04 - 2009

Eur J Pharmacol. 2009 Jan 14;602(2-3):215-22. Epub 2008 Nov 19.

The nitrosylated flurbiprofen derivative HCT1026 inhibits cytokine-induced signalling through a novel mechanism of action.

Idris AI, Ralston SH, van't Hof RJ. Rheumatic Diseases Unit, Molecular Medicine Centre, University of Edinburgh, General Western Hospital, Edinburgh, UK.

We have previously shown that the nitrosylated flurbiprofen derivative HCT1026 inhibits bone resorption, both in vivo and in vitro, and that its mechanism of action is independent of nitric oxide release and prostaglandin synthesis inhibition. Here we describe the effects of HCT1026 on osteoclast formation, activity, survival and cell signalling in vitro. HCT1026 strongly inhibited osteoclast formation, activity and survival in murine osteoclast cultures, whereas macrophages and osteoblasts were unaffected. HCT1026 induced osteoclast apoptosis, and this was partially prevented by increasing the concentration of receptor activator of nuclear factor kappa B ligand (RANKL). This suggests that HCT1026 inhibits bone resorption by inhibiting the effects of RANKL. In agreement with this we found that HCT1026 inhibited RANKL-induced activation of the nuclear factor kappa B (NFkappaB) and extracellular signal-regulated kinase (ERK) pathways in both osteoclast and macrophage cultures, whereas its parent compound flurbiprofen did not. In addition, HCT1026 also inhibited tumor necrosis factor (TNF)-, interleukin-1 (IL1)- and LPS-induced signalling, but not macrophage colony stimulating factor induced signalling. The pathways that are inhibited by HCT1026 all share a similar kinase complex upstream of the NFkappaB and ERK pathways, and this is the most likely target for the actions of HCT1026. Although the rationale for the modification of flurbiprofen with a nitric oxide donor group was to prevent gastro-intestinal toxicity, the resulting compound HCT1026 gained unexpected additional cytokine-inhibitory properties. As RANKL, TNF and IL1 are all important mediators of inflammation and joint destruction, compounds like HCT1026 could represent a novel class of anti-inflammatory compounds.

Int Immunopharmacol. 2009 Mar 30. [Epub ahead of print]

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, Vučićević D, Vučević 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.

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.