INTRODUCTION:

The league table listing the ‘top 20’ drugs includes rofecoxib (Vioxx) and celecoxib (Celebrex), two inhibitors of the prostaglandin-forming cyclooxygenase (COX) (FIG. 1),which between them commanded sales in excess of US $4 billion in the year 2000.¹ This statistic might seem surprising — after all, the therapeutic use of COX inhibitors has a venerable history dating back to the introduction of aspirin in 1898 (or even earlier if the use of salicylate-containing plant extracts is included) and, since then, the field has a record of almost continuous development. The 1940s saw the introduction of phenylbutazone, the fenamates appeared in the 1950s, indomethacin in the 1960s, the proprionates in the 1970s and the oxicams in the 1980s.

With such a long record of drug discovery in the area and such a vast range of drugs to choose from, one might be forgiven for thinking that the wellspring of chemical innovation that nurtured the field so efficiently over the past century must have long since run dry or at least begun to falter — and that the current market would have little room for new versions of what would seem to be a rather tired and well-worn formula. However, the discovery in the early 1990s of a second COX isozyme revitalized the field and stimulated a hunt for new and selective isoform inhibitors. This culminated in the introduction of Vioxx and Celebrex in Europe and North America within a mere ten years, and, at the same time, brought a fresh perspective on the unusual therapeutic profile of several existing nonsteroidal anti-inflammatory drugs (NSAIDs).But we are getting ahead of ourselves — how did this idea of the second isoform come about in the first place and what are the therapeutic advantages of these new inhibitors over the many older drugs which, after all, have seen valiant clinical service over the decades? To answer these questions, we must return to the 1970s when much of the seminal work on COX inhibition was published².

Early work³

Although the NSAIDs do not reverse the course of systemic diseases such as arthritis, they form the main stay symptomatic treatment of many inflammatory disorders and soft-tissue injuries. Approximately 50 NSAID preparations are listed in Monthly Index of Medical Specialties and, as a class; these are among the most commonly prescribed drugs. In the United Kingdom, for example, recent data (1999) indicate that 18.5 million prescriptions are written for NSAIDs each year at a cost of £170 million and this figure does not take into account the over-the-counter sales, which are considerable. Aspirin itself is still consumed in prodigious amounts around the world and new uses are continually being found for this drug.

NSAIDs are sometimes known as the aspirin-like drugs because they have an activity profile that is broadly similar to that of aspirin that is, they all possess analgesic, anti-inflammatory and antipyretic properties to some degree, and produce characteristic side effects, including gastric intolerance and depression of blood clotting through inhibitory action on platelet function. As a group, the NSAIDs are structurally diverse, with most (but not all) being carboxylic acids (FIG. 2). The main question, from the pharmacologist’s point of view, was how these apparently disparate therapeutic and side effects were mechanistically linked. There were several early suggestions, but the real breakthrough came in 1971.Vane tells us4 that the idea that the aspirin-like drugs blocked the conversion of substrate arachidonic acid to prostaglandins came to him while reviewing experiments in which aspirin blocked the release of ‘rabbit aorta contracting substance’ (RCS) from guinea-pig and dog lung. Believing that RCS was an intermediate in prostaglandin synthesis, he wrote “a logical corollary was that aspirin might well be blocking the synthesis of prostaglandins”. However, the discovery in the early 1990s of a second COX isozyme revitalized the field and stimulated a hunt for new and selective isoform inhibitors. This culminated

in the introduction of Vioxx and Celebrex in Europe and North America within a mere ten years, and, at the same time, brought a fresh perspective on the unusual therapeutic profile of several existing nonsteroidal anti-inflammatory drugs (NSAIDs).But we are getting ahead of ourselves how did this idea of the second isoform come about in the first place and what are the therapeutic advantages of these new inhibitors over the many older drugs which, after all, have seen valiant clinical service over the decades? To answer these questions, we must return to the 1970s when much of the seminal work on COX inhibition was published.

