Assessment of thein vivo biochemical efficacy of orally active leukotriene biosynthesis inhibitors

In man, the therapeutic effectiveness of specific inhibitors of leukotriene (LT) biosynthesis against allergen-induced bronchoconstriction appears to be related to thein 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 activatig 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 M plasma concentrations), and inhibited the A23187-inducedex vivo production of LTB₄ by venous blood by >90%, in concordance with its potency in canine bloodin vitro (IC₅₀=1.1 M). 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 M 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 activein 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 ofex vivo LTB₄ synthesis was also observed at this dose (49%), in accord with peak plasma concentrations of 0.5 M and anin vitro potency of 0.2–0.4 M (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 assessedin vivo in normal laboratory dogs. Such measurements, combined with bioavailability data from other species, may be useful for predicting biochemical activity in man.     

Effect of Zileuton (A-64077) on the 5-lipoxygenase activity of human whole bloodex vivo

The potency and reversibility of a new orally active 5-lipoxygenase (5-LO) inhibitor were evaluated in human volunteers. Zileuton (A-64077) 600 mg q.i.d. was administered to volunteers for 14 days in a phase I study, and blood samples were withdrawn, stimulated with ionophore A23187 and LTB₄ levels were determined using both reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA). The drug significantly inhibited (above 70%) LTB₄ biosynthesis in whole blood stimulated with A-23187 throughout the 14 days. The activity of 5-LO was also measured one week after stopping the medication and was returned to control levels. Measurement of LTB₄ levels using either RP-HPLC or RIA gave similar percentage of inhibition although RIA appeared to underestimate by half the absolute amounts of LTB₄ in the blood samples. These results show that Zileuton is a highly active and reversible 5-LO inhibitor in human.     

CGS 22745: A selective orally active inhibitor of 5-lipoxygenase

CGS 22745, and aralkyl hydroxamic acid, inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) synthesis in guinea pig leukocytes (IC₅₀=0.6M). The compound did not appreciably affect cyclooxygenase (ram seminal vesicles), 12-lipoxygenase and thromboxane synthase (human platelets) or 15-lipoxygenase (human neutrophils). CGS 22745 inhibited A23187-induced formation of LTB₄ in blood (IC₅₀'s of 4.3, 0.56 and 3.2 M for human, dog and rat, respectively). At 1 mg/kg i.v. in dogs, it caused 96% inhibition of A23187-stimulated LTB₄ formationex vivo after 5 min. Its effective biological half-life was >160 min. In dogs at 3 and 10 mg/kg p.o., CGS 22745 inhibitedex vivo A23187-stimulated LTB₄ formation at 3 hr by 48% and 97%, respectively. The inhibition persisted up to 6 hr (26% at 3 mg/kg; 49% at 10 mg/kg). CGS 22745 (3, 10 and 30 mg/kg p.o.) inhibited exudate formation, mononuclear cells and PMN accumulation in a dose-dependent manner during the late phase (48 and 72 hr) of carrageenan-induced pleurisy in the rat.     

Antiinflammatory effects of second-generation leukotriene B4 receptor antagonist,SC-53228: Impact upon Leukotriene B4- and 12(R)-HETE- mediated events

Leukotriene B₄ (LTB₄) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] are proinflammatory products of arachidonic acid metabolism that have been implicated as mediators in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB₄ and 12(R)-HETE elicit a dose-dependent migration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxyl]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid}, a first-generation LTB₄ receptor antagonist, inhibited the chemotactic actions of LTB₄ when given orally with an ED₅₀ value of 1.7 mg/kg. The second-generation LTB₄ receptor antagonist, SC-53228 [(+)-(S)-7-(3-{2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)carbonyl]phenoxy} propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid], inhibited LTB₄-induced chemotaxis when given intragastrically with an ED₅₀ value of 0.07 mg/kg. Furthermore, SC-53228 inhibited 12(R)-HETE-induced granulocyte chemotaxis with an oral ED₅₀ value of 5.8 mg/kg. When dosed orally over a range of 0.03–100 mg/kg, SC-53228 gaveC _max plasma concentrations of 0.015–41.1g/ml. SC-53228 inhibited LTB₄-primed membrane depolarization of human neutrophils with an IC₅₀ value of 34 nM. As a potent LTB₄ receptor antagonist, SC-53228 may well have application in the medical management of disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease, contact dermatitis, and psoriasis, in which LTB₄ and/or 12(R)-HETE are implicated as inflammatory mediators.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophilsin vitro. Evidence of formation of antiinflammatory compounds

