Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) selectivity of COX inhibitors

In vitro evaluations of the selectivity of COX inhibitors are based on a great variety of experimental protocols. As a result, data available on cyclooxygenase (COX)-1/COX-2/5- lipoxygenase (LOX) selectivity of COX inhibitors lack consistency. We, therefore, performed a systematic analysis of the COX-1/COX-2/5-LOX selectivity of 14 compounds with selective COX inhibitory activity (Coxibs). The compounds belonged to different structural classes and were analyzed employing the well-recognized whole-blood assay. 5-LOX activity was also tested on isolated human polymorphonuclear leukocytes. Among COX inhibitors, celecoxib and ML-3000 (licofelone) inhibited 5-LOX in human neutrophils at micromolar ranges. Surprisingly, ML-3000 had no effect on 5-LOX product synthesis in whole-blood assay. In addition, we could show that inhibition of COX pathways did not increase the transformation of arachidonic acid by the 5-LOX pathway.     

Diaryl-dithiolanes and -isothiazoles: COX-1/COX-2 and 5-LOX-inhibitory,OH scavenging and anti-adhesive activities

Three series of non-steroidal anti-inflammatory drugs (NSAIDs) inhibiting the cyclooxygenase/5-lipoxygenase (COX/5-LOX) pathways as such as formation of hydroxyl radicals and adhesion were prepared: 4,5-diaryl isothiazoles, 4,5-diaryl 3H-1,2-dithiole-3-thiones and 4,5-diaryl 3H-1,2-dithiole-3-ones. The aim of the present study was to develop substances which can intervene into the inflammatory processes via different mechanisms of action as multiple target non-steroidal anti-inflammatory drugs (MTNSAIDs) with increased anti-inflammatory potential. The current lead 11a was evaluated in COX-1/2, 5-LOX and OH scavenging in vitro assays and in a static adhesion assay where it proved to inhibit adhesion. Moreover, 11a treatment attenuated expression of macrophage adhesion molecule-1 (Mac-1) on extravasated polymorphonuclear leukocytes (PMNs) which indicates that the activation was reduced. The assays used are predictive for the in vivo efficacy of test compounds as shown for 11a in a peritonitis model of acute inflammation in mice. Thus, the novel 5-LOX/COX and OH inhibitor 11a possesses anti-inflammatory activity that, in addition to COX/5-LOX inhibition, implicates effects on leukocyte–endothelial interactions.     

Pharmacodynamic of cyclooxygenase inhibitors in humans

We provide comprehensive knowledge on the differential regulation of expression and catalysis of cyclooxygenase (COX)-1 and COX-2 in health and disease which represents an essential requirement to read out the clinical consequences of selective and nonselective inhibition of COX-isozymes in humans. Furthermore, we describe the pharmacodynamic and pharmacokinetic characteristics of major traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) and coxibs (selective COX-2 inhibitors) which play a prime role in their efficacy and toxicity. Important information derived from our pharmacological studies has clarified that nonselective COX inhibitors should be considered the tNSAIDs with a balanced inhibitory effect on both COX-isozymes (exemplified by ibuprofen and naproxen). In contrast, the tNSAIDs meloxicam, nimesulide and diclofenac (which are from 18- to 29-fold more potent towards COX-2 in vitro) and coxibs (i.e. celecoxib, valdecoxib, rofecoxib, etoricoxib and lumiracoxib, which are from 30- to 433-fold more potent towards COX-2 in vitro) should be comprised into the cluster of COX-2 inhibitors. However, the dose and frequency of administration together with individual responses will drive the degree of COX-2 inhibition and selectivity achieved in vivo. The results of clinical pharmacology of COX inhibitors support the concept that the inhibition of platelet COX-1 may translate into an increased incidence of serious upper gastrointestinal bleeding but this effect on platelet COX-1 may mitigate the cardiovascular hazard associated with the profound inhibition of COX-2-dependent prostacyclin (PGI2).     

High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders

High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify 'hit' molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer antiinflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.     

Variability in risk of gastrointestinal complications with individual non-steroidal anti-inflammatory drugs: results of a collaborative meta-analysis.

