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.     

Pharmacodynamics simulation of HOEC by a computational model of arachidonic acid metabolic network

Background: Arachidonic acid (AA) metabolic network is activated in the most inflammatory related diseases, and small-molecular drugs targeting AA network are increasingly available. However, side effects of above mentioned drugs have always been the biggest obstacle. (+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate (HOEC), a natural product acted as an inhibitor of 5-lipoxygenase (5-LOX) and 15-LOX in vitro, exhibited weaker therapeutic effect in high dose than that in low dose to collagen induced arthritis (CIA) rats. In this study, we tried to elucidate the potential regulatory mechanism by using quantitative pharmacology. Methods: First, we generated an experimental data set by monitoring the dynamics of AA metabolites’ concentration in A23187 stimulated and different doses of HOEC co-incubated RAW264.7. Then we constructed a dynamic model of A23187-stimulated AA metabolic model to evaluate how a model-based simulation of AA metabolic data assists to find the most suitable treatment dose by predicting the pharmacodynamics of HOEC. Results: Compared to the experimental data, the model could simulate the inhibitory effect of HOEC on 5-LOX and 15-LOX, and reproduced the increase of the metabolic flux in the cyclooxygenase (COX) pathway. However, a concomitant, early-stage of stimulation-related decrease of prostaglandins (PGs) production in HOEC incubated RAW264.7 cells was not simulated in the model. Conclusion: Using the model, we predict that higher dose of HOEC disrupts the flux balance in COX and LOX of the AA network, and increased COX flux can interfere the curative effects of LOX inhibitor on resolution of inflammation which is crucial for the efficient and safe drug design.     

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.     

COX-2/12-LOX dual pathway, a novel strategy for treatment of multiple myeloma?

Cyclooxygenase (COX) and lipoxygenase (LOX) metabolic enzymes are the two main pathways for arachidonic acid (AA) metabolism. Emerging reports now indicate alterations of arachidonic acid metabolism with carcinogenesis and many COX and LOX inhibitors are being investigated as potential anticancer drugs. COX-2 is frequently expressed in many tumors, such as multiple myeloma (MM), a disorder in which malignant plasma cells accumulate, generally derived from one clone in the bone marrow, and is an independent predictor of poor outcome. 12-LOX, an important member of LOX, is proved to be expressed in MM cells. We hypothesize that COX-2 and 12-LOX represent an integrated system, COX-2/12-LOX dual pathway, which much more efficiently enhances the intracellular levels of unesterified arachidonate and regulates cell proliferative, apoptosis and pro-angiogenic potential of MM. The COX-2/12-LOX dual pathway may act as a novel potential strategy for treatment of tumors co-expressing COX-2 and 12-LOX, and the agents that can simultaneously inhibit the two enzymes of COX-2 and 12-LOX may present a novel and promising therapeutic approach to these tumors.     

Synthesis and biological evaluation of isoxazolo[4,5-d]pyridazin-4-(5H)-one analogues as potent anti-inflammatory agents

In this study, eighteen new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives possessing either a 1,3,4-thiadiazole or a 1,2,4-triazole-5-thione moiety were synthesized and tested for anti-inflammatory activity in vitro (COX-1/COX-2, 5-LOX) and in vivo (rat paw edema assay). Compounds 15, 16, 25, 26 and 28-30 showed dual COX-2 (IC(50)'s in the 2.1-10.9 μM range), and 5-LOX (IC(50)'s in the 6.3-63.5 μM range) inhibitory activity. When administered orally to rats, dual COX-2/5-LOX inhibitors showed higher anti-inflammatory activity in vivo (30-45% reduction of the inflammatory response) than the reference drug ibuprofen (18%). Among dual COX-2/5-LOX inhibitors, the most potent compound (28) exhibited the best anti-inflammatory profile by inhibiting both COX-2 (IC(50)=2.1 μM) and 5-LOX (IC(50)=6.3 μM) enzymes. We investigated the binding interactions of compound 28 by an enzyme-ligand molecular modeling (docking) studies, which showed favorable binding interactions in both COX-2 and 5-LOX active sites. Furthermore, the dual acting COX-2/5-LOX compound 28 exhibited a superior gastrointestinal safety profile (ulcer index=0.25) compared to the reference drug ibuprofen (UI=7.0) when administered orally at the same molar dose. These observations suggest that isoxazolo[4,5-d]pyridazin-4(5H)-one analogs represent a new scaffold to design potent, effective, and safe anti-inflammatory agents possessing dual COX-2/5-LOX inhibitory activity.     

