Faculty Bibliography

The development of selective COX-2 inhibitors has renewed interest in the treatment of osteoarthritis with prostaglandin synthesis inhibitors. The therapeutic effects of COX inhibitors in OA may be due to their analgesic properties. However, it is now apparent that stable prostaglandins are produced by chondrocytes in OA cartilage where they may act to alter matrix synthesis and degradation. In vitro, PGEs activate metalloproteinases, but also enhance proteoglycan and type II collagen synthesis. Their net effect on matrix homeostasis in vivo remains to be determined

OBJECTIVE AND DESIGN: To compare two anti-inflammatory drugs: CSAIDS (SB203580) and hydrocortisone on iNOS and COX-2 expression. MATERIAL OR SUBJECTS: Murine macrophages and bovine chondrocytes stimulated with LPS and human OA-affected cartilage were used in this study. TREATMENT: The macrophages and chondrocytes were preincubated (30 min) with 0.1-1.0 microM CSAIDS or 10 microM of hydrocortisone before stimulating them with 1-100 microg/ml LPS. METHODS: The end products of iNOS and COX-2: nitric oxide (NO) and PGE2 were estimated by Greiss method and RIA, respectively. RESULTS: CSAIDS (1 microM) inhibited the production of NO and PGE2 (p< or =0.01) in bovine chondrocytes, but not in murine macrophages (RAW 264.7) (p< or =0.1). In fact, CSAIDS (in murine macrophages) marginally augmented nitrite accumulation (approximately 20%) at 14-24 h of LPS stimulation. Western blot analysis of COX-2 in bovine chondrocytes show decrease in COX-2 expression by hydrocortisone but not CSAIDS, although hydrocortisone and CSAIDS inhibit PGE2 accumulation. Hydrocortisone inhibited both PGE2 and NO production significantly (p< or =0.01) in murine macrophages. Furthermore, hydrocortisone significantly inhibited (p< or =0.01) PGE2 but marginally (p< or =0.05) NO in bovine chondrocytes. CONCLUSION: These experiments demonstrate differential action of CSAIDS and hydrocortisone on NO and PGE2 production in bovine chondrocytes and RAW 264.7 cells

Nitric oxide (NO) and prostaglandin E2 (PGE2) are two pleiotropic inflammatory mediators overproduced in arthritis-affected joints. The inducible isoform of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) are found both in the synovial tissue and in the cartilage. Their expression is regulated by catabolic cytokines, such as interleukin-1beta and tumor necrosis factor-alpha. These inflammatory mediators play a profound role in the pathogenic processes that arise in the pannus of rheumatoid arthritis and also interfere with cartilage homeostasis in osteoarthritis. Several drugs, including nonsteroidal anti-inflammatory drugs, immunosuppressive agents, and tetracyclines, attenuate the activity of NO and PGE2. These pleiotropic mediators are targets for pharmacologic intervention and gene therapy

The identification of COX-2 less than a decade ago has been followed by an unprecedented period of discovery and drug development. An awareness of the existence of two COX isoforms has led to potential novel insights into disease pathogenesis (arthritis, Alzheimer's disease, cancer) and the regulation of normal physiology (brain, kidney). The preliminary in vivo experience with COX-2-selective inhibitors has provided evidence for proof of concept for the COX-1 and COX-2 hypothesis, namely that the selective inhibition of COX-2-derived prostaglandins is sufficient to inhibit inflammation and is nonulcerogenic. It may be that we have moved closer to the 'better aspirin' envisioned by Sir John Vane for the treatment of degenerative and inflammatory arthritides; however, caution is still warranted. Some toxicities of current NSAIDs may result from COX-2 inhibition, as in the kidney and brain; such side effects may be shared by the selective compounds. In addition, unexpected toxicities may arise simply because new chemical compounds will be widely prescribed. Finally, since the efficacy of traditional NSAIDs derives largely from their capacity to inhibit COX-2, it may be that the COX-2 selective drugs will not prove to be therapeutically superior to available agents. Given the well-recognized toxicity of NSAIDs, however, the availability of COX-2-selective agents promises to provide significant advantage to patients with chronic diseases, such as RA and OA

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and approximately 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor L-NMMA at concentrations that inhibit nitrite accumulation by </=80% is inadequate to augment PGE2 production. However, inhibition of nitrite accumulation by >/=85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 x g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10-15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein

Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and L-N-monomethylarginine (L-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, L-NMMA at low concentrations (< or = 100 microM) inhibited the spontaneous release of No in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, L-NMMA (500 microM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO

In summary, precise classification of COX inhibitors has important clinical implications for efficacy and toxicity. However, classification of these agents clinically is difficult because there are insufficient data to predict correlations between biochemical and pharmacologic properties and the clinical effect of a given agent. In any case, specific COX-2 inhibitors are expected to show antiinflammatory and analgesic activities equivalent to those of NSAID, as well as significant reductions in the incidence of the life threatening side effects (i.e., GI bleeding) associated with COX-1 inhibition. The advantages of preferential COX-2 inhibitors may be more subtle and therefore more difficult to verify in clinical trials