Fresh heparinized blood samples were obtained from the Kuwait Central Blood Bank, after donor informed consent, and under a protocol approved by the Institutional Ethics Committee. Mononuclear cells were isolated by the ficoll gradient centrifugation method. Subsequently, highly purified monocytes (>98% purity), were isolated immunomagnetically using the Monocyte Negative Isolation kit (Invitrogen, Carlsbad, CA, USA), as previously described [21]. The viability (by trypan blue exclusion method) was >97%. Monocytes were washed twice and cultured in 96-well plate at a concentration of 5 × 10⁵ cells/ml in endotoxin-free complete culture medium (RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal calf serum). The cells were then pre-incubated with the compounds (CEE-1, its analogues, standard anti-inflammatory drugs or their solvents) for 30 min before being stimulated with 0.3 µg/ml LPS. All compounds were tested at concentrations ranging from 0.01 µM to 30 µM. After 24 h of culture in a humidified incubator at 37˚C and 5% CO₂, the supernatants were recovered by centrifugation and stored at −40˚C pending determination of the mediator contents.

In some experiments, human monocytic cell line (U937) was used. The cell line has the ability to release many inflammatory mediators including TNF-α and PGE₂ following activation with a combination of 0.3 mg/ml LPS and 50 nM phorbol myristate acetate (PMA). The cells were cultured in complete culture medium (as above) and used undifferentiated between 5 - 10 passages.

In experiments to study the toxicity of the active compounds, cells were pre-treated with CEE-1 for 24 h. At the end of the culture, cells were gently scrapped and centrifuged for 5 min at 4˚C. The pellet was re-suspended and the viability determined as described above.

The levels of the cytokines (TNF-α, IL-1β and IL-6) and prostanoids (PGE₂ and TXB₂) in the culture supernatants were determined using their respective commercially available ELISA kits obtained from R&D systems (Minneapolis, MN, USA).

The expression of mRNA for TNF-α was studied on human blood monocytes. Purified cells were re-suspended and adjusted to a concentration of 10⁶ viable cells/ml, and 1.8 ml aliquots added to each well of a 6-well plate. They were then incubated for 1 h to adhere to the plate, pre-treated with vehicle or various concentrations of CEE-1 or standard anti-inflammatory drug dexamethasone for 30 min before being stimulated with LPS (1 µg/ml). After 2 h of culture (a time previously determined as optimal for mRNA expression), the cells were washed twice and then subjected to RNA extraction. For the determination of the expression of mRNA for COX-1 and COX-2, the human monocytic cells (U937) were used. The experimental procedure used was similar to the above except that the cells were stimulated with a combination of LPS (0.3 μg/ml) and PMA (50 nM) and then cultured for 6 h (previously determined to be optimal for this cell line).

In both cases, total RNA extraction was performed using the Nucleospin^® RNAII Total RNA Isolation kit (Machery-Nagel, Duren, Germany) and following manufacturer’s instructions. The concentrations of the RNA in the samples were measured using a nano-spectrophotometer. Reverse transcription was carried out in a total volume of 20 μl using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. The qRT-PCR reaction was carried out using a TaqMan^® Gene Expression Assay GEX kit on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The Applied Biosystems assay ID numbers were [Hs00174128-m1for TNF-α, Hs00377726-m1 for COX- 1 and Hs00153133-m1 for COX-2. A ß-actin TaqMan^® control reagent kit (part # 401846) was used as control.

The relative quantitation of the products was expressed as folds of the target over endogenous control.

Aliqouts of U937 monocytes (2 × 10⁶ cells) were added to each well of a 24-well plate and pre-incubated with CEE-1 (30 µM), indomethacin (1 µM) or vehicle (as control) for 30 min before being stimulated with a combination of LPS (0.3 μg/ml) and PMA (50 nM). After 24 h culture, the cells were washed and the pellet lysed with 150 µl of RIPA lysis buffer (1X) per sample. Following thorough mixing, the lysates were placed on ice for 40 min before being centrifuged for 5 min at 10,000 g. The supernatants were collected and the protein contents determined by the Bradford protein assay method.

For the Western blotting experiments, an aliquot of 12 µg protein from each sample in 6x loading buffer was boiled for 5 min at 100˚C and electrophoresed in a 10% acrylamide gel containing 10% SDS (SDS-PAGE). Proteins were subsequently transferred to a polyvinyl difluoride (PVDF) membrane, washed and blocked with 15 ml blocking reagent (15 ml TBS Blotto A + 15 µl Tween 20) for 1 h at room temperature. Thereafter, the membrane was incubated with primary antibodies against COX-2 (mouse monoclonal IgG anti-human COX-2, diluted 1:500) overnight at 4˚C. The membrane was then washed 3 times for 5 min each, followed by incubation with secondary antibody (HRP-labeled goat anti-mouse IgG, diluted 1:2000) for 1 h at room temperature. After three further washings, the bound antibody was detected using the luminol reagent solution and exposed on X-ray film. The same blotting procedure was used for the ß-actin after striping of the membrane.

