BACKGROUND AND PURPOSEZileuton 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 APPROACHPeritoneal macrophages activated with lipopolysaccharide (LPS)/interferon g (LPS/IFNg), 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 RESULTSZileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1a (PGF1a) 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 PGF1a 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 IMPLICATIONZileuton 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. Abbreviations
BACKGROUND AND PURPOSEFrankincense, 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 APPROACHA protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E2 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 RESULTSHuman 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)H2 to PGE2 mediated by mPGES1 (IC50 = 3-10 mM). Also, in intact A549 cells, BAs selectively inhibited PGE2 generation and, in human whole blood, b-BA reduced lipopolysaccharide-induced PGE2 biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF1a and thromboxane B2. Intraperitoneal or oral administration of b-BA (1 mg·kg -1 ) suppressed rat pleurisy, accompanied by impaired levels of PGE2 and b-BA (1 mg·kg -1 , given i.p.) also reduced mouse paw oedema, both induced by carrageenan. CONCLUSIONS AND IMPLICATIONSSuppression of PGE2 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.
Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development
Based on its capacity to inhibit in vitro HIV-1 replication in T cells and the release of pro-inflammatory cytokines in monocytes, the prenylated heterodimeric phloroglucinyl α-pyrone arzanol was identified as the major anti-inflammatory and anti-viral constituent from Helichrysum italicum. We have now investigated the activity of arzanol on the biosynthesis of pro-inflammatory eicosanoids, evaluating its anti-inflammatory efficacy in vitro and in vivo. Arzanol inhibited 5-lipoxygenase (EC 7.13.11.34) activity and related leukotriene formation in neutrophils, as well as the activity of cyclooxygenase (COX)-1 (EC 1.14.99.1) and the formation of COX-2-derived prostaglandin (PG)E(2)in vitro (IC(50)=2.3-9μM). Detailed studies revealed that arzanol primarily inhibits microsomal PGE(2) synthase (mPGES)-1 (EC 5.3.99.3, IC(50)=0.4μM) rather than COX-2. In fact, arzanol could block COX-2/mPGES-1-mediated PGE(2) biosynthesis in lipopolysaccharide-stimulated human monocytes and human whole blood, but not the concomitant COX-2-derived biosynthesis of thromboxane B(2) or of 6-keto PGF(1α), and the expression of COX-2 or mPGES-1 protein was not affected. Arzanol potently suppressed the inflammatory response of the carrageenan-induced pleurisy in rats (3.6mg/kg, i.p.), with significantly reduced levels of PGE(2) in the pleural exudates. Taken together, our data show that arzanol potently inhibits the biosynthesis of pro-inflammatory lipid mediators like PGE(2)in vitro and in vivo, providing a mechanistic rationale for the anti-inflammatory activity of H. italicum, and a rationale for further pre-clinical evaluation of this novel anti-inflammatory lead.
Dual inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) is currently pursued as potential pharmacological strategy for treatment of inflammation and cancer. Here we present a series of 26 novel 2-aminothiazole-featured pirinixic acid derivatives as dual 5-LO/mPGES-1 inhibitors with improved potency (exemplified by compound 16 (2-[(4-chloro-6-{[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]amino}pyrimidin-2-yl)sulfanyl]octanoic acid) with IC50 = 0.3 and 0.4 μM, respectively) and bioactivity in vivo. Computational analysis presumes binding sites of 16 at the tip of the 5-LO catalytic domain and within a subpocket of the mPGES-1 active site. Compound 16 (10 μM) hardly suppressed cyclooxygenase (COX)-1/2 activities, failed to inhibit 12/15-LOs, and is devoid of radical scavenger properties. Finally, compound 16 reduced vascular permeability and inflammatory cell infiltration in a zymosan-induced mouse peritonitis model accompanied by impaired levels of cysteinyl-leukotrienes and prostaglandin E2. Together, 2-aminothiazole-featured pirinixic acids represent potent dual 5-LO/mPGES-1 inhibitors with an attractive pharmacological profile as anti-inflammatory drugs.
