COX-2 has been observed in colon tumors therefore specific inhibitors of COX-2 could potentially serve as chemopreventive agents (33, 34).

A recent study with celecoxib and nimesulide in intestinal polyphs in mice and colonic aberrant crypt foci (ACF) formation in rats induced by azoxymethane indicated that both agents possess strong chemopreventive activity against colon carcinogenesis (35). This finding is further supported by Reddy et al. (34, 36) who suggested that SC-58635 (a COX-2 inhibitor) significantly suppressed colonic ACF formation and crypt multiplicity, and strengthens the hypothesis that selective COX-2 inhibitors have promising therapeutic potential against colon carcinogenesis.

COX-2 inhibitors in Alzheimer's disease

Recent studies suggest that inflammatory events are associated with plaque formation in the brains of patients with Alzheimer's disease (AD). Treatment of these patients with NSAIDs slows the progression of disease. Pepeu reviewed the evidence of inflammatory mechanisms in the pathogenesis of AD. It was shown that the intracerebral injection of b-amyloid produces extensive glial reaction in the brain and induces COX-2 expression in neuronal culture (27). Moreover, experimental neurodegenerative lesions cause up-regulation of neuronal COX-2, and COX-2 inhibition can be neuroprotective in animal and cell culture models (37). Ho et al. (38) also reported an elevated expression of neuronal COX-2 in subregions of the hippocampal formation in AD and that such elevation may potentiate b-amyloid-mediated oxidative stress.

Thus, the efficacy of NSAIDs in slowing AD may be explained by inhibition of neuronal COX-2, the activity of which promotes neurodegeneration. On this hypothesis, the selective COX-2 inhibitor which penetrates the blood-brain barrier may be a good therapeutic candidate for Alzheimer's disease.

COX-2 inhibitors in delaying premature labor

Eicosanoids are important for inducing uterine contractions during labor. A significant increase in COX-2 occurs in amnion and placenta immediately before and after the start of labor. It is thus likely that COX-2 produces the oxytocic PGs that are responsible for preterm labor (31). One cause of preterm labor could be an intrauterine infection, resulting in the release of endogenous factors that increase PG production by up-regulating COX-2. These data indicate that selective COX-2 inhibition may be of use in preventing contractions in premature labor, being preferable to b-sympathomimetics (which produce maternal cardiovascular, respiratory and metabolic side effects) and indomethacin (which produces oligohydramnios and closure of the ductus arteriosus due to reduced synthesis of vasodilator PGs) (39, 40).

COX-2 inhibitors in bone resorption

Prostaglandins and IL-1 have long been known to be major mediators of osteoclast activation and bone resorption. Recent studies indicate that IL-1 causes initial PGE₂-independent bone resorption followed by induction of COX-2. A variety of other chemokines are also involved in the process (IL-6, IL-11 and TGF-b (which is stored in the bone matrix and is released during bone damage), all induce COX-2 and bone resorption.

In the study by Macial et al. (41), it was reported that PTH induces COX-2 expression in human osteoblast that is significantly altered by NS-398 (a specific COX-2 inhibitor). These studies suggest that the COX-2 inhibitors may be therapeutic agents for bone-related disorders.

CONCLUSIONS AND FUTURE TRENDS

The discovery of inducible cyclooxygenase enzyme has given a new impetus in the development of safer antiinflammatory drugs. The results of animal experiments and early clinical studies with selective COX-2 inhibitors are quite impressive and support that these selective COX-2 inhibitors will represent an effective gastrointestinal-sparing alternative to classical NSAIDs and will be beneficial in other clinical situations in which COX-2 is overexpressed, besides their therapeutic indication. Recently, various challenges posed to the COX theory that prompt a reevaluation of the original theory and a reexamination of whether the selective inhibition of COX-2 might not be as effective or as safe as anticipated. Ferreri et al. (42) reported that COX-2-deficient genetically engineered mice develop a severe nephropathy. Further data suggest that the selective inhibition of gastrointestinal COX-2 may be associated with intestinal ulceration or impaired ulcer healing (17, 19). Immunohistochemical studies suggest that there is the marked accumulation of COX-2 at the edge of an ulcer healing (18). Moreover, an important consideration is the potential consequences of inhibition of COX-2 in tissues where this enzyme has been constitutively expressed (i.e., brain and kidney). Whether or not selective inhibition of COX-2 fulfills the therapeutic potential will depend on long-term safety of selective COX-2 inhibitors.

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Address for correspondence: S.K. Kulkarni, Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, 160014, India. E-mail: skpu@yahoo.com

Methods and Findings in Experimental and Clinical Pharmacology Vol. 22, No. 5, 2000, pp. 291-298
ISSN 0379-0355 Copyright 2000 Prous Science, S.A. CCC: 0379-0355/2000 http://www.prous.com