BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the respective chemical structures of KMUP-3 and KMUP-4 according to the present invention;
FIG. 2 shows the expression of inducible nitric oxide systhase, iNOS, in the tracheal smooth muscle cell (TSM) culture exposing to the TNF-α in different time period;
FIG. 3 shows the expression of sGCα1 and sGCβ1 in the tracheal smooth muscle cell (TSM) culture exposing to the TNF-α in different time period;
FIG. 4(A) shows the expression of iNOS, in the tracheal smooth muscle cell culture exposing to the TNF-α with different concentration of KMUP-1 or KMUP-3 according to the present invention;
FIG. 4(B) shows the expression of iNOS, in the tracheal smooth muscle cell culture exposing to the TNF-α with different concentration of KMUP-1, KMUP-3, zaprinast or 8-Br-cGMP;
FIG. 5 shows the expression of sGCα1 and sGCβ1 in the tracheal smooth muscle cell culture exposing to the TNF-α with KMUP-1, KMUP-3, zaprinast or 8-Br-cGMP;
FIG. 6 shows the expression of PKA and PKG in the tracheal smooth muscle cell (TSM) culture exposing to the TNF-α in different time period;
FIG. 7(A) shows the expression of PKG in the tracheal smooth muscle cell culture exposing to the TNF-α with different concentration of KMUP-1 or KMUP-3 according to the present invention;
FIG. 7(B) shows the expression of PKA in the tracheal smooth muscle cell culture exposing to the TNF-α with different concentration of KMUP-1 or KMUP-3 according to the present invention;
FIG. 8(A) shows the expression of PKG in the tracheal smooth muscle cell culture exposing to the TNF-α with KMUP-1, KMUP-3, zaprinast or 8-Br-cGMP;
FIG. 8(B) shows the expression of PKA in the tracheal smooth muscle cell culture exposing to the TNF-α with KMUP-1, KMUP-3, zaprinast or 8-Br-cGMP;
FIG. 9(A) shows the expression of COX-2 in the tracheal smooth muscle cell (TSM) culture with or without exposing to the TNF-α in different time period;
FIG. 9(B) shows the expression of COX-2 in the tracheal smooth muscle cell culture exposing to the TNF-α with a pre-treating of KMUP-1, KMUP-3, dexamethasone or NS-398;
FIG. 10(A) shows the cGMP level in the tracheal smooth muscle cell culture exposing to TNF-α with a pre-treating of KMUP-1, KMUP-3 or zaprinast;
FIG. 10(B) shows the cAMP level in the tracheal smooth muscle cell culture with or without exposing to TNF-α in a different pre-treating of isoproterenol, KMUP-1 and KMUP-3;
FIG. 11 shows the nitrite/nitrate levels in the tracheal smooth muscle cell culture exposing to the TNF-α with a pre-treating of KMUP-1, KMUP-3, zaprinast or 8-Br-cGMP;
FIG. 12 shows the PGE2 and 6-keto-PGF1α levels in the tracheal smooth muscle cell culture exposing to the TNF-α with a pre-treating of KMUP-1, KMUP-3, dexamethasone or NS-398;
FIG. 13 shows a proposed mechanism for KMPU-1 and KMPU-3 on the TNF-α-induced inflammation in the rat tracheal smooth muscle cell according to the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for the purposes of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
The present invention discloses an anti-proinflammation substrate including one selected from the group consisting of a 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine, a 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine, a respective pharmaceutical acceptable salt thereof, and a combination thereof, where the mentioned xanthine-based compounds inhibit TNF-α-induced expression of iNOS in tracheal smooth muscle cells (TSMCs), involving sGC/cGMP/PKG expression pathway, but without the involvement of COX-2.
Please refer to the FIG. 1, which shows the respective chemical structures of the 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-3) according to the present invention.
In the following descriptions, KMUP-1 and KMUP-3 were synthesized in our laboratory. The 8-Br-cGMP, dexamethasone, indomethacin, isoproterenol, NS-398, TNF-α and zaprinast were all purchased from Sigma-Aldrich (St. Louis, Mo.). The antibodies for COX-2, iNOS and sGC were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).
The tracheal smooth muscle cell (TSM) culture is prepared from male Wistar rats purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan).
The Wistar rats were injected intraperitoneally with a lethal dose of pentobarbital. The tracheas were excised and cut longitudinally through the cartilage. Using a dissecting microscope, TSM strips were dissected from the surrounding parenchyma. The epithelium was removed from the luminal surface, and bands of TSM were gently separated from the underlying connective tissue. The TSM strips were then chopped into small sections (1 mm3) and incubated in Hank's balanced salt solution (NaCl, 138 mM; NaHCO3, 4 mM; KCl, 5 mM; KH2PO4, 0.3 mM; Na2HPO4, 0.3 mM; glucose, 1.0 mM) with 0.05% elastase type IV and 0.2% collagenase type IV (Invitrogen, Carlsbad, Calif.) for 30 min at 37° C. with gentle shaking. The solution of dissociated smooth muscle cells was centrifuged (6 min at 500 g) and the pellet was resuspended in 1:1 Dulbecco's modified Eagle's medium-Ham's F-12 medium supplemented with 10% fetal bovine serum (FBS), 0.244% NaHCO3, and 1% penicillin/streptomycin. Cells were cultured in a condition with or without KMUP-1, KMUP-3 and other negative or positive control agents, in 25 cm2 flasks at 37° C. in humidified air containing 5% CO2. The medium was changed every 2-3 days.
