5-lipoxygenase Inhibitor Research Articles

Improved Bioactivity of the Natural Product 5-Lipoxygenase Inhibitor Hyperforin by Encapsulation into Polymeric Nanoparticles.

Hyperforin, a highly hydrophobic prenylated acylphloroglucinol from the medical plant St. John's Wort, possesses anti-inflammatory properties and suppresses the formation of proinflammatory leukotrienes by inhibiting the key enzyme 5-lipoxygenase (5-LO). Despite its strong effectiveness and the unique molecular mode of interference with 5-LO, the high lipophilicity of hyperforin hampers its efficacy in vivo and, thus, impairs its therapeutic value, especially because of poor water solubility and strong plasma (albumin) protein binding. To overcome these hurdles that actually apply to many other hydrophobic 5-LO inhibitors, we have encapsulated hyperforin into nanoparticles (NPs) consisting of acetalated dextran (AcDex) to avoid plasma protein binding and thus improve its cellular supply under physiologically relevant conditions. Encapsulated hyperforin potently suppressed 5-LO activity in human neutrophils, but it failed to interfere with 5-LO activity in a cell-free assay, as expected. In the presence of human serum albumin (HSA), hyperforin was unable to inhibit cellular 5-LO activity, seemingly because of strong albumin binding. However, when encapsulated into NPs, hyperforin caused strong inhibition of 5-LO activity in the presence of HSA. Together, encapsulation of the highly hydrophobic hyperforin as a representative of lipophilic 5-LO inhibitors into AcDex-based NPs allows for efficient inhibition of 5-LO activity in neutrophils in the presence of albumin because of effective uptake and circumvention of plasma protein binding.

Effect of 5-lipoxygenase inhibitor, VIA-2291 (Atreleuton), on epicardial fat volume in patients with recent acute coronary syndrome

Background and aimsThe association between inflammation and atherosclerosis has been well described in the literature. There is now mounting evidence in support of adipose tissue as a reservoir for inflammatory markers. Epicardial adipose tissue (EAT) has been shown to associate with coronary atherosclerosis and found to be a predictor of future adverse cardiovascular events. This study, VIA-EAT, assesses the change in epicardial fat volume (EFV) after treatment with an investigational anti-inflammatory agent (VIA-2291) in a cohort of post-acute coronary syndrome (ACS) patients. MethodsThis study is derived from a post-hoc analysis of a previously conducted randomized clinical trial (NCT00358826). Patients were recruited for a prospective, double-blind, multi-center randomized trial of a 5-lipooxygenase inhibitor or placebo in a 3:1 randomization, including doses of placebo, and 25 mg, 50 mg and 100 mg of active treatment. Cardiac computed tomography was performed at baseline and at 24 weeks after treatment with VIA-2291. EAT and pericardial adipose tissue (PAT) were measured using previously published methodology. A Pearson correlation test was used to determine the relationship between change in epicardial fat and change in plaque composition. ResultsWe analyzed 54 pre- and post-treatment scans. There were no major differences between traditional cardiovascular risk factors among the 4 randomized study arms. There was a significant decrease in EAT and PAT in patients in the treatment arms vs. placebo, −3.0 ± 8.2mm3 and −3.9 ± 10.9mm3 vs. 1.7 ± 7.5mm3 and 1.4 ± 10.7mm3 (p = 0.001), respectively. The changes in EAT and PAT were more pronounced in patients taking 100 mg of the drug vs. placebo: 4.2 ± 9.6mm3, -7.6 ± 8.5mm3, p = 0.0001, respectively. In a subgroup analysis, reduction in epicardial fat volume correlated with reduction in total atherosclerotic plaque volume across all VIA treatment groups, r = 0.52 (p = 0.004). ConclusionsAfter adjustment for traditional cardiovascular risk factors including age, gender, body mass index, dyslipidemia and smoking, VIA-2291 decreases EAT and PAT in individuals with recent ACS. Treatment with the drug also appears to alter plaque volume and composition.

Induction of a 5-lipoxygenase product by daidzein is involved in the regulation of influenza virus replication.

