Fig. 1.

Anti-inflammatory activity of flurbiprofen and HCT-1026 in RAW 264.7 cell culture. (A) RAW cells were treated with different concentration of flurbiprofen (open circles; IC₅₀ = 7.2 nM) or HCT-1026 (closed circles; IC₅₀ = 40.6 nM) after induction of COX2 by 1 μg/mL of LPS. PGD₂ was measured by LC/MS after 24 h, using deuterated PGD₂ as an internal standard. Data is normalized to the amount of PGD₂ released by vehicle treated, induced cells. (B) iNOS activity, as measured by Griess assay for NO₂⁻ in culture supernatants of LPS-induced RAW cells. Cells were pretreated for 30 min with 1, 10, or 100 μM of flurbiprofen (open circles) or HCT-1026 (closed circles) followed by LPS induction and measurement of NO₂⁻ production at 24 h. The data were normalized to DMSO vehicle control experiments in LPS-induced cells. (C) The anti-inflammatory activity of HCT-1026 was compared to that of a combination of its constituent parts: flurbiprofen + organic nitrate (ISMN). Cells were pretreated for 30 min with 100 μM of drugs followed by LPS induction and measurement of NO₂⁻ production at 24 h. The data were normalized to DMSO vehicle control experiments in LPS-induced cells. For all nitrate drugs, low background NO₂⁻ production (1–3 μM) was measured in non-induced cells and subtracted from the measurements made in induced RAW cells. Treated groups were compared to control group using ANOVA with Dunnett’s post test (***, P < 0.001). The data show mean and s.e.m. from at least 3 separate experiments performed using separate cell passages.