OBJECTIVE--To compare the relative risks of serious gastrointestinal complications reported with individual non-steroidal anti-inflammatory drugs. DESIGN--Systematic review of controlled epidemiological studies that found a relation between use of the drugs and admission to hospital for haemorrhage or perforation. SETTING--Hospital and community based case-control and cohort studies. MAIN OUTCOME MEASURES--(a) Estimated relative risks of gastrointestinal complications with use of individual drugs, exposure to ibuprofen being used as reference; (b) a ranking that best summarised the sequence of relative risks observed in the studies. RESULTS--12 studies met the inclusion criteria. 11 provided comparative data on ibuprofen and other drugs. Ibuprofen ranked lowest or equal lowest for risk in 10 of the 11 studies. Pooled relative risks calculated with exposure to ibuprofen used as reference were all significantly greater than 1.0 (interval of point estimates 1.6 to 9.2). Overall, ibuprofen was associated with the lowest relative risk, followed by diclofenac. Azapropazone, tolmetin, ketoprofen, and piroxicam ranked highest for risk and indomethacin, naproxen, sulindac, and aspirin occupied intermediate positions. Higher doses of ibuprofen were associated with relative risks similar to those with naproxen and indomethacin. CONCLUSIONS--The low risk of serious gastrointestinal complications with ibuprofen seems to be attributable mainly to the low doses of the drug used in clinical practice. In higher doses ibuprofen is associated with a similar risk to other non-steroidal anti-inflammatory drugs. Use of low risk drugs in low dosage as first line treatment would substantially reduce the morbidity and mortality due to serious gastrointestinal toxicity from these drugs.     

Dynamic eicosanoid responses upon different inhibitor and combination treatments on the arachidonic acid metabolic network

The arachidonic acid (AA) metabolic network produces key inflammatory mediators which have been considered as hallmark contributors in various inflammatory related diseases. Enzymes in this network, such as 5-lipoxygenase (5-LOX), cyclooxygenase (COX), leukotriene A(4) hydrolase (LTA4H) and prostaglandin E synthase (PGES), have been used as targets for anti-inflammatory drug discovery. Multi-target drugs and drug combinations have also been developed for this network. However, how the inhibitors alter the dynamics of metabolite production and which combinatorial target intervention solutions are better needs further exploration. We did a system based intervention analysis on the AA metabolic network. Using an LC-MS/MS method, we quantitatively studied the eicosanoid metabolites responses of AA metabolic network during stimulation of Sprague Dawley rat blood samples with the calcium ionophore. Our results indicate that inhibiting the upstream rather than the downstream target of 5-LOX pathway will simultaneously alter the AA metabolism to the COX pathway (and vice versa). Therefore, single-target inhibitors cannot control all the inflammatory mediators at the same time. We also suggest that in the case of multiple-target anti-inflammatory solutions, the combination of inhibitors of the downstream enzymes may have stronger inhibition efficiency and cause less side-effects compared to the other solutions. One therapeutic strategy, LTA4H/COX inhibition solution, was found promising for the intervention of inflammatory mediator biosynthesis and at the same time stimulating the production of anti-inflammatory agents.     

Essential structural profile of a dual functional inhibitor against cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX): molecular docking and 3D-QSAR analyses on DHDMBF analogues

It is recently proposed that compounds with equal capabilities of inhibiting COX and 5-LOX, both are key enzymes involved in the arachidonic acid (AA) cascade, are expected to be safer non-steroidal anti-inflammatory drugs (NSAIDs). To dig out helpful information in designing dual functional inhibitors against the two enzymes, homology modeling, molecular dynamics (MD) simulations, automated docking, and 3D-QSAR analyses were performed in this study on 21 COX-2/5-LOX dual inhibitors, namely, 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran (DHDMBF) analogues. A 3D-model of 5-LOX was built based on the high-resolution X-ray structure of rabbit reticulocyte 15-lipoxygenase. Molecular docking was then applied to locate the binding orientations and conformations of DHDMBF analogues with COX-2 and 5-LOX, respectively, leading to highly predictive CoMFA models constructed on the basis of the binding conformations with q2 values of 0.782 and 0.634 for COX-2 and 5-LOX, respectively. In addition, CoMFA field distributions were found in good agreement with the structural characteristics of the corresponding binding sites. Both the docking simulations and QSAR analyses suggest that new potent dual inhibitors should share a structural feature with a moderately bulky group at R2 position and a rather negatively charged group around the position of the carbonyl group of DHDMBFs. Therefore, the final 3D-QSAR models and the information of the inhibitor-enzyme interaction should be useful in developing new NSAIDs as anti-inflammation drugs with favorable safety profile.     