This study was conducted to evaluate the regulation mechanism of influenza virus replication following treatment of Madin-Darby canine kidney cells with the soy isoflavone daidzein. We performed comparative qualitative and quantitative analyses of lipid peroxide between mock-infected and virus-infected cells treated with or without daidzein, as it had been reported that daidzein was an antioxidant and lipid peroxide levels increased upon virus infection. Contrary to our belief, lipid peroxides were not elevated in virus-infected cells and no decrease in lipid peroxides was observed in daidzein-treated cells. In daidzein-treated cells, 5-hydroxyeicosatetraenoic acid, the 5-lipoxygenase product derived from arachidonate, was significantly elevated compared to other lipid peroxides. Zileuton (5-lipoxygenase inhibitor) and 5-lipoxygenase knockdown reduced the daidzein-induced antiviral effect. Moreover, virus replication was regulated by treatment with 5-hydroperoxyeicosatetraenoic acid, a precursor of 5-hydroxyeicosatetraenoic acid and 5-lipoxygenase primary product. These results suggest that daidzein regulates virus replication via signal transduction through 5-lipoxygenase products.

Synthesis and structure-activity relationships of novel 5-(hydroxamic acid)methyl oxazolidinone derivatives as 5-lipoxygenase inhibitors

Oxazolidinone hydroxamic acid derivatives were synthesised and evaluated for inhibitory activity against leukotriene (LT) biosynthesis in three in vitro cell-based test systems and on direct inhibition of recombinant human 5-lipoxygenase (5-LO). Thirteen of the 19 compounds synthesised were considered active ((50% inhibitory concentration (IC50) ≤ 10 µM in two or more test systems)). Increasing alkyl chain length on the hydroxamic acid moiety enhanced activity and morpholinyl-containing derivatives were more active than N-acetyl-piperizinyl derivatives. The IC50 values in cell-based assay systems were comparable to those obtained by direct inhibition of 5-LO activity, confirming that the compounds are direct inhibitors of 5-LO. Particularly, compounds PH-249 and PH-251 had outstanding potencies (IC50 < 1 µM), comparable to that of the prototype 5-LO inhibitor, zileuton. Pronounced in vivo activity was demonstrated in zymosan-induced peritonitis in mice. These novel oxazolidinone hydroxamic acid derivatives are, therefore, potent 5-LO inhibitors with potential application as anti-allergic and anti-inflammatory agents.

Design, synthesis, molecular modelling and biological evaluation of novel 3-(2-naphthyl)-1-phenyl-1H-pyrazole derivatives as potent antioxidants and 15-Lipoxygenase inhibitors

Oxidative stress is one of the main causes of significant severe diseases. The discovery of new potent antioxidants with high efficiency and low toxicity is a great demand in the field of medicinal chemistry. Herein, we report the design, synthesis molecular modelling and biological evaluation of novel hybrids containing pyrazole, naphthalene and pyrazoline/isoxazoline moiety. Chalcones 2a–e were synthesized efficiently and were used as starting materials for synthesis of a variety of heterocycles. A novel series of pyrazoline 3a–e, phenylpyrazoline 4a–e, isoxazoline 5a–e and pyrazoline carbothioamide derivatives 6a–e were synthesized and screened for in vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and superoxide radical scavenging assay as well as 15-lipoxygenase (15-LOX) inhibition activity. Compounds 3a, 4e, 5b, 5c, 6a, 6c, and 6e showed excellent radical scavenging activity in all three methods in comparison with ascorbic acid and 15-LOX inhibition potency using quercetin as standard then were subjected to in vivo study. Catalase (CAT) activity, glutathione (GSH) and malondialdehyde (MDA) levels were assayed in liver of treated rats. Compounds 5b, 5c, and 6e showed significant in vivo antioxidant potentials compared to control group at dose of 100 mg/kg B.W. Molecular docking of compound 6a endorsed its proper binding at the active site pocket of the human 15-LOX which explains its potent antioxidant activity in comparison with standard ascorbic acid.

An Anti-Inflammatory Composition of Boswellia serrata Resin Extracts Alleviates Pain and Protects Cartilage in Monoiodoacetate-Induced Osteoarthritis in Rats.

