Department of Biochemical Pharmacology, The Medical College of St Bartholomew's Hospital, Charterhouse Square, London EC1M 6BQ, UK
Ever since the chemical synthesis of aspirin in 1899and even longer if you take into account the use of salicylic acid in the folk medicine of many culturesthe non-steroidal anti-inflammatory drugs (NSAIDs) have been a mainstay of rheumatological practice. The NSAIDs do not reverse the disease process (indeed, there are some who would argue that they may ultimately exacerbate it), but they do provide much needed relief from pain and inflammation, and it is for this reason that they are more often than not the first treatment provided for patients.
Whilst aspirin itself is still in current use, and may indeed be said to be the world's most widely consumed drug, the NSAIDs as a class have undergone several rounds of refinement and development. Looking back, one can identify different phases in their evolution: the early drugs such as aspirin and paracetamol (acetaminophen), the introduction in the 1940s of phenylbutazone, the enormous step forwards in terms of potency that we saw in the 1950s and 1960s with the discovery of the fenamates and indomethacin, the introduction of the better tolerated proprionates in the 1970s and, continuing that trend more recently, the oxicams and others. A cursory glance at Mimms reveals that there are ~50 different formulations of these drugs currently available to the UK-based physician as well as the numerous over-the-counter formulations which are available to the general public.
Throughout their history, then, we have seen an overall increase in NSAID potency and this has been accompanied, on the whole, by improved tolerability. Despite this trend, however, the classic gastrointestinal and other side-effects of the NSAIDs are just as much a feature of treatment with contemporary drugs as they were with aspirin itselfand in this context it is ironic to note that aspirin was thought initially to be a better tolerated drug in the stomach than the salicylic acid from which it was derived!
For many years, the pharmacological profile of the NSAIDs was a mystery. What was the connection between the apparently unrelated analgesic, anti-pyretic, anti-inflammatory and gastric irritant actions of these drugs, and why did they share other side-effects as well, such as prolongation of bleeding time, reduction in renal blood flow, and so on? The answer to the conundrum was the well-known series of observations made in the early 1970s by Vane and his colleagues [14] which linked both the therapeutic and side-effect actions of these drugs to the inhibition of a single enzyme, the prostaglandin-forming cyclooxygenase (COX). This discovery, and its subsequent development, revolutionized the way we think about these drugs and provided for the first time a clear explanation for the majority of both their therapeutic actions and side-effects. Indeed, once it was realized that inhibition of prostaglandin formation was a characteristic feature of all NSAIDs, it led to the notion that these drugs could be used for other clinical purposes where prostaglandins were thought to be involved, such as the closure of the ductus arteriosus in premature infants and the treatment of Bartters syndrome. Low-dose aspirin also has a prominent place in the treatment and prophylaxis of disorders such as myocardial infarction and stroke, which involve aberrant platelet aggregation, based upon its ability to inhibit platelet COX. New uses are being continually uncovered for such drugs, including, most recently, the treatment of dementia symptoms and colon cancer (reviewed in [5]).
Estimations of the relative potency of NSAIDs in different tissues and species led early workers in the field to speculate that there could be more than one form of COX (reviewed in [6]), but it was not until the early 1990s that definitive experimental proof was furnished for this idea. Elegant work from a number of laboratories [711] uncovered the presence of a new gene coding for a protein clearly related to COX which was subsequently named COX-2 to differentiate it from the original enzyme. Whilst similar to COX-1, COX-2 has a number of different properties, amongst which is the fact that it is an inducible enzyme and that it is generally present only in tissues undergoing mitogenic stimulation or inflammation. In contrast, COX-1 bears all the hallmarks of a constitutive enzyme. From these and other observations grew the notion that COX-2 was the enzyme which was most important for generating prostaglandins during pathological processes, such as those which might occur in the rheumatoid joint, whereas COX-1 was more important in producing prostaglandins for those physiological processes required for the maintenance of homeostasis, e.g. suppression of gastric acid production, regulation of platelet aggregation and renal blood flow, and so on. This notion, which is known to its practitioners in a rather Orwellian way as the `COX-1 good: COX-2 bad' hypothesis, received a further boost when the inhibitory proclivities of our current repertoire of NSAIDs was examined (cf. [12, 13]). It was found that, with a very few exceptions, all of these drugs were preferential inhibitors of COX-1, suggesting that when we treat our patients, the doses required to produce a therapeutic result (which are thought to be the result of the anti-COX-2 action) are produced at the cost of a substantial inhibition of COX-1 and that it is this latter action which is responsible for the side-effects such as gastric irritation. The hunt has therefore been on for selective inhibitors of COX-2 which, in theory at least, should not elicit such side-effects, but should be equally effective as anti-inflammatories and analgesics.
But just how selective do such drugs have to be and how do you assess this? In theory, it should be easy to say whether a drug is COX-1 or COX-2 specific, or whether it is a mixed inhibitor of both enzymes. In practice, however, this is more problematic: each laboratory has evolved its own way of conducting assays, which often differ significantly and may yield conflicting data. Amongst the common variables are the species that should be used for the assay, the most appropriate tissue source of the enzyme, whether to use cell-free preparations or cells, whether the assays should be optimized with respect to substrate and cofactors, and whether or not to allow pre-incubation of drugs with enzymes (as this, too, can alter their apparent inhibitory potency).
