The 15 invited conference speakers reported their most current findings on cyxlooxygenase (COX-1 and COX-2) inhibitor research including the latest technical developments concerning the relevance of COX-2 (inducible cyclooxygenase-2) structure and function and COX-2 inhibitors in human inflammatory diseases with particular reference to rheumatism, gastroinrestinal (GI) disorders such as colon cancer, ulcer formation and Alzheimer's disease (AD). On focus were the latest clinical and laboratory studies on the most recent generation of cyclooxygenase inhibitors or "coxibs" (etoricoxib, reofecoxib, celecoxib, etc.). Also covered were historical perspectives on the cyclooxygenases, COX-2 in renal, cardiovascular, gastointestinal complications (including H. pylori effects), oesophageal and lung carcinomas and apoptosis function, differential testing for COX-1 and COX-2 activities and cDNA array and transgenic analysis of proinflammatory events which accompany AD.

Man's Fight Against Rheumatism: From Willow Bark to Selective COX-2 Inhibitors-Sir John R. Vane, London UK (Nobel Prize in Medicine, 1982, for the discovery and pharmacology of of prostaglandins):

Sir John Vane eloquently reviewed that historically, extracts of salicylate-containing plants (i.e., myrtle, meadow sweet, willow bark, poplar, etc.) have long been used (as analgesics) to treat rheumatism. Salicylates, predominantly as ASA (acetyl salicylic acid) are currently utilized at 45,000 tons/year. During the 1960s, the contribution of the prostaglandins to inflammation, fever and pain became evident. Chemically diverse members of non-steroidal antiinflammatory drugs (NSAIDs) inhibiting the key enzymes in prostaglandin biosynthesis (which we now call cyclooxygenases or COXs) were developed. However, their side effects, especially on the stomach and GI tract, were, and still remain, a major problem. COX-1 and COX-2 have clear and distinct physiological functions. COX-1 is involved in the production of prostacyclin which when released by the gastric mucosa is cytoprotective. COX-1 in platelets produces thromboxane A, which promotes platelet aggregation and leads to thrombus formation. COX-2 is strongly induced by inflammatory stimuli such as cytokines and growth factors. COX-2/COX-1 ratios of a range of NSAIDs were discussed, and there appears to be a strong correlation between the COX-2/COX-1 ratios and epidemiological data for GI disorders. Side effects of conventional NSAIDs include perforations, ulcerations and bleeds (PUBS) that leads to the hospitalization of more than 100,000 patients a year in the USA alone. About 15% of these patients die in intensive care. Meloxicam, etodolac, celecoxib, and nimesulide are all since recognized as selective COX inhibitors. New indications are being explored for selective COX-2 inhibitors, for use in colon cancer and in Alzheimer's disease.

The Structural Basis for COX-2 Selectivity-Christopher Bayly, Merck Frosst , Dorval Quebec:

Studies of the structural basis of COX-2 selectivity have been difficult due to the flexibility of the substrate, arachidonic acid and high homology (84%) between COX-1 and COX-2 in mammalian species. The x-ray crystal structure of sheep COX-1 was described by Picot et al. (1994) and more recently for COX-2, which has a larger central pore feature. The valine 523 sidechain in COX-2 is a single methyl group smaller than the corresponding isoleucine sidechain in COX-1 and this opens up an extra side pocket in COX-2 which is available for an inhibitor. Structure-based design of COX-2 selectivity for flurbiprofen based on this data was presented. Structure-activity relationships associated with Merck's second-entry COX-2 selective clinical candidate, etoricoxib (MK-663), open up new questions regarding the structural basis for COX-2 selectivity.

COX-2 and Renal Function-Mairead A. Carroll, Nicholas R. Ferreri and John C. McGiff, Department of Pharmacology, New York Medical College, Valhalla, NY, USA:

Data were presented that low dietary salt stimulates COX-2 expression and synthesis of 20-hydroxyeicosatetraenoic (20-HETE), the principal arachidonate metabolite and vasoconstrictor produced by preglomerular microvessels, particularly in the afferent arteriole (similar mechanisms in the brain?). 20-HETE mediates both TGF and renal autoregulation and, thereby, is essential to the regulation of extracellular fluid volume. These findings impact directly on the renal (brain?) functional effects of NSAIDs and both the selective and non-selective inhibitors of COX-2. COX-2 is also critically related to differentiation and maturation of the nephron, which peaks in the first two postnatal weeks in rats and this is associated with a sharp decline in glucocorticoid plasma levels. (effects on developing brain?) Studies in adult animals have indicated the importance of glucocorticoids to arachidonate metabolism (via cytochrome P450 monooxygenases) in a key nephron segment, the medullary thick ascending limb, and this is responsible for establishing the osmotic gradient for concentrated urine excretion. Adrenalectomy induces COX-2 expression in this segment unless glucocorticoid treatment is utilized. The actions of COX-2 therefore contributes to renal function both in health and disease.