Characterization of spirostanol glycosides and furostanol glycosides from anemarrhenae rhizoma as dual targeted inhibitors of 5-lipoxygenase and Cyclooxygenase-2 by employing a combination of affinity ultrafiltration and HPLC/MS

Background : Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR).Methods: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies.Results: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC₅₀ ≤ 6.07 μM.Conclusion: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.     

Synthesis of celecoxib analogs that possess a N-hydroxypyrid-2(1H)one 5-lipoxygenase pharmacophore: biological evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity

A hitherto unknown class of celecoxib analogs was designed for evaluation as dual inhibitors of the 5-lipoxygenase/cyclooxygenase-2 (5-LOX/COX-2) enzymes. These compounds possess a SO(2)Me (11a), or SO(2)NH(2) (11b) COX-2 pharmacophore at the para-position of the N(1)-phenyl ring in conjunction with a 5-LOX N-hydroxypyrid-2(1H)one iron-chelating moiety in place of the celecoxib C-5 tolyl group. The title compounds 11a-b are weak inhibitors of the COX-1 and COX-2 isozymes (IC(50)=7.5-13.2 microM range). In contrast, the SO(2)Me (11a, IC(50)=0.35 microM), and SO(2)NH(2) (11b, IC(50)=4.9 microM), compounds are potent inhibitors of the 5-LOX enzyme comparing favorably with the reference drug caffeic acid (5-LOX IC(50)=3.47 microM). The SO(2)Me (11a, ED(50)=66.9 mg/kg po), and SO(2)NH(2) (11b, ED(50)=99.8 mg/kg po) compounds exhibited excellent oral anti-inflammatory (AI) activities being more potent than the non-selective COX-1/COX-2 inhibitor drug aspirin (ED(50)=128.9 mg/kg po) and less potent than the selective COX-2 inhibitor celecoxib (ED(50)=10.8 mg/kg po). The N-hydroxypyridin-2(1H)one moiety constitutes a novel pharmacophore for the design of cyclic hydroxamic mimetics capable of chelating 5-LOX iron for exploitation in the design of 5-LOX inhibitory AI drugs.     

Differential effects of COX inhibitors against beta-amyloid-induced neurotoxicity in human neuroblastoma cells

Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer's disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-β protein (Aβ, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Aβ and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Aβ toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Aβ neurotoxicity.     

Inhibition of sPLA2 enzyme activity by cell-permeable antioxidant EUK-8 and downregulation of p38, Akt,and p65 signals induced by sPLA2 in inflammatory mouse paw edema model

The arachidonic acid (AA) metabolic pathway, plays a vital role in the production of eicosanoids by the action of pro-inflammatory secretory phospholipase A₂ (PLA₂). Release of eicosanoids is known to be involved in many inflammatory diseases. Identification of the inhibitory molecules of this AA pathway enzyme along with the regulation of intracellular signaling cascades may be a finer choice to develop as a powerful anti-inflammatory drug. In this regard, we have screened few cell-permeable antioxidant molecules Tempo, Mito-TEMPO, N,N'-Bis(salicylideneamino)ethane-manganese(II) (EUK)-134, and EUK-8 against pro-inflammatory sPLA₂s. Among these, we found EUK-8 is a potent inhibitor with its IC₅₀ value ranges 0.7–2.0 µM for sPLA₂s isolated from different sources. Furthermore, docking studies confirm the strong binding of EUK-8 towards sPLA₂. In vivo effect of EUK-8 was studied in HSF-sPLA₂-induced edema in mouse paw model. In addition to neutralizing the edema, EUK-8 significantly reduces the phosphorylation level of inflammatory proteins such as p38 member of MAPK pathway, Akt, and p65 along with the suppression of pro-inflammatory cytokine (interleukin-6) and chemokine (CXCL1) in edematous tissue. This shows that EUK-8 not only inhibits the sPLA₂ activity, it also plays an important role in the regulation of sPLA₂-induced cell signaling cascades. Apart from the sPLA₂ inhibition, we also examine the regulatory actions of EUK-8 with other downstream enzymes of AA pathway such as 5-LOX assay in human polymorphonuclear leukocytes (PMNs) and COX-2 expression in carrageenan-λ induced paw edema. Here EUK-8 significantly inhibits 5-LOX enzyme activity and downregulates COX-2 expression. These data indicate that EUK-8 found to be a promising multitargeted inhibitory molecule toward inflammatory pathway. In conclusion, mitochondrial targeted antioxidant EUK-8 is not only the powerful antioxidant, also a potent anti-inflammatory molecule and may be a choice of molecule for pharmacological applications.     

