Cox-2: Where are we in 2003? - The role of cyclooxygenase-2 in bone repair

Prostaglandins are important mediators of bone repair, and cyclooxygenases are required for prostaglandin production. Data from animal studies suggest that both non-specific and specific inhibitors of cyclooxygenases impair fracture healing but that this is due to the inhibition of COX-2 and not COX-1. Although these data raise concerns about the use of COX-2-specific inhibitors as anti-inflammatory or anti-analgesic drugs in patients undergoing bone repair, clinical reports have been inconclusive. Because animal data suggest that the effects of COX-2 inhibitors are both dose-dependent and reversible, in the absence of scientifically sound clinical evidence it is suggested that physicians consider short-term administration or other drugs in the management of these patients.     

The COX-2 inhibitor NS-398 causes T-cell developmental disruptions independent of COX-2 enzyme inhibition

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the function of cyclooxygenases, COX-1 and COX-2, which catalyze the first step in the synthesis of inflammatory mediators (PGE2). We sought to understand the roles of cyclooxygenases and NSAIDs in T-cell development. Our data show no significant defects in T-cell development in fetal thymic organ cultures of mice disrupted in both or either COX genes or in mice disrupted in either EP-1 or EP-2 receptor genes. On the other hand, NSAIDs reproducibly caused thymocyte developmental defects. However, the specific effects of the COX-2 inhibitors were not correlated with their potency for inhibition of COX-2 activity. We focused on the NS-398 COX-2 inhibitor and showed that its effects could not be reversed by exogenous PGE2. Furthermore, NS-398 was inhibitory even when its target, COX-2, was absent. These data show that the T-cell developmental effects of NS-398 are COX-2 and PGE2 independent.     

The structure of NS-398 bound to cyclooxygenase-2

The cyclooxygenases (COX-1 and COX-2) are membrane-associated, heme-containing homodimers that generate prostaglandin H₂ from arachidonic acid (AA) in the committed step of prostaglandin biogenesis and are the targets for nonsteroidal anti-inflammatory drugs (NSAIDs). N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) was the first in a series of isoform-selective drugs designed to preferentially inhibit COX-2, with the aim of ameliorating many of the toxic gastrointestinal side effects caused by conventional NSAID inhibition. We determined the X-ray crystal structure of murine COX-2 in complex with NS-398 utilizing synchrotron radiation to 3.0 A resolution. NS-398 binds in the cyclooxygenase channel in a conformation that is different than that observed for other COX-2-selective inhibitors, such as celecoxib, with no discernible penetration into the side pocket formed in COX-2 by the isoform-specific substitutions of I434V, H513R, and I523V. Instead, the methanesulfonamide moiety of NS-398 interacts with the side chain of Arg-120 at the opening of the cyclooxygenase channel, similar to that observed for acidic, nonselective NSAIDs such as indomethacin and flurbiprofen. Our structure validates inhibitor studies that identified Arg-120 as a molecular determinant for time-dependent inhibition of COX-2 by NS-398.     

Sex differences in inflammation and inflammatory pain in cyclooxygenase-deficient mice

There are two cyclooxygenase (COX) genes encoding characterized enzymes, COX-1 and COX-2. Nonsteroidal anti-inflammatory drugs are commonly used as analgesics in inflammatory arthritis, and these often inhibit both cyclooxygenases. Recently, inhibitors of COX-2 have been used in the treatment of inflammatory arthritis, as this isoform is thought to be critical in inflammation and pain. The objective of this study was to determine the effect of COX-1 or COX-2 gene disruption on the development of chronic Freund's adjuvant-induced arthritis and inflammatory pain in male and female mice. The effect of COX-1 or COX-2 gene disruption on inflammatory hyperalgesia, allodynia, inflammatory edema, and arthritic joint destruction was studied. COX-2 knockout mice (COX-2-/-) showed reduced edema and joint destruction in female, but not male, animals. In addition, neither male nor female COX-2-/- mice developed thermal hyperalgesia or mechanical allodynia, either ipsilateral or contralateral to the inflammation. COX-1 gene disruption also reduced inflammatory edema and joint destruction in female, but not male mice, although females of both COX-/- lines did show some bony destruction. There was no difference in ipsilateral allodynia between COX-1 knockout and wild-type animals, but female COX-1-/- mice showed reduced contralateral allodynia compared with male COX-1-/- or wild-type mice. These data show that the gene products of both COX genes contribute to pain and local inflammation in inflammatory arthritis. There are sex differences in some of these effects, and this suggests that the effects of COX inhibitors may be sex dependent.     