COX-2 and the Cardiovascular System-Garret A. FitzGerald, University of Pennsylvania, Philadelphia, PA19104, USA:

COX-2 expression is highly regulated by cytokines and growth factors and represents the predominant source of prostanoid formation during the inflammatory processes. COX-2 is also highly regulated during development-it is predominantly expressed in the developing vasculature, and this may be important in developing brain vasculature function. Studies with inhibitors suggest that COX-2 is the major source of inducible prostacyclin formation in healthy humans in a variety of tissues. Data was reviewed showing that prostacyclin (a) modulates the response to thrombotic stimuli, (b) inhibits the vascular response to injury (c) inhibits leucocyte-endothelial interactions, and (d) is also generally proinflammatory in vivo to human physiology.

Preclinical Studies of COX-2 Inhibitors-Michel Pairet, Boehringer Ingelheim Pharma KG, Ingelheim am Rhein, Germany:

Data were presented that selective COX-2 inhibitors appear to offer superior GI tolerability to standard NSAIDs while they also possess a comparable anti-inflammatory and analgesic activity. With the exception a few models of acute inflammation that appear to be driven by COX-1, pharmacological data obtained in various models of acute and chronic inflammation support the hypothesis that it is almost exclusively COX-2 that is involved in the body's inflammatory response. Selective COX-2 inhibitors appear not to induce GI lesions when given at anti-inflammatory doses in preclinical studies and also appear to have better GI tolerability than standard NSAIDs in clinical studies. Other side-effects of NSAIDs and the potential advantages of selective COX-2 inhibitors are that standard NSAIDs inhibit both COX-1 and COX-2 when given at usual clinical doses. The side effects of selective COX-2 inhibitors should be no different and no greater when equivalent doses are used. Regarding the long-term safety of selective COX-2 inhibitors, new COX-2 preparations are at the forefront of drug development. Finally, it was pointed out that COX-2 is implicated not only in inflammation but also in other conditions such as preterm labour, colon cancer, and in emerging studies on the amelioration on the progression of AD. One area which will remain the sole preserve of selective COX-1 inhibition (and low-dose ASA in particular) concerns the inhibition of platelet aggregation and thereby in the prevention of cardiovascular disease.

Differential Tests for COX-1 and COX-2-Tim Warner, William Harvey Research Institute, London EC1M 6BQ, UK:

Data were reviewed concerning how NSAIDs produce their beneficial effects by inhibiting COX-2, and their deleterious effects by inhibiting COX-1. NSAID selectivity for COX-1 in comparison to COX-2 (COX1-to-COX-2 ratios) are, therefore, essential for the production of the next generation of NSAIDs with high efficacy for inflammatory disease treatment. It was stressed that it is clearly important that in developing novel COX-2-selective NSAIDs these compounds are screened against human-specific COX systems. Platelets derived from human blood were used in assays and COX-1/COX-2 selectivity of a range of NSAIDs and novel COX-2 inhibitors were established. The importance of these in vitro assays was displayed as to be essential to predict the efficacious vs. deleterious effects of NSAIDs employed in human clinical studies. For example, it was found that the singly most GI toxic NSAID, ketorolac, is many hundreds of fold selective for COX-1. It was concluded that COX-1/COX-2 comparative studies support the contention that inhibitors selective for COX-2 will be more efficacious anti-inflammatories while producing less side effects than traditional, non-selective, NSAIDs. Key Reference: Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA and Vane JR. Nonsteroid drug selectivities for cyclooxygenase-1 rather than cyclooxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci USA 1999;96:7563-7568.

