Increased dietary levels of α-linoleic acid inhibit mammary tumor growth and metastasis.

OBJECTIVE: The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma. MATERIALS AND METHODS: BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma. RESULTS: After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERß in tumor lysates. CONCLUSION: These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.

Transcellular biosynthesis of eicosanoid lipid mediators.

The synthesis of oxygenated eicosanoids is the result of the coordinated action of several enzymatic activities, from phospholipase A2 that releases the polyunsaturated fatty acids from membrane phospholipids, to primary oxidative enzymes, such as cyclooxygenases and lipoxygenases, to isomerases, synthases and hydrolases that carry out the final synthesis of the biologically active metabolites. Cells possessing the entire enzymatic machinery have been studied as sources of bioactive eicosanoids, but early on evidence proved that biosynthetic intermediates, albeit unstable, could move from one cell type to another. The biosynthesis of bioactive compounds could therefore be the result of a coordinated effort by multiple cell types that has been named transcellular biosynthesis of the eicosanoids. In several cases cells not capable of carrying out the complete biosynthetic process, due to the lack of key enzymes, have been shown to efficiently contribute to the final production of prostaglandins, leukotrienes and lipoxins. We will review in vitro studies, complex functional models, and in vivo evidences of the transcellular biosynthesis of eicosanoids and the biological relevance of the metabolites resulting from this unique biosynthetic pathway. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

In vitro pharmacological evaluation of multitarget agents for thromboxane prostanoid receptor antagonism and COX-2 inhibition.

PURPOSE: Patients with high cardiovascular risk due to ageing and/or comorbidity (diabetes, atherosclerosis) that require effective management of chronic pain may take advantage from new non-steroidal anti-inflammatory drugs (NSAIDs) that at clinical dosages may integrate the anti-inflammatory activity and reduced gastrointestinal side effects of selective cyclooxygenase-2 (COX-2) inhibitor (coxib) with a cardioprotective component involving antagonism of thromboxane A2 prostanoid (TP) receptor. METHODS: New compounds were obtained modulating the structure of the most potent coxib, lumiracoxib, to obtain novel multitarget NSAIDs endowed with balanced coxib and TP receptor antagonist properties. Antagonist activity at TP receptor (pA2) was evaluated for all compounds in human platelets and in an heterologous expression system by measuring prevention of aggregation and Gq-dependent production of intracellular inositol phosphate induced by the stable thromboxane A2 (TXA2) agonist U46619. COX-1 and COX-2 inhibitory activities were assessed in human washed platelets and lympho-monocytes suspension, respectively. COX selectivity was determined from dose-response curves by calculating a ratio (COX-2/COX-1) of IC50 values. RESULTS: The tetrazole derivative 18 and the trifluoromethan sulfonamido-isoster 20 were the more active antagonists at TP receptor, preventing human platelet aggregation and intracellular signalling, with pA2 values statistically higher from that of lumiracoxib. Comparative data regarding COX-2/COX-1 selectivity showed that while compounds 18 and 7 were rather potent and selective COX-2 inhibitor, compound 20 was somehow less potent and selective for COX-2. CONCLUSION: These results indicate that compounds 18 and 20 are two novel combined TP receptor antagonists and COX-2 inhibitors characterized by a fairly balanced COX-2 inhibitor activity and TP receptor antagonism and that they may represent a first optimization of the original structure to improve their multitarget activity.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4(+) T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 µmol/kg) and leukocyte infiltration (ED50 = 0.188 µmol/kg) with an efficacy comparable to a high dose of budesonide (1 µmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

Antiinflammatory and antioxidant effects of H2O2 generated by natural sources in Il1ß-treated human endothelial cells.