The boswellic acids, the active compounds in Boswellia serrata gum resin extract, are potent anti-inflammatory agents and are specific nonredox inhibitors of 5-Lipoxygenase (5-LOX). Here, we present the anti-osteoarthritis (OA) efficacy of LI13019F1 (also known as Serratrin®), a unique composition containing the acidic and nonacidic fractions of B. serrata gum resin. This composition strongly inhibited 5-LOX activity with the half-maximal inhibitory concentration (IC50) of 43.35 ± 4.90 μg/mL. Also, LI13019F1 strongly inhibited the leukotriene B4 (IC50, 7.80 ± 2.40 μg/mL) and prostaglandin E2 (IC50, 6.19 ± 0.52 μg/mL) productions in human blood-derived cells. Besides, LI13019F1 reduced TNF-α production with the IC50 of 12.38 ± 0.423 μg/mL. On average, 1, 2.5, and 5 μg/mL doses of LI13019F1 protected 34.62, 47.66, and 62.29% SW1353 human chondrosarcoma cells from IL-1β induced SOX-9 depletion, respectively. Further, a 28-day preclinical proof-of-concept study evaluated the pain relief efficacy of LI13019F1 in monoiodoacetate- (MIA-) induced Sprague-Dawley rats. At the end of the study, 150 and 300 mg/kg doses of LI13019F1 supplemented rats showed significant improvements (55.17 ± 5.81 g (p < 0.05), and 66.22 ± 6.30 g (p < 0.05), respectively, vs. MIA: 31.22 ± 7.15 g) in body-weight-bearing capacities. Concurrently, LI13019F1-150 and LI13019F1-300 rats substantially (p < 0.05) increased the threshold of pain sensitivity to pressure (26.98 ± 2.36 and 28.06 ± 2.72-gram force, respectively; vs. 18.63 ± 5.82 in MIA) and increased (p < 0.05) the latent time to withdraw the paw after a thermal stimulus (23.61 ± 2.73 and 28.18 ± 1.90 sec, respectively; vs. 16.56 ± 1.22 sec. in MIA). Besides, the histological observations on Safranin-O green stained articular cartilage revealed that LI13019F1 also prevented the MIA-induced structural damage of the cartilage and reduced the loss of the extracellular matrix (ECM) components in the experimental rats. In conclusion, the present observations suggest that LI13019F1, a new composition of B. serrata gum resin extracts, reduces pain and protects articular cartilage from the damaging action of MIA in a rodent model.

A new treatment for severe pulmonary arterial hypertension based on an old idea: inhibition of 5-lipoxygenase.

It has been generally accepted that severe forms of pulmonary arterial hypertension are associated with inflammation. Plasma levels in patients with severe pulmonary arterial hypertension show elevated levels of interleukins and mediators of inflammation and histologically the diseased small pulmonary arterioles show infiltrates of inflammatory and immune cells. Here, we review the literature that connects pulmonary hypertension with the arachidonic acid/5-lipoxygenase-derived leukotriens. This mostly preclinical background data together with the availability of 5-lipoxygenase inhibitors and leukotriene receptor blockers provide the rationale for testing the hypothesis that 5-lipoxygenase products contribute to the pathobiology of severe pulmonary arterial hypertension in a subgroup of patients.

5-LIPOXYGENASE INHIBITOR (ZILEUTON) PRODUCES ANTI-DEPRESSION EFFECT IN STRESS INDUCE CRS MICE MODEL

Objective: The experiment aimed to find out the effectiveness of Zileuton, a 5-LOX inhibitor on depressive behavior and neuroinflammation in vivo.&#x0D; Method: Male ICR mice (25-30g) randomly distributed Veh+Veh, CRS+Vehicle, CRS+ZIL50, and CRS+ZIL100. Zileuton was orally given in the treatment groups for 21 days after 3 weeks of stress induce CRS model. Starting from the day 1, in CRS model, mice were immobilized 8 hr/day for consecutive 21 days to induce stress. After completing the drug administration, subjected the mice for behavioral tests, and then performed histopathological &amp; Western Blotting.&#x0D; Result: Stress induces CRS model guide to the significant depressive-like behavior of the mice in behavioral tests which was united by adverse changes at the cellular/molecular level responsible for regulation of inflammatory and apoptotic processes. CRS triggered Microglial over activation in the DG of the hippocampus, which was successfully inhibited by Zileuton post-treatment at the dose of 100mg/kg than 50mg/kg. Level of TNF-α, IL- 1β, nuclear NF-κB p65, Bax, and cleaved Caspase-3 was high and Bcl-2 expression was low in the stress induce CRS -treated mice which were found to be opposite in the Zileuton (100mg/kg). However, the dose of 50mg/kg less to mimic the effects as exhibited more by the 100mg/kg dose of Zileuton.&#x0D; Conclusion: It can be concluded that selective 5-lipoxygenase inhibitor Zileuton can efficiently inhibit the depressive-like behavior/activity in CRS-induced depressive mouse model. The study is the first to show the role of 5-lipoxygenase enzyme in and Chronic Restraint Stress (CRS)-induced mice models of stress, anxiety or depression.