Apoptosis Induced with NSAIDs-Daniel L. Simmons, Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT:

Ideas were clearly elaborated that apoptosis is intrinsic and essential to development and the maintenance of cellular homeostasis. However, NSAIDs induce apoptosis in vitro in neoplastic cells and in GI tumors in vivo. Thus NSAIDS may have important clinical applications in the treatment and prevention of cancer. Relatively high concentrations (10 uM-mM) of NSAIDs are required to induce apoptosis at sites other than those involving the targets of COX-1 or COX-2. NSAIDs may in addition induce apoptosis in cells deficient in both COX-1 and COX-2. Proposed sites of NSAID interactions were reviewed and these may also include k-ras, PPARα/γ, PPARδ, IKB kinase, cGMP phosphodiesterase, gastrin binding protein signaling and the mitochondrial electron transport chain systems. Importantly, alternative explanations for the causation of apoptosis on COX-1- or COX-2-deficient cells is that other forms of COX may exist (COX-3?). NSAID-induced apoptosis may cause unwanted side effects during therapeutic use of NSAIDs such as in thinning of the cornea by diclofenac or ketorolac during treatment of ocular inflammation. It was concluded that understanding the mechanism of NSAID-induced apoptosis is therefore vitally important in multiple clinical contexts.

Gastrointestinal Effects of NSAIDs-Brendan J.R. Whittle, William Harvey Research Institute, London, UK:

Data were presented that mechanisms by which NSAIDs evoke gastrointestinal injury are multifactorial. For example, the inhibition of prostanoid production greatly augments the injury provoked by locally acting irritants, indicating that there are interactions between mechanisms of toxicity. Experimental and clinical evidence each support the idea that the beneficial actions of selective COX-2 inhibitors are associated with minimal gastrointestinal side effects

NSAIDs, H. Pylori, Ulcers and GI Complications: What Are the Connections?-Denis M. McCarthy, University of New Mexico School of Medicine, Albuquerque, NM:

While there is a good association of ASA or non-aspirin non-steroidal drug (NANSAID) use with (GI) tract disturbances, little is clear as to the exact pathogenesis of GI bleeding, perforation or fibrosis. ASA, via platelets inhibition, is the leading cause of GI bleeding-it was pointed out that the amount of ASA associated with impaired platelet aggregation in human (10mg/d) or risk of significant GI bleeding (30mg/d), are lower associated (900mg/d) with a significantly increased risk of gastric ulcer (GU) and no risk of duodenal ulcer (DU) has been established. It was concluded that DU pathogenesis is more closely linked to H. pylori infection and not to NSAID use.

Epidemiology of Gastrointestinal Side Effects of NSAIDs-Luis A. Garcia Rodriguez, CEIFE, Madrid, Spain:

Epidemiological studies have confirmed many times that upper GI tract bleeding and perforation are each strongly associated with NSAID use. A systematic review of individual and cohort studies in the literature was performed and it was concluded that aging, history of GI disturbances, predisposition to peptic ulcer disease, male gender, use of steroids, aspirin (ASA), and oral anticoagulants (to stop GI bleeding) were the strongest risk factors for upper GI tract disturbances. These risk factors need therefore to be considered and controlled for in patients receiving specific NSAIDs for the treatment of rheumatism, GI disorders such as colon cancer and Alzheimer's disease (AD).

Prostaglandins Enhance the Malignant Potential of Cultured Cells-Raymond N. DuBois, Vanderbilt University, Nashville, TN:

Experimental evidence was presented here that suggested that there was a demonstrated increase in prostaglandin endoperoxide synthase-2 or cyclooxygenase-2 (COX-2) expression in a variety of colon, skin, lung, breast, bladder, and prostate cancers. Selective COX-2 inhibitors, such as the "coxib" sereies (celecoxib, etoricoxib, etc.) blocks the growth of such cancers. Data presented here evaluated the contribution of host-derived COX-1 and COX-2 expression in tumour growth using both transgenic and pharmacologic approaches. Data were presented that showed decreases in the vascular density of tumours grafted onto COX-2 -/ - mice when compared to those grown on wild type or COX-1 -/- animals. Further studies were presented showing that endogenous and exogenous prostaglandin E2 (PGE2) affects the morphology and growth of colorectal carcinoma cells. Endogenously produced PGI2 serves as an excellent ligand for PPAR , a series of nuclear receptors that regulates the expression of several genes involved in the inflammatory response.

COX-2 in Oesophageal, Gastric, and Lung Carcinomas-Ari Ristimaki, University of Helsinki, Finland:

NSAID use is strongly associated with reduced risk of cancer, especially in the GI tract. Ristimaki and colleagues were the first to show that COX-2 gene expression (RNA and protein) are elevated in human gastric adenocarcinoma. COX-2 was also shown to be expressed in pre-neoplastic epithelium such as in the dysplasias of the stomach and in the atypical alveolar epithelium of the lung. Selective COX-2 inhibitors or disruption of COX-2 gene expression was shown to suppress the carcinogenic phenotype of transformed cells in vitro and in tumor formation of animal models in vivo. COX-2-derived prostanoids stimulate proliferative increases in the production of matrix metalloproteinases-they also promote angiogenesis, and induce immunosuppression. COX-2 expression has a prognostic value in malignant diseases, although COX-2 derived prostanoids are associated with potentially serious and unwanted side effects.

