The novel benzimidazole derivative BRP-7 inhibits leukotriene biosynthesis in vitro and in vivo by targeting 5-lipoxygenase-activating protein (FLAP).

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.

The novel anti-inflammatory agent VA694, endowed with both NO-releasing and COX2-selective inhibiting properties, exhibits NO-mediated positive effects on blood pressure, coronary flow and endothelium in an experimental model of hypertension and endothelial dysfunction.

Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.

On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo.

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 (IC(50) = 0.6µM). 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 [Ca(2+) ](i) , but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC(50) = 10µM) and reduced LTB(4) levels in the rat pleurisy model after a single oral dose of 10mg·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.

2-(4-(Biphenyl-4-ylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (HZ52)--a novel type of 5-lipoxygenase inhibitor with favourable molecular pharmacology and efficacy in vivo.

BACKGROUND AND PURPOSE: 5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) representing a potential target for pharmacological intervention with inflammation and allergic disorders. Although many LT synthesis inhibitors are effective in simple in vitro test systems, they frequently fail in vivo due to lack of efficacy. Here, we attempted to assess the pharmacological potential of the previously identified 5-LO inhibitor 2-(4-(biphenyl-4-ylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (HZ52). EXPERIMENTAL APPROACH: We evaluated the efficacy of HZ52 in vivo using carrageenan-induced pleurisy in rats and platelet-activating factor (PAF)-induced lethal shock in mice. We also characterized 5-LO inhibition by HZ52 at the cellular and molecular level in comparison with other types of 5-LO inhibitor, that is, BWA4C, ZM230487 and hyperforin. KEY RESULTS: HZ52, 1.5 mg·kg⁻¹ i.p., prevented carrageenan-induced pleurisy accompanied by reduced LTB(4) levels and protected mice (10 mg·kg⁻¹, i.p.) against PAF-induced shock. Detailed analysis in cell-based and cell-free assays revealed that inhibition of 5-LO by HZ52 (i) does not depend on radical scavenging properties and is reversible; (ii) is not impaired by an increased peroxide tone or by elevated substrate concentrations; and (iii) is little affected by the cell stimulus or by phospholipids, glycerides, membranes or Ca²âº. CONCLUSIONS AND IMPLICATIONS: HZ52 is a promising new type of 5-LO inhibitor with efficacy in vivo and with a favourable pharmacological profile. It possesses a unique 5-LO inhibitory mechanism different from classical 5-LO inhibitors and seemingly lacks the typical disadvantages of former classes of LT synthesis blockers.

Enlarging the NSAIDs family: ether, ester and acid derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents.

The development of the coxib family has represented a stimulating approach in the treatment of inflammatory disorders, such as arthritis, and for the management of acute pains, in relation to the well-known traditional Non-Steroidal Anti-inflammatory Drugs (t-NSAIDs). Prompted by the pursuit for new cyclooxygenase-2 (COX-2) inhibitors, endowed with fine tuned selectivity and high potency, in the past years we have identified novel classes of ether, ester and acid molecules characterized by the 1,5-diarylpyrrole scaffold as potentially powerful anti-inflammatory molecules (12-66). All compounds proved to exert an in vitro inhibition profile as good as that shown by reference compounds. Compounds bearing a p-methylsulfonylphenyl substituent at C5 displayed the best issues. In particular, ester derivatives proved to perform the best in vitro profile in terms of selectivity and activity toward COX-2. The cell-based assay data showed that an increase of hindrance at the C3 side chain of compounds could translate to activity enhancement. The human whole blood (HWB) test let to highlight that submitted compounds displayed 5-10 fold higher selectivity for COX-2 vs COX-1 which should translate clinically to an acceptable gastrointestinal safety and mitigate the cardiovascular effects highlighted by highly selective COX-2 inhibitors. Finally, to assess in vivo anti-inflammatory and analgesic activity three different tests (rat paw pressure, rat paw oedema and abdominal constriction) were performed. Results showed good in vivo anti-inflammatory and analgesic activities. The issues gained with these classes of compounds represent, nowadays, a potent stimulus for a further enlargement of the NSAIDs family. In this review we describe the results obtained by our research group on this topic.

