2010 - 03

Int Immunopharmacol. 2009 Jul;9(7-8):910-7. Epub 2009 Mar 31.

Comparative effects of aspirin and NO-releasing aspirins on differentiation, maturation and function of human monocyte-derived dendritic cells in vitro.

Bufan B, Mojsilović S, Vucićević D, Vucević D, Vasilijić S, Balint B, Colić M. Institute for Medical Research, Military Medical Academy, Belgrade, Serbia; Department of Microbiology and Immunology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia. bbiljana@pharmacy.bg.ac.rs

Acetylsalicilyc acid (aspirin, ASA) is a well known anti-inflammatory drug with immunomodulatory properties. NO-releasing aspirins (NO-ASA) are new compounds with anti-inflammatory properties. We studied the effects of ASA and two NO-ASA (NCX 4016 and NCX 4040) on human monocyte-derived dendritic cells (MoDC). Immature MoDC were generated in vitro from monocytes in the presence of recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Mature MoDC were obtained by adding lipopolysaccharide (LPS) in cultures of immature MoDC. As we found that ASA at 4-8 mM, NCX 4016 at 400-800 microM and NCX 4040 at 4-8 microM stimulated apoptosis of monocytes and immature MoDC, sub-apoptotic concentrations of ASA (2 mM), NCX 4016 (200 microM) and NCX 4040 (2 microM) were used in experiments. Examined substances were added at the beginning of MoDC cultivation. MoDC differentiated in the presence of examined compounds had lower expression of HLA-DR, CD80, CD40 and CD54, decreased allostimulatory activity and lower production of IL-12 p40. ASA and NCX 4016 decreased production of IL-10, whereas NCX 4040 had the opposite effect. ASA inhibited the expression of CD1a and prevented downregulation of CD14, NCX 4016 stimulated the differentiation of CD1a+CD14+ and CD1a(-)CD14+ cells, whereas NCX 4040 decreased the proportion of CD1a+CD14(-) and increased the frequency of CD1a+CD14+ cells, compared to control. Maturation, both in ASA and NO-ASA treated MoDC was characterized by decreased allostimulatory activity, lower expression of CD83, HLA-DR, costimulatory molecules and CD54 and decreased production of IL-10 and IL-12 p40. In conclusion, we confirmed that ASA impairs differentiation, maturation and function of MoDC and found that NCX 4016 and NCX 4040 exerted similar, but not identical effects at about 10- and 1000-fold lower concentrations, respectively, compared to ASA.

Biochem Pharmacol. 2009 Nov 15;78(10):1298-304. Epub 2009 Jul 2.

Nitro-aspirin inhibits MCF-7 breast cancer cell growth: effects on COX-2expression and Wnt/beta-catenin/TCF-4 signaling.

Nath N, Vassell R, Chattopadhyay M, Kogan M, Kashfi K. Department of Physiology and Pharmacology, City University of New York Medical School, 138th Street and Convent Avenue, New York, NY 10031, United States.nnath@nyit.edu

There is current evidence implicating the Wnt/beta-catenin/TCF pathway in breast cancer. We investigated the effect of para- and meta-positional isomers of nitric oxide-releasing aspirin (NO-ASA), and aspirin (ASA) on MCF-7 human breast cancer cell growth and beta-catenin/TCF signaling. The p- and m-NO-ASA isomers strongly inhibited cell growth and beta-catenin/TCF transcriptional activity compared to ASA; the IC50s for growth inhibition were 57+/-4, 193+/-10 and >5000microM, and for transcriptional inhibition they were 12+/-1.8, 75+/-6.5 and >5000microM for p-, m-NO-ASA and ASA, respectively. p-NO-ASA reduced the expression of Wnt/beta-catenin downstream target gene cyclin D1, and total cellular beta-catenin levels. COX-2 expression was induced by p-NO-ASA, protein kinase C inhibitors reversed this induction. p-NO-ASA blocked the cell cycle transition at S to G2/M phase. These studies suggest a targeted chemopreventive/chemotherapeutic potential for NO-ASA against breast cancer.

Int J Oncol. 2009 Oct;35(4):837-44.

The differential cell signaling effects of two positional isomers of theanticancer NO-donating aspirin.

Hua A, Mackenzie GG, Rigas B. Division of Cancer Prevention, Department of Medicine, Stony Brook University, Stony Brook, New York 11794-5200, USA.

We studied the mechanism by which the para and meta positional isomers of nitric oxide-donating aspirin (NO-ASA) inhibit human colon cancer cell growth. These compounds are promising chemopreventive agents and represent a broader class of novel drugs. The two isomers differ drastically in their 24-h IC50s for cell growth, which are 12 microM for p-NO-ASA and 230 microM for m-NO-ASA. We examined their effects on cell signaling cascades, including predominantly the mitogen activated protein kinases (MAPKs). The principal differences between the two isomers were: a) p-NO-ASA exerts its effect earlier than m-NO-ASA; b) the predominant effect of m-NO-ASA is on ERK1/2 and Akt; whereas that of p-NO-ASA is on JNK1/2, while both activate p38, with p-NO-ASA showing a stronger and earlier effect; c) ATF-2 is more responsive to m-NO-ASA and c-Jun to p-NO-ASA; d) both isomers seem to have similar effects on AP-1 binding, the main difference between them being the timing of the effect; p-NO-ASA's effect is early and m-NO-ASA's is late; e) p-NO-ASA has an earlier and stronger effect on p21, while m-NO-ASA's effect occurs later and is weaker; and f) cell cycle changes follow the effect on p21 expression. Our findings underscore the role of positional isomerism in modulating the pharmacological effects of drugs and have potentially important implications for the further development of these chemoprevention agents.

Br J Pharmacol. 2009 Sep;158(2):601-9. Epub 2009 Jul 23.