Specific reactive oxygen species (ROS) from different sources, might lead to different and even opposite, cellular effects. We studied the production of specific ROS resulting from the exposure of human umbilical veins endothelial cells (HUVEC) to H2O2 derived from the natural antioxidant epigallocathechin gallate (EGCG) or from the exposure to IL-1ß using a fluorogenic probe and flow cytometry, and evaluated by western blot analysis and immunocytochemistry the associated expression of transcription factors sensitive to both inflammatory and oxidative stress, such as NF-κB and Nrf2, and some downstream activated genes such as cyclooxygenase-2 (COX-2) and hemeoxygenase 1 (HO-1). The results obtained showed that exogenously-generated H2O2 induce anti-inflammatory and antioxidant effects in HUVECs counteracting the pro-inflammatory and pro-oxidant effect of IL-1ß related to the production of superoxide anions. The underlying mechanisms resulting from the extracellular production of H2O2, include (1) Nrf2 nuclear translocation and the enhanced expression of antioxidant enzymes such as HO-1, and (2) the previously unreported inhibition of NF-κB and COX-2 expression. Overall, these findings provide evidence that the production of specific reactive oxygen species finely tunes endothelial cell function and might be relevant for the reappraisal of the effects of exogenous antioxidants in the context of cardiovascular diseases.

A potential role of PUFAs and COXIBs in cancer chemoprevention.

Polyunsaturated fatty acids (PUFAs), particularly the ω-3 PUFAs and COXIBs have been associated with decreased inflammation and the prevention of tumorigenesis. ω-3 PUFAs have shown to display multiple antitumour actions, while ω-6 PUFAs and its derived eicosanoids promote the effects in cancer cell growth, angiogenesis, and invasion. ω-3 PUFAs may act by suppressing the metabolism of arachidonic acid to form proinflammatory mediators or as a precursors of novel lipid mediators with pro-resolving activity, while COXIBs are able to modulate inflammatory response by inhibiting cyclooxygenase 2 (COX-2), an inducible prostaglandin synthase overexpressed in several human cancers. As recently has been postulated, the anti-inflammation and pro-resolution processes are not equivalent. A family of lipid mediators from ω-3 PUFAs can act as agonist promoting resolution, while antinflammatory agents such as COXIBs may act as antagonists limiting the inflammatory response. The present paper reviews the current knowledge about the role of PUFAs and its derivatives (metabolites), as well as the COXIBs activity in cancer process as a sinergic therapeutic alternative for cancer treatment.

Autocrine activity of cysteinyl leukotrienes in human vascular endothelial cells: Signaling through the CysLT2 receptor.

We evaluated the autocrine activities of cysteinyl leukotrienes (cysteinyl-LTs) in HUVEC and studied the signaling and the pharmacological profile of the CysLT2 receptor (CysLT2R) expressed by ECs, finally assessing the role of the CysLT2R in permeability alterations in a model of isolated brain. Cysteinyl-LTs and their precursor LTA4 contracted HUVEC and increased permeability to macromolecules, increasing the formation of stress fibers through the phosphorylation of myosin light-chain (MLC) following Rho and PKC activation. Accordingly, in an organ model of cerebral vasculature with an intact intima, neutrophils challenge leaded to significant formation of cysteinyl-LTs and edema. Pretreatment with a selective CysLT2R antagonist prevented cytoskeleton rearrangement and HUVEC contraction, along with edema formation in the brain preparation, while leaving the synthesis of cysteinyl-LTs unaffected. We also demonstrate here that the CysLT1R antagonist zafirlukast, pranlukast, pobilukast and iralukast also possess CysLT2R antagonistic activity, which could help in reconsidering previous data on the role of cysteinyl-LTs in the cardiovascular system. The results obtained are further supporting a potential role for CysLT2R in cardiovascular disease.

Bronchodilators modulate inflammation in chronic obstructive pulmonary disease subjects.

Chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic airway inflammation and oxidative stress. Leukotriene B4 (LTB4), a potent proinflammatory mediator, is synthesized by 5-lipoxygenase (5-LO), which is activated by the presence of lipid hydroperoxides resulting from oxidative stress on biological membranes. We proposed to evaluate the effect of a four week treatment with two different bronchodilators of common practice in COPD treatment, on the production of reactive oxygen species (ROS), in particular superoxide anions, and of LTB4 by peripheral blood neutrophils obtained from COPD subjects. 24 subjects among the COPD outpatients were enrolled, and randomized to receive either formoterol (12 µg bid) or tiotropium (18 µg od). Peripheral blood neutrophils were obtained at the start and at the end of the treatment, and production of superoxide anions and of LTB4 were evaluated as previously published. The results obtained showed a decrease in the unstimulated production of superoxide by isolated neutrophils in both groups, but tiotropium only was effective in modulating the production of LTB4, while formoterol caused an increased production of superoxide in response to fMLP, when compared to values obtained before treatment. In conclusion, tiotropium showed a better antiinflammatory activity profile when compared to formoterol in a clinical setting, reducing superoxide and LTB4 production by peripheral neutrophils obtained from COPD subjects.

