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Non-steroidal anti-inflammatory drugs (NSAIDs) have repeatedly shown to be effective in tumor prevention, but important side-effects limit their wide clinical use. Nitric oxide-releasing derivatives (NO-NSAIDs) are a promising class of compounds synthesized by combining a classic NSAID molecule with an NO-releasing moiety to counteract side-effects. These new chemical entities exhibit a significantly higher activity and much lower toxicity with respect to the parental drug. In the present paper, we report the results obtained from in in vitro experimental systems aimed to evaluate the activity and mechanisms of action of the novel NO-releasing aspirin derivative, NCX 4040. The in vitro studies were carried out on a panel of human colon (LoVo, LoVo Dx, WiDr, LRWZ), bladder (HT1376, MCR), and pancreatic (Capan-2, MIA PaCa-2, T3M4) cancer cell lines. With regard to colon cancer, NCX 4040 activity was also investigated in vitro in combination with drugs currently used in clinical practice and was validated in vivo on tumor-bearing mice xenografted with the aforementioned colon cancer cell lines. The in vitro studies showed a high cytotoxic activity of NCX 4040 in all tumor histotypes and demonstrated the pivotal role of the NO component in drug activity. It was also observed that NCX 4040 exerts a pro-apoptotic activity via a mitochondria-dependent pathway. Moreover, the in vivo studies on xenografted mice further confirmed the antitumor efficacy and low toxicity of NCX 4040 in colon cancer and highlighted its role as sensitizing agent of oxaliplatin cytotoxicity.

Concerns have been raised regarding cardiovascular safety of anti-inflammatory agents, both selective cyclooxygenase (COX-2) inhibitors and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs). We describe the emerging new class of COX-inhibiting nitric oxide donors (CINOD), which have the potential to improve cardiovascular safety and spare the gastrointestinal tract. CINODs combine in the same molecule a nitric oxide (NO)-donating moiety and a well-established NSAID. Because NO protects both gastrointestinal mucosa from injury and vascular endothelial function, CINODs have the potential to be safe and effective new drugs for the treatment of pain and inflammation associated with osteoarticular disorders.

We previously showed that NCX 4040 inhibits in vitro and in vivo tumor growth and induces apoptosis in human colon cancer cell lines. On the basis of these results, NCX 4040 antitumor activity in combination with 5-fluorouracil (5-FU) or oxaliplatin was evaluated in vitro and in vivo in human colon cancer models. The cytotoxicity of different NCX 4040 and 5-FU or oxaliplatin combination schemes was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr, and LRWZ) by the sulforhodamine B assay, and apoptosis was assessed by flow cytometry. NCX 4040 and 5-FU combination was always additive in vitro regardless of the scheme used. Sequential NCX 4040→oxaliplatin treatment produced a strong synergism in three cell lines, with a ratio index ranging from 3.7 to 4. The synergistic effect was accompanied by apoptosis induction (up to 40%). In the in vivo experiments, xenografted mice were treated with the sequential combination of NCX 4040 and oxaliplatin, and apoptosis was evaluated immunohistochemically in excised tumors. Furthermore, in WiDr xenografts, this sequence caused a significantly higher reduction (∼60%) in tumor growth compared with single-drug treatments and produced extensive apoptotic cell death (15.3%), significantly higher (P < 0.01) than that observed in untreated tumors (2.7%) or in tumors treated with NCX 4040 (5.1%) or oxaliplatin (5.7%) alone. These data show that NCX 4040 sensitizes colon cancer cell lines to the effect of antitumor drugs and suggests that their combination could be useful for the clinical management of colon cancer. [Mol Cancer Ther 2006;5(4):919–26]

In more than a century of use, the benefits of aspirin have been exploited in different therapeutic areas, from inflammation to cardiovascular disease for which it is an approved treatment for acute coronary heart disease and a recommended treatment for secondary cardiovascular prevention in patients at risk. Nitric oxide (NO), a small gaseous free radical molecule, is an important messenger of cellular and biological function that demonstrates an interesting anti-inflammatory profile and vascular protective activity. In addition, NO exerts a protective action on the gastrointestinal mucosa. An elegant approach for exploiting the properties of both aspirin and NO, and for eventually increasing the overall activity of aspirin, has been to create a new molecule in which aspirin and a NO-donating group are covalently linked (NCX 4016). This molecule requires enzymatic metabolism to release both components in order to provide simultaneous actions. NCX 4016 has been shown to retain the anti-inflammatory properties of aspirin and therefore possesses the same efficacy as aspirin in animal models of pain and inflammation. In addition, the release of NO accounts for the inhibition of the release of some cytokines. In cardiovascular models, NCX 4016 shows a better efficacy than aspirin in counteracting thromboembolism, myocardial infarction and vascular reactivity and remodelling (restenosis). This review describes the pharmacological profile of the NO-donating aspirin, NCX 4016, by addressing principally the pharmacological activity in inflammation and cardiovascular models. Gastrointestinal safety is specifically addressed. The potential clinical use of NCX 4016 in vascular pathologies is also discussed.