Figure 1 | An overview of prostaglandin synthesis and metabolism⁴.

In theory, free fatty acids such as arachidonate can be formed from several sources, although phospholipid-bound arachidonate is probably the most significant pool. Phospholipases, especially cytosolic phospholipase A2 (cPLA2), are highly-regulated enzymes (by the MAP kinase (MAPK) pathway) that liberate arachidonate, which is then transformed by the cyclooxygenase (COX) complex. The mechanism of this reaction is complex 2 moles of molecular oxygen are introduced sequentially by a lipoxygenase reaction, followed by a COX reaction. This generates PGG2, a 15-hydro-peroxide prostaglandin that is reduced to PGH2, the corresponding hydroxy product. Both of these intermediates are short lived but may have independent not yet clear whether this independent activity is significant in vivo. A further battery of enzymes transforms PGH2 into a variety of products. Some of these enzymes, such as thromboxane (TX) synthase and prostacyclin (PGI2) synthase, show marked tissue localization (for example, TX synthase in platelets and PGI2 synthase in vascular endothelium). Other enzymes, such as the endoperoxide reductases and isomerases, are quite widely distributed, although in some cases they can be induced following inflammatory stimuli. In vitro at least, the endoperoxides PGG2 and PGH2 can also decay spontaneously (S) to PGF2α, PGE2 and PGD2. The evanescent products, TXA2 and PGI2, decay spontaneously to their respective inactive metabolites, TXB2 and 6-keto PGF1α. The biological activity of the other prostaglandins is curtailed following uptake into cells, by a series of metabolic enzymes that are present in some tissues (for example, the lung) at high concentrations. Inactive metabolites of these prostanoids undergo carbon chain shortening (especially in the liver) prior to secretion in the urine, in which estimates of total body prostaglandin turnover can be made by selectively monitoring these products. The most significant products from the point of view of this review are PGE2, because of its importance in inflammation, fever and pain; PGI2 because of its antiaggregatory action and possible role in hyperalgesia, and TXA2 because of its important role in platelet aggregation. Colour code: red, precursors; orange, intermediates; yellow, ‘primary’ prostaglandins that mediate most of the biological activity of this system; green, inactive or largely inactive metabolites; brown, end metabolites that are excreted primarily in the urine variations in the inhibitory potency of indomethacin against COX enzymes prepared from a range of tissueswas subsequently reported¹⁵ and the isoenzyme idea was further elaborated in several reviews^13,16.Parenthetically, one might add that the pharmaceutical industry now possessed, probably for the first time, a simple and robust in vitro technique to screen compounds for putative anti-inflammatory activity. This in itself was a significant advance, and the number of chemical abstracts dealing with potential inhibitors of the COX enzyme rose markedly, with more than 2,500 per year recorded within a decade of these ideas taking hold⁵.

Binding site of cox-1 and cox-2

Figure 3 | Comparison of the NSAID binding sites of COX1 and COX2 after Browner.

Schematic cartoon, showing the differences in the NSAID binding sites of COX1 and COX2. Note that the COX2 binding site is more accommodating and is characterized by a ‘side pocket’ that can accommodate bulky groups such as the methyl sulphonyl moiety of DuP697. COX, cyclooxygenase; NSAID, non-steroidal anti-inflammatory drug. gastrointestinal tract and elsewhere. This implies that inhibition of the healing response in various tissues might be an unwelcome side effect of these drugs. Unexpectedly, celecoxib (as well as ibuprofen, a mixed inhibitor) reduced the generation of the protective eicosanoid prostacyclin in humans, giving rise to a concern about unwanted cardiovascular side effects. Taking another tack entirely, Wallace and his colleagues have argued convincingly that inhibition of one isoform in the gastrointestinal tract can be compensated for if the other isoform is not inhibited, and that it is only mixed inhibitors of the COX isoforms that give rise to gastrointestinal damage. Interestingly, the administration of COX1 inhibitors leads to the rapid induction of COX2 in the gastric mucosa (presumably a protective response), perhaps explaining why both need to be inhibited to produce damage. Despite all these qualifications, the hypothesis has taken us a long way forward. It has led to the discovery of a family of drugs that are better tolerated than the older NSAIDs, our knowledge of the action of COX inhibitors has been greatly fortified and the advent of COX2-selective drugs has enabled us to discern the role of this enzyme in physiological or pathological processes. There have, of course, been new technical and other problems to solve. The question of the correct way of assessing and expressing the selectivity of COX1/ COX2 as some of the newer agents⁶.

