TWI386206B - A xanthine cGMP enhancing compound having an inhibitory effect on Rho-kinase activity of a lung epithelial cell line - Google Patents

Description

A xanthine cGMP enhancing compound having an inhibitory effect on Rho-kinase activity of a lung epithelial cell line

The present invention relates to a compound having a xanthine-based structure capable of reducing the physiological activity of a lung epithelial cell line, in particular, a Rho- having an increase in cyclic Guanine Monophosphate (cGMP) activity. A kinase inhibitor that inhibits cell migration by increasing cGMP to modulate the expression of Rho-kinase II/vascular endothelial growth factor (ROCK/VEGF) in lung epithelial cells.

Lung epithelial cells often play an important role in the pathogenesis of obstructive bronchial diseases such as obstructive bronchopneumonia and cancer cell metastasis. Nitric oxide synthase/cyclized ornithine (NOS/cGMP) messages in epithelial cells are involved in the regulation of airway contraction and regulation of cell growth and should be associated with obstructive bronchial disease. Epithelial cells release many regulatory substances that inhibit smooth muscle. For example, NO released rapidly through epithelial tissue can affect the contraction and growth of adjacent smooth muscle cells. There are many immunological evidences that NOS/cGMP messages are present. The characteristics of the respiratory epithelium ^1,2 .

KMUP-1(7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) is a Rho-kinase inhibitor that promotes cGMP. It has been found in previous studies that KUMP-1 can relax the tracheal contraction by activating guanyl cyclase (sGC) and NO synthase (NOS) in epithelial cells, resulting in an increase in cGMP in the cytoplasm. Effect. In addition, KMUP-1 can also participate in the activation of sGC and phosphoric acid by inhibiting the expression of inducible NO synthase (iNOS) induced by tumor necrosis factor-a (TNF-a). esterase (phosphodiester, PDE) inhibition of the mechanism, resulting cyclized tracheal smooth muscle cGMP / protein kinase G (protein kinase G) (cGMP / PKG) increases ^3. Therefore, in the present invention, KMUP-1 is considered to be involved in inhibiting proliferation, proinflammatory activity, and migration of lung epithelial cells.

In the present invention, in order to prove the efficacy of KMUP-1, we have used a number of proteins or drugs as indicators, and the characteristics and pharmacological roles of these substances are respectively described as follows: YC-1 is an sGC activator having NO-dependent cGMP enhancing activity. In different types of cells, YC-1 appears to be effective against cell proliferation, anti-angiogenesis, anti-cancer, and resistance to inflammation, and is therefore used in the present invention as a positive control compared to KMUP-1. . In terms of the effect of inhibiting the migration of lung epithelial cells, ROCKII is located downstream of the cGMP/PKG message pathway and is involved in cell migration activity. Therefore, in the present invention, the ROCK inhibitor Y27632 was also used to observe the effect of KMUP-1 on inhibiting migration of lung epithelial cells. .

For the inhibition of angiogenesis, VEGF is currently known to be an important angiogenic factor and a necessary material for tumor growth. The performance of VEGF is triggered by many factors, including hypoxia. HIF-1 regulates VEGF gene when expressed by hypoxia-inducible transcription factor, hypoxic induced factor 1 (HIF-1). YC-1 and since NO is a cGMP-dependent promoter, thus YC-1 in the vascular system also has an important function, and inhibits HIF-1a and VEGF expression in Hep3B cells ^6,7.

In addition, cyclin-dependent kinase (CDK) inhibitory proteins p21 and p27, which are involved in the inhibition of cell proliferation, are also used in the present invention to observe whether or not they are increased in the cGMP pathway. In addition to p21 and p27, phosphorylation of another protein kinase, p38, was also observed in the present invention. p38 in inflammatory cells, while the proliferation of airway structural cells and play an important role in cell survival ^8,9. Further, in order to understand the case of apoptosis, the present invention also analyzes the signal protein Bax/Bc1-2/peptone caspase 3 which is accompanied by p21 and p27 in the cell cycle.

In the present invention, there is provided a compound which inhibits the physiological activity of lung epithelial cells, KMUP-1, which affects eNOS/sGC/PKG signaling, apoptosis information Bax/Bc1-2/caspase 3, and p21 and p27 in the cell cycle. The performance, especially in the hypoxic state of ROCKII / VEGF / HIF-1a, produces inhibition of lung epithelial cell proliferation, migration and inflammation.

The present invention first proposes that KMUP-1 can be achieved by affecting cGMP-related eNOS/sGC/PKG messages, apoptosis information Bax/Bc1-2/caspase 3, and ROCKII/VEGF/HIF-1a under hypoxic conditions. It inhibits the effects of proliferation, inflammation, and migration of lung epithelial cells.

Accordingly, the present invention provides a pharmaceutical composition comprising a 7-[2- a compound of [4-(2-chlorophenyl)piperazinyl]ethyl]-1,3 dimethylxanthine, wherein the compound is a Rho-kinase inhibitor, the pharmaceutical composition having inhibition of a lung epithelial cell A physiologically active effect.

According to the above concept, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

According to the above concept, wherein the physiological activity is selected from at least one of a proliferative activity, a migration activity, and a pro-inflammatory activity.