Analyses for the iNOS and sGC Expressions
To investigate the effects of TNF-α on iNOS and sGC proteins, TSMCs were incubated with TNF-α (100 ng/ml) for 1, 3, 6, 9, 12, 18 and 24 hr, and the levels of iNOS and sGC subunit proteins were measured by immunoblotting. As shown in FIG. 2, exposure of TSMCs to TNF-α increased iNOS protein expression in a time-dependent manner, with the maximum level evident at 9 hr. In contrast to the increase expression of iNOS protein in FIG. 2, TNF-α in FIG. 3 decreased the expression of sGCα1 and sGCβ1 proteins in a time-dependent manner, with the maximal inhibition achieved at 9 hr. To investigate the effects of KMUP-1, KMUP-3, and the other PDE5 inhibitors zaprinast and exogenous 8-Br-cGMP on the TNF-α mediated pathway, TSMCs were incubated with TNF-α (100 ng/ml) and either one of the mentioned chemicals. As shown in FIG. 4(A), KMUP-1 and KMUP-3 (0.01-100 μM) inhibited TNF-α-induced increases of iNOS expression in a concentration-dependent manner. Pleaser refer to FIG. 4(B), either KMUP-1 or KMUP-3 as well as the other two PDE5 inhibitors zaprinast and exogenous 8-Br-cGMP in the same concentration (10 μM) inhibited TNF-α-induced increases of iNOS expression to a similar extent. In addition, as shown in FIG. 5, the inhibitions of TNF-α on sGCα1 and sGCβ1 proteins expressions were reversed by KMUP-1, KMUP-3, zaprinast and 8-Br-cGMP at 10 μM.
Analyses for the PKG and PKA Expressions
To investigate the effects of TNF-α on PKG and PKA proteins, TSMCs were incubated with TNF-α (100 ng/ml) for 1, 3, 6, 9, 12, 18 and 24 hr, and the levels of PKG and PKA proteins were measured by immunoblotting. As shown in FIG. 6, TNF-α (100 ng/ml) time-dependently increased PKA within 24 hr and significant decreased the expression of PKG protein between 6 to 9 hr. To investigate the effects of KMUP-1, KMUP-3, and the other PDE5 inhibitors zaprinast and exogenous 8-Br-cGMP on the TNF-α mediated pathway, TSMCs were incubated with TNF-α (100 ng/ml) and either one of the mentioned chemicals. As shown in FIG. 7(A), the increased expression of PKA protein by TNF-α was not further increased by KMUP-1 and KMUP-3 at a concentration of 0.1-100 μM. However, as can be seen in FIG. 7(B), the decreases of PKG protein expression by TNF-α were reversed by both KMUP-1 and KMUP-3. Please further refer to FIGS. 8(A) and 8(B), KMUP-1, KMUP-3, and the other chemicals of Zaprinast and 8-Br-cGMP at 10 μM also reversed TNF-α-induced decreases of PKG protein, whereas 8-Br-cAMP and zaprinast at 10 μM, similar to KMUP-1 and KMUP-3, could not affect TNF-α-induced increases of PKA protein.
Analyses for the COX-2 Expressions
To investigate the effects of TNF-α on Cox-2 protein, TSMCs were respectively incubated with or without TNF-α (100 ng/ml) for 1, 3, 6, 9, 12, 18 and 24 hr, and the levels of COX-2 proteins were measured by immunoblotting. As shown in FIG. 9(A), in the absence of TNF-α in rat TSMCs, the expression of COX-2 showed time-dependent decreases during incubation for 24 hr. In the presence of TNF-α (100 ng/ml), the expressions of COX-2 were limited to moderate decreases after 6 hr and sustained for 24 hr in comparison with non-TNF-α challenge groups. Further, to investigate the effects of KMUP-1, KMUP-3, the other chemicals, such as the anti-inflammation agent, dexamethasone, and the COX-2 inhibitor, NS-398, on the TNF-α mediated pathway, TSMCs were incubated with TNF-α (100 ng/ml) with or without a pretreatment of either one of the mentioned chemicals. As shown in FIG. 9(B), it was clear that not KMUP-1 and KMUP-3 (10 M) but dexamethasone (1 μM) and COX-2 inhibitor NS-398 (10 μM) can significantly inhibit TNF-α-induced COX-2 expression in TSMCs.
