l-663536 and zileuton

Topics: Animals; Asthma; Glomerulonephritis; Humans; Hydroxyurea; Indoles; Inflammatory Bowel Diseases; Leukotriene Antagonists; Leukotrienes; Membrane Proteins; Propionates; Psoriasis; Quinolines; Receptors, Leukotriene

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A- through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.

Topics: Animals; Benzopyrans; Carboxylic Acids; Choroidal Neovascularization; Disease Models, Animal; Eye; Eye Injuries; Hydroxyurea; Indoles; Lasers; Leucine; Leukotriene B4; Macrophages; Macular Degeneration; Male; Mice; Mice, Knockout; Neovascularization, Pathologic; Receptors, Leukotriene B4; Signal Transduction

Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES-1, are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.

Topics: Acetophenones; Animals; Anti-Inflammatory Agents; Arachidonate 5-Lipoxygenase; Disease Models, Animal; Humans; Hydroxyurea; Inhibitory Concentration 50; Intramolecular Oxidoreductases; Lipoxygenase Inhibitors; Mice; Molecular Structure; Prenylation; Prostaglandin-E Synthases; Rutaceae; Structure-Activity Relationship

Pulmonary Hypertension (PH) is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5). While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC) were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2) release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2.

Topics: Arachidonate 5-Lipoxygenase; Cell Hypoxia; Cell Line; Cell Proliferation; Endothelial Cells; Humans; Hydrogen Peroxide; Hydroxyurea; Indoles; Lipoxygenase Inhibitors; Oxygen; Pulmonary Artery

5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid to form leukotrienes. We have reported that ischemic-like injury activates 5-LOX in PC12 cells; however, the mechanisms are unknown. To determine whether ischemic-like injury activates 5-LOX mediated by oxidative stress through the p38 MAPK pathway, we transfected GFP-5-LOX into PC12 cells and induced ischemic-like injury by oxygen-glucose deprivation (OGD). We found that the transfected GFP-5-LOX was localized primarily in the nuclei and translocated to the nuclear envelope after OGD/recovery reaching a maximum 2 hr after a 2-hr exposure to OGD. The nonselective 5-LOX inhibitor caffeic acid, 5-LOX-activating protein inhibitor MK886, and selective 5-LOX inhibitor zileuton attenuated the cell injury and reduced the production of 5-LOX products, cysteinyl leukotrienes, after OGD/recovery. However, only caffeic acid inhibited OGD/recovery-induced 5-LOX translocation. OGD/recovery also increased reactive oxygen species (ROS), which was inhibited by caffeic acid only. Hydrogen peroxide, an exogenous ROS, evoked similar cell injury and 5-LOX translocation, and the inhibitors had effects on the changes after H(2)O(2) similar to those after OGD/recovery. Both OGD/recovery and H(2)O(2) increased the phosphorylated p38 MAPK level, which was inhibited by caffeic acid and the ROS scavenger edaravone, but not by MK886 or zileuton. Moreover, SB203580 (a p38 MAPK inhibitor) and edaravone inhibited the cell injury and 5-LOX translocation induced by OGD/recovery and H(2)O(2). Thus, we conclude that OGD/recovery-induced ischemic-like injury induces 5-LOX activation, which is mediated by oxidative stress through activating the p38 MAPK pathway.

Topics: 5-Lipoxygenase-Activating Proteins; Animals; Antioxidants; Antipyrine; Arachidonate 5-Lipoxygenase; Caffeic Acids; Carrier Proteins; Cell Hypoxia; Edaravone; Enzyme Activation; Enzyme Inhibitors; Glucose; Hydrogen Peroxide; Hydroxyurea; Imidazoles; Indoles; Leukotrienes; Lipoxygenase Inhibitors; MAP Kinase Signaling System; Membrane Proteins; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Phosphorylation; Pyridines; Rats; Reactive Oxygen Species; Transfection