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense.

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, ß-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 ß-BA (1 mg·kg(-1) ) suppressed rat pleurisy, accompanied by impaired levels of PGE(2) and ß-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.

The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages.

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.

Effects of zileuton and montelukast in mouse experimental spinal cord injury.

BACKGROUND AND PURPOSE: 5-lipoxygenase (5-LO) is the key enzyme in leukotriene (LT) biosynthesis from arachidonic acid (AA). Here, we examined the role of the 5-LO-product, cysteinyl-LT (Cys-LT), with a 5-LO inhibitor (zileuton) and a Cys-LT, receptor antagonist (montelukast), in the inflammatory response and tissue injury associated with spinal cord injury (SCI). EXPERIMENTAL APPROACH: SCI was induced in mice by the application of vascular clips to the dura via a two-level T6 to T7 laminectomy for 1 min. Cord inflammation was assessed histologically and by measuring inflammatory mediators (ELISA) and apoptosis by annexin V, TUNEL, Fas ligand staining and Bax and Bcl-2 expression (immunohistochemistry and western blots). Motor function in hindlimbs was assessed by a locomotor rating scale, for 10 days after cord injury. KEY RESULTS: SCI in mice resulted in tissue damage, oedema, neutrophil infiltration, apoptosis, tumour necrosis-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2) expression, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) production, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in injured tissue. Treatment of the mice with zileuton or montelukast reduced the spinal cord inflammation and tissue injury, neutrophil infiltration, TNF-alpha, COX-2 and pERK1/2 expression, PGE(2) and LTB(4) production, and apoptosis. In separate experiments, zileuton or montelukast significantly improved the recovery of limb function over 10 days. CONCLUSIONS AND IMPLICATIONS: Zileuton and montelukast produced a substantial reduction of inflammatory events associated with experimental SCI. Our data underline the important role of 5-LO and Cys-LT in neurotrauma.

The inhibitory effect of propolis and caffeic acid phenethyl ester on cyclooxygenase activity in J774 macrophages.

The effect of an ethanolic extract of propolis, with and without CAPE, and some of its components on cyclooxygenase (COX-1 and COX-2) activity in J774 macrophages has been investigated. COX-1 and COX-2 activity, measaured as prostaglandin E2 (PGE2) production, were concentration-dependently inhibited by propolis (3 x 10(-3) - 3 x 10(2) microgml(-1)) with an IC50 of 2.7 microgml(-1) and 4.8 x 10(-2) microgml(-1), respectively. Among the compounds tested pinocembrin and caffeic, ferulic, cinnamic and chlorogenic acids did not affect the activity of COX isoforms. Conversely, CAPE (2.8 x 10(-4) - 28 microgml(-1); 10(-9) - 10(-4) M) and galangin (2.7 x 10(-4) - 27 microgml(-1); 10(-9) - 10(-4) M) were effective, the last being about ten-twenty times less potent. In fact the IC50 of CAPE for COX-1 and COX-2 were 4.4 x 10(-1) microgml(-1) (1.5 x 10(-6) M) and 2 x 10(-3) microgml(-1) (6.3 x 10(-9) M), respectively. The IC50 of galangin were 3.7 microgml(-1) (15 x 10(-6) M) and 3 x 10(-2) microgml(-1) (120 x 10(-9) M), for COX-1 and COX-2 respectively. To better investigate the role of CAPE, we tested the action of the ethanolic extract of propolis deprived of CAPE, which resulted about ten times less potent than the extract with CAPE in the inhibition of both COX-1 and COX-2, with an IC50 of 30 microgml(-1) and 5.3 x 10(-1) microgml(-1), respectively. Moreover the comparison of the inhibition curves showed a significant difference (p < 0.001). These results suggest that both CAPE and galangin contribute to the overall activity of propolis, CAPE being more effective.

Celecoxib, a selective cyclo-oxygenase-2 inhibitor reduces the severity of experimental colitis induced by dinitrobenzene sulfonic acid in rats.

Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leukocyte infiltration, upregulation of the expression of intercellular adhesion molecule 1 (ICAM-1) and upregulation of P-selectin in the colon. Here, we investigate the effects of the selective cyclo-oxygenase-2 inhibitor, celecoxib, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Rats experienced hemorrhagic diarrhoea and weight loss. At 4 days after administration of DNBS, the mucosa of the colon exhibited large areas of necrosis. Neutrophil infiltration (determined by histology, as well as an increase in myeloperoxidase activity in the mucosa) was associated with upregulation of ICAM-1 and P-selectin, as well as high tissue levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) polymerase showed intense staining in the inflamed colon. Celecoxib (5 mg/kg twice a day orally) significantly reduced the degree of hemorrhagic diarrhoea and the weight loss caused by administration of DNBS. Celecoxib also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde, (iv) the increase in staining (immunohistochemistry) for nitrotyrosine, as well as (v) the upregulation of ICAM-1 and P-selectin caused by DNBS in the colon. Thus, we provide the first evidence that a selective cyclo-oxygenase-2 inhibitor celecoxib reduces the degree of colitis caused by DNBS.

The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat.

BACKGROUND: We have recently demonstrated that 17beta-estradiol (E2) inhibits the increase of inducible nitric oxide synthetase (iNOS) activity in selected model systems such as macrophages, microglia, smooth muscle cells, and proposed that this effect might be associated with an anti-inflammatory activity of this hormone. Here we investigate the effects of endogenous estrogens in rats subjected to carrageenan-induced pleurisy. MATERIALS AND METHODS: Adult female rats were ovariectomized 3 weeks before the experiments to deplete circulating estrogens. Selected inflammatory markers, landmarks of the delayed phase of carrageenan-induced pleurisy, were measured in intact (N-OVX), and ovariectomized (OVX) female rats. In addition, the effect of hormone replacement was evaluated in ovariectomized rats with intraperitoneal injection of 17beta-estradiol (E2; 50 microg/kg) 1 hr before carrageenan treatment (OVX + E2). RESULTS: Ovariectomy enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and lipid peroxidation were significantly increased in estrogens-deprived rats. The iNOS in lung samples was significantly increased by the surgery. The increase of iNOS activity was correlated with a marked enhancement in the production of TNF-alpha and IL-1beta. Immunohistochemical analysis for P-selectin and ICAM-I, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS) revealed a positive staining in lungs from carrageenan-treated rats, which was markedly enhanced in ovariectomized rats when compared to cycling rats, particularly in the estrous phase of the cycle. Estrogen replacement counteracted the effect of surgery on all of the above indicators of lung inflammation, suggesting that in the cycling rat this hormone plays a key role in the increased sensitivity to inflammatory injury observed in the OVX rat. CONCLUSION: This study demonstrates that endogenous estrogens production plays an important protective role against carrageenan-induced acute inflammation by decreasing the expression of specific markers of the delayed phase of this well-known model of acute inflammation.

Nitric oxide inhibits leucocyte migration in carrageenin-induced rat pleurisy.

OBJECTIVE AND DESIGN: The role of nitric oxide (NO) on leucocyte migration has been investigated in rat carrageenin-induced pleurisy. MATERIAL: Male Wistar rats. TREATMENT: L-arginine, NOC-18 and aminoguanidine were administered subcutaneously 1 h prior to carrageenin injection. METHODS: Leucocyte accumulation into the pleural cavity was measured 4 h after carrageenin challenge. Statistical significance was calculated by Bonferroni test. RESULTS: L-arginine (10 mg/kg) or the NO donor NOC-18 (10 mg/kg), significantly inhibited leucocyte infiltration by 31% and 20% respectively (P<0.01). On the contrary, when these compounds were given at high doses (L-arginine 300 mg/kg; NOC-18 30 mg/kg), leucocyte accumulation was increased by 22% and 33% respectively (P<0.01). Aminoguanidine, a relatively selective inhibitor of the inducible NO synthase, depending on the dose, showed a biphasic effect on cell migration. Thus, at low doses (30 and 100 mg/kg), aminoguanidine increased (by 40% and 74% respectively, P< 0.01) leucocyte infiltration which was inhibited by 41% (P < 0.01) when the drug was given at high dose (300 mg/kg). CONCLUSIONS: These results suggest that in rat carrageenin-induced pleurisy NO primarily inhibits leucocyte migration.