Nitric oxide-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs) are a promising class of compounds that cause cell cycle perturbations and induce apoptosis in cell lines from different tumors. We investigated the activity of a recently developed NO-NSAID (NCX 4040) in bladder cancer cell lines (HT1376 and MCR). Cells were treated with different drug concentrations for different exposure times. Cytostatic and cytocidal activity was tested by SRB assay and apoptosis was evaluated by TUNEL analysis, ANNEXIN V assay and fluorescence microscopy. To further investigate the cell death-inducing mechanisms of NCX 4040, we analyzed gp-170, caspase expression and mitochondrial membrane potential (Delta Psi) depolarization. NCX 4040 showed a striking cytocidal activity in both cell lines, reaching LC(50) at a 10-microM and 50-microM concentrations in HT1376 and in MCR cells, respectively, after an exposure of only 6 h followed by an 18-h washout. Apoptosis was triggered in up to 90% of cells and was associated with active caspase-3 expression and Delta Psi depolarization in both cell lines after a 6-h exposure. In conclusion, NCX 4040, which probably causes apoptosis via a mitochondrial-dependent mechanism, could prove to be a useful agent for improving bladder cancer treatment.

Organic nitrates, such as nitroglycerin, have been used in clinical practice for more than one century for the treatment of angina, even before the identification of Nitric Oxide (NO) as the so-called Endothelium Derived Relaxing Factor (EDRF). Recently, multiple functions of this molecule in biology and pathophysiology have been discovered and alterations in the NO signalling pathway have often been associated with disease progression in mammals, providing a strong rationale for the use of NO as a potential drug. To have a therapeutic benefit from NO properties, an elegant approach has been designed coupling well-known existing drugs with moieties able to slowly release NO following enzymatic metabolism. "Hybrid nitrates", in which activities of both the native drug and NO are present, have been obtained with the aim of originating safer and more active drugs. The technology consists in the choice of the appropriate chemical spacer arm carrying the nitric ester in order to obtain the best pharmacodynamic and pharmacokinetic profile. The connecting linkers already explored are of different chemical structure, ranging from aliphatic chains to heteroaromatic rings. The molecules so far obtained have already demonstrated their potential therapeutic interest in both pharmacological tests and clinical trials. In this review, we describe the approach and the possibility of generating new chemical entities, combining well-known drugs with an NO-donating moiety in order to increase activity and safety, along with examples of their activity and potential therapeutic application in different pathologies. A few significant examples of molecules in the early preclinical stage, as well as in advanced clinical development will be described.

Our work aimed at identifying the antitumoral potential of new nitric oxide (NO)-releasing non-steroidal anti-inflammatory drug (NSAID) derivatives on human prostate and bladder carcinoma cell lines. Among all molecules tested, two sulindac derivatives, NCX 1102 ((Z)-5-fluoro-2-methyl-1-[[4-(methylsulfinyl)phenyl] methylene]-1H-indene-3-acetic acid 4-(nitrooxy)butyl ester) and NCX 1105 ((Z)-5-fluoro-2-methyl-1-[[4-(methylsulfinyl)phenyl] methylene]-1H-indene-3-acetic acid 6-(nitrooxymethyl)-2-methylpyrydyl ester hydrochloride), were the most cytotoxic compounds. In contrast to its parent molecule sulindac, cell cycle analysis showed that NCX 1102 led to cell accumulation in the G2-M transition stage in all cell lines, and induced apoptosis in five out of the six cell lines. Thus, NO-NSAIDs may be useful for the elaboration of new therapeutic strategies in the management of bladder and prostate cancer.

The influence of cyclooxygenase pathway activation following thromboxane-endoperoxide (TP) receptor stimulation was studied in rat mesenteric resistance arteries (n=6 to 10 per group). We studied isolated, perfused, and pressurized mesenteric resistance arteries (mean internal diameter 214 μm) using an arteriograph, enabling us to study arteries in physiological conditions of flow and pressure. Changes in diameter were continuously recorded, and contractions measured as internal diameter reduction. Release of cyclooxygenase pathway metabolites was also assessed by enzyme immunoassay (EIA) analysis of mesenteric bed perfusions. The thromboxane A2 (TxA 2 ) analog U-46619 (1 μmol/L) induced a significant contraction (108 μm maximal diameter reduction). Inhibition by 3 chemically different cyclooxygenase inhibitors (ie, flurbiprofen, indomethacin, and aspirin) potently reduced the contraction to 27%, 25%, and 6% of control, respectively. The selective cyclooxygenase-1 inhibitor SC-58560 inhibited U-46619 contraction, whereas selective cyclooxygenase-2 inhibition (SC-58236) had no effect. Thromboxane synthase inhibition (furegrelate) did not affect U-46619-induced contraction, but it was reduced by cytosolic phospholipase A2 inhibition. Measurement of cyclooxygenase derivatives produced by the isolated mesenteric bed showed that PGE 2 was produced after TxA 2 -receptor stimulation with U-46619. Exogenous prostaglandin E 2 (in the presence of the TxA 2 receptor antagonist SQ 29 548) and U-46619 contracted mesenteric arteries with a similar potency (EC 50 : 0.30 and 0.48 μmol/L, respectively). This study provides the first evidence that TxA 2 -receptor-dependent contraction in a resistant artery involved cyclooxygenase stimulation and, at least in part, a PGE 2 formation. This mechanism of TxA 2 -dependent contraction in resistant arteries might be of importance in the understanding of diseases affecting resistant arteries and involving TxA 2 , such as hypertension.