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.     

5-Lipoxygenase as a drug target: A review on trends in inhibitors structural design,SAR and mechanism based approach

The most common inflammatory disease of the airways is asthma among children affecting around 235 million people worldwide. 5-Lipoxygenase (5-LOX) is a crucial enzyme which helps in the conversion of arachidonic acid (AA) to leukotrienes (LTs), the lipid mediators. It is associated with several inflammation related disorders such as asthma, allergy, and atherosclerosis. Therefore, it is considered as a promising target against inflammation and asthma. Currently, the only drug against 5-LOX which is available is Zileuton, while a few inhibitors are in clinical trial stages such as Atreleuton and Setileuton. So, there is a dire requirement in the area of progress of novel 5-LOX inhibitors which necessitates an understanding of their structure activity relationship and mode of action. In this review, novel 5-LOX inhibitors reported so far, their structural design, SAR and developmental strategies along with clinical updates are discussed over the last two decades.     

Impact of food polyphenols on oxylipin biosynthesis in human neutrophils

The intake of food polyphenols is associated with beneficial impacts on health. Besides anti-oxidative effects, anti-inflammatory properties have been suggested as molecular modes of action, which may result from modulations of the arachidonic acid (AA) cascade. Here, we investigated the effects of a library of food polyphenols on 5-lipoxygenase (5-LOX) activity in a cell-free assay, and in human neutrophils. Resveratrol, its dimer (ε-viniferin), and its imine analogue (IRA) potently blocked the 5-LOX-mediated LT formation in neutrophils with IC₅₀ values in low μM-range. Among the tested flavonoids only the isoflavone genistein showed potent 5-LOX inhibition in neutrophils (IC₅₀ = 0.4 ± 0.1 μM), however was ineffective on isolated 5-LOX. We exclude an interference with the 5-LOX-activating protein (FLAP) in HEK_5-LOX/±FLAP cells and suggest global effects on intact immune cells. Using LC-MS based targeted oxylipin metabolomics, we analyzed the effects of 5-LOX-inhibiting polyphenols on all branches of the AA cascade in Ca²⁺-ionophore-challenged neutrophils. While ε-viniferin causes a clear substrate shunt towards the remaining AA cascade enzymes (15-LOX, cyclooxygenase – COX-1/2, cytochrome P450), resveratrol inhibited the COX-1/2 pathway and showed a weak attenuation of 12/15-LOX activity. IRA had no impact on 15-LOX activity, but elevated the formation of COX-derived prostaglandins, having no inhibitory effects on COX-1/2. Overall, we show that food polyphenols have the ability to block 5-LOX activity and the oxylipin pattern is modulated with a remarkable compound/structural specificity. Taken the importance of polyphenols for a healthy diet and their concentration in food supplements into account, this finding justifies further investigation.     

Modification of eicosanoid profile in human blood treated by dual COX/LOX inhibitors

The arachidonic acid metabolizing enzymes, the cyclooxygenases (COXs) and lipoxygenases (LOXs), have been implicated in the development of a variety of cancers and numerous new therapeutic inhibitors are currently under investigation. However, given the interdependence of the two pathways, the effect of inhibiting one pathway with relatively selective agents can only be appreciated in the in vivo situation. Clearly then, because of their potential beneficial or deleterious effects, it is important to understand the nature and levels of the resulting arachidonic acid metabolites when treating patients with relatively selective inhibitor drugs. In this study, using reference COX-2, 5-LOX and dual COX-2/5-LOX inhibitors, we devised a protocol which permitted the simultaneous quantification of eicosanoid metabolites formed during stimulation of human peripheral venous blood samples with the calcium ionophore, A23187, in the absence and presence of lipopolysaccharide (LPS). Not surprisingly, the end products of both COX and LOX pathways were affected depending on the inhibitor, or combination of inhibitors, used and the concentrations of drug tested. In conclusion, the method described permits the rapid screening of novel compounds for potentially positive and/or negative effects upon the products of arachidonic acid metabolism.     

