Leukotriene Biosynthesis Inhibitor Research Articles

Linoleic acid peroxidation by Solanum tuberosum lipoxygenase was activated in the presence of human 5-lipoxygenase-activating protein

The present investigation describes the ability of human 5-lipoxygenase-activating protein (FLAP) to activate a plant 5-lipoxygenase. The presence of an active recombinant human FLAP in the 100 000× g membrane fraction of infected Sf9 cells led to a specific increase in 9-hydroperoxyoctadecadienoic acid (9-HPOD) synthesis (+68%) or in 5-hydroperoxyeicosatetraenoic acid (5-HPETE) synthesis (+68%), after action of Solanum tuberosum tuber 5-lipoxygenase ( S.t.LOX) on linoleic acid (natural plant lipoxygenase substrate) or on arachidonic acid. On the contrary, the presence of non-transfected membranes obtained from non-infected Sf9 cells led to an inhibition of lipoxygenase activity. MK-886, a potent inhibitor of leukotriene biosynthesis, blocked the FLAP dependent S.t.LOX activation after preincubation with FLAP transfected membranes. In conclusion, this study demonstrates that a recombinant human FLAP can stimulate a lipoxygenase other than mammalian 5-lipoxygenase ( S.t.LOX) by using different polyunsaturated fatty acids as substrates.

Design of inhibitors of leukotriene biosynthesis and their therapeutic potential

Abstract

Platelets, neutrophils, and vasoconstriction after arterial injury by angioplasty in pigs: effects of MK-886, a leukotriene biosynthesis inhibitor.

1 Leukotrienes constitute a class of potent bioactive mediators known to play a pivotal role in inflammation. Since their biosynthesis has been shown to be enhanced by platelet-neutrophil interactions, leukotrienes may be involved in these interactions and the arterial response to injury. Therefore, we investigated the effects of the selective leukotriene biosynthesis inhibitor 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid (MK-886) on the acute thrombotic and vasomotor responses after arterial injury by angioplasty. 2 Carotid arterial injury was produced by balloon dilatation in control (molecusol vehicle; n=10) and treated (MK-886, 10 mg kg(-1), i.v.; n=9) pigs. The acute thrombotic reaction following deep arterial wall injury was quantified with 51Cr labelled platelets and 111In labelled neutrophils, and the vasomotor response was determined angiographically. 3 Platelet deposition at the site of deep arterial wall injury averaged 56.4+/-11.0x10(6) platelets cm(-2) in the control group, and was significantly reduced to 18.2+/-3.8x10(6) platelets cm(-2) (P<0.005) by treatment with MK-886. Neutrophil deposition was also decreased by MK-886, from 242.8+/-36.8 to 120.9+/-20.3x10(3) neutrophils cm(-2) (P<0.01). MK-886-treated animals had a significant decrease in the postangioplasty vasoconstrictive response at the site of endothelial injury distally, from 37.5+/-3.1% in the control group to 13.5+/-2.5% (P<0.001). 4 The effects of MK-886 were associated with a profound inhibition of ex vivo leukotriene B4 (LTB4) synthesis in blood stimulated by the calcium ionophore A23187 and a significant reduction of neutrophil aggregation in whole blood (P<0.01), whereas neutrophil superoxide anion production, serum thromboxane B2 and platelet aggregation in whole blood were not influenced. 5 The relevant effects of MK-886 are primarily related to inhibition of neutrophil function and suggest an important modulatory role for leukotrienes in the pathophysiological response associated with platelet and neutrophil interactions following arterial injury in vivo.

MK-886, a leukotriene biosynthesis inhibitor, induces antiproliferative effects and apoptosis in HL-60 cells.

MK-886, a specific inhibitor of 5-lipoxygenase inhibited DNA replication in leukemic HL-60 cells in a dose-dependent manner. Addition of exogenous leukotriene B4 reversed this effect, whereas addition of leukotriene B4 failed to modulate a prostaglandin D2-induced inhibition of DNA replication. The reversal of MK-886-induced inhibition was not observed with leukotriene C4. These results suggest that the effect of MK-886 is mediated by inhibition of leukotriene B4 biosynthesis. Moreover, MK-886 not only impaired DNA replication in HL-60 cells but also decreased cell proliferation and induced apoptotic cell death. Our results suggest a crucial role of leukotriene B4 in the regulation of cell proliferation and cell survival in HL-60 cells.

Simple analogues of anthralin: unusual specificity of structure and antiproliferative activity.