So which of the many assays available (if any) should be regarded as the gold standard? Again, this is not a straightforward question for it depends upon the sort of information that you wish to acquire. The needs of the medicinal chemist whose job it is to design novel, selective inhibitors of either enzyme are best served by some type of cell-free enzyme assay run under closely defined and optimized experimental conditions. Such an approach may throw up drugs which, whilst being active in vitro, are not active in vivo because of pharmacokinetic or other considerations, but nevertheless provides the purest information about enzyme inhibition and structureactivity relationships. But, as clinicians or pharmacologists interested in the use of these drugs in man, our need is for some reliable guidance about an assay system most likely to predict the efficacy of these drugs in the clinic.
The thorny issue of which assay to pick was tackled recently by an international group of experts and the results of their deliberations are presented in this issue of the journal in a paper by Brooks et al. on page 779. Out of the many assays which have been described, the authors have selected a system generally known as the `whole-blood assay'. This technique [14, 15], originally described many years ago for investigating the pharmacology of COX-1 inhibitors, has many advantages which are discussed at length in this article. All assays have their mechanistic peccadilloes and a slightly irritating feature of this system is the necessity for incubation of samples of blood for 24 h with an inducing agent such as lipopolysaccharide to ensure adequate levels of COX-2. Nevertheless, this technique is the best available and the authors are wise to select it as the `gold standard'.
The convention for expressing COX selectivity is based upon a ratio of the concentration (or dose) required to inhibit (usually 50%) COX-2 divided by the concentration required to inhibit COX-1. Thus, a drug inhibiting both enzymes to an equal extent would have a ratio of 1.0; if it were a more selective COX-2 inhibitor, it would have a ratio of <1.0, and vice versa. This type of data is fine for laboratory work, but the authors of this report have contrived a more useful functional definition for clinical researchers and define a COX-2- selective inhibitor as one `which inhibits COX-2 but does not inhibit COX-1 across the therapeutic dose range using the whole-blood assay'. Of course, the `selectivity' of a drug depends upon a number of other features apart from its relative ability to inhibit COX-1 and COX-2. For example, the frequency of dosage and the pharmacokinetic behaviour of the drug are important. Drugs which tend to hit very high initial plasma concentrations and which must be frequently administered run the risk of inhibiting COX-1 as well as COX-2 if the selectivity ratio is not adequate, whereas another drug with the same selectivity ratio, but which slowly attains and maintains a more stable blood level, may not do so. Such potential problems can also be examined using the versatile whole-blood technique using blood samples taken following patient dosage.
With the emergence of drugs which are virtually entirely selective for COX-2, we are entering an era which may well bring about radical changes in prescribing habits. If the encouraging preliminary data with highly selective COX-2 inhibitors such as rofecoxib and celecoxib are borne out by subsequent clinical experience, then they will not only provide excellent symptomatic relief from the signs and symptoms of inflammatory and other disease, but will probably displace most existing agents, for how would it be possible to prescribe a drug with known gastric side-effects when another, equally efficacious, which did not show this type of toxicity, was readily available? But this raises the question of whether or not there will be any place left for a selective COX-1 inhibitor. It is difficult to answer this at the moment, but interestingly, a recent paper by Smith et al. [16], which compared a very highly selective COX-2 inhibitor with an equally selective COX-1 inhibitor, strongly suggested that whilst the COX-1 inhibitor was able to reduce peripheral concentrations of prostaglandins in inflammatory exudates in an animal model of hyperalgesia, it was not able to produce analgesic actions and that this was a property exhibited solely by COX-2 inhibitors which were also able to reduce prostaglandin levels in the central nervous system. If this turns out to be an invariable observation, then we must consider COX-1 inhibitors as a group of drugs possessing mainly side-effects! However, it is highly likely that low-dose aspirin will remain in the clinical armoury for the treatment and prophylaxis of stroke and infarction because of its low cost, and its unique and long-lasting action in depressing platelet aggregation, which will probably remain unsurpassed for many years.
Are there any more surprises in store for us in the COX story? Possibly: a very interesting paper appeared recently by Simmons et al. [17], one of the co-discoverers of COX-2, which suggested that apoptotic cells express a variant form of this enzyme. On the face of it, this does not seem a tremendously interesting observation from the rheumatological view point, but the real fascination for us lies in the fact that this form of COX-2 has a different cellular location, is much more sensitive to the inhibitory effects of paracetamol than the `normal' COX-2 and much less sensitive to the inhibitory actions of several other of the standard non-steroidal drugs. This study implies that there may be variant forms of COX-2 (and, who knows, perhaps COX-1 as well) which may be present in tissues and which may still hold further secrets for us to unravel. Perhaps this discovery will at last explain the anomalous observations surrounding paracetamol, a drug which for many years has been known to exhibit unusual specificity in its inhibitory profile.
Whatever the outcome of the latest study, one thing is for sure: the discovery of COX-2 and the very rapid advances in NSAID pharmacology which have been made as a result of this discovery will change for ever the pattern of usage of this popular, and some may say indispensable, group of drugs. It also raises the whole question of reclassifying the NSAIDs into separate groups. An attempt has already been made at this by Frolich [18] and it seems inevitable that some sort of new classification system will eventually emerge. When it does so it will certainly depend very heavily upon the operational definitions and choice of assay system put forward by Brooks et al. in this journal, which represents an important attempt to rationalize and standardize this field. It is up to individual workers now to use these guidelines and incorporate their thinking into their clinical and other studies.
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