Analyses for Cyclic Nucleotide Levels
Intracellular cGMP production was decreased and reaching minimal production at 9 hr in TSMCs in the presence of TNF-α (100 ng/ml). Intracellular concentrations of cAMP and cGMP in TSMC were measured to investigate the effects of KMUP-1, KMUP-3, and the other PDE5 inhibitor zaprinast or the isoproterenol thereto. As to the level of cGMP, TSMCs were pretreated with KMUP-1, KMUP-3, and the other PDE5 inhibitor zaprinast for 20 minutes and were further incubated in the presence of TNF-α (100 ng/ml) for 9 hrs. The concentrations of cGMP of each sample were measured using cGMP-[125I] radioimmunoassay kits (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.). As shown in FIG. 10(A), in the presence of KMUP-1, KMUP-3 and zaprinast (10 μM), cGMP was reversed to the basal level. As to the level of the cAMP, TSMCs were incubated with isoproterenol, KMUP-1, and KMUP-3 for 20 min, and further incubated in the absence or presence of TNF-α (100 ng/ml) for 9 hrs, where the concentrations of cAMP of each sample were measured using cAMP-[125I] radioimmunoassay kits (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.). As shown in FIG. 10(B), in the absence of TNF-α, use of KMUP-1, KMUP-3 and isoproterenol (10 μM) in the culture of TSMCs significantly increased the production of cAMP, compared to the control group. However, in the presence of TNF-α, KMUP-1 and KMUP-3 could not further increase the production of cAMP, compared to the control group.
Analyses for NO Level Measured by Nitrite/Nitrate
Exposure of TSMCs to TNF-α (100 ng/ml) for 9 hrs led to accumulation of nitrite and nitrate in the culture medium, indicating the release of NO. Please refer to the FIG. 11, TSMCs were pretreated with KMUP-1, KMUP-3, 8-Br-cGMP and zaprinast for 20 minutes, and were further incubated in the presence of TNF-α (100 ng/ml) for 9 hrs. The concentrations of nitrite and nitrate (stable breakdown product of NO) of each sample were analyzed using nitrite/nitrate colorimetric assay kits (Cayman Chemical Co.) and run in duplicate. As shown in FIG. 11, 8-Br-cGMP, KMUP-1, KMUP-3 and zaprinast (10 μM) all inhibited TNF-α-induced production of nitrite/nitrate, which represented the NO levels.
Analyses for PGs and COX-2 Activities
Incubation of TSMCs with TNF-α (100 ng/ml) increased the releases of PGs, PGE2 and 6-keto-PGF1α, a PGI2 stable metabolite, and the greatest increases occurred were measured at 24 hr. Please refer to the FIG. 12, TSMCs were pretreated with KMUP-1 (10 μM), KMUP-3 (10 μM), dexamethasone (1 μM) and zaprinast (10 μM) for 20 minutes, and were further incubated in the presence of TNF-α (100 ng/ml) for 24 hrs, where the concentrations of PGE2 and 6-keto-PGF1α (stable metabolite of PGE2) in the culture medium were measured using PGE2 and 6-keto-PGF1α EIA assay kits (Cayman Chemical Co., Ann Arbor, Mich.) in duplicate. Dexamethasone (1 μM) and NS-398 (10 μM), a selective COX-2 inhibitor, significantly inhibited TNF-α-induced PGE2 and 6-keto-PGF1α formation. However, KMUP-1 and KMUP-3 (10 μM) did not inhibit the production of PGs, resulted from activation of COX-2.
Please refer to the FIG. 13, which is a proposed mechanism for KMPU-1 and KMPU-3 on the TNF-α-induced inflammation in the rat tracheal smooth muscle cell. As shown in FIG. 13, the increased expression of iNOS by TNF-α is inhibited by KMUP-1 and KMUP-3, similar to by a iNOS inhibitor aminoguanidine; the elevated cAMP level inhibits the PKG; the elevated ONOO− level inhibits the sGC; the dark thick arrow representing the protein level is increased or decreased by the TNF-α; and the damascening thick arrow representing the protein is activated or increased by either one of the KMPU-1 or KMPU-3. To sum up, either of the KMPU-1 and KMPU-3 inhibits TNF-α-induced expression of iNOS in tracheal smooth muscle cells (TSMCs), involving sGC/cGMP/PKG expression pathway, but without the involvement of COX-2.
In conclusion, under inflammatory conditions, such as in the presence of proinflammatory TNF-α in TSM, a composition including any one of the cGMP enhancing derivatives of xanthine, KMUP-1 and KMUP-3, according to the present application modulates the cross-action between PKA and PKG, by activating sGC/cGMP/PKG pathway, without the involvement of COX-2 expression. Obviously, an anti-proinflammation composition including the xanthine-based KMUP-1 or KMUP-3 with imidazole moiety reduces the cytokine-induced pro-inflammation and limited the risk of further worsening of pulmonary dysfunction.
While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
Patent Application
20080081816