Cyclo-oxygenase-2 enzyme function was assessed as PGE₂ production occurring within 4 min of the addition of exogenous arachidonic acid (AA) in U937 monocytes that have been pre-activated for 24 h with a combination of LPS and PMA, to up-regulate COX-2 protein (as mentioned above). Pre-activated cells were washed twice and then incubated with various concentrations of CEE-1, indomethacin (1 µM), aspirin (250 μM), dexamethasone (0.3 µM) or solvent for 30 min before the addition of AA (5 µM). After 4 min, the reaction was rapidly terminated by placing the plate on ice for 1 min and centrifuging at 450 g at 4˚C for 3 min. Finally, supernatants were collected and stored at −80˚C pending PGE₂ assay.

The effect of CEE-1 on cell viability was determined in primary human monocytes by measuring the induction of apoptosis using the annexin V binding/flow cytometry method. Monocytes were cultured at a concentration of 5 × 10⁵ cells/ml in 48-well plate (in duplicate) in the presence of CEE-1 (3, 10 and 30 μM) or vehicle for 24 h. After culture, cells were centrifuged at 450 g for 5 min 4˚C and the supernatant removed. After one wash in PBS, adherent cells were recovered by treatment with 1 mM EDTA solution in PBS for 3 min at room temperature. The cells were then washed again and incubated for 15 min with a mixture of FITC-labeled monoclonal antiannexin V antibody and the vital dye 7-amino-actinomycin D (7-AAD). Total apoptotic cells (annexin-positive/7-AAD-negative and annexin-positive/7-AAD-positive, representing early apoptosis and late apoptosis/ necrosis, respectively), were then determined by flow cytometry using Cytomics FC500, flow cytometer (Beckman-Coulter, Brea, CA, USA).

The compound CEE-1 and its analogues CEE-2, BRG-14 and EMP-12 were synthesized as previously described [16,18]. The compounds were prepared fresh each time; first dissolved in dimethyl sulphoxide (DMSO) and subsequently diluted down in culture medium. The highest final solvent concentration in the wells did not exceed 0.05%—a concentration that had no significant effect on macrophage/monocyte responses.

The following drugs and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA): dexamethasone, indomethacin, DMSO, fetal calf serum, heparin, PMA, L-glutamine, LPS, aspirin, penicillin/streptomycin solution, RPMI 1640 medium, beta-mercaptoethanol, bisacrylamide N,N’-methylene-bis-acryl-amide, sodium dodecyl sulfate (SDS), bromophenol blue and tris-base. The U937 cell line was purchased from American Tissue Collection Center (Manassas, VA, USA). Minitrans blot filter paper, immuno-blot^® PVDF membrane for protein blotting (0.2 µM) and protein assay reagents were all obtained from Bio-Rad Systems, (Herculus CA, USA).

The ELISA kits for TNF-α, IL-1β, IL-6, PGE₂ and TXB₂ were all purchased from R&D Systems (Minneapolis MN, USA). Nucleospin RNA II kit was purchased from Macherey-Nagel (Duren, Germany), whereas high-capacity cDNA reverse Transcription Kit, TaqMan^® Gene Expression Master Mix, TaqMan^® Gene Expression Assay GEX kit and control reagents were purchased from Applied Biosystems (Foster City, CA, USA). Mouse monoclonal IgG anti-human COX-1 or COX-2 or ß-actin antibodies, as well as the HRP-labeled goat antimouse IgG were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Annexin V-FITC/7- AAD kit (containing mouse anti-human annexin V antibody and the vital dye 7-AAD) as well as the antibody isotype control (mouse IgG1-FITC) were obtained from Beckman-Coulter, Brea, CA, USA.

Data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). All results were expressed as the mean ± SEM. The IC₅₀ values were calculated from the concentration-response curves by non-linear regression analysis. Differences between mean values were evaluated for statistical significance using one-way ANOVA, followed by Bonferroni’s posthoc test or one-sample t-test as appropriate. Differences were considered significant when p ≤ 0.05.

As shown in Figure 1(a), pre-treatment of human blood monocytes with CEE-1 for 30 min before stimulation with LPS resulted in a strong and concentration-dependent inhibition of the release of TNF-α. The concentration causing 50% inhibition of release (IC₅₀ value) was 2.0 ± 0.4 µM, and at 30 µM almost complete inhibition was achieved. A comparison with two standard anti-inflammatory drugs—dexamethasone (a steroid) and indomethacin (a non-steroidal anti-inflammatory drug), showed that CEE-1 was about 45 times less potent than dexamethasone (IC₅₀ value ≈ 45 nM), but the efficacy (maximal effect) of both drugs were comparable (95% - 100% inhibition). As expected, indomethacin had no inhibitory effect at all concentrations tested. Similar effects were seen when the drugs were tested on human monocytic cell line (U937) following stimulation with a combination of LPS and PMA (Figure 1(b)). The IC₅₀ values were ≈4.4 µM and 87.5 nM for CEE-1 and dexamethasone, respectively.

