The anti-inflammatory activity of a novel fused-cyclopentenone phosphonate and its potential in the local treatment of experimental colitis

Abstract

A novel fused-cyclopentenone phosphonate compound, namely, diethyl 3-nonyl-5-oxo-3,5,6,6a-tetrahydro-1H-cyclopenta[c]furan-4-ylphosphonate (P-5), was prepared and tested in vitro (LPS-activated macrophages) for its cytotoxicity and anti-inflammatory activity and in vivo (DNBS induced rat model) for its potential to ameliorate induced colitis. Specifically, the competence of P-5 to reduce TNFα, IL-6, INFγ, MCP-1, IL-1α, MIP-1α, and RANTES in LPS-activated macrophages was measured. Experimental colitis was quantified in the rat model, macroscopically and by measuring the activity of tissue MPO and iNOS and levels of TNFα and IL-1β. It was found that P-5 decreased the levels of TNFα and the tested proinflammatory cytokines and chemokines in LPS-activated macrophages. In the colitis-induced rat model, P-5 was effective locally in reducing mucosal inflammation. This activity was equal to the activity of local treatment with 5-aminosalicylic acid. It is speculated that P-5 may be used for the local treatment of IBD (e.g., with the aid of colon-specific drug platforms). Its mode of action involves inhibition of the phosphorylation of MAPK ERK but not of p38 and had no effect on IκBα.

Scheme 1

Synthesis of diethyl 3-nonyl-5-oxo-3,5,6,6a-tetrahydro-1H-cyclopenta[c]furan-4-ylphosphonate (P-5).

Figure 1

Relative cytotoxicity (expressed in % of the untreated cells) of low concentration range (1–40 μM) of P-5 towards LPS-activated mouse peritoneal macrophages. N: naive control; U: untreated control; B: budesonide (10 μM, positive control). Shown is the mean of three different experiments ± S.D. ANOVA of P-5 cytotoxicity was F _10,48 = 5.32, p < 0.00001. ^∗ p < 0.05; ^∗∗ p < 0.01 compared to untreated macrophages.

Figure 2

The reduction of TNFα levels (expressed as the fraction, in %, of TNFα level secreted by untreated cells) caused by a low concentration range (1–20 μM) of P-5 as measured in LPS-activated macrophages. N: naive control; U: untreated control; B: budesonide (10 μM, positive control). Shown is the mean of three different experiments ± S.D. ANOVA of the effect of P-5 on reduction of TNFα was F _8,97 = 119.21, p < 0.00001. ^∗ p < 0.05; ^∗∗ p < 0.01; ^∗∗∗ p < 0.001, compared to untreated macrophages.

Figure 3

Cleavage of the fluorescent peptide substrate Mca-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH₂ by purified human recombinant TACE in the presence of elevated concentrations of P-5 or doxycycline.

Figure 4

The attenuating effect of P-5 on the secretion of IL-6, IL-1α, INFγ, MCP-1, MIP-1α, and RANTES in LPS-activated macrophages. Results are expressed as the fraction (in %) of the cytokine level secreted by untreated activated cells. N: naive control; U: untreated control. Shown are the mean results ± S.D. (n = 3-4). ANOVA of the effect of P-5 at a series of concentrations on IL-6 levels was F _7,33 = 115.36, p < 0.00001, on IL-1α was F _6,20 = 8.06, p < 0.001, on INFγ was F _6,19 = 4.64, p < 0.01, on MCP-1 was F _6,20 = 73.57, p < 0.00001, on MIP-1α was F _7,23 = 219.00, p < 0.00001, and on RANTES was F _5,17 = 100.54, p < 0.00001. ^∗ p < 0.05; ^∗∗ p < 0.01; ^∗∗∗ p < 0.001 compared to untreated cells.

Figure 5

Western blot and densitometry analysis of p-p38 (normalized to p38; (a)), p-ERK (normalized to ERK; (b)), and IκBα (normalized to tubulin; (c)) in activated macrophages lysates after incubation with 1 or 5 μM of P-5. C: naive control; U: untreated control. Shown are the mean ± S.D results. of 4–6 measurements. ANOVA of the effect of 1 and 5 μM of P-5 on the tested regulatory proteins were F _3,16 = 353.02 p < 0.00001 for p-38, F _3,20 = 47.19 p < 0.00001 for ERK, and F _3,16 = 97.01 p < 0.00001 for IκBα. ^∗∗ p < 0.01; ^∗∗∗ p < 0.001; compared to untreated cells.

Figure 6

MPO activity (a), iNOS expression (b), TNFα (c), and IL-1β (d) tissue levels in the colons of healthy rats, untreated DNBS-induced rats, and DNBS-induced rats treated with P-5 and 5-ASA. Results are normalized to total tissue protein. (a), (c), and (d) are expressed as a fraction (in %) of the tissue biomarker levels in the DNBS-treated group. (b) is normalized to the house keeping gene, GAPDH. Shown are the mean ± S.D. values of 3–7 rats in each group. ANOVA of the effect of P-5 on MPO activity was F _3,19 = 13.63 p < 0.0001, on iNOS expression was F _2,18 = 37.57 p < 0.00001, on TNFα level was F _3,22 = 9.00 p < 0.001, and on IL-1β level was F _3,31 = 22.61 p < 0.00001. ^∗ p < 0.05; ^∗∗ p < 0.01; ^∗∗∗ p < 0.001 compared with the DNBS group.