NCX4016 (NO‐Aspirin) has multiple inhibitory effects in LPS‐stimulated human monocytes

Department of Biomedical and Surgical Sciences, University of Verona, Medicina Interna C, Policlinico G.B. Rossi, Piazzale L.A. Scuro, 10, 37134 Verona, Italy.
British Journal of Pharmacology (Impact Factor: 4.84). 11/2001; 134(4):905-11. DOI: 10.1038/sj.bjp.0704326
Source: PubMed

ABSTRACT NCX4016 (2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester, NicOx S.A., France) is an anti-thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO.
We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A2 metabolite TXB2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes.
Both ASA and NCX4016 1 – 1000 μmol l−1 dose-dependently reduced TXB2 concentration, measured by RIA in the supernatant of 10 μg ml−1 LPS-stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 μmol l−1: −86.0±10.1%, NCX4016 300 μmol l−1: −92.2±9.0%, ASA 30 μmol l−1: −92.3±7.5%, ASA 300 μmol l−1: −97.3±1.0%, n=6, M±s.d.). Most of the activity of NCX4016 up to 100 μmol l−1 was prevented by 10 μmol l−1 ODQ, inhibitor of cyclic GMP.
NCX4016 100 – 300 μmol l−1 reduced TNF-α (NCX4016 300 μmol l−1=−77.2±19.9%, n=6) and IL-6 (NCX4016 300 μmol l−1: −61.9±15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects.
TF activity (NCX4016 300 μmol l−1: 53.7±39.9%, n=4) and immunoreactive TF (NCX4016 300 μmol l−1: −93.9±7.9%, n=7), measured in the supernatant of stimulated cells, were also dose-dependently inhibited by NCX4016 but not by ASA.
The present results indicate that NCX4016 inhibits TXA2 generation as well as cytokine release and TF in human monocytes partly via NO-dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero-thrombosis.
British Journal of Pharmacology (2001) 134, 905–911; doi:10.1038/sj.bjp.0704326

