ncx-4040 and nitroaspirin

Candida albicans biofilms play a key role in denture stomatitis, one of the most common oral pathologies in elderly people. Because biofilms are highly resistant to antifungals, new pharmacological strategies are needed. Aspirin and nitric oxide-donor molecules have both shown antibiofilm effects on C. albicans, making them promising candidates for treatment. In this study, we evaluated the antifungal/antibiofilm effect of a nitric-oxide releasing aspirin (NO-ASA) on C. albicans isolates from denture stomatitis patients in vitro. Disk diffusion assays showed that while NO-ASA had no antifungal effect, the drug potentiated fluconazole inhibition zone diameters, increasing the effect of fluconazole by 20-30% (p<0.05). The effect of NO-ASA on the morphogenesis of C. albicans was evaluated using light microscopy after inducing hyphae formation. For all clinical strains assayed, 125 μM NO-ASA significantly decreased the number of filamentous cells present (p<0.01). Adhesion to abiotic surfaces, a critical event for biofilm formation, was evaluated in 96-well polystyrene plates using crystal violet assay; 125 μM NO-ASA significantly inhibited adhesion. Biofilms were observed with scanning electron microscopy (SEM) and quantified using XTT reduction assay. NO-ASA decreased biofilm formation (IC50 ranging from 300 μM to 700 μM), consistent with SEM findings of altered biofilm microarchitecture. PGE2 and carboxy-PTIO (an NO scavenger) both blocked the antibiofilm effects of NO-ASA, suggesting that the efficacy of NO-ASA may be associated with both inhibition of PGE2 synthesis and release of NO. NO-ASA is a promising novel antibiofilm agent for treating fluconazole-resistant strains of C. albicans.

Topics: Antifungal Agents; Aspirin; Bacterial Adhesion; Biofilms; Candida albicans; Dinoprostone; Drug Resistance, Fungal; Fluconazole; Free Radical Scavengers; Humans; Inhibitory Concentration 50; Microbial Viability; Nitro Compounds; Stomatitis, Denture

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.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antigens, Differentiation; Apoptosis; Aspirin; Cell Differentiation; Cells, Cultured; Dendritic Cells; Down-Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-10; Interleukin-12 Subunit p40; Interleukin-4; Lipopolysaccharides; Lymphocyte Activation; Monocytes; Nitric Oxide; Nitro Compounds

Ovarian carcinoma is the leading cause of mortality among gynecological cancers in the world. The high mortality rate is associated with lack of early diagnosis and development of drug resistance. The antitumor efficacy and mechanism of NCX-4040, a nitric oxide-releasing aspirin derivative, against ovarian cancer is studied.. NCX-4040, alone or in combination with cisplatin (cis-diamminedichloroplatinum, cDDP), was studied in cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) cell lines as well as xenograft tumors grown in nude mice. Electron paramagnetic resonance (EPR) was used for measurements of nitric oxide and redox state. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice was used for mechanistic studies.. Cells treated with NCX-4040 (25 microM) showed a significant reduction of cell viability (A2780 WT, 34.9 +/- 8.7%; A2780 cDDP, 41.7 +/- 7.6%; p < 0.05). Further, NCX-4040 significantly enhanced the sensitivity of A2780 cDDP cells (cisplatin alone, 80.6 +/- 11.8% versus NCX-4040+cisplatin, 26.4 +/- 7.6%; p < 0.01) and xenograft tumors (cisplatin alone, 74.0 +/- 4.4% versus NCX-4040+cisplatin, 56.4 +/- 7.8%; p < 0.05), to cisplatin treatment. EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice treated with NCX-4040 and cisplatin revealed significant downregulation of pEGFR (Tyr845 and Tyr992) and pSTAT3 (Tyr705 and Ser727) expression.. The results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols. Thus NCX-4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies.