Some key residues in COX1 and COX2. The left hand panel shows a schemetic diagram of NSAID-binding site cox1.the highly conserved residues Arg120 and tyr355 stabilize the carboxylate group that is present in most NSAIDS whereas the aromatic ring structure are accommodated within the largely hydrophobic binding channel and often about into the highly conserved Tyr385.Tyr385 is close to the peroxidise site that forms a tyrosyl redical that is crucial to the introduction of molecule oxygen into the arachidonic acid substrate.Ser530 is the residue that is acetylated by aspirin note the presence of relatively bulky lle523 and the presence of Leu384 in proximity to phe503 and the presence of His513.the right hand penal depict the cox2 binding site. The highly conserved Arg120 and Tyr355 are present as before as is Tyr385 and Ser530. However residue now becomes Leu503 which being less bulky is not packed as tightly .this allows expansion of available space at the top of channel⁷.

The COX1/COX2 concept today

How does this very influential idea stack up today in the light of the results that have been published on the actions of the coxibs and other selective inhibitors of the COX2 isoform? Like all ideas, it has seen some modifications over the passage of time. At the tissue level there is an awareness that COX2 is more widespread in ‘normal’ tissue than was at first suspected and that it is not necessarily restricted to inflammatory sites. Rat brain has a large amount of the enzyme under ‘resting’ conditions, with further COX2 induced at separate sites following the administration of lipopolysaccharide⁸. Certain other tissues around the body, including the MACULA DENSA region of the kidney⁹, are also rich sources of the enzyme under ‘normal’ conditions and, probably because of this, COX2 inhibitors can cause hypertension and fluid retention in some patients¹⁰. Conversely, it has become apparent that COX1 can also be regulated under some circumstances¹¹. The central idea that it is the inhibition of COX2 that is responsible for the therapeutic actions of the drugs, whereas COX1 inhibition is responsible for the side effects, is still valid, although there are some caveats. For example, there have also been well founded reports that COX2 is present in the marginal tissue of healing ulcers and that COX2 products might contribute to the resolution of inflammation in the gastrointestinal tract and elsewhere. This implies that inhibition of the healing response in various tissues might be an unwelcome side effect of these drugs. Unexpectedly, celecoxib (as well as ibuprofen, a mixed inhibitor) reduced the generation of the protective eicosanoid prostacyclin in humans, giving rise to a concern about unwanted cardiovascular side effects. Taking another tack entirely, Wallace and his colleagues have argued convincingly that inhibition of one isoform in the gastrointestinal tract can be compensated for if the other isoform is not inhibited, and that it is only mixed inhibitors of the COX isoforms that give rise to gastrointestinal damage. Interestingly, the administration of COX1 inhibitors leads to the rapid induction of COX2 in the gastric mucosa (presumably a protective response), perhaps explaining why both need to be inhibited to produce damage.