Non-steroidal anti-inflammatory drugs (NSAIDs) are potent antitumoral agents but their side effects limit their clinical use. A novel class of drugs, nitric oxide-donating NSAIDs (NO-NSAIDs), was found to be safer and more active than classical NSAIDs. This study explored the effect of the NO-donating sulindac derivative, NCX 1102, on three human urothelial epithelial carcinoma cell lines (T24, 647V, and 1207) and primary cultures of normal urothelial cells. Cytotoxicity, antiproliferative effect, cell cycle alterations, morphological changes, and apoptosis were investigated after treatment with NCX 1102 in comparison with the native molecule. After treatment, there was a cytotoxic effect (with IC50 at 48 h of 23.1 μm on 647V, 19.4 μm on T24, and 14.5 μm on 1207) and an antiproliferative effect on all three cell lines with NCX 1102 but not with sulindac. No effect was detected on normal urothelial cells. Flow cytometric analysis showed a differential NCX 1102-induced accumulation of cells in various phases of the cell cycle, depending on cell line and concentration. NCX 1102 induced an occurrence of multinucleated cells in all cell lines and mitotic arrest in 647V and 1207. NCX 1102-treated T24 and 647V cell lines showed a significant difference of apoptotic cell amount when compared to controls. Our results demonstrated a greater antiproliferative potency of NCX 1102 compared to its parent molecule sulindac, and suggested that this new NO-NSAID may have therapeutic impact in the management of bladder cancer.

Thromboxane A2 (TxA2) is a potent proaggregating, vasoconstrictor agent produced in many physiological and pathological situations. Although mitogen-activated protein (MAP) kinases [MAPK (ERK1/2 and p38)] have been shown to be activated after endoperoxide/thromboxane receptor (TP) stimulation, no study has investigated their potential role in resistance arteries, especially in physiological conditions of pressure and flow in which the arteries can contract. Thus, responses to TP stimulation by the stable agonist U46619 were studied in isolated rat mesenteric resistance arteries (inner diameter 262 ± 5 µm) mounted in an arteriograph. Changes in diameter were recorded under physiological levels of flow (90 µl/min) and pressure (50 mm Hg). TP stimulation induced a concentration-dependent contraction (EC50 value of 1.94 ± 0.22 × 10–7M), without desensitization. U46619-induced contraction was inhibited by calcium entry blockade (nifedipine) and protein kinase C inhibition (GF109203X), but it was not affected by tyrosine kinase inhibition (tyrphostin A25). MAPKK (MEK) inhibition (PD98059) did not alter U46619-dependent contraction, although ERK1/2 MAPK were activated. By contrast, p38 MAPK inhibition (SB203580) dose-dependently inhibited the contraction, and Western blot analysis showed activation of p38 MAPK in arteries contracted with U46619. Activation of p38 MAPK by U46619 was inhibited by nifedipine and in the absence of extracellular calcium. This study brings new insights in the transduction pathway involved in the contractile response of resistance arteries to TxA2/endoperoxide receptor stimulation. This contraction requires p38 MAPK activation, but did not involve ERK1/2 MAPK activation although both were activated.

Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis. Recently, cholesterol-independent effects of statins have been described. In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells. Treatment of cells with 25 microm mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production. Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect. GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2. Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1alpha-dependent COX-2 induction. These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.

We studied the effect of endogenous and exogenous prostaglandin E(2) (PGE(2)), a metabolite of arachidonic acid through the cyclooxygenase (COX) pathway, on interleukin (IL)-1 beta-induced COX-2 expression, using primary cultures of human bronchial smooth-muscle cells (HBSMC). Treatment with exogenous PGE(2) resulted in enhanced expression of IL-1 beta-induced COX-2 protein and messenger RNA (mRNA) as compared with the effect of the cytokine per se. Inhibition of PGE(2) production with a nonselective COX inhibitor (flurbiprofen, 10 microM) resulted in a significant reduction in IL-1 beta- induced COX-2 expression, supporting a role of endogenous COX metabolites in the modulation of COX-2 expression. None of the experimental conditions used in the study affected the expression of constitutive cyclooxygenase (COX-1). Treatment with cycloheximide to inhibit translation, and with dexamethasone or actinomycin D to inhibit transcription, linked the effect of PGE(2) to the transcriptional level of COX-2 mRNA rather than to a potential effect on protein and/or mRNA stabilization. PGE(2) increased adenylate cyclase activity in a concentration dependent manner, and forskolin, a direct activator of adenylate cyclase, caused a marked increase in IL-1 beta-dependent COX-2, suggesting the existence of a causal relationship between the two events. The same results were observed with salbutamol, a bronchodilator that acts by increasing cyclic adenosine monophosphate. The effect of PGE(2) on COX-2 expression may contribute to the hypothesized antiinflammatory role of PGE(2) in human airways, providing a self-amplifying loop leading to increased biosynthesis of PGE(2) during an inflammatory event.

