Leukotriene B4 Receptor Antagonist Research Articles

Alpha-lipoic acid-mediated inhibition of LTB4 synthesis suppresses epithelial-mesenchymal transition, modulating functional and tumorigenic capacities in non-small cell lung cancer A549 cells

BackgroundLeukotriene B4 (LTB4) plays a crucial role in carcinogenesis by inducing epithelial-mesenchymal transition (EMT), a process associated with tumor progression. The synthesis of LTB4 is mediated by leukotriene A4 hydrolase (LTA4H), and it binds to the receptors BLT1 and BLT2. Dysregulation in LTB4 production is linked to the development of various pathologies. Therefore, the identification or design of inhibitors of LTB4 synthesis or receptor antagonists represents an ongoing challenge. In this context, our laboratory previously demonstrated that alpha-lipoic acid (ALA) inhibits LTA4H. The objective of this study was to evaluate the effect of ALA on the expression of canonical EMT markers and the functional and tumorigenic capacities induced by LTB4 in A549 cells. MethodsThe expression of cPLA2, 5LOX, FLAP, LTA4H, BLT1, and LTB4 production in human adenocarcinomic alveolar basal epithelial A549 cells was assessed using Western blot, RT-qPCR, and ELISA, respectively. Subsequently, the expression of canonical EMT markers was evaluated by Western blot. Functional assays were performed to assess cell viability, proliferation, invasion, migration, and clonogenicity using MTT, Western blot, Transwell assays, and colony formation assays, respectively. Results were expressed as median with interquartile range (n≥3) and analyzed using the Kruskal-Wallis or Tukey multiple comparisons tests. ResultsA549 cells express key proteins involved in LTB4 synthesis and receptor binding, including LTA4H and BLT1, and ALA inhibits the production of LTB4. Additionally, LTA4H and BLT1 were detected in lung adenocarcinoma tissue samples. LTB4 was found to induce EMT, whereas ALA treatment enhanced the expression of epithelial markers and reduced the expression of mesenchymal markers. Furthermore, ALA treatment resulted in a decrease in LTB4 levels and attenuated the functional and tumorigenic capacities of A549 cells, including their viability, migration, invasion, and clonogenic potential. ConclusionsThese findings suggest that ALA may offer therapeutic potential in the context of lung cancer, as it could be integrated into conventional pharmacological therapies to enhance treatment efficacy and mitigate the adverse effects associated with chemotherapy. Further studies are warranted to confirm the clinical applicability of ALA as an adjunctive treatment in lung cancer.

Lipid mediators in the regulation of innate and adaptive immunity.

Lipid mediators in the regulation of innate and adaptive immunity.

Leukotriene B4 Is a Major Determinant of Leukocyte Recruitment During Otitis Media.

BackgroundPathogens of otitis media (OM) induce inflammatory responses in the middle ear (ME), characterized by mucosal hyperplasia, leukocyte infiltration, and inflammatory mediators, including arachidonic acid metabolites. We studied the role of the eicosanoid leukotriene B4 (LTB4) in OM.MethodsExpression of LTB4-related genes was evaluated by gene array and single-cell RNA-Seq in MEs infected with nontypeable Haemophilus influenzae (NTHi). An inhibitor of LTB4 receptor 1 (i.e. U75302) was also used to block LTB4 responses.ResultsME expression of LTB4-related genes was observed by gene arrays and scRNA-Seq. However, not all genes involved in LTB4 generation occurred in any one specific cell type. Moreover, LTB4 receptor inhibition significantly reduced mucosal hyperplasia and virtually eliminated leukocyte infiltration.ConclusionsME expression of LTB4-related genes suggest a functional role in OM disease. The fact that LTB4-generation is spread across different cell types is consistent with a transcellular pathway of eicosanoid biosynthesis involving cell-to-cell signaling as well as transfer of biosynthetic intermediates between cells. The dramatic reduction in ME leukocyte infiltration caused by U75302 indicates that LTB4 plays a major role in ME inflammatory cell recruitment, acting via the LTB4R1 receptor. Given that there are many other chemotactic factors that occur in the ME during OM, the ability of LTB4 to activate leukocytes and stimulate their extravasation may explain the effects of inhibition. Reduction in mucosal hyperplasia due to U75302 administration may be secondary to the reduction in leukocytes since LTB4R1 is not expressed by mucosal epithelial or stromal cells. The results suggest that LTB4 receptor antagonists could be useful in treating OM.

