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 human cells and , 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 anti-inflammatory activity of CHF6001 in lipopolysaccharide (LPS)-induced lung inflammation.

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

Protectin D1 [neuroprotectin D1 (), 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 incubation in HepG2 cells, liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the formation of two main metabolites, identified as and by comparison with standards obtained through demanding total chemical syntheses.

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.

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.

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.

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.

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.

Antiinflammatory and antioxidant effects of H2O2 generated by natural sources in Il1β-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-1β 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-1β related to the production of superoxide anions.

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.

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.

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.

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.

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.

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.

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.

The pulmonary pharmacology of [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide] (2NTX-99), an anti-atherotrombotic compound with therapeutic potential in pathological conditions that target lung vasculature.

The pharmacological activity of 2NTX-99 ([4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide]) was investigated in vitro in the intact, rat pulmonary vasculature and in guinea pig airways. Rat lungs were perfused at constant flow and changes in vascular tone recorded. Challenge with the TXA₂ analogue 9,11-dideoxy-9α11α-methanoepoxy ProstaglandinF₂ (U46619, 0.

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.

Antagonism of thromboxane receptors by diclofenac and lumiracoxib.

Background And Purpose: Non-steroidal anti-inflammatory drugs (NSAIDs) are analgesic and anti-inflammatory by virtue of inhibition of the cyclooxygenase (COX) reaction that initiates biosynthesis of prostaglandins. Findings in a pulmonary pharmacology project gave rise to the hypothesis that certain members of the NSAID class might also be antagonists of the thromboxane (TP) receptor.

Experimental Approach: Functional responses due to activation of the TP receptor were studied in isolated airway and vascular smooth muscle preparations from guinea pigs and rats as well as in human platelets.

Pharmacological characterization of 2NTX-99 [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a potential antiatherothrombotic agent with antithromboxane and nitric oxide donor activity in platelet and vascular preparations.

Thromboxane (TX) A(2), prostacyclin (PGI(2)), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA(2), implemented synthesis of PGI(2), and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N(1)-(4-trans-nitrooxycyclohexyl)-N(3)-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties.

Activation of cerebral endothelium is required for mononuclear cell recruitment in a novel in vitro model of brain inflammation.

Brain inflammation is a common event in the pathogenesis of several neurological diseases. It is unknown whether leukocyte/endothelium interactions are sufficient to promote homing of blood-borne cells into the brain compartment. The role of mononuclear cells and endothelium was analyzed in a new experimental model, the isolated guinea-pig brain maintained in vitro by arterial perfusion.

eNOS, COX-2, and prostacyclin production are impaired in endothelial cells from diabetics.

The vascular endothelium is a well-recognized target of damage for factors leading to increased cardiovascular risk. Among the agents playing an important role in cardiovascular homeostasis, nitric oxide and prostacyclin represent key markers of endothelial integrity. In the present work, we report for the first time the reduced expression of both endothelial nitric oxide synthase and cyclooxygenase-2 (COX-2) proteins, as well as decreased prostacyclin production, in unstimulated human endothelial cells from insulin-dependent diabetic mothers when compared to cells from non-diabetic, control subjects.

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.

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.

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).