1. Cysteinyl-leukotrienes (cysteinyl-LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment. 2. We investigated the pharmacological profile of the cysteinyl-LT antagonists CGP 45715A (iralukast), a structural analogue of LTD4 and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips. 3. Competition curves vs [3H]-LTD4 on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [3H]-LTD4; (b) as in all the G-protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD4 (Ki1=Ki2= 16.6 nM+/-36% CV and Ki1= Ki2 = 5.7 nM+/-19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency. 4. In functional studies: (a) iralukast and CGP 57698 antagonized LTD4-induced contraction of human bronchi, with pA2 values of 7.77+/-4.3% CV and 8.51+/-1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD4 response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition. 5. Both antagonists significantly inhibited antigen-induced contraction of human isolated bronchial strips in a concentration-dependent manner, lowering the upper plateau of the anti-IgE curves. 6. In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD4 in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these compounds might be useful drugs for the therapy of asthma and other allergic diseases.

New stable leukotriene A 4 analog with acetylenic function in positions 14,15 has been synthesized. Conversion of this analog to the corresponding LTC 4 derivative and the structural and biological characterization are presented.

The reactive intermediate formed by 5-lipoxygenase metabolism of arachidonic acid, leukotriene A4, is known to be released from cells and subsequently taken up by other cells for biochemical processing. The objective of this study was to determine the relative amount of leukotriene A4 synthesized by human polymorphonuclear leukocytes (PMNL) that is available for transcellular biosynthetic processes. This was accomplished by diluting cell suspensions and measuring the relative amounts of enzymatic versus nonenzymatic leukotriene A4-derived metabolites after challenge with the Ca2+ ionophore A23187. Nonenzymatic leukotriene A4-derived metabolites were used as a quantitative index of the amount of leukotriene A4 released into the extracellular milieu. The results obtained demonstrated that in human PMNL, the relative amounts of nonenzymatic versus enzymatic leukotriene A4-derived metabolites increased with decreasing cell concentrations. After a 20-fold dilution of PMNL in cell preparations, a doubling in the amount of nonenzymatic leukotriene A4-derived metabolites was observed following challenge (from 53.9 +/- 1.3 to 110.4 +/- 8.9 pmol/10(6) PMNL, p0.01). Reduction of possible cell-cell interactions by dilution suggested that over 50% of leukotriene A4 synthesized is released from the PMNL. These data provide evidence that, in human PMNL preparations, transfer of leukotriene A4 to neighboring PMNL is taking place, resulting in additional formation of leukotriene B4 and its omega-oxidized metabolites 20-hydroxy- and 20-carboxy-leukotriene B4. Neutrophil reuptake of extracellular leukotriene A4 leads to an underestimation of the fraction of leukotriene A4 that is in fact available for transcellular metabolism when tight cell-cell interactions occur, such as during PMNL adhesion to the microvascular endothelium and diapedesis.

Cyclooxygenase (COX)-inhibiting nitric oxide (NO) donors (CINODs) are designed to inhibit COX-1 and COX-2 while releasing NO. COX inhibition is responsible for anti-inflammatory and pain-relieving effects, whereas NO donation can improve microcirculation and exert anti-inflammatory and antioxidant actions. In an in vivo mouse model of bleomycin-induced lung fibrosis, we evaluated whether a prototype CINOD compound, (S)-(5S)-5,6-bis(nitrooxy)hexyl)2-(6-methoxynaphthalen-2-yl)propanoate (NCX 466), may show an advantage over naproxen, its congener drug not releasing NO. Bleomycin (0.05 IU) was instilled intratracheally to C57BL/6 mice, which were then treated orally with vehicle, NCX 466 (1.9 or 19 mg/kg), or an equimolar dose of naproxen (1 or 10 mg/kg) once daily for 14 days. Afterward, airway resistance, assumed as lung stiffness index, was assayed, and lung specimens were collected for analysis of lung inflammation and fibrosis. NCX 466 and naproxen dose-dependently prevented bleomycin-induced airway stiffness and collagen accumulation. NCX 466, at the highest dose, was significantly more effective than naproxen in reducing the levels of the profibrotic cytokine transforming growth factor-β and the oxidative stress markers thiobarbituric acid reactive substance and 8-hydroxy-2'-deoxyguanosine. NCX 466 also decreased myeloperoxidase activity, a leukocyte recruitment index, to a greater extent than naproxen. A similar inhibition of prostaglandin E₂ was achieved by both compounds. In conclusion, NCX 466 has shown a significantly higher efficacy than naproxen in reducing lung inflammation and preventing collagen accumulation. These findings suggest that COX inhibition along with NO donation may possess a therapeutic potential in lung inflammatory diseases with fibrotic outcome.