Acceleration of Trichinella spiralis worm expulsion by leukotriene B4 receptor binding inhibition.

Helminth infection typically induces a Th2 inflammatory response that is characterized by eosinophilia, high levels of IgE and mast cells. LTB4 is generated from innate immune cells, such as neutrophils, macrophages and mast cells, in response to a range of stimuli. It mainly acts on myeloid leukocytes, inducing the activation of integrins, adhesion to endothelium walls, and chemotaxis. The objective of the present study was to determine the role of the LTB4 receptor in Trichinella spiralis expulsion. We treated mice with the LTB4 receptor antagonist before infection with T.spiralis. We observed that the number of mast cells and worm infection decreased following treatment with the BLT antagonist during the intestinal phase. We also demonstrated that blocking the LTB4 receptor inhibited neutrophil and eosinophil infiltration. Further studies are required to investigate the specific mechanism of mast cell number decrease and worm infection and the in vitro interactions between LTB4 and worm expulsion.

Convenient approach for the synthesis of ONO-LB-457, a potent leukotriene B4 receptor antagonist

Abstract This study reports a new approach for the synthesis of 5-[2-(2-carboxyethyl)-3-[6-(4-methoxyphenyl)-(5E)-hexen-1-yloxy]phenoxy]pentanoic acid V (ONO-LB-457), previously described by Konno and col. and which is considered a highly potent and orally active LTB4 receptor antagonist. This compound acts as an inhibitor of aggregation and chemotaxis, in addition to LTB4-induced human neutrophil degranulation. In this work, the preparation of ONO-LB-457 was proposed through a convergent synthesis focused on the preparation of two fragments. First, the preparation of 5-hydroxychroman-2-one (4) from 2,6-dimethoxybenzaldehyde and malonic acid, involving a Knoevenagel reaction, followed by a reduction of the olefin and intramolecular cyclization catalyzed by Lewis acid (tribromide) was achieved with an overall yield of 57%. Second, preparation of (E)-6-(4-methoxyphenyl)hex-5-en-1-yl-methanesulfonate (18) from 5-bromovaleric acid (15) involving a Wittig reaction. The desired compound V (ONO-LB-457) was obtained by nucleophilic substitution of (E)-6-(4-methoxyphenyl)hex-5-en-1-yl-methanesulfonate (18) with the ring-opened phenolic diester 14 followed by hydrolysis, in seven steps with an overall yield of about 11%.

A Research on Drug Discovery for Intracerebral Hemorrhage Focusing on Leukotriene B4 and Its Receptor

Intracerebral hemorrhage (ICH) results from blood vessels rupture in the brain, forming a blood clot in the brain parenchyma. Leakage of blood constituents causes detrimental tissue damages, ensuing long-lasting neurological deficits; however, effective therapeutic approaches are not yet developed to date. In this study, leukotriene B4 (LTB4) and its receptor leukotriene B4 receptor 1 (BLT1) are proposed as novel therapeutic targets for ICH therapy. After the onset of ICH, the LTB4 content in the brain transiently elevated. Microglia are considered as the source of LTB4 production. Thrombin, a blood constituent, activated the BV-2 microglia and increased the LTB4 secretion from the BV-2 cells. Microglia-released LTB4 promoted its own microglial activation and neutrophil-like differentiated HL-60 cell migration activity. LTB4 receptors comprised of two types: BLT1 and BLT2, with BLT1 known to be a high-affinity receptor associated with chemotaxis. BLT1 knockout mice showed decreased neutrophil invasion, attenuating sensorimotor dysfunction after ICH. Furthermore, therapeutic administration of ONO-4057, an orally active LTB4 receptor antagonist, attenuated neutrophil invasion, microglial activation, axonal fragmentation, and sensorimotor deficits induced by ICH. These results suggest that LTB4 and its receptor BLT1 can be potential promising therapeutic targets that prevent tissue damages following ICH.