Zileuton use and phenotypic features in asthma

Zileuton, a 5-lipoxygenase (5LPO) inhibitor exerts a broad influence in the arachidonic acid (AA) pathway by blocking upstream molecules that otherwise would lead to production of an array of inflammatory leukotrienes (LT) A4-E4. Hence, it has the potential to be a drug suitable to treat complicated asthmatics. Studies have shown modest response rates for zileuton in asthmatics. ObjectiveWe sought to study our hypothesis that response to zileuton varies across specific asthmatic phenotypes. MethodsWe retrospectively analyzed data from 129 patients with asthma that were prescribed zileuton at the University of Pittsburgh's Comprehensive Lung Clinic. A total of 75 patients from the above population had requisite lung function data and zileuton usage that would help assess a drug response effect. A zileuton responder was defined as having at least or greater than 5% annualized increase in post-bronchodilator FEV1% from baseline. Using a multivariate logistic regression analysis, we determined the association between responder status and the underlying phenotypic characteristics. ResultsUsing generalized estimating equations (GEE) analysis of 331 individual lung function test data-points as well as logistic regression analysis for predictors of 5% or more annualized increase in FEV1%, 21 of 75 patients (28%) met criteria for having a differential response to zileuton. Severe asthma was associated less often with responder status (OR 0.12; p 0.004). Obesity was less often associated with responder status, however did not reach significance (OR 0.46; p 0.15). ConclusionIn this retrospective study, zileuton response varies across asthmatics, with poorer response rates being associated with those with severe asthma and possibly obesity. Although prescription trends for zileuton may predominate amongst severe asthmatics, this tendency does not seem to mirror the actual likelihood to respond. As against the trivial role for zileuton per current GINA algorithms, our study brings forward a notion that zileuton may well be considered along with LTRAs (like montelukast) for non-severe asthma.

Lipoxygenase Inhibitors as Cancer Chemopreventives: Discovery, Recent Developments and Future Perspectives.

Leukotrienes (LTs) constitute a bioactive group of Polyunsaturated Fatty Acid (PUFA) metabolites molded by the enzymatic activity of lipoxygenase (LO) and have a pivotal role in inflammation and allergy. Evidence is accumulating both by in vitro cell culture experiments and animal tumor model studies in support of the direct involvement of aberrant metabolism of arachidonic acid (ACD) in the development of several types of human cancers such as lung, prostate, pancreatic and colorectal malignancies. Several independent experimental data suggest a correlation between tumoral cells viability and LO gene expression, especially, 5-lipoxygenase (5-LO). Overexpressed 5-LO cells live longer, proliferate faster, invade more effectively through extracellular matrix destruction and activate the anti-apoptotic signaling mechanisms more intensively compared to the normal counterparts. Thus, some groups of lipoxygenase inhibitors may be effective as promising chemopreventive agents. A structured search of bibliographic databases for peer-reviewed research literature regarding the role of LO in the pathogenesis of cancer was performed. The characteristics of screened papers were summarized and the latest advances focused on the discovery of new LO inhibitors as anticancer agents were discussed. More than 180 papers were included and summarized in this review; the majority was about the newly designed and synthesized 5-LO inhibitors as anti-inflammatory and anticancer agents. The enzyme's structure, 5-LO pathway, 5-LO inhibitors structure-activity relationships as well as the correlation between these drugs and a number of most prevalent human cancers were described. In most cases, it has been emphasized that dual cyclooxygenase-2/5-lipoxygenase (COX-2/5-LO) or dual 5-lipoxygenase/microsomal prostaglandin E synthase-1 (5-LO/mPGES-1) inhibitors possess considerable inhibitory activities against their target enzymes as well as potent antiproliferative effects. Several papers disclosing 5-lipoxygenase activating protein (FLAP) antagonists as a new group of 5-LO activity regulators are also subject to this review. Also, the potential of 12-lipoxygenase (12- LO) and 15-lipoxygenase (15-LO) inhibitors as chemopreventive agents was outlined to expand the scope of new anticancer agents discovery. Some peptides and peptidomimetics with anti-LT activities were described as well. In addition, the cytotoxic effects of lipoxygenase inhibitors and their adverse effects were discussed and some novel series of natural-product-derived inhibitors of LO was also discussed in this review. This review gives insights into the novel lipoxygenase inhibitors with anticancer activity as well as the different molecular pharmacological strategies to inhibit the enzyme effectively. The findings confirm that certain groups of LO inhibitors could act as promising chemopreventive agents.

