BACKGROUND AND PURPOSE Zileuton is the only 5‐lipoxygenase (5‐LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5‐LOX and ...leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo.
EXPERIMENTAL APPROACH Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan‐induced pleurisy as an in vivo model.
KEY RESULTS Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6‐keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5‐LOX inhibition, because it was also observed in macrophages from 5‐LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS‐stimulated human whole blood and suppressed PGE2 and 6‐keto PGF1α pleural levels in rat carrageenan‐induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)‐2 and did not affect COX‐2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes.
CONCLUSIONS AND IMPLICATION Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed.
5-Lipoxygenase initiates the biosynthesis of leukotrienes, lipid mediators involved in normal host defense and in inflammatory and allergic disorders. Despite an obvious gender bias in ...leukotriene-related diseases (e.g., asthma), gender aspects have been neglected in studies on leukotrienes and 5-lipoxygenase. Here, we show that leukotriene formation in stimulated whole blood or neutrophils from males is substantially lower compared with females, accompanied by changed 5-lipoxygenase trafficking. This is due to gender-specific differential activation of extracellular signal-regulated kinases (ERKs). The differences are directly related to variant male/female testosterone plus 5α-dihydrotestosterone levels, and addition of 5α-dihydrotestosterone to female blood or neutrophils reduced the high (female) LT biosynthesis capacity to low (male) levels. In conclusion, regulation of ERKs and leukotriene formation by androgens constitutes a molecular basis for gender differences in the inflammatory response, and in inflammatory diseases such as asthma.
Summary The potential of fish or fish oil as supplier for eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3) for reducing cardiovascular risk factors and supporting therapy ...of chronic inflammatory diseases, has been investigated intensively, but our knowledge about the physiological effects of the individual compounds EPA and DHA are limited. Study design In this double-blind pilot study, thirty-eight patients with defined RA were allocated to consume foods enriched with microalgae oil from Schizochytrium sp. (2.1 g DHA/d) or sunflower oil (placebo) for 10 weeks (cross-over), maintaining the regular RA medication during the study. Results In contrast to placebo, the daily consumption of DHA led to a decline in the sum of tender and swollen joints (68/66) from 13.9 ± 7.4 to 9.9 ± 7.0 (p = 0.010), total DAS28 from 4.3 ± 1.0 to 3.9 ± 1.2 (p = 0.072), and ultrasound score (US-7) from 15.1 ± 9.5 to 12.4 ± 7.0 (p = 0.160). The consumption of placebo products caused an increase of the n-6 PUFA linoleic acid and arachidonic acid (AA) in erythrocyte lipids (EL, p < 0.05). The amount of DHA was doubled in EL of DHA-supplemented patients and the ratios of AA/EPA and AA/DHA dropped significantly. We speculate that the production of pro-inflammatory/non-resolving AA-derived eicosanoids might decrease in relation to anti-inflammatory/pro-resolving DHA- and EPA-derived lipid mediators. In fact, plasma concentrations of AA-derived thromboxane B2 and the capacity of blood to convert AA to the pro-inflammatory 5-lipoxygenase product 5-hydroxyeicosatetraenoic acid were significantly reduced, while levels of the DHA-derived maresin/resolvin precursors 14-/17-hydroxydocosahexaenoic acid significantly increased due to DHA supplementation. Conclusion The study shows for the first time that supplemented microalgae DHA ameliorates disease activity in patients with RA along with a shift in the balance of AA- and DHA-derived lipid mediators towards an anti-inflammatory/pro-resolving state.
Background and purpose:
The selective inhibition of prostaglandin (PG)E
2
formation via interference with microsomal PGE
2
synthase (mPGES)‐1 could have advantages in the treatment of PGE
2
...‐associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)‐1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from
Myrtus communis
L. (myrtle) affected mPGES‐1.
Experimental approach:
The effect of MC on PGE
2
formation was investigated in a cell‐free assay by using microsomal preparations of interleukin‐1β‐stimulated A549 cells as the source of mPGES‐1, in intact A549 cells, and in lipopolysaccharide‐stimulated human whole blood. Inhibition of COX‐1 and COX‐2 activity in cellular and cell‐free assays was assessed by measuring 12(S)‐hydroxy‐5‐cis‐8,10‐trans‐heptadecatrienoic acid and 6‐oxo PGF
1α
formation.
Key results:
MC concentration‐dependently inhibited cell‐free mPGES‐1‐mediated conversion of PGH
2
to PGE
2
(IC
50
= 1 µmol·L
−1
). PGE
2
formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX‐2 activity in A549 cells nor isolated human recombinant COX‐2 was significantly affected by MC up to 30 µmol·L
−1
, and only moderate inhibition of cellular or cell‐free COX‐1 was evident (IC
50
> 15 µmol·L
−1
).