Lessons for drug discovery

So what lessons can we draw from this story? There are many ways of discovering new drugs, but it is clear that the rapid and successful discovery and marketing of celecoxib and rofecoxib owed little to the high-tech approach that most feel is the way forward for the pharmaceutical industry in this post-genomic era. It can be argued, with some justice, that this was a unique case, in that several potential lead compounds were already apparent from the literature. But at the very least, the episode shows that highly successful drugs can still be discovered (and substantial sales achieved) by organizations that are flexible in their approach to finding new medicines. It stresses the key role of the skilled and alert scientist backed by management with the vision and resources to exploit such findings as they arise. The manner in which COX2 was identified also reminds us that we seldom know where the cutting edge of our field is at any one time. It highlights the extraordinary value of the anomalous observation and warns us to be wary of ignoring compounds that have therapeutic profiles that do not quite ‘fit’. Molecular biology was key in the discovery and characterization of the COX2 isozyme, but the outcome of the initial transgenic ‘hypothesis testing’ studies were, perhaps, not so clear, and those who rely heavily on transgenic disease models would be right to regard this as a cautionary tale. The other point that emerges clearly is the significance of structural data, with its important lesson that even small and apparently unimportant changes in sequence might make a huge difference to the pharmacology of the enzyme.

The future of COX pharmacology

Are there any more ‘anomalous’ COX inhibitors out there and can we expect any further surprises from the COX system? Perhaps, we do not have to look far for drugs with unusual inhibitory mechanisms. In fact, the oldest NSAID of all must surely fall into this category. Clinically, salicylic acid is almost equipotent with aspirin as an anti-inflammatory drug but shows up with much lower IC50values in all in vitro assays. The discovery of COX2 has not helped in this instance, as the drug is a weak inhibitor of both isoforms in vitro. A key observation relating to this matter was published in 1972 by Hamberg, who measured the excretion of prostaglandin metabolites in volunteers taking indomethacin, aspirin or salicylic acid. Indomethacin was the most potent, but both aspirin and salicylate (4 × 600 mg per day) had a similar maximum effect on prostaglandin output. Although the numbers were small, there was a tantalizing hint of a latency of effect with the latter drug, indicating a mode of action other than simple COX inhibition. Several attempts have been made to solve the discrepancy between the potency of salicylic acid in vitro and in vivo. Early studies focused on the possibility that salicylate metabolites, such as the dihydroxy gentisic acid, were active inhibitors of COX a notion that has received recent support.A further suggestion that has been widely canvassed is that salicylate inhibits transcription of COX genes by preventing phosphorylation of IκBα (inhibitor α of nuclear factor-κB (NF-κB)), thereby inhibiting NF-κB activation^12,13, or by inhibiting COX2 gene transcription. Tumour necrosis factor (TNF)-stimulated inducible nitric oxide synthase (iNOS) gene expression and adhesion molecule expression in HUVECs has been reported to be inhibited by both aspirin and sodium salicylate. However, this is by no means universally agreed as a potential mechanism for controlling COX. Effects on other signaling systems, such as extracellular signal-regulated kinase and c-JUN N-terminal kinase in human neutrophils and fibroblasts have also been claimed, as well as other adenosine-dependent effects of these drugs that are apparently maintained in Nfκb-null mice¹. What this all means mechanistically and whether any of these studies are truly relevant to therapeutic dosing is still not entirely clear. Ironically, it seems that although being the simplest from a structural viewpoint, the salicylates might turn out to be among the most complex drugs of all. The matter is still very relevant, for there is sound evidence from contemporary double-blind clinical trials that willow-bark extracts can ameliorate the pain of osteoarthritis, and this effect is almost certainly due to conjugated salicylate in the preparation. It is an area that will certainly repay a further inspection.

COX-3 inhibitors

Simmons also recently co-discovered COX-3 in 2002 and analyzed this new isozyme's relation to acetaminophen (paracetamol), arguably the most widely used analgesic drug in the world. (Chandrasekharan et al 2002). The authors postulated that inhibition of COX-3 could represent a primary central mechanism by which these drugs decrease pain and possibly fever. The clinical ramifications and knowledge of COX isozymes are therefore rapidly expanding and could perhaps offer significant hope for future treatments of pain, inflammation and fever¹⁵.

Paracetamol Phenacetin Phenazone (antipyrine)

COX-1, COX-2 and COX-3– the end of the story?