Antinociceptive effects of NCX-701 (nitro-paracetamol) in neuropathic rats: enhancement of antinociception by co-administration with gabapentin.

Curros-Criado MM, Herrero JF. Departamento de Fisiología, Campus Universitario, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.

BACKGROUND AND PURPOSE: Neuropathic pain is characterized by a poor response to classic analgesics. In the present study, we have assessed the antinociceptive activity of NCX-701 (nitro-paracetamol) in neuropathic rats, after systemic and intrathecal (i.t.) administration. In addition, we analysed the possible benefit of the combination of NCX-701 and gabapentin, a well-known potent analgesic, in the treatment of neuropathic pain. EXPERIMENTAL APPROACH: The antinociceptive effects of i.v. and i.t. NCX-701 and paracetamol were studied in spinal cord neuronal responses from neuropathic adult male Wistar rats, using the recording of single motor units technique. The effect of i.v. and i.t. NCX-701 in combination with i.v. gabapentin was studied by isobolographic analysis. KEY RESULTS: The experiments showed that NCX-701, but not paracetamol, dose-dependently reduced the nociceptive responses evoked by noxious mechanical and electrical stimulation, after i.v. (ID(50) 542 +/- 5 micromol kg(-1) for noxious mechanical stimulation) or i.t. (ID(50) 932 +/- 16 nmol kg(-1)) administration. The combined administration of i.v. or i.t. NCX-701 and i.v. gabapentin induced a more intense antinociceptive effect than any of the two drugs given alone. The isobolographic analysis showed a synergistic effect. CONCLUSIONS AND IMPLICATIONS: NCX-701 is an effective antinociceptive compound in situations of neuropathy-induced sensitization, with an action mainly located in the spinal cord. The combination of NCX-701 and gabapentin induces a synergistic enhancement of the depression of nociceptive responses evoked by natural noxious stimulation. The use of NCX-701 alone or in combination with gabapentin might open up new and promising perspectives in the treatment of neuropathic pain.

J Immunol. 2010 Jan 11. [Epub ahead of print]

NCX 4040, a Nitric Oxide-Donating Aspirin, Exerts Anti-Inflammatory Effects through Inhibition of I{kappa}B-{alpha} Degradation in Human Monocytes.

Ricciotti E, Dovizio M, Di Francesco L, Anzellotti P, Salvatore T, Di Francesco A, Sciulli MG, Pistritto G, Monopoli A, Patrignani P. Department of Medicine and Center of Excellence on Aging, School of Medicine, "G. d'Annunzio" University and.

NO-donating aspirins consist of aspirin to which a NO-donating group is covalently linked via a spacer molecule. NCX 4040 and NCX 4016 are positional isomers with respect to the -CH(2)ONO(2) group (para and meta, respectively) on the benzene ring of the spacer. Because positional isomerism is critical for antitumor properties of NO-donating aspirins, we aimed to compare their anti-inflammatory effects with those of aspirin in vitro. Thus, we assessed their impacts on cyclooxygenase-2 activity (by measuring PGE(2) levels), protein expression, and cytokine generation(IL-1beta, IL-18, TNF-alpha, and IL-10) in human whole blood and isolated human monocytes stimulated with LPS. Interestingly, we found that micromolar concentrations of NCX 4040, but not NCX 4016 or aspirin, affected cyclooxygenase-2 expression and cytokine generation. We compared the effects of NCX 4040 with those of NCX 4016 or aspirin on IkappaB-alpha stabilization and proteasome activity in the LPS-stimulated human monocytic cell line THP1. Differently from aspirin and NCX 4016, NCX 4040, at a micromolar concentration range, inhibited IkappaB-alpha degradation. In fact, NCX 4040 caused concentration-dependent accumulation of IkappaB-alpha and its phosphorylated form. This effect was not reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase, thus excluding the contribution of NO-dependent cGMP generation. In contrast, IkappaB-alpha accumulation by NCX 4040 may involve an inhibitory effect on proteasome functions. Indeed, NCX 4040 inhibited 20S proteasome activity when incubated with intact cells but not in the presence of cell lysate supernatants, thus suggesting an indirect inhibitory effect. In conclusion, NCX 4040 is an inhibitor of IkappaB-alpha degradation and proteasome function, and it should be taken into consideration for the development of novel anti-inflammatory and chemopreventive agents.

Diabetes Care. 2010 Mar 18. [Epub ahead of print]


Gresele P, Marzotti S, Guglielmini G, Momi S, Giannini S, Minuz P, Lucidi P, Bolli GB, Bolli GB. Section of Internal and Cardiovascular Medicine*

AbstractObjective. Acute, short-term hyperglycemia enhances high shear-stress-induced platelet activation in type-2-diabetes-mellitus (T2DM). Several observations suggest that platelets in T2DM are resistant to inhibition by aspirin. Our aim was to assess comparatively the effect of aspirin, a nitric oxide-donating agent (NCX 4016), their combination, or placebo on platelet activation induced by acute hyperglycemia in T2DM. Research Design and Methods. In a double-blind, placebo-controlled, randomized trial, 40 T2DM patients were allocated to aspirin 100mg, o.i.d., NCX 4016 800 mg, b.i.d., both of them, or placebo, for 15 days. On day 15, one hour after the morning dose, a four-hour hyperglycemic clamp (plasma glucose 13.9mmol/l) was performed and blood samples were collected before and immediately after it for platelet activation and cyclooxygenase-1 (COX-1) inhibition studies. Results. Acute hyperglycemia enhanced shear stress-induced platelet activation in placebo-treated patients (basal closure time 63+/-7.1sec, after hyperglycemia 49.5+/-1.4sec, -13.5+/-6.3sec, P<0.048); p="NS">