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.

Neuroprotective effect of flurbiprofen and HCT-1026 on N2a cells in co-culture with RAW 264.7 cells. N2a cells were cultured on the bottom of 24 well plates, while RAW cells were cultured on inserts within the 24 well plates. Addition of 1 μg/ml LPS to the co-culture significantly increased the cytotoxicity of RAW cells towards N2a cells as measured 48 h later by the MTT assay. The co-cultures were pretreated for 30 min with 1, 10, or 100 μM of flurbiprofen or HCT-1026 followed by LPS induction (1 μg/ml). Pretreatment with flurbiprofen or HCT-1026 significantly attenuated the neurotoxicity of inflammatory factors towards N2a cells in a concentration (μM) dependent manner. Data show mean and s.d. from two separate cell passages compared to RAW/LPS: p < 0.05 for all groups (* p < 0.01) by one-way ANOVA with Tukey’s post test.

Effect of drugs on aversive memory in the STPA test. Male C57BL/6 mice received 1 mg/kg scopolamine or water by i.p. injection at t = −30 min (1^st injection), 4.5 μmol/kg of drug or water i.p. at t = −20 min (2^nd injection), and training was initiated at t = 0 min. Retention of aversive memory was tested 48 h later in the absence of any drugs or scopolamine. Latency to enter the dark chamber is shown at 48 h for treatment groups. Data show mean and s.e.m. (N = 8–11). Statistical significance (* p < 0.01) assessed by one-way ANOVA with Tukey post test.

Anti-inflammatory activity of flurbiprofen and HCT-1026 in primary rat cortical astrocyte cultures. (A) iNOS activity as measured by Griess assay for NO₂⁻ in culture supernatants of primary astrocytes. Cells were pretreated 1 h with 100 μM flurbiprofen or HCT-1026 followed by induction by LPS (1 μg/ml)/IFN-γ (100 U/ml) and measurement of NO₂⁻ production at 48 h. (B) TNF-α & IL-6 production was measured by ELISA 48 h after induction by LPS/IFN-γ. Treated groups were compared to LPS/IFN-γ control group using ANOVA with Tukey’s post test (*, P <0.05; **, P < 0.001).

Effect of drug treatment on the level of Aβ produced by N2a/APPsw cells. (A) N2a/APPsw cells were incubated with ISMN, flurbiprofen (flurb), ISMN + flurbiprofen, or HCT-1026 (all drugs 100 μM). Levels of Aβ_1–42 were measured 24 h later by ELISA and the data were normalized to DMSO vehicle treated control. The average level of Aβ_1–42 in DMSO treated group was ~500 pg/mL. Treated groups were compared to control group using ANOVA with Dunnett’s post test (*, P < 0.05) (B) Concentration-response for relative levels of Aβ_1–42 after treatment of N2a/APPsw cells with flurbiprofen (closed circles), or HCT-1026 (open circles). (C) Quantification of Aβ fragments (Aβ_1–42, Aβ_1–40, Aβ_1–38, Aβ_1–37) produced by N2a/APPsw cell treated with drugs or vehicle by IP-MALDI-TOF analysis; the y-axis is scaled relative to 100% for each Aβ fragment in the DMSO vehicle control group, to demonstrate the raising or lowering of peptide levels in response to drugs. The data show mean and s.e.m. from at least 4 separate experiments performed using separate cell passages.

Effect of HCT-1026 on spatial memory in a 6-arm RWM. Male Long-Evans rats were administered scopolamine (1mg/kg, i.p.) 30 min before and HCT-1026 (open circles; 4.5 μmol/kg) or saline (crosses) 20 min before training/testing on each day: Day 1: blocks 1 & 2; Day 2: blocks 3 & 4; 4 trials within each block; 5 min inter-block interval; each trial 60 s or less). Short-term and long-term spatial memory was measured by the total number of errors made in finding the hidden platform in the RWM in each trial. For each group of animals (N = 8), the mean and s.e. are shown: the statistical significance between groups (p = 0.02) was assessed using a repeated-measure generalized linear model.