This study investigated endogenous mediators involved in mosquito allergy-associated itching in mice. An intradermal injection of an extract of mosquito salivary gland elicited marked scratching in sensitized mice. The 5-lipoxygenase inhibitor zileuton (100 mg/kg), the 5-lipoxygenase activating peptide inhibitor MK-886 (10 mg/kg), and the glucocorticoid betamethasone 17-valerate (3 mg/kg) inhibited the scratching. The scratching was not affected by the cyclooxygenase inhibitors indomethacin and ketoprofen, the TP prostanoid receptor antagonist SQ-29548, the leukotriene B(4) antagonist ONO-4057, the cysteinyl leukotriene antagonist pranlucast, the leukotriene D(4) antagonist MK-571, the platelet-activating factor antagonist CV-3988, the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester, the H(2) histamine-receptor antagonist cimetidine, the H(1) histamine-receptor antagonist terfenadine plus cimetidine, and cypoheptadine that blocks the 5-HT(1/2) serotonin receptors. Zileuton (100 mg/kg) inhibited the increased activity of the cutaneous nerve branch induced by an intradermal injection of the extract, suggesting the peripheral action. Zileuton and MK-886 (10 and 100 microM) did not affect high K(+)-induced increase in intracellular Ca(2+) concentration in cultured dorsal root ganglion neurons. The results suggest that 5-lipoxygenase metabolite(s) other than leukotriene B(4) and cysteinyl leukotrienes are involved in mosquito allergy-associated itching.

Topics: Animals; Arachidonate 5-Lipoxygenase; Betamethasone Valerate; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Chromones; Cimetidine; Culicidae; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Hydrazines; Hydroxyurea; Hypersensitivity; Indoles; Indomethacin; Ketoprofen; Leukotriene B4; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred ICR; NG-Nitroarginine Methyl Ester; Phenylpropionates; Phospholipid Ethers; Propionates; Pruritus; Quinolines; Terfenadine

Macrophage (MØ) phagocytosis via the Fc receptor for immunoglobulin G (Fc gammaR) requires the spleen tyrosine kinase (Syk) and serves an important antimicrobial function. We have reported previously that Fc gammaR-mediated ingestion and Syk activation in MØ are amplified by and depend on the proinflammatory lipid mediator leukotriene B4 (LTB4). Although Fc gammaR-mediated ingestion is also important for antigen uptake, there is no information about LTB4 regulation of these processes in dendritic cells (DCs). In this study, we compared murine bone marrow (BM)-derived DCs to MØ from BM, peritoneum, and the pulmonary alveolar space. Neither phagocytosis nor Syk activation in DCs was influenced by exogenous LTB4. Unlike the various MØ populations, Syk activation in DCs was likewise unaffected by pharmacologic or genetic strategies to inhibit endogenous LTB4 synthesis or to block the high-affinity LTB4 receptor BLT1. DCs were refractory to regulation by LTB4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in Fc gammaR signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.

Topics: Animals; Antigen Presentation; Bone Marrow Cells; Calcium Signaling; Cells, Cultured; Dendritic Cells; Endocytosis; Enzyme Activation; Erythrocytes; Hydroxyurea; Immunoglobulin G; Indoles; Intracellular Signaling Peptides and Proteins; Leukotriene B4; Macrophages; Macrophages, Alveolar; Macrophages, Peritoneal; Mice; Mice, Inbred CBA; Mice, Knockout; Opsonin Proteins; Phagocytosis; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Purinergic P2 Receptor Antagonists; Rats; Rats, Wistar; Receptors, IgG; Receptors, Leukotriene B4; Sheep; Syk Kinase

5-Leukotriene pathway inhibitors, Accolate, MK-886, and Zileuton, were evaluated as chemopreventive agents in female strain A mice. The mice were administered by injection vinyl carbamate (2 x 16 mg/kg) to induce lung tumors. Two weeks later, they received in their diet Accolate (270 and 540 mg/kg), MK-886 (30 mg/kg), Zileuton (600 and 1200 mg/kg), or combinations containing the lower concentration of two agents. Thirteen weeks later, Accolate, Zileuton (only the high concentration), and combinations of Zileuton with either Accolate or MK-886 reduced lung tumor multiplicity. At week 43, MK-886, Accolate, and Zileuton reduced lung tumor multiplicity by 37.8, 29.5, and 28.1%, respectively. They also decreased the size of the tumors and the yield of carcinomas. These results demonstrate that leukotriene inhibitors prevent lung tumors and slow the growth and progression of adenomas to carcinoma; leukotriene inhibitors warrant further consideration for potential use in humans.