Topics: Animals; Antineoplastic Agents; Aspirin; Cell Line, Tumor; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Mice; Mice, Nude; Molecular Structure; Nitro Compounds; Ovarian Neoplasms; Sensitivity and Specificity; Statistics as Topic; Sulfhydryl Compounds; Xenograft Model Antitumor Assays

Cellular defense mechanisms that respond to damage from oxidative and electrophilic stress, such as from quinones, represent a target for chemopreventive agents. Drugs bioactivated to quinones have the potential to activate antioxidant/electrophile responsive element (ARE) transcription of genes for cytoprotective phase 2 enzymes such as NAD(P)H-dependent quinone oxidoreductase (NQO1) but can also cause cellular damage. Two isomeric families of compounds were prepared, including the NO-NSAIDs (NO-donating nonsteroidal anti-inflammatory drugs) NCX 4040 and NCX 4016; one family was postulated to release a quinone methide on esterase bioactivation. The study of reactivity and GSH conjugation in model and cell systems confirmed the postulate. The quinone-forming family, including NCX 4040 and conisogenic bromides and mesylate, was rapidly bioactivated to a quinone, which gave activation of ARE and consequent induction of NQO1 in liver cells. Although the control family, including NCX 4016 and conisogenic bromides and mesylates, cannot form a quinone, ARE activation and NQO1 induction were observed, compatible with slower SN2 reactions with thiol sensor proteins, and consequent ARE-luciferase and NQO1 induction. Using a Chemoprevention Index estimate, the quinone-forming compounds suffered because of high cytoxicity and were more compatible with cancer therapy than chemoprevention. In the Comet assay, NCX 4040 was highly genotoxic relative to NCX 4016. There was no evidence that NO contributes to the observed biological activity and no evidence that NCX 4040 is an NO donor, instead, rapidly releasing NO3- and quinone. These results indicate a strategy for studying the quinone biological activity and reinforce the therapeutic attributes of NO-ASA through structural elements other than NO and ASA.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Benzoquinones; Catalysis; Cell Line, Tumor; Cell Survival; Chemoprevention; Cytoprotection; Esterases; Glutathione; Liver; Luciferases; Metabolic Detoxication, Phase II; Mice; Models, Biological; NAD(P)H Dehydrogenase (Quinone); NADPH Dehydrogenase; Nitro Compounds; Response Elements; Swine

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.

Topics: Adenocarcinoma; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Caspase 9; Cell Cycle; Cell Line, Tumor; Cytotoxins; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Nitric Oxide Donors; Nitro Compounds; Pancreatic Neoplasms; S-Nitroso-N-Acetylpenicillamine; Salicylates

Although nonsteroidal antiinflammatory drugs (NSAIDs) are ineffective in treating acute respiratory distress syndrome (ARDS), inhalational NO has proved to be useful. NO-donating NSAIDs may therefore be more effective in treating ARDS than NSAIDs alone. Because oxidant stress is central to the pathophysiology of ARDS, the effect of nitroaspirins (NCX 4016, NCX 4040, and NCX 4050) compared with morpholinosydnonimine (SIN-1; an NO donor) and aspirin (ASA) on superoxide (O2*-) formation and gp91phox (an active catalytic subunit of NADPH oxidase) expression in pig pulmonary artery vascular smooth muscle cells (PAVSMCs) and endothelial cells (PAECs) was investigated.. Cultured PAVSMCs and PAECs were incubated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1alpha (with or without NO-ASA, SIN-1, or ASA) for 16 hours, and O2*- release was measured by use of the reduction of ferricytochrome c. The expression of gp91(phox) was assessed by use of Western blotting. LPS, TNF-alpha, and IL-1alpha all stimulated the formation of O2*- and expression of gp91(phox) in both PAVSMCs and PAECs, an effect inhibited by NADPH oxidase inhibitors, diphenyleneiodonium, and apocynin. SIN-1, NCX 4016, and NCX 4050 but not ASA alone inhibited the formation of O2*- and expression of gp91(phox).. LPS and cytokines promote the formation of O2*- in PAVSMCs and PAECs through an augmentation of NADPH oxidase activity, which in turn is prevented by NO. Thus, NO may play a protective role in preventing excess O2*- formation, but its negation by O2*- may augment the progress of ARDS. The inhibitory effect of nitroaspirins suggests that they may be therapeutically useful in treating ARDS through the suppression of NADPH oxidase upregulation and O2*- formation.

Topics: Acetophenones; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Drug Evaluation, Preclinical; Endothelial Cells; Endothelium, Vascular; Enzyme Induction; Epoprostenol; Glycoproteins; Guanylate Cyclase; Interleukin-1; Lipopolysaccharides; Male; Molsidomine; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Onium Compounds; Pulmonary Artery; Respiratory Distress Syndrome; Superoxides; Sus scrofa; Tumor Necrosis Factor-alpha