Microglia-released leukotriene B4 promotes neutrophil infiltration and microglial activation following intracerebral hemorrhage

Intracerebral hemorrhage (ICH) from blood vessel rupture results in parenchymal hematoma formation and neuroinflammation, ultimately leading to neurodegeneration. Several lines of evidence suggest that the severity of ICH-induced neural damage is exacerbated by infiltration of T-cells, monocytes, and especially neutrophils into the hematoma. Neutrophil migration is regulated by chemokines, formyl peptides, and leukotriene B4 (LTB4), a metabolite of arachidonic acid. In this study, we demonstrate that LTB4 is a key signaling factor promoting microglial activity and leukocyte infiltration into hematoma and thus a potentially critical determinant of ICH pathogenesis and clinical outcome. Lipidomic analysis revealed markedly increased LTB4 concentration in the hematoma-containing brain tissues 6–24 h after experimental ICH in mice. Expression of 5-lipoxygenase, a rate-limiting enzyme for LTB4 production, was upregulated in activated microglia and neutrophils within the hematoma following ICH. Treatment of cultured BV-2 microglia with thrombin, which is abundant in hematoma, promoted activation, proinflammatory cytokine expression, and LTB4 secretion. Further, conditioned medium from thrombin-stimulated BV-2 cells potentiated the transwell migration of neutrophil-like cells, a response blocked by a LTB4 receptor antagonist. These results suggest that arachidonic acid conversion to LTB4 following ICH contributes to neuroinflammation and ensuing neural tissue damage by inducing microglial activation and neutrophil recruitment.

TMIC-57. PRO-TUMORAL EFFECTS OF INTRA-TUMORAL NEUTROPHILS IN THE GLIOBLASTOMA MICROENVIRONMENT

Abstract While macrophage enrichment and lymphocyte depletion have been described in glioblastoma, intratumoral neutrophils and their effect on glioblastoma have been under-characterized. While tumor-associated neutrophils (TANs) were initially regarded as passive bystanders due to their short-lived nature, investigation of TANs in other cancer types revealed pro-tumoral roles. Therefore, we sought to characterize TANs in the glioblastoma microenvironment using transcriptomic analysis and define their oncologic effects. Flow cytometric analysis of patient samples for neutrophils (CD11b+/CD15+/CD66b+) revealed higher percentages of TANs in glioblastoma compared to low-grade gliomas (1.76% [n=13] vs. 0.33% [n=6], p=0.03). Using the Transwell migration assay with glioblastoma tumor conditioned-media (CM), we found that recruitment of circulating neutrophils to tumor sites is mediated by leukotriene-B4 chemoattraction and that this interaction can be blocked with the addition of LtB4 receptor antagonist, LY293111. TANs were morphologically activated, unlike circulating neutrophils from GBM patients (P< 0.05) and, while not intravascular, were close to blood vessels. We performed single-cell RNA sequencing of isolated TANs and found a distinct transcriptomic profile relative to circulating neutrophils from these patients, particularly upregulated osteopontin. Osteopontin concentration was significantly higher in TAN CM than in patient-matched peripheral blood neutrophil CM (3.2ng/mL [n=3] vs. 0.02ng/mL [n=3], p< 0.05). Because osteopontin is linked to GBM stem cell-like phenotype maintenance and TANs localized to the perivascular niche where GBM stem cells reside, we investigated TAN-GBM stem cell interactions and osteopontin as a potential mediator. We found TAN CM increased proliferation and stem cell markers (Nanog, Oct4, Sox2) of stem cell-containing GBM neurospheres (p< 0.01). These effects were blocked by osteopontin-neutralizing antibodies (p< 0.01). Our work defines neutrophil-mediated pro-tumoral effects and their mechanisms and identifies a novel approach to target GBM stem cells—by disrupting the immune cell mediators that create their supportive microenvironment in the perivascular niche.

Pro-tumoral Effects of Intra-tumoral Neutrophils in the Glioblastoma Microenvironment