Conclusions and implications:
MC is the first natural product to inhibit mPGES‐1 that efficiently suppresses PGE
2
formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non‐steroidal anti‐inflammatory drugs.
BACKGROUND AND PURPOSE
Frankincense, the gum resin derived from
Boswellia
species, showed anti‐inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed ...to be responsible for these effects but their anti‐inflammatory efficacy
in vivo
and their molecular modes of action are incompletely understood.
EXPERIMENTAL APPROACH
A protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E
2
synthase‐1 (mPGES1) as a BA‐interacting protein. Cell‐free and cell‐based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan‐induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs
in vivo
.
KEY RESULTS
Human mPGES1 from A549 cells or
in vitro
‐translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H
2
to PGE
2
mediated by mPGES1 (IC
50
= 3–10 µM). Also, in intact A549 cells, BAs selectively inhibited PGE
2
generation and, in human whole blood, β‐BA reduced lipopolysaccharide‐induced PGE
2
biosynthesis without affecting formation of the COX‐derived metabolites 6‐keto PGF
1α
and thromboxane B
2
. Intraperitoneal or oral administration of β‐BA (1 mg·kg
−1
) suppressed rat pleurisy, accompanied by impaired levels of PGE
2
and β‐BA (1 mg·kg
−1
, given i.p.) also reduced mouse paw oedema, both induced by carrageenan.
CONCLUSIONS AND IMPLICATIONS
Suppression of PGE
2
formation by BAs via interference with mPGES1 contribute to the anti‐inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti‐inflammatory properties.
Background and Purpose
Leukotrienes (LTs) are inflammatory mediators produced via the 5‐lipoxygenase (5‐LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and ...cardiovascular diseases. We recently identified the benzimidazole derivative BRP‐7 as chemotype for anti‐LT agents by virtual screening targeting 5‐LOX‐activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP‐7 as an inhibitor of LT biosynthesis.
Experimental Approach
We analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell‐free assays. The effectiveness of BRP‐7 in vivo was evaluated in rat carrageenan‐induced pleurisy and mouse zymosan‐induced peritonitis.
Key Results
BRP‐7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5‐LOX co‐localization with FLAP. Neither the cellular viability nor the activity of 5‐LOX in cell‐free assays was affected by BRP‐7, indicating that a functional FLAP is needed for BRP‐7 to inhibit LTs, and FLAP bound to BRP‐7 linked to a solid matrix. Compared with the FLAP inhibitor MK‐886, BRP‐7 did not significantly inhibit COX‐1 or microsomal prostaglandin E2 synthase‐1, implying the selectivity of BRP‐7 for FLAP. Finally, BRP‐7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels.
Conclusions and Implications
BRP‐7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti‐inflammatory effectiveness in vivo, with promising potential for further development.
BACKGROUND AND PURPOSE Leukotrienes (LTs) are pro‐inflammatory mediators produced by 5‐lipoxygenase (5‐LO). Currently available 5‐LO inhibitors either lack efficacy or are toxic and novel approaches ...are required to establish a successful anti‐LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant‐derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis.
EXPERIMENTAL APPROACH We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell‐free assays (neutrophil homogenates or recombinant human 5‐LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan‐induced pleurisy.
KEY RESULTS Tryptanthrin potently reduced LT‐formation in human neutrophils (IC50= 0.6 µM). However, tryptanthrin is not a redox‐active compound and did not directly interfere with 5‐LO activity in cell‐free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in Ca2+i, but it modified the subcellular localization of 5‐LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC50= 10 µM) and reduced LTB4 levels in the rat pleurisy model after a single oral dose of 10 mg·kg−1.
CONCLUSIONS AND IMPLICATIONS Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.
Background and Purpose
1,4‐Benzoquinones are well‐known inhibitors of 5‐lipoxygenase (5‐LOX, the key enzyme in leukotriene biosynthesis), but the molecular mechanisms of 5‐LOX inhibition are not ...completely understood. Here we investigated the molecular mode of action and the pharmacological profile of the novel 1,4‐benzoquinone derivative 3‐((decahydronaphthalen‐6‐yl)methyl)‐2,5‐dihydroxycyclohexa‐2,5‐diene‐1,4‐dione (RF‐Id) in vitro and its effectiveness in vivo.