Zileuton ameliorates depressive-like behaviors, hippocampal neuroinflammation, apoptosis and synapse dysfunction in mice exposed to chronic mild stress

Our previous study has found that zileuton, a selective 5-lipoxygenase (5LO) inhibitor, abrogated lipopolysaccharide-induced depressive-like behaviors and hippocampal neuroinflammation. Herein, we further extended our curiosity to investigate effects of zileuton on stress-induced depressive-like behaviors. Our data indicated that zileuton significantly ameliorated depressive-like behaviors in mice subjected to chronic mild stress (CMS), as shown in the tail suspension test, forced swimming test and novelty-suppressed feeding test. The further studies indicated that zileuton suppressed hippocampal neuroinflammation, evidenced by lower levels of TNF-α, IL-1β and nuclear NF-κB p65 as well as decreased number of Iba1-positive cells. It also significantly ameliorated hippocampal apoptosis, indicated by deceased number of TUNEL-positive cells, deceased ratio of cleaved caspase-3/procaspase-3 and increased ratio of Bcl-2/Bax. More importantly, zileuton increased the level of synaptic proteins PSD-95 and SYN and the number of NeuN+/BrdU+ cells in the hippocampus. Over all, zileuton alleviated CMS-induced depressive-like behaviors, neuroinflammatory and apoptotic responses, abnormalities of synapse and neurogenesis in the hippocampus, suggesting that it might has beneficial effects on depression.

Participation of 5-lipoxygenase and LTB4 in liver regeneration after partial hepatectomy

Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.

Pharmaceutical analysis combined with in-silico therapeutic and toxicological profiling on zileuton and its impurities to assist in modern drug discovery

The obligatory testing of drug molecules and their impurities to protect users against toxic compounds seems to provide interesting opportunities for new drug discovery. Impurities, which proved to be non-toxic, may be explored for their own therapeutic potential and thus be a part of future drug discovery. The essential role of pharmaceutical analysis can thus be extended to achieve this purpose. The present study examined these objectives by characterizing the major degradation products of zileuton (ZLT), a 5-lipoxygenase (5-LOX) inhibitor being prevalently used to treat asthma. The drug sample was exposed to forced degradation and found susceptible to hydrolysis and oxidative stress. The obtained Forced Degradation Products (FDP’s) were resolved using an earlier developed and validated Ultra-High-Pressure Liquid Chromatography Photo-Diode-Array (UHPLC-PDA) protocol. ZLT, along with acid-and alkali-stressed samples, were subjected to Liquid-chromatography Mass-spectrometry Quadrupole Time-of-flight (LC/MS-QTOF) studies. Major degradation products were isolated using Preparative TLC and characterized using Q-TOF and/or Proton nuclear magnetic resonance (1HNMR) studies. The information obtained was assembled for structural conformation. Toxicity Prediction using Komputer Assisted Technology (TOPKAT) toxicity analyses indicated some FDP’s as non-toxic when compared to ZLT. Hence, these non-toxic impurities may have bio-affinity and can be explored to interact with other therapeutic targets, to assist in drug discovery. The drug molecule and the characterized FDP’s were subjected to 3-Dimensional Extra Precision (3D-XP)-molecular docking to explore changes in bio-affinity for the 5-LOX enzyme (PDB Id: 3V99). One FDP was found to have a higher binding affinity than the drug itself, indicating it may be a suitable antiasthmatic. The possibility of being active at other sites cannot be neglected and this is evaluated to a reasonable extent by Prediction of Activity Spectra for Substances (PASS). Besides being antiasthmatic, some FDP’s were predicted antineoplastic, antiallergic and inhibitors of Complement Factor-D.