Stimulated recovery of perturbed haematopoiesis by inhibition of prostaglandin production — promising therapeutic strategy

Inhibitors of prostaglandin production, designated as classical non-steroidal anti-inflammatory drugs (NSAIDs) and acting on the base of non-selective inhibition of cyclooxygenases, have been found in numerous studies to potentiate recovery of perturbed haematopoiesis by removing the negative feedback control mediated by prostaglandins. However, classical NSAIDs show pronounced undesirable gastrointestinal side effects, which limits the possibility of their utilization for various pathophysiological states including myelosuppression. Specific cyclooxygenase-2 (COX-2) inhibitors, targeted at selective inhibition of this inducible cyclooxygenase isoform and having much better gastrointestinal side effect profile, have been found in recent studies to retain the haematopoiesis-stimulating effects of classical NSAIDs. These results suggest that the indication spectrum of selective COX-2 inhibitors may be extended to the indication of myelosuppression of various etiology. Combining the anti-tumour and haematopoiesis-stimulating activities in a single COX-2 inhibitor may have a positive clinical impact.     

Molecular dynamics simulations of arachidonic acid complexes with COX-1 and COX-2: insights into equilibrium behavior

The cyclooxygenase (COX) enzymes are responsible for the committed step in prostaglandin biosynthesis, the generation of prostaglandin H(2). As a result, these enzymes are pharmacologically important targets for nonsteroidal antiinflammatory drugs, such as aspirin and newer COX-2 selective inhibitors. The cyclooxygenases are functional homodimers, and each subunit contains both a cyclooxygenase and a peroxidase active site. These enzymes are quite interesting mechanistically, as the conversion of arachidonic acid to prostaglandin H(2) requires two oxygenation and two cyclization reactions, resulting in the formation of five new chiral centers with nearly absolute regio- and stereochemical fidelity. We have used molecular dynamics (MD) simulations to investigate the equilibrium behavior of both COX-1 and COX-2 enzyme isoforms with bound arachidonate. These simulations were compared with reference simulations of arachidonate in solution to explore the effect of enzyme on substrate conformation and positioning in the active site. The simulations suggest that the substrate has greater conformational freedom in the COX-2 active site, consistent with the larger COX-2 active site volume observed in X-ray crystal structures. The simulations reveal different conformational behavior for arachidonate in each subunit over the course of extended equilibrium MD simulations. The simulations also provide detailed information for several protein channels that might be important for oxygen and water transport to or from active sites or for intermediate trafficking between the cyclooxygenase and peroxidase active sites. The detailed comparisons for COX-1 versus COX-2 active site structural fluctuations may also provide useful information for design of new isozyme-selective inhibitors.     

Cardiovascular and gastrointestinal effects of COX-2 inhibitors and NSAIDs: achieving a balance

Conventional 'nonselective' nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain and inflammation; however, the potential gastrointestinal risks associated with their use can be a cause for concern. In response to the adverse effects that can accompany nonselective NSAID use, selective cyclo-oxygenase (COX)-2 inhibitors were developed to target the COX-2 isoenzyme, thus providing anti-inflammatory and analgesic benefits while theoretically sparing the gastroprotective activity of the COX-1 isoenzyme. Data from large-scale clinical trials have confirmed that the COX-2 inhibitors are associated with substantial reductions in gastrointestinal risk in the majority of patients who do not receive aspirin. However, some or all of the gastrointestinal benefit of COX-2 inhibitors may be lost in patients who receive low, cardioprotective doses of aspirin, and recent evidence suggests that some of these agents, at some doses, may be associated with an increased risk for cardiovascular adverse events compared with no therapy. The risks and benefits of conventional NSAIDs and of COX-2 inhibitors must be weighed carefully; in clinical practice many patients who might benefit from NSAID or COX-2 therapy are likely to be elderly and at relatively high risk for gastrointestinal and cardiovascular adverse events. These patients are also more likely to be taking low-dose aspirin for cardiovascular prophylaxis and over-the-counter NSAIDs for pain. Identifying therapies that provide relief from arthritis related symptoms, confer optimum cardioprotection, and preserve the gastrointestinal mucosa is complex. Factors to consider include the interference of certain NSAIDs with the antiplatelet effects of aspirin, differences in the adverse gastrointestinal event rates among nonselective NSAIDs and selective COX-2 inhibitors, emerging data regarding the relative risks for cardiovascular events associated with these drugs, and the feasibility and cost of co-therapy with proton pump inhibitors.     

Targeting PGE2 receptor subtypes rather than cyclooxygenases: a bridge over troubled water?