Background & Aims Proton pump inhibitors (PPIs) and nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used classes of drugs, with the former frequently coprescribed to reduce gastroduodenal injury caused by the latter. However, suppression of gastric acid secretion by PPIs is unlikely to provide any protection against the damage caused by NSAIDs in the more distal small intestine. Methods Rats were treated with antisecretory doses of omeprazole or lanzoprazole for 9 days, with concomitant treatment with anti-inflammatory doses of naproxen or celecoxib on the final 4 days. Small intestinal damage was blindly scored, and changes in hematocrit were measured. Changes in small intestinal microflora were evaluated by denaturing gradient gel electrophoresis and reverse-transcription polymerase chain reaction. Results Both PPIs significantly exacerbated naproxen- and celecoxib-induced intestinal ulceration and bleeding in the rat. Omeprazole treatment did not result in mucosal injury or inflammation; however, there were marked shifts in numbers and types of enteric bacteria, including a significant reduction (∼80%) of jejunal Actinobacteria and Bifidobacteria spp. Restoration of small intestinal Actinobacteria numbers through administration of selected ( Bifidobacteria enriched) commensal bacteria during treatment with omeprazole and naproxen prevented intestinal ulceration/bleeding. Colonization of germ-free mice with jejunal bacteria from PPI-treated rats increased the severity of NSAID-induced intestinal injury, as compared with mice colonized with bacteria from vehicle-treated rats. Conclusions PPIs exacerbate NSAID-induced intestinal damage at least in part because of significant shifts in enteric microbial populations. Prevention or reversal of this dysbiosis may be a viable option for reducing the incidence and severity of NSAID enteropathy.

Summary The cytochrome P-450 enzyme system was studied in juvenile Acipenser naccarii (317.6±77 g initial live weight) reared at 23°C and at a standard diet over a period of two month before using for experiments (final weight 546.3±148.8 g). While determining normal levels of cytochrome P-450 in liver tissue its induction was also studied under exposure to P-naphthoflavone (BNF; single dose at 80 mg/Kg body weight 52 hrs prior to sampling). Control fish showed total cytochrome P-450 levels in liver microsomes around 0.40±0.12 nmoles/mg proteins (n=12) while in BNF exposed fish the values increased significantly (0.55±0.11nmoles/mg proteins; n=12). Ethoxyresorufin-O-deethylase (EROD) in control fish was 0.015±0.02 nmoles/mg proteins/min (n=12); it was found to be NADPH dependent and inducible by BNF. Western Immunoblotting and enzyme-linked-immunosorbent (ELISA) analyses of liver microsomes from untreated and BNF-exposed fish, using an antibody to rat cytochrome P-450 1A1 isoform (P-450 1A1) (Nelson et al.,1996) suggested the presence of a constitutive and BNF-inducible cytochrome P-450 homologous to rat P-450 protein. Measurement of cytochrome P-450-dependent EROD activity and P-450 1A1 expression levels in A. naccarii may be a useful parameter in monitoring environmental pollution.

Nonsteroidal anti-inflammatory drugs (NSAIDs) remain the most commonly used medications for the treatment of the symptoms of many chronic inflammatory diseases, including osteoarthritis. Unfortunately, the toxicity of NSAIDs substantially limits their long-term use. Some newer NSAIDs, namely selective cyclooxygenase (COX)-2 inhibitors, exhibit greater gastrointestinal safety, and concomitant use of anti-secretory drugs can also reduce NSAID-induced gastropathy. However, NSAIDs also adversely affect the cardiovascular system. A new class of anti-inflammatory drugs, COX-inhibiting nitric oxide donators (CINODs), has been designed to exert similar anti-inflammatory effects as NSAIDs, but with an improved safety profile. CINODs release nitric oxide, providing protective effects in the gastrointestinal tract and attenuating the detrimental effects on blood pressure normally associated with NSAIDs. We provide an outline of the rationale for CINODs and their activity, in addition to an overview of the pre-clinical and clinical profile of the most advanced CINOD, naproxcinod.

Background: Topical nitric oxide-releasing dexamethasone (NCX1021) may avoid the negative effects of dexamethasone phosphate. Aims: To obtain more information on the role of nitric oxide in glaucoma and to compare a nitric oxide-releasing dexamethasone with dexamethasone phosphate with regard to intraocular pressure (IOP) and ocular haemodynamics in an experimental rabbit model. Methods: Six rabbits were treated with dexamethasone phosphate 0.1% in the right eye and with NCX1021 in the left eye for 5 weeks. The parameters considered were IOP, nitric oxide marker levels in aqueous humour, ocular haemodynamics of ophthalmic artery (by means of colour Doppler imaging), expression of endothelial nitric oxide synthase (eNOS)in ciliary processes and histology of ciliary bodies. Results: Dexamethasone increased IOP levels, NCX1021 did not. Nitrite and cyclic guanosine monophosphate levels in aqueous humour were lowered by dexamethasone and increased by NCX1021. Resistivity index of the ophthalmic artery was increased, eNOS expression was reduced and ciliary bodies showed histological lesions in dexamethasone-treated eyes, not in NCX1021-treated ones. Conclusions: NCX1021 may avoid the IOP increase, impairment of ocular blood flow and the morphological changes in the ciliary bodies possibly induced by corticosteroid treatment.