NADPH Oxidase Controls Neutrophil Infiltration in the Lung in Early Response to Fungal Cell Walls Via LTB4

The leukocyte NADPH oxidase generates superoxide, the precursor to reactive oxygen species important for host defense and immunoregulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by serious bacterial and fungal infections and aberrant inflammation. Aspergillus pneumonia is frequent, and associated with pyogranulomatous inflammation. Of note, even sterile fungal cell walls elicit increased neutrophilic inflammation in CGD mice, demonstrating that that NADPH oxidase can limit inflammation independent of its antimicrobial effects. Leukotriene B4 (LTB4) is a potent inflammatory mediator, acting as a chemoattractant and activating other polymorphonuclear leukocyte (PMN) functions. PMN themselves are prominent source of LTB4, and sense LTB4 through its receptor BLT1, which stimulates additional LTB4 synthesis by autocrine and paracrine routes. This leads to feed forward amplification of LTB4 production locally and formation of PMN clusters. Ca2+ acts as an important second messenger in PMN, including regulation of LTB4 synthesis. Activated human CGD PMN have increased intracellular Ca2+ levels compared to PMN from healthy donors due to electrogenic effects of the NADPH oxidase (Geiszt M. et al JBC 1997). Intracellular Ca2+ overload in PMN is speculated to contribute to increased PMN pro-inflammatory activity, but specific pathways and in vivo relevance are not well defined. Therefore, we therefore investigated the production of LTB4 by murine CGD PMN and the role of LTB4 in PMN recruitment and lung hyperinflammation following pulmonary challenge with zymosan, a sterile fungal cell wall preparation. Zymosan-stimulated PMN from Cybb-null mice, a model for X-linked CGD, produced higher LTB4 in vitro compared to wild type (WT), even in the presence of SOD and catalase to counteract oxidative degradation. Higher LTB4 production correlated with higher levels of intracellular Ca2+ in stimulated X-CGD PMN. The increased LTB4 produced by X-CGD PMN was dependent on cell density and the LTB4 receptor BLT1, consistent with a feed-forward loop that amplified LTB4 production. Zymosan-stimulated X-CGD PMN also formed larger and more numerous clusters in vitro compared to WT PMN. Cluster formation was abrogated by inhibiting LTB4 synthesis with the 5-lipoxygenase (5-LO) inhibitor zileuton or by the BLT1 receptor antagonist U75302, demonstrating that cluster formation was LTB4 and BLT1 dependent. We next examined whether LTB4 regulated the response to zymosan in the lung. Following intranasal administration of zymosan, X-CGD mice had higher PMN numbers in bronchoalveolar lavage (BAL), larger PMN foci by lung histology, and increased lung LTB4 as compared to WT mice. These differences were evident by 8 hr and progressed over the first 24 hr post-challenge, in contrast to WT mice where inflammation plateaued at 8 hr. Treatment with zileuton or U75302 just prior to zymosan significantly reduced lung PMN numbers for both WT and X-CGD mice at 8 hr post challenge, and lung PMN remained substantially reduced in X-CGD mice at 24 hrs. Moreover, delaying administration of zileuton to 8 hr after zymosan challenge also significantly decreased PMN inflammation in X-CGD but not WT mice at 24 hrs, indicating that ongoing synthesis of LTB4 otherwise continued to promote PMN recruitment in CGD mice. These results demonstrate that PMN production of LTB4 in response to fungal cell walls is limited by NADPH oxidase via its effects on intracellular Ca2+. In oxidase-deficient CGD mice, LTB4 plays a major role in driving excessive PMN recruitment to the lung in the early response to zymosan. This study is the first to implicate LTB4 in promoting neutrophilic lung inflammation in response to fungal cell walls in CGD, likely by an amplified feed-forward loop involving increased production of LTB4 by CGD PMN. LTB4 could be a potential therapeutic target to ameliorate CGD hyperinflammation. Disclosures No relevant conflicts of interest to declare.

Zileuton, a 5-Lipoxygenase Inhibitor, Exerts Anti-Angiogenic Effect by Inducing Apoptosis of HUVEC via BK Channel Activation.