Critical role of COX-1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone.

We aimed at evaluating the relative contribution of cyclooxygenase (COX) -1 and COX-2 to the synthesis of prostacyclin in endothelial cells under static conditions in the presence or absence of exogenous arachidonic acid and/or altered intracellular redox balance. Selective inhibitors of either COX-1 (SC560 and FR122047) or COX-2 (SC236) concentration dependently (1-300 nM) reduced basal and interleukin (IL) -1beta-induced prostacyclin production in human umbilical vein endothelial cells by 70% or more; compound selectivity was confirmed using a whole-blood assay (IC(50) COX-1/COX-2: 13 nM/930 nM for SC-560; 9 microM/457 nM for SC-236). The observed concomitant formation of isoprostane appeared to be associated with COX enzyme activity, while formation of COX-1/COX-2 heterodimers was detected by immunoprecipitation. In the presence of arachidonic acid and 12-hydroperoxy-eicosatetraenoic acid, either exogenous or provided by platelet activation, or after glutathione depletion, COX-1 inhibition but not COX-2 inhibition concentration dependently decreased prostacyclin production. Both isoforms appear to contribute to basal prostacyclin production by endothelial cells, with COX-2 providing the hydroperoxide tone required for COX-1 activity. Conversely, in the case of intracellular glutathione depletion or enhanced availability of arachidonic acid and hydroperoxides, selective COX-2 inhibition did not significantly affect the production of endothelial prostacyclin. These findings contribute to a better understanding of the effects of cyclooxygenase inhibitors on prostacyclin production.

Rapid Metabolization of Protectin D1 by ß-Oxidation of Its Polar Head Chain.

Protectin D1 [neuroprotectin D1 (NPD1), PD1] has been proposed to play a key role in the resolution of inflammation. Aside from its ω-monohydroxylated metabolite, little has been reported on its metabolic fate. Upon NPD1 incubation in HepG2 cells, liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the formation of two main metabolites, identified as 2,3-dinor-NPD1 and 2,3,4,5-tetranor-NPD1 by comparison with standards obtained through demanding total chemical syntheses. These data represent the first evidence of ß-oxidation occurring in specialized proresolving mediators and show that the biotransformation of NPD1 by human hepatoma cells is extremely rapid and faster than that of leukotriene (LTE4). Unlike LTE4, the main metabolic process occurs from the polar head chain of NPD1. It may limit NPD1 systemic circulation and prevent its urinary excretion, making difficult its detection and quantitation in vivo. Interestingly, tetranor-NPD1, but not dinor-NPD1, maintained the bioactivity of the parent NPD1, inhibiting neutrophil chemotaxis in vitro and neutrophil tissue infiltration in vivo.

CHF6001 Inhibits NF-κB Activation and Neutrophilic Recruitment in LPS-Induced Lung Inflammation in Mice.