Topics: Animals; Anticarcinogenic Agents; Carcinogens; Dose-Response Relationship, Drug; Female; Hydroxyurea; Indoles; Leukotriene Antagonists; Lipoxygenase Inhibitors; Lung Neoplasms; Mice; Mice, Inbred A; Phenylcarbamates; Sulfonamides; Tosyl Compounds; Urethane

The 5-LO inhibitor, WY-50295 tromethamine (T) prevented leukotriene release (LTB4 production) in calcium ionophore stimulated, purified human and rat neutrophils. However, whereas WY-50295T inhibited both in vitro and ex vivo rat whole blood leukocyte LTB4 formation (IC50= 40 microM and oral ED50 of 18 mg/kg, respectively), it did not inhibit LTB4 production in calcium ionophore stimulated human whole blood at concentrations to 200 microM. To reduce binding of WY-50295T to serum albumin, 250 microM of a naphthalene sulfonic acid (> 99.9% binding to albumin primarily at the carboxylic site) and 250 microM sulfanilamide (binding to nonspecific sites) separately or in combination were preincubated in whole blood prior to addition of WY-50295T; however, WY-50295T still did not inhibit 5-LO and free drug blood levels were unchanged. When purified human neutrophils in the presence of fatty acid saturated albumin (fraction V) was employed, the 5-LO inhibitory activity of WY-50295T was prevented. Zileuton (5 microM) inhibited LTB4 production by 99% in the presence of these albumins. Also, rat albumin presented WY-50295T to purified rat neutrophils more effectively than human albumin (i.e. WY-50295T was more active in the presence of rat albumin). These results suggest that the high affinity binding of WY-50295T to human albumin and possibly the reduction of drug uptake (passive diffusion) using purified human vs rat neutrophils may account for the inactivity of WY-50295T in the human whole blood assay.

Topics: Animals; Binding Sites; Dose-Response Relationship, Drug; Eicosanoids; Guinea Pigs; Humans; Hydroxyurea; Indoles; Leukocytes; Leukotriene Antagonists; Lipoxygenase Inhibitors; Male; Naphthaleneacetic Acids; Neutrophils; Quinolines; Rats; Serum Albumin

In 16HBE transformed human bronchial epithelial cells, histamine stimulated interleukin (IL)-8 mRNA and protein secretion, and this histamine stimulation was inhibited by the H1-receptor antagonist diphenhydramine (DPH), by the inhibitor of 5-lipoxygenase-activating protein (FLAP) MK-886, by the 5-lipoxygenase inhibitor Zileuton, and by dexamethasone. Histamine stimulated bronchial epithelial cell production of leukotriene B4 (LTB4), and this production was inhibited by FLAP inhibitors MK-886 and L-655,238 and Zileuton. Histamine stimulated IL-8 luciferase reporter gene activity that was inhibited with DPH, dexamethasone, MK-886 and L-655,238, and Zileuton. The inhibition of IL-8 transcription and protein secretion by FLAP inhibitors and Zileuton was reversed with exogenous LTB4. There was increased IL-8 nuclear factor-kappaB (NF-kappaB) DNA-binding activity after histamine stimulation, and this was inhibited by DPH and MK-886. Cytoplasmic phospholipase A2 mRNA levels were also potently induced by histamine. Thus histamine stimulation of bronchial epithelial cells involves binding at H1 receptors, production of LTB4, activation of NF-kappaB and increased expression of IL-8.