Abstract INTRODUCTION The glioblastoma microenvironment contains immune cells, particularly well-defined macrophage populations; however, intratumoral neutrophils and their effects on GBM biology are under-characterized. While tumor-associated neutrophils (TANs) were initially thought as passive bystanders due to their short-lived nature, current investigation of TANs in other cancer types revealed distinct pro-tumoral roles. Therefore, we sought to characterize TANs in the glioblastoma microenvironment and define their oncologic effects. METHODS Following informed consent, patient-derived GBM samples were collected for flow cytometry of TANs, which were then used to produce conditioned media (CM) for in Vitro studies on tumor cell proliferation and ELISA quantification of TAN-secreted factors. Single-cell RNA sequencing was performed on TANs to identify pro-tumoral factors. RESULTS Flow cytometric analysis (CD11b+/CD15+ /CD66b+) indicated higher percentages of TAN-infiltration to glioblastoma compared to low-grade gliomas (1.76% [n = 13] vs 0.33% [n = 6], P = .03). Using the Transwell migration assay with glioblastoma tumor CM, we found recruitment of circulating neutrophils to tumor sites is mediated by leukotriene-B4 and that this chemoattraction can be blocked with an LtB4 receptor antagonist, LY293111. We then performed single-cell RNA sequencing of isolated TANs and identified upregulation of osteopontin. Osteopontin is linked to GBM stem cell-like phenotype maintenance, thus, we decided to investigate osteopontin as a possible driver of pro-tumoral signaling. Osteopontin concentration was significantly higher in TAN CM than in patient-matched peripheral blood neutrophil CM (3.2 ng/mL [n = 3] vs 0.02 ng/mL [n = 3], P < .05). In Vitro, TAN CM led to significantly increased GBM cell proliferation and increased stem marker expression (Nanog, Oct4, Sox2) when incubated with neurospheres from an established GBM line. Pro-tumoral effects were lost in presence of osteopontin-neutralizing antibodies. CONCLUSION This study elucidates a possible mechanism of neutrophil-mediated pro-tumoral signaling. We found that neutrophils are recruited to tumor sites and play a biologically relevant role in GBM cellular proliferation and maintenance of a stem cell phenotype via osteopontin secretion.

Leukotriene-B4 modulates macrophage metabolism and fat loss in type 1 diabetic mice.

Serum levels of leukotriene-B4 (LTB4) are increased in type 1 diabetes (T1D) and it mediates systemic inflammation and macrophage reprogramming associated with this condition. Herein, we investigated the involvement of LTB4 in adiposity loss, hyperlipidemia, and changes in macrophage metabolism in a mouse model of streptozotocin-induced T1D. LTB4 receptor (BLT1) antagonist u75302 was employed to block LTB4 effects. As expected, hypoinsulinemia in T1D was associated with hyperglycemia, low levels of glucagon, hyperlipidemia, significant body fat loss, and increased white adipose tissue expression of Fgf21, a marker for lipolysis. With the exception of hyperglycemia and hypoglucagonemia, blockade of LTB4 signaling reverted these parameters in T1D mice. Along with hyperlipidemia, macrophages from T1D mice exhibited higher lipid uptake and accumulation. These cells also had enhanced glycolysis and oxidative metabolism and these parameters were dependent on the mitochondrial uncoupling respiration, as evidenced by elevated expression of oxidation markers carnitine palmitoyltransferase and uncoupling protein 1. Interestingly, all these parameters were at least partially reverted in T1D mice treated with u75302. Altogether, these findings suggest that in T1D mice LTB4/BLT1 is involved in the fat loss, hyperlipidemia, and increased macrophage lipid uptake and metabolism with an important involvement of mitochondrial uncoupling activity. These previously unrecognized LTB4/BLT1 functions may be explored in future to therapeutically alleviate severity of hyperlipidemia and systemic inflammation in T1D.

β2-Microglobulin, interleukin-31, and arachidonic acid metabolites (leukotriene B4 and thromboxane A2) are involved in chronic renal failure-associated itch-associated responses in mice

Chronic renal failure (CRF) is a progressive disease with severe pruritus and dry skin as the major symptoms. However, the mechanisms of CRF-induced pruritus remain unclear. In this study, 5/6 nephrectomized (NPCT) mice were used as a mouse model of CRF. Serum concentrations of blood urea nitrogen and creatinine in 5/6 NPCT mice were increased. The stratum corneum water content in the skin of 5/6 NPCT mice was decreased. These findings suggest that 5/6 nephrectomy in mice results in a phenotype resembling human CRF. 5/6 NPCT mice showed spontaneous scratching, which was inhibited by μ-opioid receptor antagonist, suggesting that the scratching is an itch-related response. The number of cutaneous mast cells was not altered in 5/6 NPCT mice compared with sham-operated mice. The H1 histamine-receptor antagonist, proteinase-activated receptor 2-neutralizing antibody, and 5-HT3-receptor antagonist did not inhibit spontaneous scratching in 5/6 NPCT mice; therefore, the role of mast cells and serotonin in spontaneous scratching appears to be minimal. The anti-allergy agent azelastine, BLT leukotriene (LT) B4 receptor antagonist, and TP thromboxane (TX) A2 receptor antagonist inhibited spontaneous scratching in 5/6 NPCT mice, suggesting that LTB4 and TXA2 are involved in CRF-induced pruritus. Interestingly, in the skin of 5/6 NPCT mice, levels of two newly identified pruritogens (β2-microglobulin and interleukin-31) were increased. Taken together, these findings suggest that 5/6 NPCT mice are useful for the study of itching in CRF. In addition to LTB4 and TXA2, it is also suggested that β2-microglobulin and interleukin-31 are involved in itching associated with CRF-induced pruritus.