Although non steroidal antiinflammatory drugs (NSAIDs) have been shown to be effective as chemopreventive agents, important side-effects limit their clinical use. A promising novel class of drugs, nitric oxide-donating NSAIDs (NO-NSAIDs), has been found to be more active than classical NSAIDs. This study explored the effect of the NO-donating aspirin derivative, NCX 4040, on three human pancreatic adenocarcinoma cell lines (Capan-2, MIA PaCa-2 and T3M4). NCX 4040 activity was compared with that of NCX 4016 (an NO(2)-positional isomer of NCX 4040), SNAP (a standard NO-releasing molecule), NCX 4042 (denitrated analog of NCX 4040), and aspirin. NCX 4040 showed a striking cytocidal activity in all cell lines, already inducing significant percentages of apoptotic cells at 10 muM in Capan-2 cell lines. This study focused on the biological mechanisms of sensitivity and resistance to NCX 4040, highlighting that the cytotoxic action of this drug may be due to the hyperexpression of Bax, its translocation to the mitochondria, the release of Cytochrome C, and the activation of caspases-9 and -3, overall in a p53-independent manner. Moreover, the use of a specific COX-2 inhibitor (NS 398) in the experimental models showed that COX-2 hyperexpression could partially explain the resistance mechanisms to NCX 4040.

Active suppression of tumor-specific T lymphocytes can limit the immune-mediated destruction of cancer cells. Of the various strategies used by tumors to counteract immune attacks, myeloid suppressors recruited by growing cancers are particularly efficient, often resulting in the induction of systemic T lymphocyte dysfunction. We have previously shown that the mechanism by which myeloid cells from tumor-bearing hosts block immune defense strategies involves two enzymes that metabolizel-arginine: arginase and nitric oxide (NO) synthase. NO-releasing aspirin is a classic aspirin molecule covalently linked to a NO donor group. NO aspirin does not possess direct antitumor activity. However, by interfering with the inhibitory enzymatic activities of myeloid cells, orally administered NO aspirin normalized the immune status of tumor-bearing hosts, increased the number and function of tumor-antigen-specific T lymphocytes, and enhanced the preventive and therapeutic effectiveness of the antitumor immunity elicited by cancer vaccination. Because cancer vaccines and NO aspirin are currently being investigated in independent phase I/II clinical trials, these findings offer a rationale to combine these treatments in subjects with advanced neoplastic diseases.

1 The concept that nitric oxide (NO) release can be beneficial in inflammatory conditions has raised more attention in the recent years, particularly with the development of nitric oxide-releasing anti-inflammatory drugs. There is considerable evidence that NO is capable of enhancing the anti-inflammatory benefits of conventional anti-inflammatory drugs. 2 Since hydrocortisone is the most widely used anti-inflammatory drug for the treatment of skin inflammation, we compared the anti-inflammatory effects of hydrocortisone to an NO-releasing derivative of hydrocortisone, NCX 1022, in a murine model of irritant contact dermatitis, induced by epidermal application of benzalkonium chloride. 3 Topical pre- and post-treatment with NCX 1022 (3 nmol) in C57BL6 mice not only reduced ear oedema formation in a dose-dependent manner, but also was significantly more effective than the parent compound during the initial stages of inflammation (from 1 to 5 h). NCX 1022, but not hydrocortisone, significantly inhibited granulocyte recruitment (tissue myeloperoxidase activity). Histological samples of mouse ears treated with NCX 1022 showed significant reduction in both the number of infiltrated cells and disruption of the tissue architecture compared to hydrocortisone-treated tissues. 4 With intravital microscopy, we observed that both pre- and post-treatments with NCX 1022 were more effective than hydrocortisone in terms of inhibiting benzalkonium chloride-induced leukocyte adhesion to the endothelium, without affecting the flux of rolling leukocytes or venule diameter. 5 These results suggest that by releasing NO, NCX 1022 modulates one of the early events of skin inflammation: the recruitment of leukocytes to the site of inflammation. Overall, we have shown that NO-hydrocortisone provided faster and greater protective effects, reducing major inflammatory parameters (leukocyte adhesion and recruitment, oedema formation, tissue disruption) compared to its parental compound.