Enzymatic transformation of then-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B₄ (LTB₄), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of othern-6 fatty acids on the formation of LTB₄ by human neutrophils and to determine if thesen-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 M) and different concentrations (0–100 M) of then-6 fatty acids linoleic acid (LA) and dihomo-gammalinolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB₄ was dose dependently inhibited by both LA (IC₅₀=45 M) and DGLA (IC₅₀=40M). This inhibition of LTB₄ formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB₄ formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB₄ formation (IC₅₀=7.5 M and IC₅₀=0.2 M). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB₄ formation (IC₅₀=0.75 M). The results show that the addition of LA and DGLA to neutrophils results in an inhibition of LTB₄ formation and simultaneously to the formation of 13-HODE and 15-HETrE, that also inhibits LTB₄ formation. Therefore, dietary supplementation or topical application of LA and DGLA or preferentially their respective 15-LO products, may have a therapeutic effect in inflammatroy diseases in which LTs are suspected to play a pathogenic role.     

Effects of a 5-lipoxygenase inhibitior (L-651,392) on primary and late pulmonary responses to ascaris antigen in the squirrel monkey

Allergic squirrel monkeys when exposed to an aerosol ofAscaris suum either develop a reproducible, immediate bronchoconstriction or an immediate bronchoconstriction followed by a reproducible late response. Pretreatment of ascaris-challenged squirrel monkeys with a potent, selective, orally active 5-lipoxygenase inhibitor, L-651,392 (4-bromo-2,7-dimethoxy-3,4-phenothizin-3-one), at a dose of 5 mg/kg p.o. resulted in near complete inhibition of the increases in pulmonary resistance (R _L) and decreases in dynamic compliance (Cdyn) normally observed following exposure to the antigen. A lower dose (1 mg/kg p.o.) of L-651-392 produced only a significant inhibition of the decreases in Cdyn. In monkeys known to develop dual responses to antigen, L-651,392 (5 mg/kg p.o.) significantly attenuated the immediate response and markedly inhibited the late response. These results suggest an important role for leukotrienes in primary and late phase allergen-induced bronchoconstriction.     

Cytokine-Induced Neutrophil Chemoattractant in a Rat Model of Lipopolysaccharide-Induced Acute Lung Injury

To elucidate the role of major chemotactic factors, cytokine-induced neutrophil chemoattractant (CINC), leukotriene B₄ (LTB₄) and C5a in lipopolysaccharide (LPS)-induced acute lung injury in rat, we employed three reagents: anti-CINC-1 antibody, an LTB₄ receptor antagonist (ONO-4057) and an anti-complementary agent (K-76COONa). Rats were divided into five groups: (1) control group; (2) LPS group, which received intratracheal instillation of LPS (100 g/kg); (3) Anti-CINC group, which received intratracheal coinstillation of LPS with anti-CINC-1 antibody (1 mg/kg); (4) LTB₄-Ra group, which received intravenous ONO-4057 (10 mg/kg) prior to intratracheal LPS; (5) Anti-C5a group, which received intravenous K-76COONa (100 mg/kg) prior to intratracheal LPS. The number of neutrophils in bronchoalveolar lavage (BAL) fluids 6 h after LPS instillation was significantly reduced in the Anti-CINC group, however, no reduction was found in either the LTB₄-Ra group or Anti-C5a group. The levels of CINC-1, CINC-2 and CINC-3 in BAL fluids were significantly higher in the LPS group than in the saline-instilled control group. In vitro, the production of CINC-1 and CINC-3 from LPS-stimulated macrophages was significantly elevated compared to unstimulated macrophages 6 h later. The increase in CINC-2 production was markedly less than that of CINC-1 or CINC-3. These results indicate that CINCs, especially CINC-1 and CINC-3 play an important role in the recruitment of neutrophils to the lung in LPS-induced acute lung injury.     