Fifty-nine simple analogues of the antipsoriatic agent, anthralin, have been prepared by modifying the positions of the 1,8-hydroxyl groups, replacement of the hydroxyl groups, substitution at the oxygen functions, introduction of additional functional groups into various positions of the anthracenone nucleus, or removal of particular structural elements. The compounds were evaluated for their antiproliferative action against human keratinocytes and inhibition of the generation of leukotriene B4 in polymorphonuclear leukocytes, which may be useful to resolve the proliferative and inflammatory aspects of psoriasis, respectively. Even though many anthracenones were more potent inhibitors of leukotriene biosynthesis than anthralin, none of the compounds was substantially more effective as this drug in suppressing keratinocyte cell growth. There is an absolute requirement for two hydroxyl groups peri to a hydrogen bond acceptor such as a keto or an imino group for high potency. In addition to further delineating the nature of the pharmacophore for this class of compounds, also naphthalenedione with a peri hydroxyl group was identified as a pharmacophore with antiproliferative activity against keratinocyte growth.

A test system for leukotriene synthesis inhibitors based on the in-vitro differentiation of the human leukemic cell lines HL-60 and Mono Mac 6.

Differentiation of HL-60 cells along the granulocytic lineage by DMSO in the presence of transforming growth factor-beta and low concentrations of 1,25-dihydroxyvitamin D3 leads to the upregulation of 5-lipoxygenase activity in 100,000 g supernatants and intact cells to levels which are comparable to normal granulocytes. Similarly, differentiation of the human monocytic cell line Mono Mac 6 by 1,25-dihydroxyvitamin D3 and transforming growth factor-beta strongly upregulates the 5-lipoxygenase pathway. Here, we describe an assay system for leukotriene biosynthesis inhibitors which is based on the in-vitro differentiation of HL-60 and Mono Mac 6 cells. Different leukotriene biosynthesis inhibitors like the nonredox type inhibitor ZM 230487, the redox type inhibitor BW A4C and the FLAP inhibitor MK886 were tested and the results were compared with an assay system based on normal human granulocytes. ZM 230487, BWA4C and MK886 showed similar potencies in these cell lines as compared to normal leukocytes. Thus, the in-vitro differentiation of HL-60 and Mono Mac 6 cells provides an excellent model for the screening of drugs affecting the 5-lipoxygenase pathway.

Modulation of endotoxin-induced inflammation in the bovine teat using antagonists/inhibitors to leukotrienes, platelet activating factor and interleukin 1β

A leukotriene biosynthesis inhibitor (MK886), a platelet-activating factor (PAF)-receptor antagonist (WEB 2086), a recombinant human interleukin-1 receptor antagonist (IL-1ra) and a polyclonal antibody to recombinant bovine IL-1β (anti-rBoIL-1β) was used to investigate the involvement of leukotrienes, PAF and IL-1β during endotoxin-induced inflammation in the bovine teat cistern. Endotoxin alone was infused into one teat cistern and endotoxin in combination with an inhibitor/antagonist was infused into another teat cistern of the same animal. Teat cistern samples were taken before infusion and at 3.5 and 7 h after infusion, and the numbers of neutrophils were counted. Saline infusion was used as control. The inhibitors/antagonists were also tested in combination with leukotriene B 4 (LTB 4), PAF and rBoIL-1β, respectively. MK886 or WEB 2086 significantly reduced the accumulation of neutrophils mainly between 3.5 and 7 h after infusion, indicating roles for leukotrienes, probably LTB 4, and PAF in neutrophil accumulation during endotoxin-induced inflammation. As WEB 2086 also reduced cell accumulation between 0 and 3.5 h, PAF was implicated also in the early influx of neutrophils. WEB 2086 almost completely inhibited PAF-induced cell accumulation between 0 and 3.5 h. LTB 4 did not induce significant cell accumulation in the teat cistern. IL-1ra did not affect endotoxin-induced neutrophil accumulation whereas anti-rBoIL-1β reduced total cell accumulation and, to some degree, accumulation between 0 and 3.5 h after infusion. Infusion of IL-1ra significantly inhibited cell accumulation induced by rBoIL-1β. Anti-rBoIL-1β also significantly reduced neutrophil accumulation induced by rBoIL-1β, but to a lesser degree. The results suggest roles for leukotrienes, most likely LTB 4, and PAF, and to a lesser extent IL-1β, during endotoxin-induced neutrophil migration into the bovine teat cistern. The potential of the inhibitors/antagonists as therapeutic agents for bovine mastitis should be investigated.

Attenuation of early and late phase allergen-induced bronchoconstriction in asthmatic subjects by a 5-lipoxygenase activating protein antagonist, BAYx 1005.