Synthesis and structure-activity of antisense peptides corresponding to the region for CaM-binding domain of the inducible nitric oxide synthase.

Nitric oxide synthase (NOS) catalyses the conversion of L-arginine to nitric oxide (NO) which plays an important role in the regulation of cellular functions and intracellular communications. Three distinct isoforms of NOS have so far been identified, two constitutive and one inducible. All three mammalian isoforms of NOS contain putative CaM-binding domains with the canonical composition. In this paper we report the synthesis and the inhibitory activity on rat neuronal and lung inducible NOS of antisense peptides corresponding to the antisense strand read in 3' to 5' (CALM 1) or 5' to 3' (CALM 2) direction of the region encoding for the CaM-binding domain of the inducible NOS isoform (residues 503-522). CALM 1 inhibited, at all the concentrations tested (0.01-1 mM), both the inducible and constitutive NOS (IC(50) 98 microM and 56 microM, respectively), while CALM 2 (0.01-1 mM) was ineffective on both isoforms. The acetylation of CALM 1 at its amino terminal (CALM 8) completely abolished its inhibitory activity. We also synthesized and analysed the activity of amino terminal truncated analogues (CALM 3-7) of CALM 1, which selectively inhibited the inducible isoform, although less potently than the parent compound. The pentapeptides (CALM A-D) deriving from the cleavage of CALM 1 were ineffective, except the pentapeptide CALM C corresponding to the residues 513-517, which was as potent as the parent compound (IC(50) 65 microM).

Prostaglandins and nitric oxide as molecular targets for anti-inflammatory therapy.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most used drugs worldwide, in spite of their renal and gastric side effects. Medicinal plants may represent a useful source of new effective therapeutic agents, particularly considering the new findings concerning the mediators of inflammation, such as prostaglandins and nitric oxide. In fact, the discovery of two isoforms of the enzyme cyclooxygenase, which catalyzes the conversion of arachidonic acid to prostaglandins, has opened new interesting perspectives in the treatment of inflammatory diseases. As cyclooxygenase, also nitric oxide synthase, the enzyme which converts L-arginine to nitric oxide, exists in two isoforms. It appears that the constitutive isoforms of both enzymes (cyclooxygenase-1 and constitutive nitric oxide synthase) have a regulatory-physiological role, whereas the inducible isoforms (cyclooxygenase-2 and inducible nitric oxide synthase) are involved in inflammation. A number of medicinal plants have been screened for their ability to inhibit cyclooxygenase-2 and/or inducible nitric oxide synthase activity and/or expression.

17beta-estradiol antiinflammatory activity in carrageenan-induced pleurisy.

We have recently demonstrated that 17beta-estradiol (E2) opposes cytokine-dependent increase of inducible nitric oxide synthase (iNOS) activity in rat smooth muscle cells and proposed that this effect might be associated to an antiinflammatory activity of this hormone. In the present study, we examine the E2 effects on a well-known in vivo model of inflammation. We show that, in carrageenan treatment of ovariectomized rats, prior exposure to E2 significantly attenuated inflammatory response as measured by histological examination and exudate production. The effect was visible with a single injection of a physiological dose of E2 1 h before the carrageenan treatment and was blocked by coadministration of the estrogen receptor antagonists ICI 182,780 or tamoxifen. This latter observation suggests that the effect is receptor mediated. The mechanisms by which estradiol has beneficial effects in this model of inflammation are unclear: we show that in hormonally treated rats there is a decrease in polymorphonuclear cells migration as shown by cell counting and myeloperoxidase measurement. In addition, E2 pretreatment opposes carrageneen-induced high lipid peroxidation maintaining malondialdehyde activity at control levels. E2 treatment decreases NO production and the activity of iNOS with consequent diminished nitrite synthesis and nitrosine accumulation. Finally, immunohistochemical analysis for poly (ADP-ribose) synthase revealed a positive staining in lungs from carrageenan-treated rats that was blocked by estradiol treatment. We conclude that E2 attenuates the degree of inflammation and tissue damage associated with carrageenan-induced pleurisy in the rat.