Topics: Bronchi; Cell Line; Cell Line, Transformed; Epithelial Cells; Histamine; Humans; Hydroxyurea; Indoles; Interleukin-8; Leukotriene B4; Lipoxygenase Inhibitors; NF-kappa B; Phospholipases A; Phospholipases A2; Polymerase Chain Reaction; Quinolines; RNA, Messenger; Transfection

Prostaglandins and leukotrienes differentially regulate the production of interleukin-1 (IL-1) in monocytes. It was, therefore, decided to investigate the effects of some 5-lipoxygenase inhibitors compared with standard IL-1 synthesis inhibitors on the production of IL-1 by human synovial tissue explants in organ culture. Human synovial (from hip/knee arthroplasty) or porcine tibio-tarsal joint synovial explants were incubated in organ culture in Dulbecco's Modified Eagle's Medium + 5% foetal calf serum in the presence of the test compounds or solvents (controls), or media alone for 1-5 days. Total bioactive IL-1 was assayed in the medium (following serial dilution or with polyethylene glycol 8000 added in some assays to remove inhibitors) using the D-10 T-cell bioassay. Some assays of interleukins 1 alpha, 1 beta, 6 or 8 were performed by ELISA. Of the 5-lipoxygenase inhibitors investigated, MK-886(3-(1-(4-chlorobenzyl)-3-tert-butyl-thio-5-isopropylindol-2- yl)-2,2 -dimethyl propanoic acid), L-656,224 ((7-chloro-2-[4-methoxypenyl]methyl)-3 -methyl-5-propyl-4-benzofuranol), PF-5901 and tepoxalin were the most potent inhibitors of IL-1 production. While the PF-5901 was effective at 5-30 microM and tepoxalin was effective at 1-10 microM, the others were the most potent having minimal inhibitory activity in the range of 0.01-0.1 microM. The presumed IL-1-synthesis inhibitors, tenidap and IX-207,887, were inactive at concentrations of 30-50 microM. Leukotriene B4 (1-100 ng/mL-1) added to MK-886 (5 microM)-treated cultures reversed the inhibitory effects of the latter on IL-1, confirming the role of 5-lipoxygenase products in the regulation of IL-1 production. Addition of polyethylene glycol 8000 to MI-886-treated cultures eliminated the inhibitory effects of this drug, suggesting that this drug exerts its effects by promoting production of IL-1 inhibitors. MK-886 also inhibited synovial production of two other pleiotrophic cytokines which it regulates, IL-6 and IL-8. The results suggest that some 5-lipoxygenase inhibitors may be usefully employed in regulating production of those interleukins involved in joint cartilage destruction.

Topics: Animals; Benzofurans; Enzyme-Linked Immunosorbent Assay; Hip Joint; Humans; Hydroxyurea; Indoles; Interleukin-1; Knee Joint; Lipoxygenase Inhibitors; Naphthalenes; Organ Culture Techniques; Oxindoles; Pyrazoles; Quinolines; Structure-Activity Relationship; Swine; Synovial Membrane; Tarsus, Animal; Thiazoles; Thiophenes

alpha-naphthylisothiocyanate (ANIT) administration to rats results in periportal hepatic inflammation and injury. Glutathione (GSH) appears to be necessary for the liver injury to occur. The leukotrienes (LTs) are metabolites of arachidonic acid and potent mediators of inflammation that have been implicated in certain liver injury models. Inasmuch as GSH is a cofactor for the synthesis of cysteinyl-LTs and since inflammation is a prominent component of ANIT injury, we hypothesized that LTs are involved in producing the hepatic insult that results from ANIT administration. To test this hypothesis, rats were treated with one of several inhibitors of LT biosynthesis, A63162, Zileuton or MK-886. Each of these agents prevented the formation of LTB4 in Ca++ ionophore-stimulated whole blood from rats treated with the inhibitors. A63162 attenuated the hepatic parenchymal injury caused by ANIT and resulted in a modest decrease in ANIT-induced cholestasis. In contrast, neither Zileuton nor MK-886 attenuated liver injury. AT-125 (Acivicin) inhibits gamma-glutamyl transferase (GGT), the enzyme that catalyzes the formation of LTD4 from LTC4. AT-125 pretreatment did not prevent ANIT-induced hepatic parenchymal insult. It did, however, ameliorate the cholestasis caused by ANIT. In conclusion, the partial protection afforded by A63162 and AT-125 likely results from effects unrelated to the formation of LTs, since Zileuton and MK-886 inhibited LT synthesis without affording protection. The lack of protection by Zileuton and MK-886 in the face of LT synthesis inhibition suggests that LTs are not necessary for the expression of injury after ANIT administration.

