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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.

The design of compounds that are able to inhibit cyclooxygenase (COX) and to release nitric oxide (NO) should give rise to drugs endowed with an overall safer profile for the gastrointestinal and cardiovascular systems. Herein we report a new class of pyrrole-derived nitrooxy esters (11a-j), cyclooxygenase-2 (COX-2) selective inhibitors endowed with NO releasing properties, with the goal of generating new molecules able to both strongly inhibit this isoform and reduce the related adverse side effects. Taking into account the metabolic conversion of nitrooxy esters into corresponding alcohols, we also studied derivatives 12a-j. All compounds proved to be very potent and selective COX-2 inhibitors; nitrooxy derivatives displayed interesting ex vivo NO-dependent vasorelaxing properties. Compounds 11c, 11d, 12c, and 12d were selected for further in vivo studies that highlited good anti-inflammatory and antinociceptive activities. Finally, two selected compounds (11c and 12c) tested in human whole blood (HWB) assay proved to be preferential inhibitors of COX-2.

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.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.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) .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.

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.

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.

A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers (6, 7, and 8) has been synthesized with the aim to assess if in the previously reported 1,5-diarylpyrrole derivatives (5) the replacement of the acetic ester moiety with an alkoxyethyl group still led to new, highly selective and potent COX-2 inhibitors. In the in vitro cell culture assay, all the compounds proved to be potent and selective COX-2 inhibitors. In the human whole blood (HWB) assay, compound 8a had a comparable COX-2 selectivity to valdecoxib, while it was more selective than celecoxib but less selective than rofecoxib. The potential anti-inflammatory and antinociceptive activities of compounds 7a, 8a, and 8d were evaluated in vivo, where they showed a very good activity against both carrageenan-induced hyperalgesia and edema in the rat paw test. In the abdominal constriction test compound 7a, 8a, and 8d were able to reduce the number of writhes in a statistically significant manner. Furthermore, the affinity data of these compounds have been rationalized through enzyme docking simulations in terms of interactions with a crystallographic model of the COX-2 binding site by means of the software package Autodock 3.0.5, GRID 21, and MacroModel 8.5 using the complex between COX-2 and SC-558 (1b), refined at a 3 A resolution (Brookhaven Protein Data Bank entry: 6cox ).

The important role of cyclooxygenase-2 (COX-2) in the pathogenesis of inflammation and side effect limitations of current COX-2 inhibitor drugs illustrates a need for the design of new compounds based on alternative structural templates. We previously reported a set of substituted 1,5-diarylpyrrole derivatives, along with their inhibitory activity toward COX enzymes. Several compounds proved to be highly selective COX-2 inhibitors and their affinity data were rationalized through docking simulations. In this paper, we describe the synthesis of new 1,5-diarylpyrrole derivatives that were assayed for their in vitro inhibitory effects toward COX isozymes. Among them, the ethyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-[3-fluorophenyl]-1H-pyrrol-3-acetate (1d), which was the most potent and COX-2 selective compound, also showed a very interesting in vivo anti-inflammatory and analgesic activity, laying the foundations for developing new lead compounds that could be effective agents in the armamentarium for the management of inflammation and pain.

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).

Animal work suggests that with certain doses of aspirin the antithrombotic effect exerted via the inhibition of the proaggregatory platelet thromboxane A2 (TXA2) may be neutralised by the concomitant vascular reduction of the antiaggregatory prostacyclin (PGI2). Such a situation might result not only in therapeutic ineffectiveness but also in a thrombotic tendency. A patient with a bleeding disorder characterised by a mildly prolonged bleeding time and defective platelet-release reaction due to a congenital deficiency of cyclo-oxygenase provided an opportunity for studying this problem. Her platelets did not aggregate with arachidonic acid, but they did so with a synthetic endoperoxide analogue. Thrombin added to her platelet-rich plasma and whole blood did not generate thromboxane B2 (TXB2). Washed platelets, when incubated with 14C-arachidonic acid, did not produce the cyclo-oxygenase metabolites. A biopsy specimen of her vein did not generate PGI2, as measured both by platelet-aggregation inhibition and radioimmunoassay of 6-keto-PGF1 alpha. Clinically, the patient had a mild bleeding tendency but no thrombotic problems. The findings suggest that in man aspirin therapy, even at doses which inhibit PGI2 formation, would only impair haemostasis mildly without producing a thrombotic tendency.