The acylphloroglucinol hyperforin (Hyp) from St. John's wort possesses anti-inflammatory and anti-carcinogenic properties which were ascribed among others to the inhibition of 5-lipoxygenase. Here, we investigated whether Hyp also interferes with prostanoid generation in biological systems, particularly with key enzymes participating in prostaglandin (PG)E2 biosynthesis, i.e., cyclooxygenases (COX)-1/2 and microsomal PGE2 synthase (mPGES)-1 which play key roles in inflammation and tumorigenesis. Similar to the mPGES-1 inhibitors MK-886 and MD-52, Hyp significantly suppressed PGE2 formation in whole blood assays starting at 0.03–1 μM, whereas the concomitant generation of COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, thromboxane B2, and 6-keto PGF1α was not significantly suppressed up to 30 μM. In cell-free assays, Hyp efficiently blocked the conversion of PGH2 to PGE2 mediated by mPGES-1 (IC50 = 1 μM), and isolated COX enzymes were not (COX-2) or hardly (COX-1) suppressed. Intraperitoneal (i.p.) administration of Hyp (4 mg kg−1) to rats impaired exudate volume and leukocyte numbers in carrageenan-induced pleurisy associated with reduced PGE2 levels, and Hyp (given i.p.) inhibited carrageenan-induced mouse paw edema formation (ED50 = 1 mg kg−1) being superior over indomethacin (ED50 = 5 mg kg−1). We conclude that the suppression of PGE2 biosynthesis in vitro and in vivo by acting on mPGES-1 critically contributes to the anti-inflammatory efficiency of Hyp.
The severity and course of inflammatory processes differ between women and men, but the biochemical mechanisms underlying these sex differences are elusive. Prostaglandins (PG) and leukotrienes (LT) are lipid mediators linked to inflammation. We demonstrated superior LT biosynthesis in human neutrophils and monocytes, and in mouse macrophages from females, and we confirmed these sex differences in vivo where female mice produced more LTs during zymosan-induced peritonitis versus males. Here, we report sex differences in PG production in neutrophils during acute inflammation. In the late phase (4–8 hrs) of mouse zymosan-induced peritonitis and rat carrageenan-induced pleurisy, PG levels in males were higher versus females, seemingly due to higher PG production in infiltrated neutrophils. Accordingly, human neutrophils from males produced more PGE2 than cells from females. Increased PG biosynthesis in males was accompanied by elevated cyclooxygenase (COX)-2 expression connected to increased nuclear factor-kappa B activation, and was abolished when LT synthesis was pharmacologically blocked, suggesting that elevated PG production in males might be caused by increased COX-2 expression and by shunting phenomena due to suppressed LT formation. Conclusively, our data reveal that the biosynthesis of pro-inflammatory PGs and LTs is conversely regulated by sex with consequences for the inflammatory response.
The microsomal prostaglandin E 2 synthase (mPGES)-1 is one of the terminal isoenzymes of prostaglandin (PG) E 2 biosynthesis. Pharmacological inhibitors of mPGES-1 are proposed as an alternative to nonsteroidal anti-inflammatory drugs. We recently presented the design and synthesis of a series of pirinixic acid derivatives that dually inhibit mPGES-1 and 5-lipoxygenase. Here, we investigated the mechanism of mPGES-1 inhibition, the selectivity profile, and the in vivo activity of ␣-(n-hexyl)-substituted pirinixic acid [YS121; 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid)] as a lead compound. In cell-free assays, YS121 inhibited human mPGES-1 in a reversible and noncompetitive manner (IC 50 ϭ 3.4 M), and surface plasmon resonance spectroscopy studies using purified in vitro-translated human mPGES-1 indicate direct, reversible, and specific binding to mPGES-1 (K D ϭ 10 -14 M). In lipopolysaccharide-stimulated human whole blood, PGE 2 formation was concentration dependently inhibited (IC 50 ϭ 2 M), whereas concomitant generation of the cyclooxygenase (COX)-2-derived thromboxane B 2 and 6-keto PGF 1␣ and the COX-1-derived 12(S)-hydroxy-5-cis-8,10-transheptadecatrienoic acid was not significantly reduced. In carrageenan-induced rat pleurisy, YS121 (1.5 mg/kg i.p.) blocked exudate formation and leukocyte infiltration accompanied by reduced pleural levels of PGE 2 and leukotriene B 4 but also of 6-keto PGF 1␣ . Taken together, these results indicate that YS121 is a promising inhibitor of mPGES-1 with anti-inflammatory efficiency in human whole blood as well as in vivo.Prostaglandins (PGs) are potent lipid mediators that promote inflammatory reactions but also possess homeostatic functions (Funk, 2001). Their biosynthesis involves oxygenation of arachidonic acid by cyclooxygenase (COX)-1 or -2 to PGH 2 and further conversion by PG synthases to the respective PGs (Funk, 2001). Inhibition of COX-1 and -2 by nonsteroidal anti-inflammatory drugs and selective suppression of COX-2 by coxibs are common and effective strategies for the therapy of inflammatory disorders, fever, and pain, but their long-term use is associated with severe side effects (Rainsford, 2007). Unselective COX-1/2 inhibitors may cause gastric toxicity, whereas an increased cardiovascular risk in This work was supported in part by Carl Zeiss GmbH (to C.P.). Article, publication date, and citation information can be found at
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