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 metabolic effects of inhibitors of 5-lipoxygenase and of cyclooxygenase 1 and 2 are an advancement in the efficacy and safety of anti-inflammatory therapy

Chronic treatment of inflammatory diseases with non-steroidal anti-inflammatory drugs is effective but not always devoid of serious side effects. In particular, the use of traditional non-steroidal aspirin-like drugs has been associated with a high incidence of gastrointestinal bleedings. The development of a new class of drugs, the selective cyclooxygenase type 2 (COX-2) inhibitors, has generated much expectation on the possibility to have safer compounds. After the initial enthusiasm of the scientific community, a re-evaluation of some large, randomized double-blind clinical studies performed with two of these compounds, has disclosed that the late serious gastrointestinal complications are not significantly reduced in comparison with non-selective inhibitors and that cardiovascular concerns might arise particularly if theses drugs are utilized in patients with underlying heart diseases. A new promising class of drugs to control inflammatory diseases is in advanced clinical development. The balanced inhibitors of 5-lipoxygenase (5-LOX) and of cyclooxygenase (both types 1 and 2) block the formation of all the enzymatically arachidonic acid-derived metabolites, both prostaglandins (like COX inhibitors) and leukotrienes (LT); these drugs have been shown to possess a very good anti-inflammatory efficacy without serious side effects. Licofelone, previously known as ML3000, is the molecule in the most advanced phase of clinical development (phase III) among this class of compounds; it is a potent, competitive, and well balanced inhibitor of 5-LOX and COX pathways. The drug has been shown to possess analgesic, anti-inflammatory, antipyretic antibronchocostrictory and antiplatelet properties at doses which are safe for the gastrointestinal tract. Moreover, the newly performed preclinical studies, here briefly reviewed, appear to indicate that the compound seems particularly suitable to protect the articular cartilage and the synovial space in degenerative joint disease and to exert a relevant antithrombotic activity. Preliminary results of clinical studies of licofelone in osteoarthritis indicate that the drug has a comparable or slightly better efficacy than that of naproxen but possesses a much better gastrointestinal safety. This latter important aspect has been also evaluated by an endoscopic study in normal volunteers randomly assigned to a 4-week treatment with licofelone, placebo or naproxen. The results indicate that no ulcers occurred in either licofelone group or the placebo group, while ulcers with unequivocal depth were present in 20% of the naproxen-treated subjects.     

5-LOX inhibitor modulates the inflammatory responses provoked by Helicobacter pylori infection

BACKGROUND: Arachidonic acid metabolites have been considered as pivotal mediators in Helicobacter pylori-induced inflammatory response, which are mainly metabolized by two distinct enzymes: cyclooxygenase (COX) and lipoxygenase (LOX). While COX has become well known to play a key role in either carcinogenesis or inflammation related to H. pylori infection, little is known regarding the implication of LOX in H. pylori infection. In this study, we evaluated the roles of 5-LOX and its metabolites in H. pylori-induced host responses and further a potential beneficial action of specific LOX inhibitors against H. pylori infection. MATERIALS AND METHODS: Expressions of cytosolic phospholipase A(2) (cPLA(2)), COX-2, and 5-LOX after H. pylori infection were evaluated by immunofluorescence staining and Western blotting. Synthesis of LOX metabolites was measured with reversed-phase high-performance liquid chromatography. For analyzing the influence of 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA) and geraniin, on H. pylori-induced inflammatory responses, RNase protection assay and RT-PCR were performed. RESULTS: H. pylori stimulated the translocation of cPLA(2) from cytoplasm to nucleus and increased the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) as a predominant form of 5S-HETE in gastric epithelium. NDGA exerted a strong suppression activity of H. pylori-induced 5-LOX signaling. The administration of LOX inhibitors was related with down-expression of proinflammatory mediators such as interleukin-8 and tumor necrosis factor-alpha in both H. pylori-infected gastric epithelial cells and macrophage cells. CONCLUSION: LOX modulation with its specific inhibitors could impose significant anti-inflammatory responses after H. pylori infection, based on the fact that H. pylori infection provoked gastric inflammation through metabolizing arachidonic acid by the 5-LOX pathway.     

