ABSTRACT
Title
Anti-inflammatory Effects of the CINOD NCX 429 in Human Monocytes and Macrophages
Authors
D Federici-Canova1, A Amoruso1, LG Fresu1, J Padron2, M Bolla2, S Brunelleschi1
1Dept. Scienze Mediche, University of Piemonte Orientale “A. Avogadro”, Novara (Italy); 2NicOx Research Institute, Bresso, Italy
1Dept. Scienze Mediche, University of Piemonte Orientale “A. Avogadro”, Novara (Italy); 2NicOx Research Institute, Bresso, Italy
Abstract
Cyclooxygenase-inhibiting nitric oxide (NO) donors (CINODs) are investigational anti-inflammatory compounds designed to provide the anti-inflammatory effects of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) along with NO donation. CINODs exert their analgesic and anti-inflammatory via the NSAIDs component, and show protective action of NO on gastric mucosa and on vascular, cardiac and renal function in animal models. NCX 429 is a naproxen-based CINOD that showedpharmacological effects in the murine macrophage cell line, RAW 264.7.
We decided to study the ability of NCX 429 to affect oxy-radical production and pro-inflammatory cytokine release, as well as PPARgexpression and activity, and to modulate matrix metalloproteinase (MMP)-9 activity,in human monocytes and monocyte-derived macrophages (MDM) from healthy donors, in comparison to naproxen. Human monocytes were isolated from heparinised venous blood of healthy donors by standard techniques. Cytokine release was measured by ELISA kit: cells were pre-treated for 1 h with NCX 429 or naproxen (10-9 – 10-4 M) and then stimulated by phorbol-12-myristate 13-acetate (PMA) 10-7 M for 24 h. To evaluatesuperoxide anion (O2-) production, human monocytes were stimulated, in the absence or presence of drugs, by PMA 10-6 M for 30 min. O2- production was evaluated by the superoxide dismutase (SOD)-inhibitable cytochrome C reduction. MMP-9 activity was evaluated by zymography; expression of PPARgby Western Blot.
In the range 10-9 - 10-4 M, NCX 429 dose-dependently inhibited (IC50 = 870 nM) PMA-induced IL-6 release in monocytes; at the highest concentration, the inhibition by NCX 429 amounted to 62 + 8%, significantly higher than the 36 + 4% inhibition afforded by naproxen. In MDM, 10-4 M NCX 429 inhibited by 52 + 11 % PMA-induced IL-6 release, while naproxen had no effect. No differences between drugs have been observed in PMA-induced TNF-arelease. A stronger effect of NCX 429 was also observed by evaluating O2- production in monocytes and MDM. At 10-6 M, NCX 429 inhibited PMA-induced O2- production by about 24% in monocytes and 60% in MDM, whereas naproxen had no effect. For PPARgprotein expression, cells were treated with drugs under study at a concentration of 10-4 M for 6 hr; rosiglitazone and 15-deoxy-D12,14-prostaglandin J2 (at 10-6 M and 10-5 M, respectively) were used as positive controls. NCX 429 and naproxen similarly increased the protein expression of PPARgin monocytes and MDM. However, NCX 429, but not naproxen, significantly inhibited MMP-9 activity in lipopolysaccharide (LPS)-challenged monocytes.
In conclusion, NCX 429 demonstrates significant anti-inflammatory properties in human monocytes and macrophages, superior and different to those shown by naproxen. The NO donating feature provides the adjunctive pharmacodynamics effects on inflammatory pathways. These data suggest that NCX 429 could have a beneficial role in inflammatory diseases necessitating long-term treatment to be further explored.
We decided to study the ability of NCX 429 to affect oxy-radical production and pro-inflammatory cytokine release, as well as PPARgexpression and activity, and to modulate matrix metalloproteinase (MMP)-9 activity,in human monocytes and monocyte-derived macrophages (MDM) from healthy donors, in comparison to naproxen. Human monocytes were isolated from heparinised venous blood of healthy donors by standard techniques. Cytokine release was measured by ELISA kit: cells were pre-treated for 1 h with NCX 429 or naproxen (10-9 – 10-4 M) and then stimulated by phorbol-12-myristate 13-acetate (PMA) 10-7 M for 24 h. To evaluatesuperoxide anion (O2-) production, human monocytes were stimulated, in the absence or presence of drugs, by PMA 10-6 M for 30 min. O2- production was evaluated by the superoxide dismutase (SOD)-inhibitable cytochrome C reduction. MMP-9 activity was evaluated by zymography; expression of PPARgby Western Blot.
In the range 10-9 - 10-4 M, NCX 429 dose-dependently inhibited (IC50 = 870 nM) PMA-induced IL-6 release in monocytes; at the highest concentration, the inhibition by NCX 429 amounted to 62 + 8%, significantly higher than the 36 + 4% inhibition afforded by naproxen. In MDM, 10-4 M NCX 429 inhibited by 52 + 11 % PMA-induced IL-6 release, while naproxen had no effect. No differences between drugs have been observed in PMA-induced TNF-arelease. A stronger effect of NCX 429 was also observed by evaluating O2- production in monocytes and MDM. At 10-6 M, NCX 429 inhibited PMA-induced O2- production by about 24% in monocytes and 60% in MDM, whereas naproxen had no effect. For PPARgprotein expression, cells were treated with drugs under study at a concentration of 10-4 M for 6 hr; rosiglitazone and 15-deoxy-D12,14-prostaglandin J2 (at 10-6 M and 10-5 M, respectively) were used as positive controls. NCX 429 and naproxen similarly increased the protein expression of PPARgin monocytes and MDM. However, NCX 429, but not naproxen, significantly inhibited MMP-9 activity in lipopolysaccharide (LPS)-challenged monocytes.
In conclusion, NCX 429 demonstrates significant anti-inflammatory properties in human monocytes and macrophages, superior and different to those shown by naproxen. The NO donating feature provides the adjunctive pharmacodynamics effects on inflammatory pathways. These data suggest that NCX 429 could have a beneficial role in inflammatory diseases necessitating long-term treatment to be further explored.