BACKGROUND: The cysteinyl leukotrienes (LTC4, LTD4 and LTE4) have been implicated in the pathogenesis of allergen-induced airway responses. The effects of pretreatment with BAYx 1005, an inhibitor of leukotriene biosynthesis...

Inhibition of allergen-induced airway obstruction and leukotriene generation in atopic asthmatic subjects by the leukotriene biosynthesis inhibitor BAYx 1005.

BACKGROUND: Leukotriene receptor antagonists significantly blunt allergen-induced bronchoconstriction in asthmatic subjects. Inhibitors of leukotriene synthesis should theoretically provide similar protection, but conflicting results have been obtained when synthesis inhibitors have...

Leukotrienes in ulcerative colitis: Results of a multicenter trial of a leukotriene biosynthesis inhibitor, MK-591

BACKGROUND & AIMS: Leukotrienes (LTs) are believed to be important in the pathogenesis of ulcerative colitis (UC). The aim of this study was to determine whether inhibition of LT biosynthesis with a 5- lipoxygenase inhibitor (MK-591) induces remission in patients with mild to moderate UC.METHODS: One hundred eighty-three patients with mild to moderately active UC enrolled in this randomized parallel group, double- blind study. Patients received placebo or MK-591 at a dose of 12.5, 50, or 100 mg twice daily for 8 weeks. A subset of patients underwent rectal dialysis to determine LTB4 concentration.RESULTS: MK-591 reduced LTB4 concentrations in rectal dialysate at the final determination. The median percent of baseline LTB4 concentration for 100 mg taken twice daily was 1.4% (n = 4); for 50 mg taken twice daily, 16.5% (n = 6); for 12.5 mg taken twice daily, 12% (n = 6); and for placebo, 78% (n = 6). There was no correlation between reduction of LTB4 and remission. Patients in remission at week 8 were as follows: placebo, 9 of 44 (20.5%); 100 mg taken twice daily, 11 of 43 (25.6%); 50 mg taken twice daily, 8 of 49 (16.3%); and 12.5 mg taken twice daily, 4 of 47 (8.5%) (P > 0.10).CONCLUSIONS: MK-591 markedly inhibited LT biosynthesis, but it did not differ significantly from placebo in clinical efficacy. Inhibition of LT biosynthesis was not effective as a single therapeutic modality in active UC.(Gastroenterology 1997 Mar;112(3):725-32)

Measurement characteristics of two asthma symptom diary scales for use in clinical trials

The measurement characteristics of two asthma symptom diary scales developed for use as health outcome measures in clinical trials of asthma therapy were investigated. A daytime diary scale was designed to capture the frequency and inconvenience of daytime asthma symptoms and their effects on activities, and a nocturnal asthma symptom diary scale was designed to capture awakenings with asthma symptoms. The internal consistency, reliability, validity and responsiveness of both asthma diary scales were assessed in 346 adult asthma patients in two placebo-controlled clinical trials of an investigational asthma therapy, a leukotriene biosynthesis inhibitor. The daytime symptom scale showed sufficient internal consistency (0.90-0.92), and the daytime and nocturnal symptom scales showed sufficient test retest reliability (0.69-0.87). Construct validity was demonstrated by generally moderate-to-strong correlations for changes in the diary scales with changes in other measures of asthma status, such as forced expiratory volume in one second (FEV1), peak expiratory flow (PEF), and puffs of beta-agonist inhaler. Both scales demonstrated significant responsiveness to change in asthma due to therapy in one of the clinical trials. Based on these results, the daytime and nocturnal asthma symptom diary scales show measurement characteristics appropriate for use as asthma outcome measures in clinical trials of asthma therapy.

Synthesis of indolylalkoxyiminoalkylcarboxylates as leukotriene biosynthesis inhibitors

A series of substituted indolylalkoxyiminoalkylcarboxylates were found to be potent leukotriene biosynthesis inhibitors. The structure-activity relationships were investigated. Representative potent inhibitors identified were the quinolyl 3a (A-86885) and pyridyl 3b (A-86886) congeners with in vitro IC 50s of 21 and 9 nM and in vivo leukotriene inhibition in the rat with oral ED 50s of 0.9 and 1.7 mg/kg, respectively.

Pigments of Fungi, 68. – Phlebiachrysoic Acids, New Inhibitors of Leukotriene Biosynthesis from Phlebia chrysocrea (Basidiomycetes)

AbstractFive yellow 2‐hydroxy‐5‐methoxybenzoquinones with C15 and C17 alkyl side chains were isolated from fruitbodies of the wood‐rotting fungus Phlebia chryoscrea. The compounds, named phlebiachrysoic acids A–E are strong inhibitors of leukotriene biosynthesis and are responsible for the red colour reaction when the fruitbodies are touched with aqueous alkali.