Syntheses and characterization of nimesulide derivatives for dual enzyme inhibitors of both cyclooxygenase-1/2 and 5-lipoxygenase

Cyclooxygenase-1/2 (COX-1/2) and 5-lipoxygenase (5-LOX) are enzymes in two different pathways in the inflammatory process. In the present study, a variety of new nimesulide derivatives were synthesized through incorporation of a 5-LOX pharmacophore into nimesulide followed with some structural modifications, which were then characterized for dual enzyme inhibitors for these two types of enzymes. Their structure-activity relationships (SARs) were studied, and compound 20f was found to be an excellent dual enzyme inhibitor. Its binding conformation and interaction mode were studied with molecular docking experiments. Compound 20f could become a lead compound for further development for potential anti-inflammatory drugs.     

Synthesis and biological evaluation of a novel class of rofecoxib analogues as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs)

A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substituents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure-activity relationships identified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of COX-2 (COX-2 IC50 = 1.4 microM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC50 = 0.28 microM; 15-LOX IC50 = 0.32 microM), inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a) possesses dual COX-2 (IC50 = 2.7 microM) and 5-LOX (IC50 = 0.30 microM) inhibitor actions. Further in vivo studies employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA), but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of the COX and LOX enzymes.     

α-lipoic acid ameliorates inflammation state and oxidative stress by reducing the content of bioactive lipid derivatives in the left ventricle of rats fed a high-fat diet

Lipid mediators derived from arachidonic acid (AA) are implicated with the occurrence of inflammation and oxidative stress. The current knowledge of AA metabolism focuses on searching for the therapeutic strategy to subvert affected AA metabolism. The aim of our study was to evaluate the potential protective effect of chronic α-lipoic acid (α-LA) supplementation on myocardial inflammation state and oxidative stress in obesity-related cardiovascular dysfunction. The experiment was carried out on male Wistar rats receiving a standard or a high-fat diets with intragastric α-LA administration for 8 weeks. Plasma and myocardial AA concentrations were determined using the gas-liquid chromatography (GLC). The Western blot technique was used to examine the expression of proteins from the inflammatory pathway. The content of selected cytokines, inflammatory mediators, and oxidative stress indicators was detected by the ELISA, colorimetric, and multiplex assay kits. Our results revealed that α-LA caused a notable reduction in AA content, mainly in the phospholipid fraction with a simultaneous diminishment in the synthesis of pro-inflammatory mediators, i.e., prostaglandin E2, leukotrienes B4 and C4 by decreasing the expression of COX-2 and 5-LOX. α-LA also augmented the level of antioxidative SOD2 and GSH and decreased the level of lipid peroxidation products, which improved oxidative system impairment in the left ventricle tissue. The data clearly showed that α-lipoic acid has a significant role in inflammation and oxidative stress development ameliorating the risk of cardiac obesity induced by high-fat feeding.     

Altered brain levels of arachidonic acid-derived inflammatory eicosanoids in a rodent model of anorexia nervosa

Increasing evidence underline the role of inflammation in the behavioral, emotional and cognitive dysregulations displayed in anorexia nervosa (AN). Among the inflammatory mediators acting at both peripheral and central levels, growing attention receives a class of lipids derived from arachidonic acid (AA), called eicosanoids (eiCs), which exert a complex, multifaceted role in a wide range of neuroinflammatory processes, peripheral inflammation, and generally in immune system function. To date, little is known about their possible involvement in the neurobiological underpinnings of AN. The present study evaluated whether the activity-based model of AN (ABA) may alter AA-metabolic pathways by changing the levels of AA-derived eiCs in specific brain areas implicated in the development of the typical anorexic-like phenotype, i.e. in prefrontal cortex, cerebral cortex, nucleus accumbens, caudate putamen, amygdala, hippocampus, hypothalamus and cerebellum. Our results point to brain region-specific alterations of the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 epoxygenase (CYP) metabolic pathways rendering altered levels of AA-derived eiCs (i.e. prostaglandins, thromboxanes and hydroxyeicosatetraenoic acids) in response to induction of and recovery from the ABA condition. These changes, supported by altered messenger RNA (mRNA) levels of genes coding for enzymes involved in eiCs-related methabolic pathways (i.e., PLA₂, COX-2, 5-LOX and 15-LOX), underlie a widespread brain dysregulation of pro- and anti-inflammatory eiC-mediated processes in the ABA model of AN. These data suggest the importance of eiCs signaling within corticolimbic areas in regulating key neurobehavioral functions and highlight eiCs as biomarker candidates for monitoring the onset and development of AN, and/or as possible targets for pharmacological management.