Topics: 1-Naphthylisothiocyanate; Acetamides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Calcimycin; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Glutathione; Hydroxyurea; Indoles; Inflammation; Leukotriene Antagonists; Leukotrienes; Lipoxygenase Inhibitors; Liver; Liver Diseases; Male; Phenyl Ethers; Rats; Rats, Sprague-Dawley

BAY x1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid), an inhibitor of leukotriene synthesis, was evaluated, both in vitro and in vivo, for inhibition of antigen-induced airway contraction in the sensitised guinea-pig. Antigen (ovalbumin 0.001-10 micrograms/ml) challenge of tracheae in the presence of pyrilamine and indomethacin induced contractile responses which were inhibited by BAY x1005 with an IC50 value of 0.36 (0.2-0.8) microM. Using the same test system BAY x1005 (1 microM), ICI D2138 (0.3 microM) or AA 861 (1 microM) had similar inhibitory activities, whereas MK 886, MK 591, and Zileuton (A64077) all tested at 1 microM and REV 5901 (10 microM) had no significant effect. Using tracheae from non-sensitised (control) guinea-pigs the calcium ionophore A23187 (1 microM) induced a maximal contraction which was significantly inhibited by BAY x1005 at 1 microM, whereas MK 886 was only active at 3 microM. BAY x1005 tested at 10 microM and 3 microM had no effect against leukotriene D4- or KCl-induced contractions of guinea-pig tracheae respectively. In the anaesthetised ovalbumin sensitised guinea-pig BAY x1005 caused a dose-related inhibition of ovalbumin-induced bronchoconstriction, with approximate ID50 values of 0.85 mg/kg i.v. and 6.3 mg/kg p.o. In the same model MK 886, MK 591, AA 861 and ICI D2138 each given at 10 mg/kg p.o. had no significant inhibitory activity against antigen challenge. Six hours after administration BAY x1005 (10 mg/kg p.o.) was still effective against the antigen-induced response.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 5-Lipoxygenase-Activating Proteins; Administration, Oral; Animals; Benzoquinones; Bronchoconstriction; Carrier Proteins; Guinea Pigs; Hydroxyurea; In Vitro Techniques; Indoles; Injections, Intravenous; Leukotriene Antagonists; Lipoxygenase Inhibitors; Male; Membrane Proteins; Ovalbumin; Pyrans; Quinolines; Quinolones; Trachea

We found that intrinsic neutrophil 5-lipoxygenase activity was necessary for human neutrophil adherence and chemotaxis in vitro and human neutrophil-mediated acute edematous injury in isolated perfused rat lungs given interleukin 8 intratracheally. Treatment with either Zileuton (a specific reversible competitive inhibitor of 5-lipoxygenase) or MK886 (a specific irreversible inhibitor of the 5-lipoxygenase activator protein) prevented stimulated neutrophil adherence and chemotaxis (but not superoxide anion production) in vitro. Zileuton- or MK886-inhibited neutrophil chemotaxis was not restored by adding leukotriene B4 in vitro. Perfusion with neutrophils and either Zileuton or MK886, or with MK886-pretreated neutrophils (without adding MK886 to the perfusate), also prevented lung injury (reflected by lung weight gain and lung Ficoll retention) and perfusate leukotriene B4 increases in isolated rat lungs given interleukin 8 intratracheally. Again, adding leukotriene B4 to the perfusate did not damage interleukin 8-treated isolated lungs perfused with Zileuton-inhibited neutrophils. We conclude that intrinsic 5-lipoxygenase activity is required for neutrophil adherence and chemotaxis and neutrophil-mediated lung injury.