Selected ion-monitoring (SIM) analysis proved more reliable and sensitive than biological assay for the detection of estrogen residues in food and permitted the simultaneous determination of several estrogens.

Summary A polymer bound nicotinic acid preparation (P61/9) was administered orally to rats at various doses. Plasma levels of free nicotinic acid were determined by a newly developed analytical procedure which utilizes the multiple selected ion monitoring technique. The pharmacological activity of P61/9 was evaluated as the decrease of plasma free fatty acid (FFA) and triglyceride (TG) levels. With plasma levels of nicotinic acid as low as 170 ng/ml an active effect on FFA and TG has been observed. When compared with free nicotinic acid, P61/9 is shown to produce a more prolonged biological effect.

The stimulation of human lung parenchymal fragments with A-23187 induces formation of leukotrienes as well as of PGD 2 : LTE 4 is the compound found in larger amount and independently of the intensity of the stimulus the % of metabolized precursor which is converted to leukotrienes or PGD 2 is remarkably similar. However, under conditions of IgE-AntilgE challenge the predominant conversion of arachidonic acid occurs to PGD 2 , LTB 4 is almost negligible and LTD 4 and E 4 together represent less Ln 30% of the oxidative products of arachidonic acid. Whether PGD 2 and leukotrienes derive from the same or different subset of cells is unresolved.

Thromboxane B2 (TXB2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formed from the endogenous and exogenous arachidonate during human platelet incubation, was evaluated by selected ion monitoring (SIM). TXB2 formed from endogenous substrate accounted for about one third of the total, whereas the great part of 12-HETE derived from exogenous arachidonate. These data indicate that under the tested conditions the pool of arachidonate that acts as substrate for cyclo-oxygenase is different from the pool that acts as substrate for lipoxygenase and that the arachidonate released from phospholipids is preferentially utilized by cyclo-oxygenase.

Isoproterenol and salbutamol caused relaxation of guinea-pig trachea and generation of PGE 2 . The phenomenon appears to be specific for β-adrenergic agonists since the relaxant activity induced by atropine, theophylline and papaverine was not associated with generation of prostaglandin-like material from tracheal smooth muscles. Indomethacin reduced only slightly the relaxation of the tracheal tissue induced by isoproterenol and salbutamol. The possibility is discussed that the increased arachidonic acid metabolism during trachea β-adrenergic relaxation represents a metabolic event involving phospholipid methylation and membrane fluidity.

The elucidation of the structure of SRS-A (slow reacting substance of anaphylaxis) has led to the discovery of Leukotrienes (LTs), a family of arachidonate metabolites produced by the enzyme 5-lipoxygenase (1) (2). A stereospecific synthesis brings about formation of two classes of LTs, one major class composed of the sulfidopeptide leukotrienes, LTC4, LTD4 and LTE4, and another class represented solely by LTB4. A variety of stimuli, cleaving arachidonate from membrane phospholipids, provide the necessary substrate for the synthesis of prostaglandins (PGs), thromboxanes (TX) and LTs (3). PGs and to a minor extent TX are formed ubiquitarily, whereas the synthesis of LTs seems to require a considerable cellular specificity (4) (5): all of them are formed by normal and asthmatic human lung where they may play a role as potent bronchoconstrictors as well as primary mediators of airway hyperreactivity (6).