BACKGROUND The aim of our study was to explore the anti-tumoral potential of the Nitric Oxide-Donating Non-Steroidal Anti-Inflammatory Drugs (NO-NSAID) NCX1102 (nitrosulindac), on three human prostatic epithelial cell lines at varying degree of transformation (PNT1A, LNCaP, and PC3). METHODS Cytotoxicity, anti-proliferative effects, cell-cycle alterations, morphological changes, and apoptosis were investigated after treatment with nitrosulindac in comparison to the native molecule sulindac. Involvement of the polyamine pathway in the action of nitrosulindac was also examined. RESULTS Nitrosulindac but not sulindac exerted a cytotoxic effect on all cell lines and an anti-proliferative effect on LNCaP and PC3 cells only. Nitrosulindac differentially altered the cell cycle, induced mitotic arrest and displayed a pro-apoptotic activity in all cell lines. Finally, the polyamine pathway does not seem to be involved in the mechanism of nitrosulindac action. CONCLUSIONS Our results demonstrate the anti-proliferative and proapoptotic activity of nitrosulindac on prostate cancer cell lines and suggest its potential interest for new strategies in the management of prostate cancer. © 2004 Wiley-Liss, Inc.

For decades, aspirin has represented the elective treatment for inflammatory-based pathologies, due to its antipyretic and anti-inflammatory activities [1].

Cysteine-containing leukotrienes (cysteinyl-LTs) are potent bronchoconstrictors and play a key role in asthma. We found that histamine and LTD 4 markedly constrict strips of human bronchi (HB) with similar efficacy. However, in human airway smooth-muscle (HASM) cells, LTD 4 , at variance with histamine, elicited only a small, transient change in intracellular calcium ion concentration. HASM cells express both Ca 2 1 -dependent and -independent isoforms of protein kinase C (PKC) (i.e., PKC- a and PKC- « ). Western blot analysis showed that PKC- a is activated by histamine and, to a lesser extent, by LTD 4 , whereas only LTD 4 translocates PKC- « . This translocation was specifically inhibited by the LTD 4 antagonist pobilukast. Phorbol-dibutyrate ester (PDBu) (a PKC activator) contracted HB strips to the same extent in the presence as in the absence of extra- and intracellular Ca 2 1 . In the absence of Ca 2 1 , LTD 4 contracted HB strips to the same extent as did PDBu, suggesting the involvement of a Ca 2 1 -independent PKC in LTD 4 -mediated signal transduction. PDBu-induced desensitization and the PKC inhibitor H7 abolished the slow and sustained LTD 4 -triggered contraction of HB strips in the absence of Ca 2 1 , although H7 did not greatly affect the response in the presence of the ion. Thus, in human airways, we identified a novel LTD 4 transduction mechanism linked to bronchial smooth-muscle contraction, which is partly independent of Ca 2 1 and involves the activation of PKC- « .

Cysteinyl-containing leukotrienes (cysteinyl-LTs) produce bronchoconstriction, mucus hypersecretion, and pro-inflammatory effects (Dahlen et al., 1980; Dahlen et al., 1981; Foster C von Sprecher et al., 1991; von Sprecher et al., 1996).

The renin-angiotensin system is an important modulator of arterial blood pressure and inhibitors of the angiotensin-converting enzyme (ACE-Is) and are currently used in the treatment of hypertension. The pleiotropic actions exerted by angiotensin II (AngII) on the functionality of the vessel wall may have pro-atherosclerotic outcomes; evidence for an anti-atherosclerotic effect of ACE-Is has been presented and an antioxidant effect has been attributed to thiol-containing ACE-Is, like Captopril. The present study has been undertaken to investigate the effect of Delapril, a lipophilic ACE-I, on the development of atherosclerosis in cholesterol-fed rabbits. While it did not correct hyperlipidemia, Delapril dose dependently inhibited the development of atherosclerosis, expressed as aortic area covered by lesions (23.3+/-4.1, 21.3+/-2.4 and 18.5+/-3.3% with Delapril at the daily dose of 5, 10 and 20 mg/kg, respectively, versus 38.2%+/-6.4 for control animals) and its effect was similar to that of Captopril (14.5+/-5.1% at the daily dose of 25 mg/kg). Furthermore, Delapril partially and dose dependently restored endothelium-dependent relaxation, which is impaired in vessels from hypercholesterolemic animals (51.80+/-12.18, 59.74+/-5.16, 69.13+/-8.70 maximal percent relaxation versus 48.26+/-3.05% for the untreated control and 67.67+/-6.72% for Captopril-treated animals). An antioxidant mechanism is unlikely to explain this data, since Delapril does not contain thiol groups. These observations suggest that Delapril may represent an effective pharmacological approach for the treatment of atherosclerosis during its early phases.

Sulphido-peptide leukotrienes (cys-LT, LTC4, D4 and E4), are products of the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism and are intimately involved in the genesis of inflammatory responses, due to their vasopermeant properties that cause plasma extravasation and diapedesis of white cells into perivascular tissues [1]. The generation of cys-LT exhibits remarkable cellular specificity; polymorphonuclear leukocytes (PMNL) generate predominantly the unstable epoxide LTA4 which is then transformed to leukotriene B4 (LTB4) [2], whereas mast cells and eosinophils show preferential generation of cys-LT.

A variety of inflammatory cells synthesize cys-leukotrienes (cys-LTs) C4, D4 and E4 in response to biological and non-biological stimuli1: eosinophils, basophils and mast cells2,3 are able to synthesize cys-LTs from arachidonic acid but cys-LTs can also be produced through transcellular metabolism from neutrophil-derived LTA4, by vascular endothelial cells4-6 and platelets7.