Topics: Animals; Arachidonate 5-Lipoxygenase; Cell Adhesion; Chemotaxis, Leukocyte; Cytochrome c Group; Dose-Response Relationship, Drug; Humans; Hydroxyurea; In Vitro Techniques; Indoles; Interleukin-8; Kinetics; Leukotriene Antagonists; Leukotriene B4; Lipoxygenase Inhibitors; Lung; Male; Neutrophils; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Superoxides; Tetradecanoylphorbol Acetate

The involvement of leukotrienes (LTs) in antigen-induced airway hyper-reactivity (AHR) was characterized pharmacologically by using several 5-lipoxygenase (5-LO) inhibitors and LTD4 antagonists in guinea pigs. AHR was evidenced by consistent and significant increases in sensitivity to bronchoconstriction induced by i.v. methacholine in anesthetized and ventilated animals 24 hr after a single ovalbumin aerosol challenge, but maximum methacholine-induced bronchoconstriction did not increase. Animals were pretreated with maximum doses of WY-50,295 tromethamine (WY-50,295), LY-171,883, MK-886 or zileuton, based upon inhibition of antigen-induced LT-dependent bronchoconstriction. WY-50,295, having a long duration of action, was the only compound that prevented AHR when given once before antigen challenge. However, LY-171,883 and MK-886 prevented AHR when a second dose was given 4 hr after challenge. Zileuton, having a short duration of action, failed to prevent AHR when given before and after challenge. The prevention of AHR did not result from functional antagonism (bronchodilation) by any compound. In bronchoalveolar lavage studies, neither WY-50,295 nor MK-886 inhibited the influx of eosinophils into the airways 24 hr after antigen challenge. The results provide pharmacological evidence that LTs play an important role in the pathogenesis of antigen-induced AHR in guinea pigs. Furthermore, the effectiveness of 5-LO inhibitors and LTD4 antagonists in this model depends upon a long duration of drug action and appears to result from inhibition of a direct airway effect of LTs rather than inhibition of eosinophil influx into the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetophenones; Animals; Antigens; Bronchoconstriction; Eosinophilia; Guinea Pigs; Hydroxyurea; Indoles; Leukotriene D4; Leukotrienes; Lipoxygenase Inhibitors; Male; Naphthaleneacetic Acids; Quinolines; Tetrazoles

In man, the therapeutic effectiveness of specific inhibitors of leukotriene (LT) biosynthesis against allergen-induced bronchoconstriction appears to be related to the in vivo biochemical efficacy of these compounds, as measured by inhibition of whole blood LTB4 generation (upon A23187 stimulus) and, particularly, urinary LTE4 excretion. Accordingly, we have assessed the ability of two clinically documented LT biosynthesis inhibitors, zileuton and MK-886, and the structurally novel 5-lipoxygenase activating protein antagonist, MK-0591, to inhibit the production of these inflammatory arachidonic acid metabolites in laboratory dogs. Zileuton (2 mg/kg) was extremely bioavailable in dogs (> 10 microM plasma concentrations), and inhibited the A23187-induced ex vivo production of LTB4 by venous blood by > 90%, in concordance with its potency in canine blood in vitro (IC50 = 1.1 microM). Despite this degree of inhibition in whole blood, urinary LTE4 excretion was reduced by only 52%, a profile of activity similar to that seen in clinical studies. MK-886 was less well absorbed, with plasma concentrations of 3 microM being achieved only at 25 mg/kg. These levels resulted in < 45% inhibition of LTB4 production, but a significant (p < 0.05) 47% inhibition of urinary LTE4 excretion. MK-0591 was similarly bioavailable (compared with MK-886), but 10-fold more active in vivo as a 2 mg/kg dose resulted in 41-62% inhibition of urinary LTE4 excretion (p < 0.05 vs controls; n = 4, 28). Significant inhibition of ex vivo LTB4 synthesis was also observed at this dose (49%), in accord with peak plasma concentrations of 0.5 microM and an in vitro potency of 0.2-0.4 microM (IC50) in whole blood from these animals. At higher dose (10 mg/kg), MK-0591 inhibited LTE4 excretion by 69%, with 88% inhibition of the LT biosynthetic capacity of whole blood. These data demonstrate that the biochemical efficacy of structurally diverse leukotriene biosynthesis inhibitors can be assessed in vivo in normal laboratory dogs. Such measurements, combined with bioavailability data from other species, may be useful for predicting biochemical activity in man.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Depression, Chemical; Dogs; Dose-Response Relationship, Drug; Hydroxyurea; Indoles; Leukotriene Antagonists; Leukotriene E4; Leukotrienes; Lipoxygenase Inhibitors; Male; Quinolines