Summary Tiaramide hydrochloride (THC), a benzothiazolinone derivative, displays a direct bronchodilating activity associated with anti-anaphylactic properties. The smooth muscle relaxing capacity of this compound is not related to increased levels of cyclic AMP. The anti-anaphylactic activity has been investigated using isolated lungs from ovoalbumin sensitized guinea-pigs. THC prevents histamine and TXA 2 release from lungs during antigen exposure. In addition to this THC antagonizes the direct effect of histamine on TXA 2 generation in guinea-pig lungs without affecting the cyclo-oxygenase system. Our results indicate that THC can control the anphylactic reaction through at least two different mechanisms: 1) inhibition of mediator release, 2) direct antagonism of their spasmogenic activity.

Since the discovery of prostaglandins from pig and sheep lungs by Bergstrom (1), the description of their pharmacological activity upon bronchial smooth muscle (2) and pulmonary vessels (3) has stimulated investigation on the role of these lipidic compounds in the physiopathology of the respiratory system. Interest in the prostaglandin field increased remarkably when, in 1975, Thromboxane-A2 (TXA2) was identified, and its potent vasoconstrictor and thrombogenic activity was established (4). The potential clinical importance of TXA2 began to be more considered when it was demonstrated that this “ephemeral substance”, which is made by platelets, could be also generated in the lungs as a main metabolite of arachidonic acid (AA) (5). In fact release of prostaglandins and mainly rabbit-aorta contracting substance (RCS), now identified as a mixture of TXA2 and a small amount of endoperoxides was shown by Piper and Vane, when studying chemical mediators in anaphylactic guinea-pig lungs (6). TXA2 is a dangerous agent which displays a powerful contracting capacity on the bronchial smooth muscle. Intravenous injection of TXA2 in anaesthetized guinea-pigs brings about a sustained increase in the airways resistence, an effect which is 40 folds higher than

Leukotriene C4 (LTC4), one of the major components of the slow-reacting substance of anaphylaxis (SRS-A), is a potent constrictor of bronchial smooth muscle in many species including humans. Here we report the identification and characterization of specific binding sites for LTC4 in membranes from human lung parenchyma. At 4°, 3H-LTC4 binding is specific, saturable Bmax = 32–41 pmoles mg prot.), rapid (equilibrium being attained within 15 min), reversible and of high affinity (Kd = 3.6−7 × 10−8 M). The binding sites are sensitive to heat and probably possess a protein moiety, being inactivated upon trypsinization. CaCl2 affects both the association and the dissociation rate and dose-dependently enhances the binding of 3H-LTC4 at equilibrium; maximal enhancement (4-fold) occurred at 10−2 M CaCl2. Unlabelled LTC4 is able to compete with 3H-LTC4 for its binding sites with an IC50 of 7.8 × 10−8 M. The addition of 10−2 M CaCl2 increases the potency of LTC4 in inhibiting the binding (2.2-fold); both the competition curves are monophasic, indicating the existence of a homogeneous class of binding sites. In the presence of CaCl2, LTD4, LTE4 and the SRS-A antagonist FPL 55712 can inhibit 3H-LTC4 specific binding, being, however, less potent than LTC4 ( ic 50 s = 2.2 × 10−6, 2.4 × 10−5 and 2.4 × 10−5 M, for LTD4, LTE4 and FPL 55712, respectively). FPL 55712 displayed a competitive mechanism; its affinity, however, was lower if absorption to glass was not prevented. The present studies indicate that specific binding sites for 3H-LTC4 exist in human lung parenchyma, and that a receptor-mediated process might be involved in the bronchoconstriction induced by LTC4.