Inhibitors of phosphodiesterase 4 (PDE4) are potent anti-inflammatory agents, inhibiting the production of inflammatory mediators through the elevation of intracellular cAMP concentrations. We studied the activity of a novel PDE4 inhibitor, CHF6001, both in vitro in human cells and in vivo, using bioluminescence imaging (BLI) in mice lung inflammation. Mice transiently transfected with the luciferase gene under the control of an NF-κB responsive element (NF-κB-luc) have been used to assess the in vivo anti-inflammatory activity of CHF6001 in lipopolysaccharide (LPS)-induced lung inflammation. BLI as well as inflammatory cells and the concentrations of pro-inflammatory cytokines were monitored in bronchoalveolar lavage fluids (BALF) while testing in vitro its ability to affect the production of leukotriene B4 (LTB4), measured by LC/MS/MS, by LPS/LPS/N-formyl--methionyl--leucyl-phenylalanine (fMLP)-activated human blood. CHF6001 inhibited the production of LTB4 in LPS/fMLP-activated human blood at sub-nanomolar concentrations. LPS-induced an increase of BLI signal in NF-κB-luc mice, and CHF6001 administered by dry powder inhalation decreased in parallel luciferase signal, cell airway infiltration, and pro-inflammatory cytokine concentrations in BALF. The results obtained provide in vitro and in vivo evidence of the anti-inflammatory activity of the potent PDE4 inhibitor CHF6001, showing that with a topical administration that closely mimics inhalation in humans, it efficiently disrupts the NF-κB activation associated with LPS challenge, an effect that may be relevant for the prevention of exacerbation episodes in chronic obstructive pulmonary disease subjects.

Arachidonic Acid and Docosahexaenoic Acid Metabolites in the Airways of Adults With Cystic Fibrosis: Effect of Docosahexaenoic Acid Supplementation.

Cystic fibrosis (CF) is an autosomal recessive disorder, caused by genetic mutations in CF transmembrane conductance regulator protein. Several reports have indicated the presence of specific fatty acid alterations in CF patients, most notably decreased levels of plasmatic and tissue docosahexaenoic acid (DHA), the precursor of specialized pro-resolving mediators. We hypothesized that DHA supplementation could restore the production of DHA-derived products and possibly contribute to a better control of the chronic pulmonary inflammation observed in CF subjects. Sputum samples from 15 CF and 10 chronic obstructive pulmonary disease (COPD) subjects were collected and analyzed by LC/MS/MS, and blood fatty acid were profiled by gas chromatography upon lipid extraction and transmethylation. Interestingly, CF subjects showed increased concentrations of leukotriene B4 (LTB4), prostaglandin E2 (PGE2), and 15-hydroxyeicosatetraenoic acid (15-HETE), when compared with COPD patients, whereas the concentrations of DHA metabolites did not differ between the two groups. After DHA supplementation, not only DHA/arachidonic acid (AA) ratio and highly unsaturated fatty acid index were significantly increased in the subjects completing the study (p < 0.05) but also a reduction in LTB4 and 15-HETE was observed, together with a tendency for a decrease in PGE2, and an increase in 17-hydroxy-docosahexaenoic acid (17OH-DHA) levels. At the end of the washout period, LTB4, PGE2, 15-HETE, and 17OH-DHA showed a trend to return to baseline values. In addition, 15-HETE/17OH-DHA ratio in the same sample significantly decreased after DHA supplementation (p < 0.01) when compared with baseline. In conclusion, our results show here that in CF patients, an impairment in fatty acid metabolism, characterized by increased AA-derived metabolites and decreased DHA-derived metabolites, could be partially corrected by DHA supplementation.

Discovery of the First in Vivo Active Inhibitors of the Soluble Epoxide Hydrolase Phosphatase Domain.

The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities that are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity, located in the N-terminal phosphatase domain of sEH (sEH-P). Herein we report the discovery and optimization of the first inhibitor of human and rat sEH-P that is applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.

Nonsteroidal Anti-Inflammatory Drugs: Exploiting Bivalent COXIB/ TP Antagonists for the Control of Cardiovascular Risk.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are some of the most widely prescribed or dispensed over the counter analgesics and antipyretics that act by inhibiting prostaglandins and thromboxane synthesis. After the identification of a second isoform of COX, the pharmaceutical research focused on developing COX-2- selective drugs (COXIBs) considered as second generation NSAIDs that would retain the anti-inflammatory and analgesic activities of traditional NSAID without blunting the gastrointestinal cytoprotection sustained by COX1-derived products such as PGE2. However, while several clinical trials confirmed a gastrointestinal safer profile of COXIBs vs unselective COX inhibitors, increasing evidence for potential cardiovascular risk associated with COXIBs rapidly emerged. Today, there are no really safe NSAIDs to be used in chronic pain and anti-inflammatory treatments, as an adequate therapy associated with a minimal gastrointestinal damage and cardiovascular toxicity is yet to be developed. OBJECTIVE: Here, we present evidences that combining the anti-aggregating and antiatherotrombotic activities of a thromboxane receptor antagonist with the antiinflammatory activity of a COXIB we could obtain a new multitarget drug providing protection against the harmful activities mediated by the COXIB component, yet exploiting its recognized therapeutic advantages as a gastrointestinal-safer anti-inflammatory drug. We also summarize recent progress achieved in this field of research and possible new strategies to obtain a new bivalent compound. CONCLUSION: This possible third-generation NSAID with a safer pharmacological profile, will have all the pharmacological characteristics for the long-term therapy of chronic disorders such as inflammatory diseases or selected forms of cancer.