Effect of a leukotriene B4 receptor antagonist on leukotriene B4-induced neutrophil chemotaxis in cavine dermis

Leukotriene B₄ (LTB₄) is a proinflammatory product of arachidonic acid metabolism that has been implicated as a mediator in a number of inflammatory diseases. When injected intradermally into the cavine, LTB₄ elicits a dose-dependent immigration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-l-benzopyran-2-carboxylic acid, a potent LTB₄ receptor antagonist inhibited the chemotactic actions of LTB₄ when coadministered into the dermal site and when given intravenously or orally with ED₅₀ values of 200 ng, 0.5 mg/kg, and 0.6 mg/ kg respectively. This compound may well have application in disease states, such as inflammatory bowel disease and psoriasis, where LTB₄ is implicated as a proinflammatory mediator.     

Pharmacological profile of a novel,orally active leukotriene B4 antagonist,SM-15178

SM-15178, a new hydroxyacetophenone derivative, was evaluated to determine its antiinflammatory activity and antagonistic activity against leukotriene B₄ (LTB₄). SM-15178 inhibited [³H]LTB₄ binding to its receptors on human neutrophils (IC₅₀=0.30M). It inhibited LTB₄-induced chemotaxis of human neutrophils (IC₅₀ =0.72M) with little inhibitory effect against C5a or FMLP-induced chemotaxis at concentrations up to 30M. The compound alone did not cause human neutrophil chemotaxis at concentrations up to 10M. LTB₄-induced chemotaxis of mouse and rat neutrophils and guinea pig eosinophils was also inhibited by the compound, with IC₅₀ values of 0.55, 0.52, and 0.58 M, respectively. In an in vivo study, SM-15178, given orally, significantly prevented LTB₄-induced transient leukopenia. It also suppressed LTB₄-induced bronchoconstriction in the guinea pig almost completely when given orally at a dose of 40 mg/kg. Furthermore, orally given SM-15178 suppressed arachidonic acid-induced neutrophil infiltration in mouse ears and Arthus reaction-induced paw edema in the mouse in a dose-dependent manner. These results suggest that SM-15178 is a selective and orally active LTB₄ antagonist and that it might be effective for the treatment of some types of inflammatory diseases.     

Measurement of Cyclooxygenase Inhibition in vivo: A Study of Two Non-Steroidal Anti-Inflammatory Drugs in Sheep

The anti-inflammatory effects of the non-steroidal anti-inflammatory drugs phenylbutazone (PBZ) and flunixin meglumine (FM) and the relationship between the effects and drug concentration in vivo were studied using a subcutaneous tissue-cage model in sheep. Intracaveal injection of carrageenan induced prostaglandin (PG) E₂production in tissue-cage exudate (maximal concentration, 101 nM) with significant increases in white blood cell (WBC) numbers, skin temperature over the inflamed cage and exudate leukotriene B₄ (LTB₄) concentration (P < 0.05). Intravenous PBZ, 4.4 mg kg^–1 produced mild inhibition of exudate PGE₂ generation (10%), but greater inhibition of serum TXB₂ (75.3%). The IC₅₀ for TXB₂ was 36.0 M. Phenylbutazone did not alter effects on skin temperature, WBC numbers or exudate LTB₄ concentrations. Intravenous FM, 1.1 mg kg^–1, significantly inhibited carrageenan-induced exudate PGE₂ formation (E_max, 100%, IC₅₀, <0.4 nM) and serum TXB₂ generation (E_max, 100%, IC₅₀, 17 nM) for up to 32 h. Flunixin meglumine significantly inhibited the rise in skin temperature but had a limited effect on exudate WBC. Phenylbutazone and FM have distinct effects on carrageenan-induced cyclooxygenase (COX-2) and platelet COX (COX-1). Flunixin meglumine was a more potent COX inhibitor than PBZ and was more selective for the inducible form of COX in vivo.     