Leptin Mediates In Vivo Neutrophil Migration: Involvement of Tumor Necrosis Factor-Alpha and CXCL1.

Leptin directly activates macrophages and lymphocytes, but the role of leptin in neutrophil activation and migration is still controversial. Here, we investigate the in vivo mechanisms of neutrophil migration induced by leptin. The intraperitoneal injection of leptin (1 mg/kg) induces a time- and concentration-dependent neutrophil influx. We did not observe the enhancement of lipid bodies/droplets in neutrophils, after leptin treatment, as we had observed previously in peritoneal macrophages. The participation of leukotriene B4 (LTB4) in neutrophil recruitment triggered by leptin was investigated using different strategies. Leptin-induced neutrophil recruitment occurs both in the absence of 5-lipoxygenase activity in 5-lipoxygenase (5-LO)−/− mice and after the administration of either 5-LO inhibitor (Zileuton) or the LTB4 receptor antagonist (U-75302). Moreover, no direct induction of LTB4 by leptin could be observed. Neutrophil influx could not be prevented by the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, contrasting with the leptin-induced signaling for lipid body formation in macrophage that is mTOR-dependent. Leptin administration led to tumor necrosis factor-alpha (TNFα) production by the peritoneal cells both in vivo and in vitro. In addition, neutrophil recruitment was inhibited in tumor necrosis factor receptor 1 (TNFR1−/−) mice, indicating a role for TNF in leptin-induced neutrophil recruitment to the peritoneal cavity. Leptin-induced neutrophil influx was PI3Kγ-dependent, as it was absent in PI3Kγ−/− mice. Accordingly, leptin induced the peritoneal cells to produce CXCL1, both in vivo and in vitro, and the neutrophil influx was ablated after using an antibody against CXCL1. Our results establish TNFα/TNFR1- and CXCL1-dependent signaling as important pathways for leptin-induced neutrophil migration in vivo.

Eosinophil transendothelial migration induced by the bronchoalveolar lavage fluid of acute eosinophilic pneumonia.

Acute eosinophilic pneumonia (AEP) is characterized by a massive pulmonary infiltration of eosinophils. Mechanisms regulating the selective accumulation of eosinophils in AEP have not been fully established. The objective of this study was to evaluate the mechanisms of eosinophil accumulation in alveolar spaces through examination of bronchoalveolar lavage fluid (BALF) from AEP patients (AEP-BALF). Eosinophils were isolated from the blood of healthy subjects and were placed on a human pulmonary microvascular endothelial cell monolayer cultured on Transwell filters (Coster, Cambridge, MA, USA). A saline control solution or BALF from patients with AEP, sarcoidosis or hypersensitivity pneumonitis was applied to the lower compartment, and the transendothelial migration of the eosinophils was evaluated. The concentrations of cytokines and chemokines in BALF were also measured. Transmigration of eosinophils across endothelial cells was only induced by the AEP-BALF. This transmigration was blocked by anti-β2 integrin mAb. The concentrations of eotaxin-2 and monocyte chemotactic protein (MCP)-4, which are CC chemokine receptor (CCR) 3 ligands, were elevated in the AEP-BALF, and anti-CCR3 mAb or anti-MCP-4 mAb inhibited the AEP-BALF-induced transmigration of eosinophils. Furthermore, the concentration of leukotriene (LT) B4 was increased in the AEP-BALF, and an LTB4 receptor antagonist partially suppressed the AEP-BALF-induced transmigration of eosinophils. These findings suggest that CCR3 ligands including eotaxin-2 and MCP-4, and LTB4 play a role in the accumulation of eosinophils in AEP.

Involvement of Leukotriene B4 Released from Keratinocytes in Itch-associated Response to Intradermal Interleukin-31 in Mice.