The arachidonic acid metabolism through 5-lipoxygenase (5-LO) pathways is involved in modulating both tumorigenesis and angiogenesis. Although anti-carcinogenic activities of certain 5-LO inhibitors have been reported, the role of zileuton, a well known 5-LO inhibitor, on the endothelial cell proliferation and angiogenesis has not been fully elucidated. Here, we report that zileuton has an anti-angiogenic effect, and the underlying mechanisms involved activation of the large-conductance Ca2+-activated K+ (BK) channel. Our results show that zileuton significantly prevented vascular endothelial growth factor (VEGF)-induced proliferation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as in vivo. However, such anti-angiogenic effect of zileuton was abolished by iberiotoxin (IBTX), a BK channel blocker, suggesting zileuton-induced activation of BK channel was critical for the observed anti-angiogenic effect of zileuton. Furthermore, the anti-angiogenic effect of zileuton was, at least, due to the activation of pro-apoptotic signaling cascades which was also abolished by IBTX. Additionally, zileuton suppressed the expression of VCAM-1, ICAM-1, ETS related gene (Erg) and the production of nitric oxide (NO). Taken together, our results show that zileuton prevents angiogenesis by activating the BK channel dependent-apoptotic pathway, thus highlighting its therapeutic capacity in angiogenesis-related diseases, such as cancer.

Extra Virgin Olive Oil Minor Compounds Modulate Mitogenic Action of Oleic Acid on Colon Cancer Cell Line.

Experimental and clinical findings suggest that olive oil has a protective effect, whereas oleic acid consumption induces colorectal cancer (CRC). Considering this apparent contradiction and that olive oil is a complex mix of fatty acids, mainly oleic acid and minor compounds such as phenolic compounds, lignans, hydrocarbons, and triterpenes, we study its effects on intestinal epithelial cell growth. Our results show that oleic acid (1-100 μM) but not elaidic acid induced DNA synthesis and Caco-2 cell growth (2-fold higher than cells without growth factors, p < 0.05). These effects were inhibited by 5-lipoxygenase inhibitors as well as the leukotriene antagonist (p < 0.05), suggesting the implication of this pathway in this mitogenic action. Hydroxytyrosol, oleuropein, pinoresinol, squalene, and maslinic acid (0.1-10 μM) reverted DNA synthesis and Caco-2 cell growth induced by oleic acid. These effects were not the consequence of the cell cycle arrest or the impairment of cell viability with the exception of hydroxytyrosol and maslinic acid that induced cell detachment and apoptosis (35.6 ± 2.3 and 43.2 ± 2.4%, respectively) at the higher concentration assayed. Oleuropein effects can be related with hydroxytyrosol release as a consequence of oleuropein hydrolysis by Caco-2 cells (up to 25%). Furthermore, hydroxytyrosol modulates the arachidonic acid cascade, and this event can be associated with its antimitogenic action. In conclusion, oleic acid and oleic acid in the presence of olive oil representative minor components have opposite effects, suggesting that the consumption of seed oils, high oleic acid seed oils, or olive oil will probably have different effects on CRC.

First report of antioxidant 1H-benzochromenone from muricid gastropod Chicoreus ramosus as dual inhibitors of pro-inflammatory 5-lipoxygenase and carbolytic enzymes

Chromene derivatives with manifold structural framework and pharmacological properties were ubiquitous in the mollusks of marine origin. A previously undescribed 1H-benzochromenone was isolated through bioassay-guided chromatographic purification of the organic extract of the marine gastropod mollusk Chicoreus ramosus. The compound was characterised as 6-(2′,2′-dimethyl)-3′-en-1′-yl-1′-oxy)-3-hydroxy-1H-benzo[c]chromene-2(10aH)-one based on integrated spectroscopic analysis. The antioxidant studies by employing the stable free radicals reported that the antioxidant activity (IC50 1.4–1.6 mM) was comparable to α-tocopherol (IC50 1.4–1.7 mM). The attenuating potential of the studied compound against pro-inflammatory 5-lipoxygenase (IC50 2.12 mM) was significantly greater than that exhibited by anti-inflammatory drug ibuprofen (IC50 4.4 mM), whereas its inhibitory properties against carbolytic α-amylase (IC50 ∼0.72 mM) was comparable with that displayed by acarbose (IC50 0.43 mM). The present study recognised the potential of 1H-benzochromenone derivative isolated from C. ramosus as important pharmaceutical lead with anti-diabetic and anti-inflammatory potentials to reduce the risk of hyperglycaemia and inflammatory pathologies.

Effect of Zileuton Treatment on Sinonasal Quality of Life in Patients with Aspirin-Exacerbated Respiratory Disease.