The effect of leukotriene-B4 receptor antagonist,SC-41930, on acetic acid-induced colonic inflammation

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, is a potentin vitro leukotriene-B₄ (LTB₄) receptor antagonist. LTB₄ levels are elevated in colonic tissue of inflammatory bowel disease (IBD) patients which may account for the high degree of neutrophil (PMN) infiltration. The guinea pig acetic acid-induced colonic inflammation model has characteristics of IBD including PMN infiltration, edema, ulceration and necrosis. The model was used to evaluate the effect of SC-41930. SC-41930 was given orally, 30 min before and after intrarectal administration of 3% acetic acid. The PMN marker enzyme, myeloperoxidase, was measured along with histological evaluation to assess inflammation. Both parameters showed significantly less inflammation in SC-41930 treated animals with an oral ED₅₀ of 20 mg/kg. These study results with an LTB₄ receptor antagonist indicate a role for LTB₄ in colonic inflammation and that an LTB₄ receptor antagonist may be beneficial for treatment of IBD.     

Granulocyte chemotaxis in the canine trachea: Inhibition by lipid mediator antagonists and systemic inhibitors

Inflammation of the airways contributes to the multicomponent disease known as asthma. The primary cells that infiltrate the airways in response to antigen exposure are PMNs and eosinophils, cells that can release cellular components, and damage the airways. We adapted a double-ballon endotracheal tube to study the cellular response to threede novo synthesized lipid mediators (LTB₄, PAF-acether and 15 HETE) found in respiratory fluids following antigen exposure.In random repeat challenges in groups of 7 dogs using mongrel dogs at 240 min following exposure to 10^–6 M agonists, the PMN content of the perfused fluid was 870±240, 1632±883, 515±395, and 1575±214 cells/ml/5 high power fields for vehicle, LTB₄, PAF, and 15 HETE respectively. Eosinophils that infiltrated the lumen at 240 min were 162±23, 608±287, 502±23, 115±14 cells/ml/5 HPF for vehicle, LTB₄, PAF, and 15 HETE respectively. Thus LTB₄ and PAF-acether significantly (p<0.05) increased eosinophils, and LTB₄ and 15 HETE increased PMNs (p<0.05). After determining the agonist response for the 3 agonists we included 2 specific antagonists in the perfusate. The LTB₄ antagonist U-75,302 10^–5 M, and the PAF antagonist L 652,731 10^–5 M in chambers containing LTB₄ and PAF-acether respectively blocked significantly the influx of PMNs and eosinophils compared to vehicle (p<0.01). Methylprednisolone 5 mg/kg i.m.-18 hrs blocked eosinophilia to PAF and LTB₄. Oral U-78,517F a Trolox amine lazaroid, active as an inhibitor of lipid peroxidation, 30 mg/kg-18 hrs significantly blocked eosinophilia to PAF-acether and LTB₄ directed chemotaxis compared to vehicle (p<0.05) but not 15 HETE. Specificity was shown for each antagonist since the PAF and LTB₄ antagonists did not block the opposite agonist. Use of this novelin vivo chemotaxis model allows the additional advantage of studying chemotaxis in living tissue.     

Lovastatin inhibits antigen-induced airway eosinophilia without affecting the production of inflammatory mediators in mice

Objective and design   Statins have been proposed as a novel treatment of respiratory diseases. To determine the beneficial effects of statins on allergic bronchial asthma, the effect of systemic treatment with lovastatin on antigen-induced airway inflammation was investigated. Subjects  Male BALB/c mice were used. Treatments  Mice were sensitized and repeatedly challenged with ovalbumin (OA) antigen to induce asthmatic response. Animals were also treated with lovastatin (4 mg/kg/day, i.p.) once a day prior to and during the antigen inhalation period. Methods   Inflammatory cell counts and levels of interleukin (IL)-4, IL-13, eotaxin, thymus and activation-regulated chemokine and leukotriene B₄ (LTB₄) in bronchoalveolar lavage (BAL) fluids were measured. Results  Significant increases in eosinophils and levels of the T helper 2 cytokines, chemokines and LTB₄ in BAL fluids in association with the increments of total and OA-specific immunoglobulin E (IgE) in sera were observed in the repeatedly antigen-challenged mice. The airway eosinophilia was ameliorated by lovastatin, whereas it had no significant effect on the levels of these inflammatory mediators or IgE. Conclusion  Lovastatin may be beneficial for the treatment of allergic inflammatory diseases in the airways, such as allergic bronchial asthma.     