Expression and Regulation of COX-2 in the CNS; Relevance to Alzheimer's Disease-Nicolas G. Bazan, Victor L. Marcheselli & Walter J. Lukiw, Louisiana State University, New Orleans:

Data were presented that brain COX-2 is overexpressed in response to ischaemia, trauma, seizures, and neurodegeneration such as in AD. Using cDNA microarray technologies and CA1 human hippocampus from AD patients and carefully age-matched controls this laboratory was the first to show upregulated expression of lipid signaling inflammatory genes in AD (such as that for COX-2) when compared to normally aging brain (Alzheimer Reports 3:161-167, 2000). DNA sequence analysis and deletion-mutation experiments also revealed that the promoter for the inducible human COX-2 contained AP1, AP1-like, AP2 , NFKB, HIF-1, SP1, STAT1(GAS) and SNN transcription factor consensus sequences. In fact, comparative analysis of transcription factor-DNA binding consensus sequences, within the immediate promoters of eight other genes implicated in the pathogenesis of Alzheimer's disease discloses several analogies in the use of proinflammatory transcription factor signaling (Alzheimer Report 3:233-245, 2000). Using LAU-8080, a specific intracellular platelet-activating factor antagonist, we found that this bioactive phospholipid is a key intermediate in triggering AP1, NF- B, STAT1 activation and cFOS and COX-2 gene expression during experimental epileptogenesis. PAF was identified as a transcriptional activator of COX-2. Data also reviewed that modulation of COX-2 expression is involved in synaptic plasticity as well as in neurodegneration and experimental stroke, neurotrauma and epileptogenesis.

The Role of COX-2 Dependent Cell Cycle Activities in the Clinical Progression of Alzheimer's Disease Dementia-Giulio Maria Pasinetti, Neuroinflammation Research Center, Mount Sinai School of Medicine, New York, NY 10029, USA:

This lecture pointed out, and quite correctly so, that there are tremendous resources being devoted by industry and academia for the testing of anti-inflammatory drugs (such as the "coxib" NSAIDs series) for the treatment and prevention of AD. Data were presented that the neuroprotective mechanisms of action of NSAIDs are mediated in part by inhibition of COX-2 activity in the brain and the suppression of deleterious inflammatory activities therein. COX-2 activities were found to be elevated in AD brains. Transgenic mouse models with neuronal overexpression of human COX-2 strongly suggested that COX-2 potentiates the intensity of beta-amyloid (Aβ) presence and excitotoxic neurodegeneration. Utilizing cDNA microarray techniques, it was found that ~25 differentially (>2 fold) regulated genes were identified and were related to clusters of specific gene expression variations in the brain. One of these clusters was found to have patterns that correlated with genetic variation involved in the regulation of the cell cycle. In the AD brain it was found that there was a coincidental elevation of CDK4 and COX-2 expression in the hippocampal formation of AD during the conversion from questionable cognitive deficit to mild AD dementia. Such results are applicable to the design of NSAIDs treatment trials for AD. These insightful studies provide pharmacologically useful data into COX-2 dependent mechanisms critical to the discovery and validation of NSAIDS as anti-inflammatory targets in early AD.

Place of Selective COX-2 Inhibitors in Therapy-Chris Hawkey, School of Medical and Surgical Sciences, Nottingham, UK:

NSAIDs, as well as damaging the GI tract, impair GI ulcer healing by mechanisms that are not fully understood. Data presented here suggested that these mechanisms may be similar to those which induce apoptosis of malignant cells. Data was shown that GI injury is evident within a few hours of ASA ingestion and that about 20% of patients taking full dose aspirin or NSAIDs chronically have endoscopic ulcers and related GI tract disorders. As reported by Luis A. Garcia Rodriguez's group, important risk factors for GI complications include age, past history, NSAID selection, NSAID dose and anti-coagulants used for ulcer bleeding. The advent of selective COX-2 inhibitors that spare GI tract prostaglandin synthesis is a major conceptual and therapeutic advance. "Coxibs" thereby appear to cause no more GI problems than placebo and toxicity to the rest of the GIl tract appears reduced. Outstanding questions are whether the "Coxibs" inhibit GI tract healing or provoke the relapse of GI ulceration or associative colitis-these are important considerations when using NSAIDS for anti-rheumatism or AD therapy.—Walter J. Lukiw, LSU Neuroscience Center and Department of Ophthalmology


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