N-Acetylcysteine enhances the action of anti-inflammatory drugs as suppressors of prostaglandin production in monocytes

The anti-inflammatory effect of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with inhibition of cyclooxygenase (COX), the rate-limiting enzyme responsible for the synthesis of prostaglandins. Since oxygen free radicals can act as second cellular messengers, especially to modulate the metabolism of arachidonic acid and the prostaglandin tract, it seems plausible that antioxidants might affect the production of prostaglandin by activated cells. This research is focused on the effect of the antioxidant N-acetylcysteine (NAC) on the inhibition of prostaglandin E(2) formation in activated monocytes by specific and non-specific COX inhibitors. We found that lipopolysaccharide-induced prostaglandin E(2) formation was significantly reduced by rofecoxib and by diclofenac, two NSAIDs. Addition of NAC to each of these drugs enhanced the effect of the NSAIDs. These results suggest that one might expect either a potentiation of the anti-inflammatory effect of COX inhibitors by their simultaneous administration with NAC, or obtaining the same anti-inflammatory at lower drug levels.     

Effect of licofelone against NSAIDs-induced gastrointestinal ulceration and inflammation

The present study was aimed to evaluate the effect of licofelone, a dual inhibitor of cycloxygenase1/2-5-lipoxygenase against indomethacin-induced gastric damage in rats and mice in order to assess the role of leukotrienes if any, in non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrointestinal inflammation. Acute pretreatment with licofelone reversed the indomethacin-induced gastric ulceration, neutrophil adhesion in mesentery venules, neutrophil count in blood, lipid peroxides and vascularity in the stomachs of mice and rats. Further, chronic pretreatment of licofelone also prevented indomethacin-induced gastric morphological changes and cellular infiltration in mesentery venules. Moreover, acute administration of indomethacin elevated leukotriene B4 levels in gastric mucosa, which was reversed by pretreatment with licofelone The results suggest that licofelone offered gastroprotection against NSAIDs-induced gastropathy through its effect on leukotrienes and by inhibiting extravasation of neutrophils.     

Computer-aided, rational design of a potent and selective small peptide inhibitor of cyclooxygenase 2 (COX2)

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors. Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX1 and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

Regulation of inflammation in cancer by eicosanoids

Inflammation in the tumor microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenases (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.     

The protective effect of licofelone on experimental osteoarthritis is correlated with the downregulation of gene expression and protein synthesis of several major cartilage catabolic factors: MMP-13, cathepsin K and aggrecanases

This study sought to evaluate the levels of mRNA expression and protein synthesis of MMP-13, cathepsin K, aggrecanase-1 (ADAMTS-4), aggrecanase-2 (ADAMTS-5) and 5-lipoxygenase (5-LOX) in cartilage in the experimental anterior cruciate ligament (ACL) dog model of osteoarthritis (OA), and to examine the effects of treatment with licofelone, a 5-lipoxygenase (LOX)/cyclooxygenase (COX) inhibitor, on the levels of these catabolic factors. Sectioning of the ACL of the right knee was performed in three experimental groups: group 1 received no active treatment (placebo group); and groups 2 and 3 received therapeutic concentrations of licofelone (2.5 or 5.0 mg/kg/day orally, respectively) for 8 weeks, beginning the day following surgery. A fourth group consisted of untreated dogs that were used as normal controls. Specimens of cartilage were selected from lesional areas of OA femoral condyles and tibial plateaus, and were processed for real-time quantitative PCR and immunohistochemical analyses. The levels of MMP-13, cathepsin K, ADAMTS-4, ADAMTS-5 and 5-LOX were found to be significantly increased in OA cartilage. Licofelone treatment decreased the levels of both mRNA expression and protein synthesis of the factors studied. Of note was the marked reduction in the level of 5-LOX gene expression. The effects of the drug were about the same at both tested dosages. In vivo treatment with therapeutic dosages of licofelone has been found to reduce the degradation of OA cartilage in experimental OA. This, coupled with the results of the present study, indicates that the effects of licofelone are mediated by the inhibition of the major cartilage catabolic pathways involved in the destruction of cartilage matrix macromolecules. Moreover, our findings also indicate the possible auto-regulation of 5-LOX gene expression by licofelone in OA cartilage.     