Prostaglandins (PG) are synthesized by the sequential action of phosholipases, cyclooxygenases (COX)-1 and COX-2, and specific terminal synthases, and exert their diverse biological effects through several membrane receptors. In particular, PGE2 is involved in many normal and pathological pathways that are mediated by four different E prostanoid receptors (EP1-4). Selective COX-2 inhibitors (Coxibs) have analgesic and antipyretic effects that are indistinguishable from those of nonsteroidal anti-inflammatory drugs (NSAIDs), but some possess hazardous cardiovascular side effects. Recent results indicate that EP1 and EP4 antagonists might prove useful for inhibiting the unwanted actions of COX-2. Has the time come for research to examine earnestly the selective antagonism of EP subtypes rather than further the development of direct COX-2 inhibitors?     

Short-term administration of non-selective and selective cox-2 nsaids do not interfere with bone repair in rats

Selective cyclooxygenase-2 non-steroidal anti-inflammatory drugs are known to inhibit bone repair, especially when long-term administration is required due to chronicle inflammatory diseases. In order to evaluate the action of this drug in bone repair during short-term administration, 48 rats underwent surgical bone defects in their tibias, being randomly distributed into three groups: (Group 1) negative control; (Group 2) animals treated with celecoxib, and (Group 3) animals treated with ketoprofen, both experimental groups at 1 mg/kg dose, beginning 1 h before the surgical procedure and after every 12 h for the following 3 days, or until the day of sacrifice. The animals were killed after 48 h, 7, 14, and 21 days. The tibias were removed for morphological, morphometric, and immunohistochemistry analysis for COX-2. No statistical significant differences were observed in the quality of bone repair and quantity of formed bone among the groups. COX-2 immunoreactivity of the celecoxib treated specimens was more intense in the first analyzed period, and no longer observed in the periods of 14 and 21 days. Such results suggest that the administration of the analyzed drugs in short periods does not interfere with the process of bone repair in the tibia of rats.     

Synthesis, molecular modelling and enzymatic evaluation of (+/-)3,5-diphenyl-2-thioxoimidazolidin-4-ones as new potential cyclooxygenase inhibitors

A series of substituted (+/-)3,5-diphenyl-2-thioxoimidazolin-4-ones was synthesized in order to design new type-2 cyclooxygenase (COX-2) inhibitors. This study has led to molecules which completely inhibit human recombinant COX-2 at 50 microM. Molecular modelling highlighted drug interactions with the active site of both cyclooxygenases and suggested modifications to enhance the selectivity of the compounds. In human blood, COX-2 expression was then induced by LPS, and the inhibitory potency of these drugs was disappointing. This weak activity was attributed to a poor aqueous stability of these imidazolidinones substituted by two aryl in position 3 and 5 (15 min < t(1/2) < 130 min). The improvement of the stability of this heterocycle could generate a novel template to treat COX-associated diseases such as arthritis, rheumatoid polyarthritis and cancer.     

Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-1

Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E₂. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E₂ production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH₂, or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.     

The role of statins as potential targets for bone formation

Inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme have recently been shown to stimulate bone formation in rodents both in vitro and in vivo. In bone cells, these inhibitors increase the gene expression of bone morphogenetic protein-2, which is an autocrine-paracrine factor for osteoblast differentiation.The findings that statins increase bone formation and bone mass in rodents suggest a potential new action for these compounds, which may be beneficial in patients with established osteoporosis where marked bone loss has occurred. Recent clinical data suggest that they may reduce the risk of fracture in patients taking these drugs.     

The COX-2 pathway is essential during early stages of skeletal muscle regeneration

Skeletal muscle regeneration comprises several overlapping cellular processes, including inflammation and myogenesis. Prostaglandins (PGs) may regulate muscle regeneration, because they modulate inflammation and are involved in various stages of myogenesis in vitro. PG synthesis is catalyzed by different isoforms of cyclooxygenase (COX), which are inhibited by nonsteroidal anti-inflammatory drugs. Although experiments employing nonsteroidal anti-inflammatory drugs have implicated PGs in tissue repair, how PGs regulate muscle regeneration remains unclear, and the potentially distinct roles of different COX isoforms have not been investigated. To address these questions, a localized freeze injury was induced in the tibialis anterior muscles of mice chronically treated with either a COX-1- or COX-2-selective inhibitor (SC-560 and SC-236, respectively), starting before injury. The size of regenerating myofibers was analyzed at time points up to 5 wk after injury and found to be decreased by SC-236 and in COX-2^–/– muscles, but unaffected by SC-560. In contrast, SC-236 had no effect on myofiber growth when administered starting 7 days after injury. The attenuation of myofiber growth by SC-236 treatment and in COX-2^–/– muscles is associated with decreases in the number of myoblasts and intramuscular inflammatory cells at early times after injury. Together, these data suggest that COX-2-dependent PG synthesis is required during early stages of muscle regeneration and thus raise caution about the use of COX-2-selective inhibitors in patients with muscle injury or disease. prostaglandins; nonsteroidal anti-inflammatory drugs; muscle growth; inflammation; satellite cells     

