HYPOTHESIS

Hypothesis

COX-1, COX-2, and COX-3 and the future treatment of chronic inflammatory disease
Derek A Willoughby, Adrian R Moore, Paul R Colville-Nash A new generation of non-steroidal anti-inflammatory drugs has been described that selectively targets the inducible isoform of cyclo-oxygenase, cyclo-oxygenase 2 (COX-2). This isoform is expressed at sites of inflammation, which has led to the speculation that its inhibition could provide all the benefits of current nonsteroidal anti-inflammatory drugs, but without their major side-effects on the gastrointestinal system (which are due to inhibition of COX-1). We have shown that COX-2 (identified by use of specific antibodies) is induced during the resolution of an inflammatory response, inhibition of COX-2 resulting in persistence of the inflammation due to the prevention of the synthesis of a range of anti-inflammatory prostanoids. We propose that there is a third isoform of this enzyme family, COX-3, a proposal that will have implication for the prescription of both existing and new generation anti-inflammatory drugs, and might represent a new therapeutic target. Vane1 described the mode of action of aspirin-like drugs to be due to the inhibition of cyclo-oxygenase (COX). The enzyme that was originally used was COX-1, a constitutive isoform involved in a range of physiological functions. Inhibition of gastric constitutively expressed COX-1 resulted in most of the unwanted side-effects seen in patients. Non-steroidal inflammatory drugs (NSAIDs) treated the symptoms of inflammatory disease without affecting the underlying disease; indeed many NSAIDs accelerated cartilage breakdown in rheumatoid arthritis.2 Nevertheless, for many patients this family of drugs improved quality of life, if the patient escaped the unpleasant side-effects on the gastrointestinal tract. With the more recent discovery of a second isoform of COX, COX-2, the inducible isoform of the prostaglandin-forming enzyme that is expressed at sites of inflammation, we have been eagerly awaiting the new class of NSAIDs. 37 Studies that use in vitro methods show inhibitory activity of putative anti-inflammatory drugs on either COX-1 or COX-2.8 On the basis of these tests, many of the original NSAIDs were found to have inhibitory activity against both isoformsie, were dual inhibitors of COX-1 and COX2. The more selective COX-2 inhibitors had fewer gastrointestinal side-effects and thus, after 100 years of NSAIDs, we see the first major step forwarda new class of NSAIDs giving pain relief and reduction of the other classic signs of inflammation (heat, redness, swelling). This new generation of anti-inflammatory drugs, which are selective COX-2 inhibitors, are predicted to be, and largely are, free of gastrointestinal side-effects.
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Department of Experimental Pathology, William Harvey Research Institute, St Bartholomews and Royal London Hospital Schools of Medicine & Dentistry, Charterhouse Square, London EC1M 6BQ, UK (Prof D A Willoughby BSc, A R Moore PhD, P R Colville-Nash PhD) Correspondence to: Prof D A Willoughby (e-mail: d.a.willoughby@mds.qmw.ac.uk)

Is the discovery of COX-2 the end of this story? Will these drugs stay with us like aspirin for another century, or are there hopes of even more dramatic therapies arising from this group of COX enzymes? Many pharmaceutical companies use simple tests to identify anti-inflammatory activity, and this point is important to recognise. A classic example of a test, which has been used for decades, is the carrageenan rat paw oedema model. Putative anti-inflammatory drugs are generally tested for their effect on paw swelling at a single time point, 3 h after injection of the irritant. 9 This dependence on a brief window in time to predict anti-inflammatory activity and mode of action could be misleading. Thus, if concentrations of COX-2 are raised at this time point, leading to the production of proinflammatory prostanoids such as PGE2, but before or after this time point there are other enzyme systems and mediators active, these results cannot reflect an overall mechanism or process. Indeed, different types of experimental inflammation are recognised as being brought about by a sequential release of pharmacologically-active mediators such as histamine, 5-hydroxytryptamine, kinins, and prostaglandins.10 When the window of assessment is at a very early stage, anti-inflammatory activity with antihistamines is detected, this being the initial mediator of the acute inflammatory response. 10 Such a basis for the development of new therapeutics is, thus, less than satisfactory. A further important factor during the evolution of these models of inflammation is the changing cellular migration pattern, initially polymorphonuclear-cell dominated then switching to a mononuclear dominated reaction. This switch in cell populations is brought about by various chemotactic factors and adhesion-molecule expression, plus the apoptosis of the polymorphonuclear leucocytes leading to the mononuclear cell dominance. Thus, to examine inflamed tissue or exudates for enzyme activity at different time points is almost akin to examining a different tissue. Willoughby and colleagues11 described the effect of some selective COX-2 inhibitors and some dual

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HYPOTHESIS

Peak of inflammation

Resolution Onset

Cox-1 + Cox-2

Cox-1

? Cox-3 + Heme-oxygenase 1

Time FIgure 1: Hypothetical time course showing the expression of COX enzymes during the onset and resolution of an inflammatory response.