Anti-inflammatory activity of macrolide antibiotics.

The effect of four macrolide antibiotics (roxithromycin, clarithromycin, erythromycin, and azithromycin) on the generation of some mediators and cytokines involved in the inflammatory process has been studied both in vivo and in vitro. Rat carrageenin pleurisy was used as a model of acute inflammation, and the macrolides were administered (10, 20, and 40 mg/kg p.o.) 1 h before the carrageenin challenge. Exudate volume and leukocyte accumulation were both dose-dependently reduced by roxithromycin, clarithromycin and erythromycin in either normal or adrenalectomized animals. Furthermore, in normal rats, prostaglandin (PG)E(2), nitrate plus nitrite, and tumor necrosis factor-alpha levels in pleural exudate were significantly reduced by these macrolides. Roxithromycin appeared more effective than erythromycin and clarithromycin, whereas azithromycin only slightly affected the inflammatory reaction. None of the macrolides were able to modify leukotriene B(4) exudate levels. In vitro experiments have shown that the four macrolides (5-80 microM) reduced in a concentration-dependent manner the production of 6-keto-PGF(1alpha), NO(2)(-), tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 by lipopolysaccharide-stimulated J774 macrophages. In J774 cells, the inhibition of 6-keto-PGF(1alpha) and NO(2)(-) production by roxithromycin and erythromycin was not dependent on direct inhibition of cyclooxygenase-2 and inducible nitric oxide synthase activity because it appears to be related to the inhibition of cyclooxygenase-2 and inducible nitric oxide synthase protein expression. In conclusion, the present study shows that macrolide antibiotics have anti-inflammatory activity, which likely depends on their ability to prevent the production of proinflammatory mediators and cytokines, and suggest that these agents, particularly roxithromycin, can exert therapeutic effects independently of their antibacterial activity.

Cyclooxygenase-2-dependent generation of 8-epiprostaglandin F2alpha by lipopolysaccharide-activated J774 macrophages.

OBJECTIVE: The generation of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) by arachidonic acid (AA)- and lipopolysaccharide (LPS)- stimulated J774 macrophages has been investigated. MATERIAL: Murine monocyte/macrophage J774 cell line. METHODS: Cells were incubated with AA or LPS and the amount of 8-epi-PGF2alpha, 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha) and prostaglandin E2 (PGE2) released in the incubation media measured by radioimmunoassay (RIA) or, in some experiments, by enzyme immunoassay (EIA). The effect of dexamethasone (DXM), cycloheximide (CXM) and 5,5 dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU), a cyclooxygenase-2 (COX-2) selective inhibitor, on LPS-induced generation of AA metabolites was assessed. RESULTS: AA induced a significant production of 6-ketoPGF1alpha and PGE2, whereas LPS caused a concentration- and time-dependent increase of 8-epi-PGF2alpha, 6-keto-PGF1alpha and PGE2. DXM (2 microM) as well as CXM (1 microM) significantly decreased (p<0.001; n = 4) the LPS-stimulated production of 8-epi-PGF2alpha (by 86% and 82%, respectively), 6-ketoPGF1alpha (by 78% and 74%, respectively) and PGE2 (by 83% and 78%, respectively). Immunostimulated production of AA metabolites was also inhibited by DFU (IC50 0.3+/-0.04 microM; 0.16 +/- 0.02 microM and 0.11 +/- 0.05 microM for 8-epi-PGF2alpha, 6-keto-PGF1alpha and PGE2, respectively. CONCLUSIONS: These results demonstrate the role of COX-2 in the generation of 8-epi-PGF2alpha by LPS-stimulated J774 macrophages. The relevance of these findings requires further elucidation.

Role of IL-6 in the pleurisy and lung injury caused by carrageenan.