Apart from TNF-α, CEE-1 also inhibited the release of other pro-inflammatory cytokines. As shown in Figure 2, it significantly inhibited, in a concentration-dependent manner, the release of IL-1β and IL-6. The drug was at least 10 times more potent in inhibiting the production of IL-1β than IL-6 (IC₅₀ values of 0.25 μM and 3.0 μM, respectively).

In order to determine the structural requirement for activity, we synthesized and tested 3 analogues of CEE-1 (CEE-2, EMP-12 and BRG-14) that incorporated different modifications on the enhydrazone pharmacophore. CEE-2 is structurally very close to CEE-1 except that the ethyl-group attached to the carboxyl moiety was replaced with a methyl-group. EMP-12 is a 2,4-dinitro-substituted CEE-1, while BRG-14 is an analogue of CEE-1 that lacks the hydrazino (NH-NH) moiety. As shown in Fi-

gure 3, CEE-2 had almost identical activity with the parent compound (CEE-1) in inhibiting TNF-α release from activated human monocytes (IC₅₀ ≈ 3.2 µM). In contrast, EMP-12 and BRG-14 had very weak or no antiinflammatory activity, thus indicating that the presence of the hydrazino functional group as well as an unsubstituted aromatic ring linked to this hydrazino group were critical requirements for anti-inflammatory activity.

To study the effect of CEE-1 on the release of prostanoids from monocytes, undifferentiated U937 cells were used. As shown in Figure 4, treatment of the cells with the drug for 30 min prior to stimulation with a combination of LPS (0.3 μg/ml) and PMA (50 nM) resulted in a pronounced concentration-dependent inhibition of PGE₂ production. The IC₅₀ value was 2.4 ± 0.6 μM, and at 30 µM, the inhibition of was almost complete (95%). Similarly, both dexamethasone and indomethacin also produced strong inhibitory effects, with IC₅₀ values of 75 nM and 32 nM, respectively. Thus, even though CEE-1 was about 30 and 76 times less potent than dexamethasone and indomethacin, respectively, it has a comparable efficacy (maximal effect) of almost 100% inhibition. Similar results were obtained with the release of TXB₂ (a stable surrogate of TXA₂) (data not shown).

In order to determine the mechanism by which CEE-1 inhibited the production of TNF-α and prostanoids, the effect of the drug on the expression of mRNA for both TNF-α and COX were studied by the qRT-PCR method. As shown in Figure 5(a), pre-treatment of human blood monocytes with CEE-1 for 30 min before stimulation with LPS (1 μg/ml) produced a concentration-dependent inhibition of the up-regulation of mRNA for TNF-α. However, only at the higher concentration of 30 µM did the inhibition (56.8 ± 6.4%) reach statistical significance (p < 0.05). In comparison, dexamethasone (0.3 µM) completely abolished the response. A similar effect was also seen in the inhibition of COX-2 mRNA up-regulation by CEE-1 in U937 cells stimulated with a combination of LPS (0.3 µg/ml) and PMA (50 nM), (Figure 5(b)). As would be expected, dexamethasone (0.3 μM), but not indomethacin (1 μM) was effective in inhibiting COX-2 mRNA up-regulation. Stimulation with the above protocol did not result in any significant up-regulation of COX-1 mRNA expression over the time frame of the experiments, neither did pre-treatment with CEE-1 or other drugs have any significant effect on the basal level.

To determine if the effect on COX-2 mRNA expression was reflected in COX-2 protein expression, the Western blotting analysis was used to quantify COX-2 protein in whole cell lysates. As shown in Figure 6(a), activation of U937 cells with the combination of LPS and PMA, resulted in increased expression of the COX-2 protein after 24 h of culture. The increase was essentially prevented by prior treatment of the cells with CEE-1 (30 µM), but not indomethacin (1 µM).

To investigate whether CEE-1 also acted by inhibiting the function of the COX-2 enzyme, the effect of the drug on the conversion of exogenous AA into PGE₂ in cells pre-activated for 24 h to up-regulate COX-2 protein was investigated. As shown in Figure 6(b), the drug had no effect on PGE₂ production at concentrations of 10 µM and 30 µM. As expected, the two NSAIDs, indomethacin (1 μM) and Aspirin (250 μM), both produced significant inhibitory effects of 81% (p < 0.001) and 59% (p < 0.01), respectively, while the steroid dexamethasone (0.3 µM) had no effect.

To verify that the anti-inflammatory activity of CEE-1 was not due to toxicity to the cells, we analyzed the effect of the drug on apoptosis of human primary monocytes using the annexin V binding/flow cytometry method. As shown in Figure 7, culturing the cells with

CEE- 1 at the concentrations of 3, 10 or 30 µM for 24 h did not significantly increase total apoptosis (both early and late) when compared to vehicle-treated cells.