Arachidonic acid induces DNA-fragmentation in human polymorphonuclear neutrophil granulocytes.

We analyzed DNA-fragmentation in human polymorphonuclear neutrophil granulocytes (PMNs) from healthy donors after addition of exogenous arachidonic acid (AA) and eicosapentaenoic acid (EPA) by flow cytometry (propidium iodide staining of DNA and DNA strand break detection). The PMNs were incubated from 30 min up to 48 hours in RPMI 1640 which was supplemented with different concentrations of AA or EPA (5-40 microM). In contrast to EPA the addition of AA led to a significant increase in apoptosis up to 67.8% compared to the RPMI-control whereas the addition of EPA led to an inhibition of DNA-fragmentation. When the cells were incubated with MK 886 (1 microM, inhibitor of leukotriene biosynthesis) an increase in DNA-fragmentation (up to 63.3%) was observed. Conversely, in the presence of indomethacin (1 microM, inhibitor of prostanoid synthesis) an inhibition in DNA-fragmentation (up to 60.9%) occurred. Furthermore, preincubation of PMNs with pentoxifylline (1mM, phosphodiesterase inhibitor) reduced AA-stimulated DNA-fragmentation up to 43.4%. These data provide evidence for the involvement of AA and distinct AA metabolites in the regulation of apoptosis in human PMNs.

Characterization of A-93178, an iminoxy-quinoline inhibitor of leukotriene biosynthesis.

In recent years a number of leukotriene modulators have shown promise in the treatment of asthma.1, 2 Most of these have been Cys-LT1 antagonists.2 Inhibitors of both cysteinyl leukotrienes and LTB4 however are likely to be more effective given the pro-inflammatory characteristics of LTB4. In addition, these inhibitors would also inhibit the formation of the mediators formed from double lipoxygenation such as lipoxins.3 Thus far, most bioavailable inhibitors of 5-lipoxygenase have been related to zileuton or ZD-2138.4 An alternative to 5-lipoxygenase inhibition is the antagonism of the 5-lipoxygenase activating protein (FLAP).5 Three compounds with this mechanism have reached the clinic but all three have apparently been withdrawn.4 We have synthesized a number of leukotriene inhibitors that are not direct inhibitors of 5-lipoxygenase with optimized pharmacokinetic and pharmacological characteristics. The best of these is A-93178.

N-omega-carbethoxypentyl-4-quinolones: a new class of leukotriene biosynthesis inhibitors.

6-[(4-Quinolinyl)oxy]hexanoic acids and the corresponding esters were designed and synthesized as inhibitors of the production of arachidonic acid metabolites. The inhibitory activities were assayed in vitro by evaluation of serum leukotriene B4 and thromboxane B2 production. While all 6-[(4-quinolinyl)oxy]hexanoic acids and their esters proved to be inactive, the N-alkyl-4-quinolones, obtained as by-products in their synthesis, were found to be a new class of leukotriene biosynthesis inhibitors.

Mechanism of the contractile effect of diaspirin-cross-linked hemoglobin in rat isolated aorta strip denuded of endothelium as revealed using an oil-immersion procedure