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Available from: Alessandro Lechi, Sep 28, 2015
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    • "In this study, aspirin did not significantly reduce TNFα release from LPS-stimulated monocytes or macrophages. This is consistent with a similar study in which aspirin failed to have an effect even at a 30-fold higher concentrations than was used in the present study [50]. A further study did report an inhibitory effect of aspirin on TNFα release from LPS-stimulated monocytes but this was at concentrations of 5–10 mM [49], which may not be representative of pharmacologically relevant plasma concentrations [51]. "
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    ABSTRACT: The cytoprotective nature of nitric oxide (NO) led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNFalpha release from lipopolysaccharide (LPS)-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-kappaB) activation. Peripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 muM), their respective furazan NO-free counterparts (B16, B15; 10 muM), aspirin (10 muM), existing nitroaspirin (NCX4016; 10 muM), an NO donor (DEA/NO; 10 muM) or dexamethasone (1 muM), in the presence and absence of LPS (10 ng/ml; 4 h). Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNFalpha release and by lactate dehydrogenase (LDH) assay for cell necrosis. To assess NF-kappaB activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-kappaB, IkappaBalpha (assessed by western blotting) and nuclear localisation (assessed by immunofluorescence) of the p65 subunit of NF-kappaB were determined. B8 significantly reduced TNFalpha release from LPS-treated macrophages to 36 +/- 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNFalpha release to 28 +/- 5, and 49 +/- 9% of control, respectively. The B8 effect was equivalent in magnitude to that of dexamethasone, but was not shared by 10 muM DEA/NO, B7, the furazans, aspirin or NCX4016. LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic IkappaBalpha and nuclear translocation of the p65 NF-kappaB subunit was inhibited by the active NO-furoxans. Here we show that furoxan-aspirin, B8, significantly reduces TNFalpha release from both monocytes and macrophages and suggest that inhibition of NF-kappaB activation is a likely mechanism for the effect. This anti-inflammatory action highlights a further therapeutic potential of drugs of this class.
    Journal of Inflammation 02/2008; 5(1):12. DOI:10.1186/1476-9255-5-12 · 2.02 Impact Factor
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    • "NCX4016 induced inhibition of capsase-1 is mediated through S-nitrosylation of a sulphydryl group (Dimmeler et al., 1997), therefore, other NO-NSAIDs, such as S-nitroso-diclofenac (see Figure 6a) may be more effective inhibitors of capsase-1 through transnitrosation reactions. Nitroaspirins also inhibit the release of TNF-a from lipopolysaccharide-stimulated macrophages (Minuz et al., 2001), although it is difficult to determine whether this is a direct effect of NO or through inhibition of other cytokines. Regardless, this property would likely provide benefits in the treatments of a range of inflammatory diseases, above that of conventional aspirin (Fiorucci and Del Soldato, 2003). "
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    ABSTRACT: During the 1980s, the free radical, nitric oxide (NO), was discovered to be a crucial signalling molecule, with wide-ranging functions in the cardiovascular, nervous and immune systems. Aside from providing a credible explanation for the actions of organic nitrates and sodium nitroprusside that have long been used in the treatment of angina and hypertensive crises respectively, the discovery generated great hopes for new NO-based treatments for a wide variety of ailments. Decades later, however, we are still awaiting novel licensed agents in this arena, despite an enormous research effort to this end. This review explores some of the most promising recent advances in NO donor drug development and addresses the challenges associated with NO as a therapeutic agent. British Journal of Pharmacology (2007) 151, 305–321; doi:10.1038/sj.bjp.0707224
    British Journal of Pharmacology 06/2007; 151(3):305-21. DOI:10.1038/sj.bjp.0707224 · 4.84 Impact Factor
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    ABSTRACT: Resumen La incidencia de la obesidad y de la resistencia a la insulina con sus complicaciones asociadas, como la hipertensión arterial y el aumento de la morbi-mortalidad cardiovascular, alcanzan hoy en día proporciones epidémicas y representan un problema mayor de salud pública. En los últimos años se ha demostrado que la administración de insulina, además de sus efectos metabólicos, posee efectos cardiovasculares importantes. El sistema nervioso simpático y el sistema L-arginina -óxido nítrico son los mediadores centrales de estas acciones cardiovasculares de la insulina. Mostramos, gracias a estudios realizados en animales y en humanos, que no sólo un déficit de la síntesis del óxido nítrico (NO), sino también un aumento exagerado en su producción representan un defecto subyacente central de las anomalías metabólicas, cardiovasculares y del sis-tema nervioso simpático que caracterizan a la insulino resistencia. Mostramos cómo estos resultados estable-cen el fundamento científico para la utilización de sustancias farmacológicas capaces de liberar de manera pro-longada cantidades fisiológicas de NO o de inhibidores de su sobreproducción como futuros tratamientos para la resistencia a la insulina y sus complicaciones asociadas. Palabras clave: insulino resistencia, óxido nítrico, endotelio, hipertensión arterial, sistema nervioso autónomo, mitocondria Abstract Defective nitric oxide homeostasis. Common underlying mechanism between insulin resist-ance, sympathetic overactivity and cardiovascular morbidity and mortality. Obesity, insulin resistance and associated cardiovascular complications are reaching epidemic proportions worldwide and repre-sent a major public health problem. Over the past decade, evidence has accumulated indicating that insulin administration, in addition to its metabolic effects, also has important cardiovascular actions. The sympathetic nervous system and the L-arginine-nitric oxide pathway are the central players in the mediation of insulin's car-diovascular actions. Based on recent animal and human research, we demonstrate that both defective and aug-mented NO synthesis represent a central defect triggering many of the metabolic, vascular and sympathetic abnormalities characteristic of insulin-resistant states. These observations provide the rationale for the use of pharmaceutical drugs releasing small and physiological amounts of NO and/or inhibitors of NO overproduction as a future treatment for insulin resistance and associated comorbidities.
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