So far it is generally agreed that the constitutively expressed COX-1 enzyme is the predominant form in the GI tract, kidney and platelets, providing a rich source of physiologically important PGs. It is also evident that COX-2 is expressed at inflammatory sites by leukocytes as well as by activated mesenchymal cells. The conceptual framework and the properties of selective COX-2 inhibitors have been demonstrated as effective NSAIDs in blocking the signs and symptoms of inflammation.

Although this beneficial effect of specific COX-2 inhibitors is evident, some data suggest that in certain models PGs may be unexpectedly beneficial in the resolution of inflammation or tissue injury. Gilroy and Willoughby described the effect of selective COX-2 inhibitors on carrageenan pleurisy in the rat over a time course ranging from 0–48 h after injection of the irritant. This investigation produced surprising results and showed that efficacy by COX-2 inhibitors strongly depends on the time course of the inflammatory process: onset of inflammation, peak inflammation and resolution¹⁵.

If this hypothesis is really true, a marker for disease activity is urgently needed in order to determine the appropriate time for the use of selective COX-2 inhibitors and if a COX-3 enzyme really exists, this fact will lead to a generation of new anti-inflammatory drugs with a new therapeutic target.

REFERENCE:

1. Analysts Rx Product Desk Reference (Merrill Lynch, London, 2001).

2. Smith, M. J. and Dawkins, P. D. Salicylate and enzymes. J. Pharm. Pharmacol. 23, 729–744 (1971).

3. Collier, H. O. J. A pharmacological analysis of aspirin. Adv. Pharmacol. Chemother. 7, 333–405 (1969).

4. Smith, J. B. and Willis, A. L. Aspirin selectively inhibits prostaglandin production in human platelets. Nat. New Biol. 231, 235–237 (1971).

5. Collier, J. G. and Flower, R. J. Effect of aspirin on human seminal prostaglandins. Lancet 2, 852–853 (1971).

6. Luong, C. et al. Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2. Nature Struct. Biol. 3, 927–933 (1996).

7. Bazan, N. and Flower, R. Lipid signals in pain control. Nature 420, 135–138 (2002).

8. Cao, C., Matsumura, K., Yamagata, K. and Watanabe, Y. Induction bylipopolysaccharide of cyclooxygenase-2 mRNA in rat brain; its possible role in the febrile response. Brain Res. 697, 187–196 (1995).

9. Harris, R. C. et al. Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction. J. Clin. Invest. 94, 2504–2510 (1994).

10. Whelton, A., White, W. B., Bello, A. E., Puma, J. A. and Fort, J. G. Effects of celecoxib and rofecoxib on blood pressure and edema in patients > or =65 years of age with systemic hypertension and osteoarthritis. Am. J. Cardiol. 90, 959–963 (2002).

11. Smith, C. J., Morrow, J. D., Roberts, L. J., 2nd and Marnett, L. J. Induction of prostaglandin endoperoxide synthase-1 (COX-1) in a human promonocytic cell line by treatment with the differentiating agent TPA. Adv. Exp. Med. Biol. 400A, 99–106 (1997).

12. Pierce, J. W., Read, M. A., Ding, H., Luscinskas, F. W. and Collins, T. Salicylates inhibit I kappa B-alpha phosphorylation, endothelial-leukocyte adhesion molecule expression, and neutrophil transmigration. J. Immunol. 156, 3961–3969 (1996).

13. Fernandez de Arriba, A. et al. Inhibition of cyclooxygenase-2 expression by 4-trifluoromethyl derivatives of salicylate, triflusal, and its deacetylated metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid. Mol. Pharmacol. 55, 753–760 (1999).

14. Talley J.J., Prog. Med. Chem. 36 (1999) 201–234.

15. Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS, Simmons DL. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci USA 2002; 99:13926-31.

Received on 19.01.2010 Modified on 28.03.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 272-277