(COX-1 and COX-2) inhibitors on carrageenan pleurisy in the rat over a time course ranging from 048 h after injection of the irritant. This investigation gave surprising results in that the COX-2 inhibitors showed anti-inflammatory activity early in the onset of the inflammatory response, coincident with the expression of COX-2 protein, as did the older dual inhibitors such as indometacin. However, by 6 h the COX-2 inhibitors were without effect although the dual inhibitors still showed efficacy. At this point the COX-2 protein, as shown by western blotting, was no longer present. Even more surprising, at 48 hthe time of near complete resolution of inflammation in this model there was a second substantial increase in COX-2protein expression detected by western blotting. This COX-2 protein did not produce the proinflammatory prostaglandin PGE 2 in an ex vivo biochemical assay with exogenously supplied arachidonic acid, nor could this be detected at this time in vivo. By contrast, anti-inflammatory prostaglandins (PGD2 and PGF2 ) plus a member of the were cyclopentenone family (15deoxy1214PGJ2) produced in vivo at this time. When the same group12 suppressed this newly formed COX by treatment either with COX-2 selective inhibitor or the COX-2 dual inhibitor 2448 h after application of the irritant, these agents now showed a different effect in that the inflammatory response was not inhibited but continued and did not resolve, as seen in the untreated animals.12 The recognition of the enzyme protein was by western blot, by means of an antibody that was raised to a small epitope on the COX-2 protein. We postulate that this large expression of protein is not COX-2 but may represent a third isoform of COX, COX-3, which unlike COX-1 and COX-2 does not produce proinflammatory prostanoids but produces anti-inflammatory members of that family (figure). The expression of a catalytic variant of COX-2 or a third COX enzyme has been shown in vitro in transformed macrophages treated with high doses of NSAIDs (apart from aspirin and paracetamol) for 48 h.13 This enzyme (again identified by an antibody against COX-2) is sensitive to inhibition by paracetamol but less sensitive to other NSAIDs compared with COX-2 induced by lipopolysaccharide. However, this scenario is clearly different from that which exists in the resolution phases of inflammation in an untreated

animal. Furthermore, the COX-2 like protein in the transformed macrophages produced PGE 2 in response to exogenous arachidonic acid, unlike the COX-2-like protein in samples from the 48 h pleurisy in which PGE 2 synthesis was not seen in response to exogenous arachidonic acid nor could PGE2 be detected in the pleural exudates at this time. Perhaps, therefore, there exists yet another isoform of COX that might be expressed under certain circumstances. One potential mechanism for the induction of this COX-2like protein in the transformed cells is via the activation of a group of orphan nuclear transcription factors, the peroxisome proliferator-activated receptors (PPARs).14 This group of receptors might be activated by various chemicals known as peroxisome proliferators, including drugs such as the fibrate-based compound clofibrate and NSAIDs14 an activation that induces expression of a COX-2 like protein. Again, unlike COX-2 induced by other stimuli, this COX-2 does not produce cellular PGE2 although, again, exogenous arachidonic acid will result in PGE2 synthesis. Notably, one of the proposed anti-inflammatory prostanoids proposed to be important in the resolution of the carrageenan pleurisy is 15deoxy1214PGJ. Inhibition of the production of 15deoxy1214PGJ2 results in an exacerbation of the model, a process than can be reversed by its This agonist is a exogenous replacement.12 naturally occurring PPAR agonist, and activation of this system in macrophages is associated with antiinflammatory effects in macrophages. 1517 If this hypothesis is true, expression of this third inducible isoform of COX could result in the typical periods of remission seen in many clinical cases of chronic inflammatory disease such as rheumatoid arthritis. If this hypothesis is further proved in man, an urgent need for markers of disease activity would be needed, thus making it possible to stop prescribing these and similar drugs (dual or COX-2 inhibitors) at periods when the patients should naturally be moving into remission. Indeed, prescribing these drugs could retard normal periods of remission. Furthermore, if this third isoform, COX-3, is found to have these anti-inflammatory effects in human beings, could we promote the production of this enzyme and use the naturally occurring factors it produced to modulate the inflammatory response? The production of cyclopentenone prostaglandins by COX-3 at 48 h in the carrageenan pleurisy12 provide a link to the induction of other endogenous anti-inflammatory mechanisms such as the stress protein heme-oxygenase 1. 15 We have previously proposed that modulation of this naturally produced heat-shock protein could represent a unique therapeutic mechanism. Experimentally, an increase in concentration of this inducible enzyme has antiinflammatory activity that is equivalent to or greater than that seen with steroids in inflammatory models. 18 This pathway has, as yet, also to be specifically targeted therapeutically in human beings. If these hypotheses are proven we hope that some of the pharmaceutical companies would consider these alternative therapeutic targets rather than following what will inevitably be a rush to produce COX-2 inhibitors, thus producing a series of me too therapeutic agents.

Arbitrary inflammatory response

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References
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