Utility of novel 2-furanones in synthesis of other heterocyclic compounds having anti-inflammatory activity with dual COX2/LOX inhibition

Inflammation is associated with the development of several diseases comprising cancer and cardiovascular disease. Agents that suppress cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, besides chemokines have been suggested to minimise inflammation. Here, a variety of novel heterocyclic and non-heterocyclic compounds were prepared from novel three furanone derivatives. The structures of all synthesised compounds were confirmed by elemental and spectral analysis including mass, IR, and ¹H-NMR spectroscopy. Anti-inflammatory activities of these synthesised compounds were examined in vitro against COX enzymes, 15-LOX, and tumour necrosis factor-α (TNF-α), using inhibition screening assays. The majority of these derivatives showed significant to high activities, with three pyridazinone derivatives (5b, 8b, and 8c) being the most promising anti-inflammatory agents with dual COX-2/15-LOX inhibition activities along with high TNF-α inhibition activity.     

Computer-Aided,Rational Design of a Potent and Selective Small Peptide Inhibitor of Cyclooxygenase 2 (COX2)

Abstract

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors.

Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX' and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

NSAID-induced gut inflammation and vasoconstriction: Causes and potential reversal with beta-CGRP - A hypothesis

Traditional non-steroidal anti-inflammatory drugs, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitors control inflammation. While these drugs are formulated to reduce one of the cardinal signs of inflammation by reducing prostaglandin levels at the site of inflammation, COX-1 inhibitors induce inflammation in the stomach as well as the small bowel. The COX-2 inhibitors, a large portion of the non-steroidal anti-inflammatory drug market, provide a gastro-intestinally safer class of drugs. However, COX-2 inhibitors induce vasoconstriction via actions in renal and cardiovascular tissues. Since COX-2 inhibitors also have anticancer potential, it is worthwhile to design drug formulations that will not cause hypertension or cardiovascular damage. An attempt has thus been made in this article to formulate a hypothesis to circumvent the COX inhibitors induced inflammation and vasoconstriction through COX independent activation of calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide found throughout the vascular and sensory nervous system.     

In vitro antiproliferative activity against human colon cancer cell lines of representative 4-thiazolidinones. Part I

The characterization of two cyclooxygenase isoforms (COX), the rate-limiting enzyme for the synthesis of prostaglandins (PGs) from arachidonic acid, has allowed the development of COX-2 selective inhibitors as non-steroidal anti-inflammatory drugs (NSAIDs) with significant gastric tolerability. However, PGs are also important in cancer pathogenesis. Thus, there is an increasing interest in studying COX-2 inhibitors as potential drugs aimed at the prevention and treatment of cancer, especially colorectal cancer. The purpose of this study was to determine the inhibitory effects of some representative 4-thiazolidinones, already widely investigated as potential NSAIDs, on the growth of five human colon carcinoma cell lines with a different COX-2 expression, and to correlate them with COX-2 inhibitory properties. Our results preliminarily revealed that 2-phenylimino derivative 3 and 2,4-thiazolidindione 4 were the most active compounds. In particular, 3 mainly inhibited the HT29 cell line characterized by a high COX-2 expression, whereas 4 showed antiproliferative properties on all tested cell lines, suggesting molecular targets other than COX-2 inhibition.     

Potentiation of antinociceptive effect of NSAIDs by a specific lipooxygenase inhibitor, acetyl 11-keto-beta boswellic acid

The present study was aimed to assess the combined effects of cyclooxygenase and 5-lipoxygenase (COX/5-LOX) inhibitors in different animal models of nociception. Naproxen, nimesulide and rofecoxib are well-established antinociceptive agents acting via COX inhibition. AKBA (acetyl-keto-beta-boswellic acid) is a 5-LOX inhibitor. AKBA (50-200 mg/kg) produced a dose dependent and significant antinociceptive effect in different animal models of nociception. Based on the earlier reports from our laboratory, sub effective doses of all the three COX Inhibitors were selected and they were administered (naproxen, 5 mg/kg; nimesulide, 1 mg/kg; and rofecoxib, 1 mg/kg) with AKBA (100 mg/kg). This produced a more significant antinociceptive effect as compared to per se effect observed in all the three models of nociception. However, the effect of combination of nimesulide with AKBA was more pronounced as compared to naproxen and rofecoxib and their combination with AKBA. The present finding provided an evidence for the potentiation of antinociceptive effect of NSAIDs with AKBA. Such a combination may help to reduce the therapeutic doses of conventional NSAIDs and also reduce side effects (gastric, cardiac and renal) that are popularly associated with the NSAIDs.