Cysteinyl-leukotrienes receptor activation in brain inflammatory reactions and cerebral edema formation: a role for transcellular biosynthesis of cysteinyl-leukotrienes.

We studied the effect of intravascular activation of human neutrophils on the synthesis of cysteinyl leukotrienes (cysLT) and the formation of cerebral edema in guinea-pig brains. Challenge with the chemotactic formylated tripeptide fMLP (0.1 microM) of neutrophil-perfused brain in vitro resulted in blood-brain barrier disruption associated with a significant increase of cysLT. Both events were completely prevented by neutrophil pretreatment with a specific 5-lipoxygenase (5-LO) inhibitor. Perfusion with the 5-LO metabolite leukotriene B4 (10 nM), together with neutrophils treated with the 5-LO inhibitor, did not restore the alteration in permeability observed upon perfusion with untreated and activated neutrophils. The dual cysLT1-cysLT2 receptor antagonist BAYu9773 was more potent and more effective than a selective cysLT1 antagonist in preventing the brain permeability alteration induced by neutrophil activation. RT-PCR showed significant expression of cysLT2 receptor mRNA in human umbilical vein endothelial cells. Intravital microscopy in mice showed that inhibition of leukotriene synthesis significantly reduced firm adhesion of neutrophils to cerebral vessels without affecting rolling. These data support the hypothesis that neutrophil and endothelial cells cooperate toward the local synthesis of cysLT within the brain vasculature and, acting via the cysLT2 receptor on endothelial cells, may represent a contributing pathogenic mechanism in the development of cerebral inflammation and edema.

Evaluation of the effects of anti-thromboxane agents in platelet-vessel wall interaction.

We evaluated the capacity of anti-aggregating agents to influence thromboxane A(2) and prostacyclin formation, arachidonic acid-endoperoxide redirection, platelet aggregation and vessel tone, in isolated rabbit aorta incubated with homologous platelets. Picotamide (N,N'bis(3-pyridinylmethyl)-4-methoxy-isophthalamide), the only dual thromboxane A(2)-synthase inhibitor/receptor antagonist in clinical use, inhibited arachidonic acid-induced platelet aggregation with low potency, increased 180-fold by aorta presence. It inhibited thromboxane A(2) formation in platelets and, in aorta presence, increased prostacyclin formation. Ozagrel (OKY-046, (E)-3-(4-(1-imidazolylmethyl)phenyl)-2-propenoic acid), a pure thromboxane A(2)-synthase inhibitor, behaved similarly to picotamide, although the aorta caused a higher (600-fold) shift. The potency of the antagonist SQ 29,548 (1S-(1 alpha,2 beta(5Z),3 beta,4 alpha))-7-(3((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid) was unaffected by aorta. In coincubation experiments, arachidonic acid-challenge increased thromboxane A(2)-dependent vessel tone; picotamide increased prostacyclin and reduced thromboxane A(2) formation and vasoconstriction. Ozagrel mimicked picotamide; aspirin (acetylsalicylic acid) reduced aorta contractility, thromboxane A(2) and prostacyclin formation. SQ 29,548 reduced vasoconstriction without affecting eicosanoids. We demonstrate the importance of redirection of eicosanoids in the mechanism of action of thromboxane A(2) inhibitors/antagonists within platelet-vascular wall interactions. These findings bear relevance in the development of novel anti-thrombotic drugs.

Leukotriene modifiers: novel therapeutic opportunities in asthma.