Mucosal damage induced by preferential COX-1 and COX-2 inhibitors: role of prostaglandins and inflammatory response

Nonsteroidal anti-inflammatory drugs (NSAID) are well known to induce gastric mucosal damage including bleeding, ulceration and perforation in humans and animals too. These effects are related with the inhibition of the enzyme cyclooxygenase, which is the main established mechanism of action for these drugs. Fasted rats were given piroxicam, preferential COX-1 inhibitor (10-20 mg/kg) or meloxicam, preferential COX-2 inhibitor (7.5-15 mg/kg) orally. Six or nine hours (h) later, respectively, the stomach was excised, the severity of the damage assessed and myeloperoxidase (MPO) activity measured, as well as prostaglandin PGE(2) content. Furthermore, in order to assess the effects of these oxicams over previously damaged gastric mucosa, 1 ml of 0.6 N HCl was administered p.o. followed, 1 h after, of the correspondent dose of each NSAID, and the same parameters were determined. Oral administration of both drugs dose-dependently caused acute gastric haemorrhage erosions. Myeloperoxidase activity was significantly increased by piroxicam administration. In addition, PGE(2) content was significantly reduced. The association between the administration of the acid and NSAID caused a worsening of the damage and, while myeloperoxidase activity did not modify by both piroxicam and meloxicam, PGE(2) levels were reduced. These results suggest that the PG derived from both COX-1 and COX-2 pathway plays a beneficial role in the gastroprotection, and thus caution should be exercise in the clinical use of preferential COX-2 inhibitors.     

Identification of human cyclooxegenase-2 inhibitors from Cyperus scariosus (R.Br) rhizomes

Cyperus scariosus (R.Br) belongs to the family Cyperaceae and it has a diverse medicinal importance. To identify human cyclooxegenase-2 (COX-2) inhibitors from C. scariosus, the rhizome powder was exhaustively extracted with various solvents based on the increasing polarity. Based on the presence and absence of secondary metabolites, we have selected the methanolic extract to evaluate the anti-oxidant and anti-inflammatory activity. The same extract was further subjected to gas chromatography-mass spectroscopy (GC-MS) analysis to identify the active compounds. Binding affinities of these compounds towards anti-inflammatory protein COX-2 were analyzed using molecular docking interaction studies. Phytochemical analysis showed that methanol extract is positive for all secondary metabolites. The antioxidant activity of the C. scariosus rhizomes methanolic extract (CSRME) is half to that of ascorbic acid at 50 µg/ml. The anti-inflammatory activity of CSRME is higher than that of diclofenac sodium salt at high concentration, which is evident from the dose dependent inhibition of bovine serum albumin denaturation at 40 µg/ml–5 mg/ml. GC-MS analysis showed the presence of nine compounds, among all N-methyl-1-adamantaneacetamide and 1,5,diphenyl-2H-1,2,4- triazine form a hydrogen bond interactions with Ser-530 and Tyr-385 respectively and found similar interactions with crystal structure of diclofenac bound COX-2 protein. Benzene-1, 2-diol, 4-(4-bromo-3 chlorophenyl iminomethyl forms hydrogen bond interactions with Thr-199 and Thr-200 as similar to crystallized COX-2 protein with valdecoxib. Collectively our results suggest that CSRME contains medicinally important anti-inflammatory compounds and this justifies the use of this plant as a folklore medicine for preventing inflammation associated disorders.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Insights from the docking analysis of biologically active compounds from plant Litsea Genus as potential COX-2 inhibitors

Litsea spp of Laural family are traditionally used as herbal medicine for treating inflammation including gastroenterologia, oedema and rheumatic arthritis. Therefore, it is of interest to investigate and understand the molecular principles for such actions. Here, we have illustrated the binding of thirteen Litsea derived biologically active compounds against the inflammation associated target COX (cyclo-oxygenase) -2 enzymes. We compared the binding information of these compounds with a selected number of already known COX-2 inhibitors. The comparison reflected that some of these compounds such as linderol, catechin, 6''-hydroxy-2'',3'',4'' - trimethoxy-chalcone and litseaone have better or equivalent binding features compared to already known inhibitory compounds namely celecoxib, acetylsalicylic acid, rofecoxib. Therefore, all these small compounds reported from plant Litsea spp were found to possess potential medicinal values with anti-inflammatory properties.