We assessed the effect of 5-lipoxygenase inhibitors on the contraction of human bronchial ring segments passively sensitized with IgE in vitro. Contraction was induced by increasing doses of anti-IgE (0.01-0.5 micrograms/ml) in the presence of the antihistamine mepyramine. Zileuton and MK-886 (10 microM) significantly inhibited the dose-related contraction to anti-IgE as compared with solvent (DMSO). At the highest dose of anti-IgE used, contraction reached 10.3 and 9.6% of the maximal carbachol contraction, respectively, as compared with 30.3% for solvent. Maximal carbachol contraction was unaffected. These results suggest a potential beneficial effect of 5-lipoxygenase inhibitors in anaphylactic reactions in man.

Topics: Antibodies, Anti-Idiotypic; Bronchoconstriction; Humans; Hydroxyurea; Immunoglobulin E; In Vitro Techniques; Indoles; Leukotrienes; Lipoxygenase Inhibitors; Pyrilamine

The intrapleural injection of carrageenan in the rat induces exudate formation, cellular influx and leukotriene generation in the pleural cavity. We have demonstrated that the inflammatory response (exudate volume, and LTB4 levels) is increased in situ by the intrapleural administration of calcium ionophore A 23187 (100 nmol/rat) at 4, 16, 24, 48, and 72 h after the injection of carrageenan and that the A 23187-induced increase is dose-dependent. The oral administration of A-64077 and MK-886, two 5-lipoxygenase inhibitors (5-LOIs), at 10 mg/kg causes marked decreases in LTB4 release at the above-mentioned time intervals. However, A 23187-induced augmented exudate formation is not affected by the treatment with 5-LOIs. The results suggest that the use of 5-LOIs to inhibit LTB4 biosynthesis may be beneficial in various LTB4-dependent pathological conditions.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Calcimycin; Carrageenan; Dose-Response Relationship, Drug; Hydroxyurea; Indoles; Leukotriene B4; Lipoxygenase Inhibitors; Pleura; Radioimmunoassay; Rats