Cysteinyl leukotrienes (Cys-LT) are powerful proinflammatory autacoids that cause long-lasting bronchoconstriction, plasma leakage, increased mucus production; their biological activity suggests a prominent role in the etiopathology of asthma and several Cys-LT receptor antagonists and synthetase inhibitors have been developed as new antiasthmatic drugs. Zafirlukast was discovered by a mechanism-based approach to drug discovery; early structure-activity relationship analyses of the prototype SRS-A antagonist FPL-55712, lead to the identification of an indole-containing lead compound that was more specific than FPL-55712. Modifications were made on the lipid-like tail, indole backbone and acidic head region of this lead compound, resulting in potent and selective leukotriene receptor antagonists such as ICI-198615 and 204219 (zafirlukast). On the basis of successful results in preclinical asthma models, zafirlukast was recommended for clinical development and became the first leukotriene-modifier to be approved for the treatment of asthma. Leukotriene biosynthesis inhibitors (LSI) also represent a promising approach to the treatment of asthma and may theoretically provide a broader protection than Cys-LT receptor antagonists by inhibition of the synthesis of the two major leukotrienes, the Cys-LT and the chemotactic LTB4. The LSI BAY X-1005 is the result of a broad chemistry program that identified 15-HETE as an endogenous inhibitor of leukotriene synthesis and REV 5901 as a lead prototypic quinoline-based 5-lipoxygenase (5-LO) inhibitor. Clinical studies demonstrated the effectiveness of BAY X-1005 in experimental conditions such as allergen provocation and cold-air induced asthma. However, no consistent treatment effect in the overall asthma population (mild to moderately severe asthmatics) lead to discontinuation of its development.

Cyclooxygenase-1 and prostacyclin production by endothelial cells in the presence of mild oxidative stress.

This study aimed at evaluating the relative contribution of endothelial cyclooxygenase-1 and -2 (COX-1 and COX-2) to prostacyclin (PGI(2)) production in the presence of mild oxidative stress resulting from autooxidation of polyphenols such as (-)-epigallocatechin 3-gallate (EGCG), using both endothelial cells in culture and isolated blood vessels. EGCG treatment resulted in an increase in hydrogen peroxide formation in human umbilical vein endothelial cells. In the presence of exogenous arachidonic acid and EGCG, PGI(2) production was preferentially inhibited by a selective COX-1 inhibitor. This effect of selective inhibition was also substantially reversed by catalase. In addition, EGCG caused vasorelaxation of rat aortic ring only partially abolished by a nitric oxide synthase inhibitor. Concomitant treatment with a selective COX-1 inhibitor completely prevented the vasorelaxation as well as the increase in PGI(2) accumulation in the perfusate observed in EGCG-treated aortic rings, while a selective COX-2 inhibitor was completely uneffective. Our data strongly support the notions that H(2)O(2) generation affects endothelial PGI(2) production, making COX-1, and not COX-2, the main source of endothelial PGI(2) under altered oxidative tone conditions. These results might be relevant to the reappraisal of the impact of COX inhibitors on vascular PGI(2) production in patients undergoing significant oxidative stress.

Designing multitarget anti-inflammatory agents: chemical modulation of the lumiracoxib structure toward dual thromboxane antagonists-COX-2 inhibitors.

A series of lumiracoxib derivatives were designed to explore the influence of isosteric substitution on balancing COX-2 inhibition and thromboxane A(2) prostanoid (TP) receptor antagonism. The compounds were synthesized through a copper-catalyzed coupling procedure and characterized for their pK(a) values. TP receptor antagonism was assessed on human platelets; COX-2 inhibition was determined on human isolated monocytes and human whole blood. TPα receptor binding of the most promising compounds was evaluated through radioligand binding assays. Some of the isosteric substitutions at the carboxylic acid group afforded compounds with improved TP receptor antagonism; of these, a tetrazole derivative retained good COX-2 inhibitory activity and selectivity. The identification of this tetrazole acting as a balanced dual-acting compound in human whole blood, along with SAR analysis of the synthesized lumiracoxib derivatives, might contribute to the rational design of a new class of cardioprotective anti-inflammatory agents.