Background Aspirin-exacerbated respiratory disease (AERD) is a subtype of chronic sinusitis comprised of asthma, chronic rhinosinusitis with nasal polyps, and a non-IgE hypersensitivity to cyclooxygenase-1 inhibitors. AERD is typically refractory to medical and often surgical management and causes significant quality-of-life concerns for patients. Objective This study aimed to retrospectively assess the rhinologic disease-specific outcomes as well as quality-of-life metrics in a cohort of AERD patients who medically manage their condition with zileuton, a 5-lipoxygenase inhibitor. Methods Retrospective review of 45 patients at a tertiary care center with diagnosis of AERD who underwent functional endoscopic sinus surgery (FESS) with at least 6 months of pre- and postoperative clinical data were included in the study. Zileuton cohort chosen based on intention-to-treat after initial FESS. Background data collected included patient demographics, surgery information, and zileuton initiation dates. Zileuton and control cohorts were followed for 2.8 and 2.4 years, respectively. Outcomes measured were rhinosinusitis disability index (RSDI) scores, antibiotics use, corticosteroid use, otolaryngology visits, and time to revision surgery. Results RSDI scores, antibiotics use, corticosteroid use, otolaryngology visits, and time to revision surgery had no statistically significant difference between cohorts. However, those taking zileuton tended to undergo fewer revision surgeries during the study. Conclusion Zileuton therapy for AERD patients shows no statistical benefit for rhinologic quality-of-life symptoms but may be useful in diminishing frequency of surgical intervention. In addition, pulmonary symptoms may be improved by treatment but were not assessed. Future study is warranted.

Myxochelin- and Pseudochelin-Derived Lipoxygenase Inhibitors from a Genetically Engineered Myxococcus xanthus Strain

Precursor-directed biosynthesis was used to introduce selected aryl carboxylic acids into the pseudochelin pathway, which had recently been assembled in Myxococcus xanthus. Overall, 14 previously undescribed analogues of the natural products myxochelin B and pseudochelin A were generated and structurally characterized. A subset of 10 derivatives together with their parental molecules were evaluated for their activity toward human 5-lipoxygenase. This testing revealed pseudochelin A as the most potent 5-lipoxygenase inhibitor among the naturally occurring compounds, whereas myxochelin A is the least active. Replacement of the catechol moieties in myxochelin B and pseudochelin A affected the bioactivity to different degrees.

Lens cholesterol biosynthesis inhibition: A common mechanism of cataract formation in laboratory animals by pharmaceutical products.

CJ-12,918, a 5-lipoxygenase (5-LO) inhibitor, caused cataracts during a 1-month safety assessment studies in rats whereas the structurally similar ZD-2138 was without effect. For CJ-12,918 analogs, blocking different sites of metabolic liability reduced (CJ-13,454) and eliminated (CJ-13,610) cataract formation in both rats and dogs. Using this chemical series as a test set, models and mechanisms of toxicity were first explored by testing the utility of ex vivo rat lens explant cultures as a safety screen. This model overpredicted the cataractogenic potential of ZD-2138 due to appreciably high lens drug levels and was abandoned in favor of a mechanism-based screen. Perturbations in lens sterol content, from a decline in lathosterol content, preceded cataract formation suggesting CJ-12,918 inhibited lens cholesterol biosynthesis (LCB). A 2-day bioassay in rats using ex vivo LCB assessments showed that the level of LCB inhibition was correlated with incidence of cataract formation in animal studies by these 5-LO inhibitors. Thereafter, this 2-day bioassay was applied to other pharmaceutical programs (neuronal nitric oxide synthase, sorbitol dehydrogenase inhibitor, squalene synthetase inhibitor and stearoyl-CoA desaturase-1 inhibitors/D4 antagonists) that demonstrated cataract formation in either rats or dogs. LCB inhibition >40% was associated with a high incidence of cataract formation in both rats and dogs that was species specific. Bioassay sensitivity/specificity were further explored with positive (RGH-6201/ciglitazone/U18666A) and negative (tamoxifen/naphthalene/galactose) mechanistic controls. This body of work over two decades shows that LCB inhibition was a common mechanism of cataract formation by pharmaceutical agents and defined a level of inhibition >40% that was typically associated with causing cataracts in safety assessment studies typically ≥1month.