WY-50,295 tromethamine demonstrates significant 5-lipoxygenase inhibitory activity with IC50 values ranging from 0.055 microM in rat peritoneal exudate cells, to 0.16 microM in mouse macrophages, 1.2 microM in human peripheral neutrophils and 8.1 microM in rat blood leukocytes. This activity appeared selective for 5-lipoxygenase as concentrations up to 10 microM in rat peritoneal exudate cells, and 1 microM in mouse macrophages did not effect prostaglandin generation. In non-cellular enzyme assays, WY-50,295 tromethamine displayed inhibitory activity against a soluble 5-lipoxygenase from guinea pig peritoneal exudate cells (IC50 = 5.7 microM), while it was essentially inactive against 12-lipoxygenase, 15-lipoxygenase, or prostaglandin H synthetase at concentrations up to 500 microM, or against human phospholipase A2 at concentrations up to 50 microM. In purified human blood neutrophils the inhibitory activity was reversible but did not appear dependent upon substrate concentration. IN contrast, in the guinea pig cell-free 5-lipoxygenase assay changing the arachidonic acid substrate concentration from 5 to 500 microM produced a concentration-dependent reduction in inhibitory activity. WY-50,295 tromethamine inhibited the release of peptidoleukotrienes from fragmented guinea pig lung with an IC50 of 0.63 microM. When administered p.o. with a 4 h pretreatment time, WY-50,295 tromethamine inhibited ex vivo leukotriene B4 production in rat blood leukocytes with an ED50 of 19.6 mg/kg. Against an ovalbumin-induced leukotriene dependent bronchoconstriction in anesthetized sensitized guinea pigs, WY-50,295 tromethamine inhibited the ovalbumin-induced bronchoconstriction with an i.v. ED50 of 2.5 mg/kg (5 min pretreatment) and a p.o. ED50 of 7.3 mg/kg (4 h pretreatment). Significant activity was also evident with an 18 h pretreatment. Thus WY-50,295 tromethamine is an potent and selective 5-lipoxygenase inhibitor in a number of in vitro systems. Additionally the compound is orally efficacious and has a long duration of action in an allergic bronchoconstriction model. This data suggests that WY-50,295 tromethamine may have utility in the treatment of asthma and other leukotriene-dependent pathologies.

Topics: Administration, Oral; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Asthma; Female; Guinea Pigs; Humans; Hydroxyurea; Indoles; Leukocytes; Leukotriene B4; Lipoxygenase Inhibitors; Macrophages; Male; Mice; Naphthaleneacetic Acids; Prostaglandin-Endoperoxide Synthases; Quinolines; Rabbits; Rats; Rats, Wistar

The abilities of WY-50,295 tromethamine, a 5-lipoxygenase inhibitor, to inhibit antigen-induced leukotriene (LT) release from guinea pig lung fragments, and to prevent LTD4 or antigen-induced contraction of isolated guinea pig tracheal muscle were compared with the activities of zileuton and MK-886 (two selective 5-lipoxygenase inhibitors), and LY171883 (a LTD4 receptor antagonist). In fragmented guinea pig lung, WY-50,295 tromethamine inhibited antigen-induced LT release with an IC50 of 0.63 microM, and was 4.6- and 5.2-fold more potent than zileuton and MK-886, respectively. WY-50,295 tromethamine differed from these 5-lipoxygenase inhibitors, however, in that WY-50,295 tromethamine competitively antagonized LTD4-induced tracheal contractions (pA2 = 6.06) at concentrations that inhibited LT release. LY171883 was an effective LTD4 receptor antagonist (pA2 = 6.96), that only inhibited antigen-induced LT release at higher concentrations (IC50 = 7.9 microM). WY-50,295 tromethamine almost completely inhibited antigen-induced leukotriene-dependent tracheal contractions, whereas high concentrations of zileuton, MK-886, or LY171883 produced only partial inhibition. This partial inhibition was likely to result from 'breakthrough' 5-lipoxygenase activity, because combinations of zileuton plus MK-886 or zileuton plus LY171883, were more effective than zileuton, MK-886, or LY171883 alone. The greater efficacy of WY-50,295 tromethamine in the antigen-challenged guinea pig trachea is likely to result from its combined abilities to prevent LT biosynthesis and block LTD4 receptors.

Topics: Acetophenones; Animals; Guinea Pigs; Hydroxyurea; In Vitro Techniques; Indoles; Leukotriene Antagonists; Lipoxygenase Inhibitors; Lung; Male; Muscle Contraction; Muscle, Smooth; Naphthaleneacetic Acids; Quinolines; SRS-A; Tetrazoles; Trachea

The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.

Topics: Animals; Guinea Pigs; Hydroxyurea; In Vitro Techniques; Indoles; Leukotriene B4; Lipoxygenase Inhibitors; Lung; Male; Muscle Contraction; Muscle, Smooth; Ovalbumin; Phenols; Trachea

Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Cell Compartmentation; Cell Membrane; Cytosol; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; In Vitro Techniques; Indoles; Leukotrienes; Neutrophils; Substrate Specificity