A recent study suggests that interleukin-31 (IL-31) exerts its effect via indirect mechanisms rather than through direct stimulation of cutaneous nerves. However, the underlying peripheral mechanisms of IL-31-induced itch in the skin remain unclear. Therefore, the present study investigated the peripheral mechanisms underlying IL-31-induced itch in mice. IL-31-induced itch-related response was inhibited by anti-allergic drugs (tranilast and azelastine), but not by an H1 histamine receptor antagonist (terfenadine). Furthermore, a 5-lipoxygenase inhibitor (zileuton), but not a cyclooxygenase inhibitor (indomethacin), and a leuko-triene B4 (LTB4) receptor antagonist (CMHVA) attenuated the action of IL-31. IL-31 receptor-immunoreactivity was observed in the epidermis and primary sensory neurones. IL-31 receptor mRNA was expressed in mouse keratinocytes and dorsal root ganglia neurones. IL-31 increased the production of LTB4 in mouse keratinocytes. These results suggest that IL-31 elicits itch not only through direct action on primary sensory neurones, but also by inducing LTB4 production in keratinocytes.

Trans-Basement Membrane Migration of Eosinophils Induced By LPS-Stimulated Neutrophils from Human Peripheral Blood in Vitro

In the airways of severe asthmatics, an increase of neutrophils and eosinophils is often observed despite high-dose corticosteroid therapy. We previously reported that interleukin-8-stimulated neutrophils induced trans-basement membrane migration (TBM) of eosinophils, suggesting the link between neutrophils and eosinophils. Concentrations of lipopolysaccharide (LPS) in the airway increase in severe asthma. As neutrophils express Toll-like receptor (TLR)4 and can release chemoattractants for eosinophils, we investigated whether LPS-stimulated neutrophils modify eosinophil TBM. Neutrophils and eosinophils were isolated from peripheral blood of healthy volunteers and severe asthmatics. Eosinophil TBM was examined using a modified Boyden's chamber technique. Eosinophils were added to the upper compartment, and neutrophils and LPS were added to the lower compartment. Migrated eosinophils were measured by eosinophil peroxidase assays. LPS-stimulated neutrophils induced eosinophil TBM (about 10-fold increase), although LPS or neutrophils alone did not. A leukotriene B4 receptor antagonist, a platelet-activating factor receptor antagonist or an anti-TLR4 antibody decreased eosinophil TBM enhanced by LPS-stimulated neutrophils by almost half. Neutrophils from severe asthmatics induced eosinophil TBM and lower concentrations of LPS augmented neutrophil-induced eosinophil TBM. These results suggest that the combination of neutrophils and LPS leads eosinophils to accumulate in the airways, possibly involved the pathogenesis of severe asthma.

Modulation of Leukotriene Signaling Affecting Cell Growth in Chronic Myeloid Leukemia: A Key Pathway to Cure?

▪Background: Recent data suggest that tyrosine kinase inhibitor (TKI)-insensitive leukemic stem cells often prevail in chronic myeloid leukemia (CML) patients subjected to long-term TKI treatment. To achieve cure additional aberrant pathways in these patients, such as leukotriene (LT) signaling, may need to be targeted. We have previously shown that human CML cells have an increased capacity to produce LT and that LT can stimulate normal myelopoiesis (Tornhamre ExpHem 2003). Chen later noted a striking up-regulation of ALOX5/5LO (catalyzing an initial step in LT formation) in CML mice, and that inhibition of this enzyme improved survival of these animals by a magnitude similar to that induced by imatinib (Chen NatureGen 2009). Here wexve assessed the effect of LT modulating agents on the growth of human CML cells in vitro.Materials and methods: Human CML cells, derived from the three cell lines K562, Kcl22 and KU812, were grown in microtitre plate cultures, in the presence of RPMI 1640. The MTT technique was used to evaluate the number of viable cells at 72 hours. Protein expression was assessed by Western blot and immunocytochemistry.Results: Several specific modulators of LT-signaling were capable of inducing dose-dependent growth inhibitory effects on CML cells. Thus, the cysteinyl-LT receptor antagonist montelukast, a drug with approved clinical use in human asthma, was shown to reduce the growth of all three tested CML cell lines. In Fig. 1 this is examplified by K562 cells, where also additive effects between montelukast and imatinib are indicated. All cell lines expressed the cysLT1-receptor. Furthermore, the LTB4 receptor antagonist etalocib, as well as the 5-LO activating protein (FLAP) inhibitors licofelone and Bay-X-1005, also executed inhibitory effects at concentrations considered as physiological. As expected, addition of the TKIs imatinib, nilotinib or dasatinib to the cultures also generated dose-dependent inhibitory effects on the growth of all tested CML cell lines. An LD50 of approximately 0.5 µM, 10 nM and 0.1 nM was noted for imatinib, nilotinib and dasatinib, respectively, for K562 cells at 72 hours.Conclusions: We demonstrate that several compounds, known to specifically inhibit leukotriene signaling by different mechanisms, were at clinically relevant concentrations capable of significantly suppressing the growth of human CML cells. Some of these compounds are already in clinical practice for non-hematological disorders. They could provide an additional therapeutic possibility in the quest to cure CML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Trans-basement membrane migration of eosinophils induced by LPS-stimulated neutrophils from human peripheral blood in vitro.