Diaspirin-cross-linked hemoglobin (DCLHb) is a chemically modified hemoglobin (Hb) (i.e., alpha-subunits are cross-linked by a covalent bond) currently being tested as a potential oxygen-carrying blood substitute. It was examined for possible vasoactive properties, using the rat isolated aorta strip denuded of endothelium. In this experimental model, DCLHb (1.6-155 microM) was found to be inactive as a vasoconstrictor when added to the Krebs medium but to elicit contractile responses once the Krebs medium containing DCLHb was replaced by mineral oil, a procedure that favors the sequestration of a fixed amount of DCLHb within a substantially reduced volume of extracellular fluid. The contractile activity of DCLHb in our experimental model (i.e., prior exposure of tissues to drugs in the Krebs medium followed by replacement of the Krebs medium by mineral oil) was mimicked by methemoglobin and metmyoglobin, but not by cytochrome c, albumin, hemin, hematin, Fe2+, and a variety of hemorphins. It was abolished by indomethacin, SQ-29548 (prostaglandin H2-thromboxane A2 receptor antagonist), thiourea, or N-2-mercaptopropionylglycine (MCPG), reduced partially by verapamil, but not affected by dazmegrel, MK-886 (leukotriene biosynthesis inhibitor), dimethylsulfoxide, vitamin C or E, deferoxamine, NG-nitro-L-arginine, naloxone, and a variety of other drug receptor antagonists (e.g., prazosin) and protease inhibitors (e.g., pepstatin). Rat aorta strips denuded of endothelium exhibited contractile responses to arachidonic acid added in the Krebs medium (i.e., with no mineral oil added afterwards). Such contractile activity was reduced by SQ-29548, thiourea, or MCPG. Addition of U-46619 (prostaglandin H2-thromboxane A2 mimetic) to the Krebs medium also elicited contractile responses in rat aorta strips denuded of endothelium. Such contractile activity was reduced by SQ-29548, thiourea, or verapamil but not by MCPG. Within the limitations of our experimental approach, these results suggest that (1) the contractile activity of DCLHb in rat aorta strips denuded of endothelium following replacement of the Krebs medium by mineral oil involves the participation of a secondary mediator, which could be a vasoconstrictor metabolite of arachidonic acid; (2) the participation of reactive oxygen species, potential degradation products of DCLHb (e.g., heme, Fe2+, hemorphins), or other mediators in the contractile activity of DCLHb is unlikely; and (3) Ca2+ entry into target cells might be involved in the process by which DCLHb elicits its contractile activity in our experimental model.

Assessment of the physical stability of lyophilized MK-0591 by differential scanning calorimetry

Abstract MK-0591, a potent indirect leukotriene biosynthesis inhibitor, is poorly absorbed when administered orally as a crystalline sodium salt, primarily because of its low aqueous solubility (5 μg/mL). However, the lyophilized X-ray amorphous form with much higher aqueous solubility is very well absorbed and physically stable for long time periods. To better understand the physical stability of the amorphous state, conditions at which the compound will crystallize from the amorphous state were investigated in the context of its glass transition temperature. The physical stability of X-ray amorphous MK-0591 was evaluated by DSC with enhanced sensitivity using the crystallization exotherm at ca. 185°C (10°C/min) to detect crystallization in the solid matrix. No crystal formation was observed at 30°C for 6 months, 60°C for 6 months or 30°C at 75% RH for 6 months. This prolonged physical stability was attributable to two factors: its high glass transition temperature (ca. 125°C) and liquid crystal formation in aqueous solutions at concentrations greater than 60 mg/mL. Crystallization could not be induced after isothermally holding the X-ray amorphous MK-0591 at 120°C for 17 h. Seeding with crystalline MK-0591 (10%) also failed to induce crystallization at 50°C for 6 months or at 30°C at 75% RH for 6 months. Water plasticizes lyophilized MK-0591, lowering the Tg and inducing the onset of crystallization to 100°C. Crystallization at room temperature does not occur with equilibration at high relative humidites probably because of the additional stability imparted to the system by the formation of a lyotropic liquid crystalline phase. The behaviour of amorphous MK-0591, with its high Tg in the solid state and its liquid crystalline properties in concentrated aqueous solution, provided sufficient physical stability to permit its use in oral formulations.

Synthesis and resolution of 2-(cyclohexyl-4-(2-quinolylmethoxy)phenyl)methoxyiminopropionic acid, leukotriene biosynthesis inhibitors

The synthesis and resolution of 2-(E)-(cyclohexyl-4-(2-quinolylmethoxy)-phenyl)methoxyiminopropionic acid 1, a potent leukotriene biosynthesis inhibitor is described. Dibenzoyltartaric acid was used as the chiral auxiliary for the resolution of the hydroxylamine intermediates used in the synthesis. A difficult chromatographic separation was made more practical by changing the order of elution of diastereomers by selection of the natural or unatural tartaric acid auxiliary.

An automated sample preparation and high performance liquid chromatographic method for the determination of MK-591, a novel leukotriene biosynthesis inhibitor, in human plasma

A high performance liquid chromatography assay utilizing an automated sample preparation procedure for the determination of a novel leukotriene biosynthesis inhibitor, (MK-591), in human plasma has been developed. After aliquoting plasma samples and adding internal standard manually, the BenchMate™ Workstation executed protein precipitation and solid-phase extraction. Following evaporation to dryness, the residue was reconstituted and chromatographed isocratically on a cyano-phase analytical column. MK-591 and the internal standard were separated from each other and from endogenous plasma substances and detected with an absorbance detector. The assay has been validated in the concentration range 10–1000 ng ml −1 and has the sensitivity and specificity necessary to quantify plasma concentrations from several clinical studies.