Effect of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf on inflammatory cytokine and mediator induction from alveolar macrophages of chronic bronchitic rats

Objective: The study was to evaluate the effect of triterpene acids of Eriobotrya japonica (thunb.) lindl. leaf (TAL) on inflammatory cytokine and mediator expression in alveolar macrophages (AM) of chronic bronchitic (CB) rats. Methods: CB was induced by endotracheal instillation of lipopolysaccharide (LPS) followed by Bacillus Calmette Guerin (BCG) injection via the caudal vein one week later. Treatment groups received TAL at there different doses (50, 150, or 450 mg/kg daily i. g.), Ketotifen fumarate (5 mg/kg daily i. g.) or dexamethasone (1.2 mg/kg daily i. g.) for two weeks, 7 days after LPS injection. AM were then isolated and incubated for 24 h. IL-1, TNF-α and PGE2 levels in cultured supernatants were measured by thymocyte co-stimulating assay and radioimmunoassay. Immunocytochemistry staining and western-blot were used for intracellular location and activation of p65 subunit of NF-kB. LTB₄ level was analyzed by reverse-phase high performance liquid chromatography (RP-HPLC). Results: The levels of TNF-α, IL-1, NF-kB, PGE2 and LTB₄ expression in AM of TAL groups were significantly decreased compared to the CB group (P < 0.05 or P < 0.01), in a dose dependent manner. Conclusion: TAL inhibited NF-kB activation in AM from CB rats and led to down regulation of TNF-α, IL-1, PGE₂ and LTB₄ expression, which might be a mechanism for its anti-inflammatory effects in CB rats. Received 17 November 2005; returned for revision 19 March 2006; returned for final revision 14 July 2006; accepted by G. Wallace 30 August 2006     

Inflammation of guinea pig dermis

Neutrophil (PMNL) infiltration is a prominent feature of human psoriasis. Psoriatic skin lesions contain abnormally high amounts of leukotriene B₄ (LTB₄), itself a potent PMNL chemoattractant both in vivo and in vitro. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-propyl-2H-1-benzo-pyran-2-carboxylic acid}, an orally active LTB₄ receptor antagonist, was tested topically in models of skin inflammation induced by 200 nmol of the calcium ionophore A23187 or 200g phorbol-12-myristate-13-acetate (PMA) applied topically to the guinea pig ear as assessed by ear weight, levels of the PMNL marker enzyme myeloperoxidase (MPO), and histological examination (PMA model) at 4 and 18 h respectively. When coapplied topically with A23187 or PMA, SC-41930 significantly inhibited epidermal inflammation with ED₅₀ values of 0.6 and 4 mg, respectively. SC-41930 treatment also was associated with lowered dermal LTB₄ levels in both models. The PMA-induced skin inflammation model also was assessed histologically and revealed acanthosis, edema, PMNL infiltration, and rete ridge prominence as long as 96 h after a single application that was completely inhibited by SC-41930 topical coapplication. Furthermore, oral treatment (40 mg/kg) significantly reduced edema and inflammatory cell infiltration in both models. These models possess many of the characteristics of human psoriasis, and agents such as SC-41930 that demonstrate activity in these models may well have therapeutic utility in the treatment of human psoriasis.     

Pharmacologic properties of FPL 55712 administered by aerosol

FPL 55712 was investigated by the aerosol route of administration for efficacy at protecting against leukotriene-induced bronchoconstrictions in guinea pigs and for mediator release inhibitory activity in passively sensitized rats. In the studies to investigate leukotriene antagonism; anesthetized, spontaneously breathing guinea pigs were pretreated with propranolol and were exposed via tracheal cannula to aerosols generated by a Monaghan nebulizer. Subsequently, the animals were artificially ventilated and challenged with LTD₄ or LTE₄ (25 g/kg, i.v.). FPL 55712 produced a concentration-dependent inhibition of LTD₄ and LTE₄-induced bronchoconstriction (IC₅₀'s 0.5% and 0.8%, respectively). Although the biologic half-life of FPL 55712, administered intravenously, was very short (1.7 minutes against LTD₄ and 1.2 minutes against LTE₄) after aerosol administration the biological half-life was surprisingly long (120 minutes against LTD₄ and 90 minutes against LTE₄). Aerosolized FPL 55712 also possessed weak antiallergic activity in comparison to disodium cromoglycate when measured as an inhibitor of IgE-mediated anaphylactic bronchoconstriction in rats (IC₅₀'s of 2.0% and 0.01%, respectively). Thus, these studies demonstrate that, when administered by aerosol, FPL 55712 is effective at protecting against leukotriene-induced bronchoconstrictions, exhibits a long duration of action and also possesses weak antiallergic activity.     

Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid

Two groups of six adults with persistent asthma, who were identical clinically, received 0.1 or 4 g of purified eicosapentaenoic acid ethyl ester (EPA) daily for 8 weeks. Both doses increased significantly the generation of leukotriene B₅ (LTB₅) from EPA by polymorphonuclear (PMN) and mononuclear leukocytes, while only the high dose decreased leukocyte arachidonic acid (AA) and the generation of LTB₄ and prostaglandin E₂ from AA. Only the high dose led to inhibition of PMN leukocyte chemotaxis to multiple stimuli by a mean of 57–70% (P<0.01), without altering monocyte chemotaxis, the production of plateletactivating factor by mononuclear leukocytes, or the IgE-dependent release of histamine from basophils. Both doses of EPA increased the responses of T lymphocytes to phytohemagglutinin by a mean of 73% or more (P<0.01) without modifying the numbers of helper and suppressor T lymphocytes. EPA affects the functions of several types of leukocytes critical to inflammation and immunity.     

Ebselen is a specific inhibitor of LTB4-mediated migration of human neutrophils

Ebselen is a seleno-organic anti-inflammatory compound with glutathione peroxidase-like activity that has the unique characteristic of mediating the isomerization of 5-HETE and LTB₄ to their biologically inactive trans isomers, both directly in fluid phase and indirectly through metabolic pathways in stimulated peripheral blood leukocytes. LTB₄ is an inflammatory mediator with potent chemotactic activity for neutrophilic leukocytes. We studied the effects of ebselen on the chemotactic and chemokinetic responses with human-blood-derived neutrophils. With the use of 120-m-thick 5-m-pore durapore filters and low BSA concentrations (0.05%) in the chemotaxis buffers, ebselen was evaluated for its effect on both chemotactic and chemokinetic responses to LTB₄, C5a, and fMLP. Ebselen at 3–20 M concentrations inhibited both chemotactic and chemokinetic responses to optimal concentrations of LTB₄ without altering chemotactic responses to C5a or fMLP. Likewise, ebselen at 20 M specifically inhibited LTB₄-stimulated transendothelial migration of neutrophils, while not altering responses to C5a nor fMLP.     

Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid

Exposure of human neutrophils to 5(S),12(R) dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (leukotriene B₄, LTB₄) resulted in a time- and concentration- (10^–9-10^–6 M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB₄. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB₄ prior to contact with cytochalasin B. LTB₄-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N, N-dietliylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB₄-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) andN-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB₄-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive, Neutrophils pretreated with LTR₄ or 5(S), 12(R), 20-trihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB₄, an -oxidation metabolite of LTB₄) were desensitized to the subsequent exposure to LTB₄. Cross-desensitization was also demonstrated between LTB₄ and 20-OH-LTB₄. The stimulus specific nature of LTB₄-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) orN-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB₄-pretreated neutrophils. Enzyme release from LTB₄-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB₄-induced degranulation.     

The effect of methotrexate on lipoxygenase metabolism in neutrophils from rats:In vitro andex vivo studies

Studies were undertaken to examine the effect of methotrexate (MTX) administeredin vivo or addedin vitro upon the production of the 5-lipoxygenase (5-LO) metabolites of arachidonic acid (AA) by rat neutrophils. Peptone-induced peritoneal exudate cells were stimulated by A23187 and the cell suspensions assayed for leukotriene B₄ (LTB₄), the all-trans isomers of LTB₄ and 5-hydroxyeicosatetraenoic acid (5-HETE) using high-pressure liquid chromatography. MTX addedin vitro to rat cells was weakly inhibitory; however, no inhibition of LTB₄ production was seen followingin vivo administration of MTX by oral, subcutaneous or intraperitoneal routes. On the basis of these findings, inhibition of 5-LO metabolism does not appear to explain the anti-inflammatory effects of MTX.