In the airways of severe asthmatics, an increase of neutrophils and eosinophils is often observed despite high-dose corticosteroid therapy. We previously reported that interleukin-8-stimulated neutrophils induced trans-basement membrane migration (TBM) of eosinophils, suggesting the link between neutrophils and eosinophils. Concentrations of lipopolysaccharide (LPS) in the airway increase in severe asthma. As neutrophils express Toll-like receptor (TLR)4 and can release chemoattractants for eosinophils, we investigated whether LPS-stimulated neutrophils modify eosinophil TBM.Neutrophils and eosinophils were isolated from peripheral blood of healthy volunteers and severe asthmatics. Eosinophil TBM was examined using a modified Boyden's chamber technique. Eosinophils were added to the upper compartment, and neutrophils and LPS were added to the lower compartment. Migrated eosinophils were measured by eosinophil peroxidase assays.LPS-stimulated neutrophils induced eosinophil TBM (about 10-fold increase), although LPS or neutrophils alone did not. A leukotriene B4 receptor antagonist, a platelet-activating factor receptor antagonist or an anti-TLR4 antibody decreased eosinophil TBM enhanced by LPS-stimulated neutrophils by almost half. Neutrophils from severe asthmatics induced eosinophil TBM and lower concentrations of LPS augmented neutrophil-induced eosinophil TBM.These results suggest that the combination of neutrophils and LPS leads eosinophils to accumulate in the airways, possibly involved the pathogenesis of severe asthma.

Impairment of neutrophil migration to remote inflammatory site during lung histoplasmosis.

Histoplasma capsulatum (Hc) induces a pulmonary disease in which leukotrienes promote activation and recruitment of effectors cells. It is also well-recognized that leukotriene B4 (LTB4) and platelet-activating factor (PAF) induce leukocyte recruitment to inflammatory sites. We investigated the impact of pulmonary Hc infection on PMN migration to a remote inflammatory site. Our results show that pulmonary Hc infection impairs LTB4- or PAF-stimulated PMN recruitment to air pouch. Yet, remote inflammation did not modify PMN numbers in the bronchoalveolar lavage fluid (BALF) of Hc-infected mice. Interestingly, the concomitant administration of PAF and LTB4 receptor antagonists inhibited PMN recruitment to both BALF and the remote site, demonstrating cooperation between both mediators. Along that line, our results show that PAF-elicited PMN chemotaxis was abrogated in 5-lipoxygenase-deficient animals. These results suggest caution in the indiscriminate use of anti-inflammatory drugs during infectious diseases.

ChemInform Abstract: Functionalized Organozinc Reagents in Medicinal Chemistry: Discovery of Novel Drug Candidates

AbstractReview: [43 refs.

Functionalized Organozinc Reagents in Medicinal Chemistry: Discovery of Novel Drug Candidates

Discovery, optimization, and early development of drug candidates are very challenging and require a versatile synthetic chemistry toolbox. Organozinc chemistry is a powerful methodology for carbon–carbon bond formation which tolerates several functional groups that are critical for maintaining good physicochemical properties of drug molecules. Here we demonstrate that organozinc chemistry offers facile solutions to otherwise challenging medicinal chemistry problems commonly encountered during lead optimization. In this review, we summarize several examples in which utilization of organozinc-mediated couplings enabled us to rapidly explore a medicinal chemistry hypothesis using readily available intermediates. This chemistry allowed us to make rapid decisions with limited chemistry resources. 1 Introduction 2.1 Alpha4 Integrin Antagonists 2.2 CRTH2 Antagonists 2.3 Glucokinase (GK) Activators 2.4 Leukotriene B4 (LTB4) Receptor Antagonists 2.5 Janus Activated Kinase 3 (JAK3) Inhibitors 2.6 Synthesis of Novel Scaffolds 3 Conclusions 4 Experimental Methods