Experimental Approach
Mechanistic investigations in cell‐free assays using 5‐LOX and other enzymes associated with eicosanoid biosynthesis were conducted, along with cell‐based studies in human leukocytes and whole blood. Molecular docking of RF‐Id into the 5‐LOX structure was performed to illustrate molecular interference with 5‐LOX. The effectiveness of RF‐Id in vivo was also evaluated in two murine models of inflammation.
Key Results
RF‐Id consistently suppressed 5‐LOX product synthesis in human leukocytes and human whole blood. RF‐Id also blocked COX‐2 activity but did not significantly inhibit COX‐1, microsomal PGE2 synthase‐1, cytosolic PLA2 or 12‐ and 15‐LOX. Although RF‐Id lacked radical scavenging activity, reducing conditions facilitated its inhibitory effect on 5‐LOX whereas cell stress impaired its efficacy. The reduced hydroquinone form of RF‐Id (RED‐RF‐Id) was a more potent inhibitor of 5‐LOX as it had more bidirectional hydrogen bonds within the 5‐LOX substrate binding site. Finally, RF‐Id had marked anti‐inflammatory effects in mice in vivo.
Conclusions and Implications
RF‐Id represents a novel anti‐inflammatory 1,4‐benzoquinone that potently suppresses LT biosynthesis by direct inhibition of 5‐LOX with effectiveness in vivo. Mechanistically, RF‐Id inhibits 5‐LOX in a non‐redox manner by forming discrete molecular interactions within the active site of 5‐LOX.
Abstract
The biosynthesis of leukotrienes (LTs) is initiated by the transformation of free arachidonic acid to LTA
4
by 5-lipoxygenase (5-LO). Subsequent enzymatic conversion of LTA
4
yields LTB
4
...and the cysteinyl-LTs C
4
, D
4
and E
4
. LTs have prominent functions in pathophysiology and are connected to numerous disorders including bronchial asthma, allergic rhinitis, inflammatory bowel and skin diseases, rheumatoid arthritis, cancer, osteoporosis and cardiovascular diseases. Pharmacological and genetic interruption of the 5-LO pathway or blockade of LT receptors, serving as means for intervention with LTs, may be of therapeutic value for certain related disorders. Natural or plant-derived substances were among the first 5-LO inhibitors identified in the early 1980 s. To date, a huge number of diverse plant-derived compounds have been reported to interfere with 5-LO product synthesis. However, many investigations have addressed the efficacy of a given compound solely in cellular test systems and analysis of direct interference with 5-LO has been neglected. In the first part of this review, the biology and molecular pharmacology of the 5-LO pathway is summarized in order to understand its overall regulation and complexity as well as to comprehend the possible points of attack of compounds that eventually lead to inhibition of 5-LO product formation in intact cells. In the second part, natural compounds that interfere with 5-LO product formation are compiled and grouped into structural classes, and the underlying molecular mechanisms and structure-activity relationships are discussed.
Background and purpose:
Licofelone is a dual inhibitor of the cyclooxygenase and 5‐lipoxygenase (5‐LO) pathway, and has been developed for the treatment of inflammatory diseases. Here, we ...investigated the molecular mechanisms underlying the inhibition by licofelone of the formation of 5‐LO products.
Experimental approach:
The efficacy of licofelone to inhibit the formation of 5‐LO products was analysed in human isolated polymorphonuclear leukocytes (PMNL) or transfected HeLa cells, as well as in cell‐free assays using respective cell homogenates or purified recombinant 5‐LO. Moreover, the effects of licofelone on the subcellular redistribution of 5‐LO were studied.
Key results:
Licofelone potently blocked synthesis of 5‐LO products in Ca2+‐ionophore‐activated PMNL (IC50=1.7 μM) but was a weak inhibitor of 5‐LO activity in cell‐free assays (IC50≫10 μ
M). The structures of licofelone and MK‐886, an inhibitor of the 5‐LO‐activating protein (FLAP), were superimposable. The potencies of both licofelone and MK‐886 in ionophore‐activated PMNL were impaired upon increasing the concentration of arachidonic acid, or under conditions where 5‐LO product formation was evoked by genotoxic, oxidative or hyperosmotic stress. Furthermore, licofelone prevented nuclear redistribution of 5‐LO in ionophore‐activated PMNL, as had been observed for FLAP inhibitors. Finally, licofelone as well as MK‐886 caused only moderate inhibition of the synthesis of 5‐LO products in HeLa cells, unless FLAP was co‐transfected.
Conclusions and implications:
Our data suggest that the potent inhibition of the biosynthesis of 5‐LO products by licofelone requires an intact cellular environment and appears to be due to interference with FLAP.
British Journal of Pharmacology (2007) 152, 471–480; doi:10.1038/sj.bjp.0707416; published online 20 August 2007
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