In the present study we used IL-6 knockout mice (IL-6KO) to evaluate the role of IL-6 in the inflammatory response caused by injection of carrageenan into the pleural space. Compared with carrageenan-treated IL-6 wild-type (IL-6WT) mice, carrageenan-treated IL-6KO mice exhibited a reduced degree of pleural exudation and polymorphonuclear cell migration. Lung myeloperoxidase activity and lipid peroxidation were significantly reduced in IL-6KO mice compared with those in IL-6WT mice treated with carrageenan. Immunohistochemical analysis for nitrotyrosine and poly(A)DP-ribose polymerase revealed a positive staining in lungs from carrageenan-treated IL-6WT mice. No positive staining for nitrotyrosine or PARS was found in the lungs of the carrageenan-treated IL-6KO mice. Staining of lung tissue sections obtained from carrageenan-treated IL-6WT mice with an anti-cyclo-oxygenase-2 Ab showed a diffuse staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase was found mainly in the macrophages of the inflamed lungs from carrageenan-treated IL-6WT mice. The intensity and degree of the staining for cyclo-oxygenase-2 and inducible nitric oxide synthase were markedly reduced in tissue sections obtained from carrageenan-treated IL-6KO mice. Most notably, the degree of lung injury caused by carrageenan was also reduced in IL-6KO mice. Treatment of IL-6WT mice with anti-IL-6 (5 microg/day/mouse at 24 and 1 h before carrageenan treatment) also significantly attenuated all the above indicators of lung inflammation. Taken together, our results clearly demonstrate that IL-6KO mice are more resistant to the acute inflammation of the lung caused by carrageenan injection into the pleural space than the corresponding WT mice.

Leptin increases serotonin turnover by inhibition of brain nitric oxide synthesis.

Leptin administration inhibits diencephalic nitric oxide synthase (NOS) activity and increases brain serotonin (5-HT) metabolism in mice. We evaluated food intake, body-weight gain, diencephalic NOS activity, and diencephalic content of tryptophan (TRP), 5-HT, hydroxyindoleacetic acid (5-HIAA), and 5-HIAA/5-HT ratio after intracerebroventricular (ICV) or intraperitoneal (IP) leptin injection in mice. Five consecutive days of ICV or IP leptin injections induced a significant reduction in neuronal NOS (nNOS) activity, and caused a dose-dependent increase of 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio. Diencephalic 5-HT metabolism showed a significant increase in 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio 3 hours after a single leptin injection. This effect was maintained for 3 hours and had disappeared by 12 hours after injection. After a single IP leptin injection, the peak for 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio was achieved at 6 hours. Single injections of ICV or IP leptin significantly increased diencephalic 5-HT content. Leptin-induced 5-HT increase was antagonized by the coadministration of L-arginine only when the latter was ICV injected, whereas D-arginine did not influence leptin effects on brain 5-HT content. Finally, in nNOS-knockout mice, the appetite-suppressant activity of leptin was strongly reduced, and the leptin-induced increase in brain 5-HT metabolism was completely abolished. Our results indicate that the L-arginine/NO pathway is involved in mediating leptin effects on feeding behavior, and demonstrate that nNOS activity is required for the effects of leptin on brain 5-HT turnover.

Regulation of prostaglandin production by inhibition of poly (ADP-ribose) synthase in carrageenan-induced pleurisy.

The aim of the present study was to investigate the effect of two inhibitors (3-aminobenzamide and nicotinamide) of poly (ADP-ribose) synthetase on the production of the inflammatory mediator prostaglandins in a model of acute inflammation, carrageenan-induced pleurisy, where prostaglandins are known to play a crucial role. The results show that the poly (ADP-ribose) synthetase inhibitors, 3-aminobenzamide (2.5, 5 and 10 mg/kg) and nicotinamide (12.5, 25 and 50 mg/kg), inhibit the inflammatory response (pleural exudate formation, polymorphonuclear cell infiltration and prostaglandin production). The present results demonstrate that inhibition of poly (ADP-ribose) synthetase exerts potent anti-inflammatory effects. Part of these anti-inflammatory effects may be related to a reduction of prostaglandin production during the inflammatory process.