Quantitation of cysteinyl leukotriene production and smooth muscle contraction upon immunological challenge of isolated human bronchi was evaluated. Analysis of picomole amounts of leukotriene C 4 , D 4 , and E 4 was achieved using HPLC separation and enzyme immunoassay quantitative determination. The aim of the study was to correlate the contraction of airway smooth muscle and cysteinyl leukotriene production with and without 5-lipoxygenase inhibition. In human isolated bronchial tissue treated with indomethacin and pyrilamine to make their contractile responses leukotriene dependent only, the novel 5-lipoxygenase inhibitor 5,6-Dihydroxy-2-( N , N -Dimethylhydrazino)-1,2,3,4-tetrahydro-naphtalene bromide (CHF 1909) caused a concentration-dependent inhibition of the immunologically induced contraction, showing an IC 50 value of 13 ± 2.2 μM (mean ± CV). At the concentration of 30 (μM, this compound caused more than 90% inhibition of the maximal bronchoconstriction in vitro , and inhibited cysteinyl leukotriene production by 90% as well. Contemporary measurement of immunologically induced contraction and production of cysteinyl leukotrienes in isolated human bronchi provided a direct correlation between smooth muscle contraction and synthesis of leukotriene C 4 , D 4 , and E 4 .

Asthma is a complex disease with the following characteristics: (1) airway obstruction that is reversible (but not completely so in some patients) either spontaneously or with treatment; (2) airway inflammation; and (3) increased airway responsiveness to a variety of stimuli (National Heart, Lung, and Blood Institute; National Asthma Education Program; Guidelines for the diagnostic and management of asthma, March 11, 1991). Recent observations in both human subjects and animal models suggest that the abnormalities of asthma are manifestations of airway inflammation that is a key factor even in patients with asthma at a clinically early stage (Laitinen et al., 1993).

real-time reverse transcription-polymerase chain reaction . To clarify the role of HMGB1 in healing of gastric ulcer, the mice were intraperitoneally administered mouse recombinant HMGB1 (rHMGB1), anti-HMGB1 neutralizing antibodies or nonspecific chicken IgY after the induction of ulcer.Results: At day 4 after ulcer induction, mean ulcer sizes did not differ among the 4 groups. Endothelial cells and inflammatory cells such as macrophages in the ulcerous region showed immunoreactivity for TLR2 and TLR4 and RAGE proteins. By day 9, the ulcer size in RAGE KO mice and TLR4 KO mice was 40% smaller than that in wild-type mice, while there was no difference in ulcer sizes between wild-type mice and TLR2 KO mice. Deficiency of RAGE and TLR4 markedly inhibited the increase in expression of TNF-αmRNA in ulcerous tissue. Administration of rHMGB1 delayed the healing of gastric ulcer (ulcer index: 1.39 ± 0.30 mm2 in the vehicle-treated mice and 4.52 ± 0.35 mm2 in rHMGB1-treated mice), accompanied by increase in expression of TNF-α mRNA and MPO activities in ulcerous tissue, while administration of anti-HMGB1 antibody enhanced ulcer healing by day 9 (ulcer index: 2.90 ± 0.86 mm2 in non-specific IgY-treated mice, 1.46 ± 0.80 mm2 in neutralizing antibody for HMGB1-treated mice). Conclusion: These results suggest that HMGB1 is a critical factor that delays healing of gastric ulcer not via TLR2 but via TLR4 and RAGE.

Nitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity. In vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks. In the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO2 group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all. NCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.

BackgroundDespite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, inin vitrohuman colon cancer models.MethodsThe effect on tumor growth was evaluated in four human colon cancer cell lines (LoVo, LRWZ, WiDr and LoVo Dx) by sulforhodamine B assay, oxidative stress by immunohistochemistry, apoptosis by laddering assay, mitochondrial membrane potential (ΔΨm) by flow cytometry, and apoptosis- and chemoresistance-related markers by western-blot and real-time method, respectively. Prostaglandin E2levels were determined by ELISA.ResultsNCX 4040 produced a higher cytotoxic effect in all the cell lines than that produced by other NO donors tested. In particular, in LoVo and LRWZ cells, NCX 4040 induced a cytocidal effect and apoptosis through p53 and NAG-1 expression, an early ΔΨmcollapse, and a sequential release of cytoplasmatic cytochrome c and caspase -9 and -3 active forms. 8-hydroxyguanine lesions, indicative of oxidative stress, were also observed. Conversely, in WiDr line, the drug caused a cytocidal effect, albeit not through apoptosis, and a concomitant increase in COX-2 activity. In LoVo Dx line, characterized by high levels drug resistance and DNA repair-related markers, only a cytostatic effect was observed, again in concomitance with the increase in COX-2 enzyme activity.ConclusionThis study highlights the multiplicity of mechanisms involved in sensitivity or resistance to NCX 4040 and could provide useful indications for tailored therapy by identifying potentially drug-responsive tumors.