When chopped human lung is stimulated with the Ca++ ionophore A23187 (0.25-10 microM) leukotriene (LT) B4, LTD4 and LTE4 are found in the incubation medium according to different patterns. LTD4 is released promptly and its levels increase up to 45 min after the stimulus (A23187, 10 microM) and decline later (120 min); LTE4 formation follows a sigmoidal shape and continues to accumulate even 2 hr after the challenge; LTB4 levels reach a plateau at 45 min. LTC4 was undetectable in most experiments but it was found to accumulate when reduced glutathione (10 mM) was added. Addition of exogenous LTC4 to unstimulated fragments of human lung shows that an effective interconversion to LTD4 and LTE4 takes place: A23187 stimulates formation of LT and cyclo-oxygenase products dose dependently; a statistically significant formation of LT occurs at A23187 concentration of 1 microM whereas thromboxane B2 (TXB2) and 6-K-prostaglandin F1 alpha levels increased significantly only at 2.5 microM A23187. Pretreatment with U-60257 (100 microM) prevented formation of LT without a concomitant increase of TXB2 levels. Indomethacin (1.5 microM) blocked the release of 6-K-prostaglandin F1 alpha and TXB2 without a shift of arachidonic acid towards LT-like activity. Addition of exogenous LTC4 did not trigger synthesis of TXB2 or 6-K-prostaglandin F1 alpha. Our results indicate that reduced glutathione levels and the activity of the enzymes involved in LT biosynthesis and/or metabolism play an important role in controlling the pattern of LT release from human lung.

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2α, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2α, and TXB2, show considerable increase.

Ca++-ionophore and antigen challenge of human lung fragments caused a massive release of leukotrienes, whose relative proportions varied considerably and were dependent on the experimental conditions. A broncho-selective beta 2-adrenoceptor agonist fenoterol was able to prevent the antigen-induced leukotriene release completely, whereas the Ca++-ionophore induced leukotriene release was only moderately inhibited. The HPLC data were confirmed by SRS-A bioassay.

Anthocyanins are a group of naturally occurring phenolic compounds related to the colouring of plants, flowers and fruits. These pigments are important as quality indicators, chemotaxonomic markers and for their antioxidant activities. Here we have investigated the therapeutic efficacy of anthocyanins contained in a blackberry extract on (i) circulatory failure, (ii) multiple organ dysfunction and (iii) activity of the inducible isoforms of nitric oxide (NO) synthase (iNOS) and cyclooxygenase (COX-2) in anaesthetised rats with endotoxic shock. In a model of endotoxic shock induced by lipopolysaccharide (LPS, E. coli, 10 mg/kg, i. v.) in the rat, pretreatment with anthocyanins present in the blackberry extract (5 mg/kg, i. v. 30 min before LPS) prevented the hypotension induced by LPS. Endotoxaemia also caused rises in the serum levels of (i) glutamyl oxaloacetic transaminase (GOT), glutamyl pyruvic transaminase (GPT), alkaline phosphates and bilirubin (hepatic dysfunction) (ii) creatinine (renal dysfunction), (iii) amylase and lipase (pancreatic injury), (iiii) NOx and 6-keto-PGF1α. Anthocyanins attenuated the hepatic and pancreatic injury, the renal dysfunction and decreased NOx and 6-keto-PGF1α levels. Endotoxaemia for 6 h resulted in a substantial increase in iNOS and COX activity in rat lung, which was attenuated in rats pretreated with anthocyanins. Moreover, anthocyanins (0.02 - 0.32 mg/mL) inhibited in vitro iNOS and COX activity from lung of LPS-treated rats. Polymorphonuclear (PMN) infiltration (myeloperoxidase activity), lipid peroxidation (malondialdehyde levels) as well as tissue injury (histological examination) induced by LPS in rat lung and ileum was reduced by anthocyanins (5 mg/kg, i. v. 30 min before LPS). Furthermore, endotoxaemia induced the formation of nitrotyrosine and poly(ADP-ribose) synthetase (PARS) activation as determined by immunohistochemical analysis of lung and ileum tissues. The degree of staining was lowered by anthocyanin treatment. These results indicate that the anthocyanins contained in the blackberry extract exert multiple protective effects in endotoxic shock.

Rat and bovine aorta rings incubated with 10−5 M dexamethasone release proteins which inhibit rat dextran oedema. These proteins seem to be related to vasocortin, derived from the peritoneal fluid of dexamethasone-treated rats, and may contribute to the control that glucocorticoids exert on vascular tonus and permeability.