According to the above concept, wherein the migration activity is a transfer activity of one of the cancer cells.

According to the above concept, the physiological activity is achieved by increasing cGMP in lung epithelial cells and inhibiting Rho-kinase activity.

The invention further provides a method for inhibiting physiological activity of a lung epithelial cell comprising a pharmaceutically effective amount of 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,6 a compound of dimethylxanthine which is administered to a mammal in need thereof, wherein the compound is synthesized from xanthine and is a Rho-kinase inhibitor.

According to the above concept, wherein the compound further comprises a pharmaceutically effective carrier.

According to the above concept, wherein the physiological activity is selected from at least one of a proliferative activity, a migration activity, and a pro-inflammatory activity.

According to the above concept, wherein the migration activity is a transfer activity of one of the cancer cells.

According to the above concept, the compound inhibits the physiological activity of the lung epithelial cells by increasing cGMP and inhibiting Rho-kinase activity in the lung epithelial cells.

In another aspect, the present invention provides a method of preparing a pharmaceutical composition, wherein the pharmaceutical composition comprises a 7-[2-[4-(2-chlorophenyl)piperazinylethyl]-1,3-dimethyl a compound of radix astragali.

According to the above concept, the pharmaceutical composition has an effect of inhibiting physiological activity of one of the lung epithelial cells, and the physiological activity is selected from at least one of a proliferative activity, a migration activity, and a pro-inflammatory activity.

According to the above concept, wherein the migration activity is a transfer activity of one of the cancer cells.

According to the above concept, the pharmaceutical composition further comprises a pharmaceutically effective carrier.

The present invention will be fully understood by the following description of the preferred embodiments and the accompanying drawings. In the example.

The present invention provides a KMUP-1 compound having a physiological activity for reducing lung epithelial cells. The structure and synthesis of the KMUP-1 compound of the present invention are disclosed in U.S. Patent No. 6,979,687, the disclosure of which is hereby incorporated herein. The following is a detailed description of the test results confirming the pharmacological activity of KMUP-1.

Pharmacological test

Cell viability NCI-H441 cells obtained from the American Type Culture Collection (ATCC) were cultured in RPMI 1640 medium, and 2 mM glutamic acid, penicillin/streptomycin, and 10% fetal bovine serum were added. The cells were cultured in a 37 ° C incubator in a state of normal oxygen (20% oxygen) and anoxic (1% oxygen) in a 5% carbon dioxide incubator. In order to achieve anoxic state, a pre-analyzed mixed gas (95% nitrogen - 5% carbon dioxide) was injected into the incubator.

In the cell survival and proliferation test, H441 cells were cultured in a 24-well plate at a density of 10 ⁵ cells per culture, and treated with different concentrations of KMUP-1 for various times, and cell viability was obtained by MTT assay. All experimental data are expressed as mean ± standard error, and the number of samples is n=4. For unpaired and paired samples, the statistical significance was analyzed in an independent and paired t-test. One-way ANOVA or two-way ANOVA was used when the control group was compared to more than one experimental group. When the ANOVA analysis showed statistical differences, the results were further subjected to Dunnett's or Tukey analysis, where P < 0.05 indicates significantness. The experimental values were analyzed via SigmaStat: Version 2.03, plotted with SigmaPlot: Version 8.0 (Systat Software, Point Richmond, CA) and executed using an IBM computer.

Please refer to the first figure, which is the different concentrations (1.0, 10 and 100 μM) of KMUP-1 of the present invention after 24, 48 and 72 hours of treatment in the normal (Fig. 1A) and hypoxic (Fig. 1B) states. Inhibition of H441 cell viability. KMUP-1 (10, 10, 100 μM) inhibited the survival of H441 cells under normal and hypoxic conditions. As shown in the first figure (A) and the first figure (B), the high concentration of KMUP-1 ( 10 μM) significantly inhibited the survival rate of H441 cells.

2. Cell cycle distribution The cells after trypsin were collected, washed with PBS, and the cells were suspended in PBS containing 75% alcohol, and the cells were placed at 4 ° C for 30 minutes. Before the analysis, the cells were washed and suspended again with PBS, followed by 0.05 mg/ml propidium iodide, 1.0 mM ethylenediaminetetraacetic acid (EDTA), 0.1% surfactant Triton X- in PBS. 100 and 1 mg/ml RNA-degrading enzyme RNase A in propidium iodide solution for 30 minutes. Next, the cell suspension was passed through a nylon filter and analyzed by flow cytometry (Coulter Epics XL-MCL, Beckman Coulter, USA).

Please refer to the second figure, which is the effect of different concentrations of KMUP-1 (0.01, 0.1, 1.0, 10 and 100 μM) on the cell cycle distribution ratio (%). Under normal conditions, the results of flow cytometry analysis showed the effect of KMUP-1 on cell cycle progression, and the distribution of cell cycle was affected by KMUP-1 concentration (0.01, 0.1, 1.0, 10, 100 μM), showing concentration correlation. . The results of the second graph show that the ratio of G0/G1 phase increases with the concentration of KMUP-1, and the ratio of S phase to G2/M phase decreases with the concentration of KMUP-1.

Please refer to the third to third (A) to third (C), which show the effect of the KMUP-1 (1.0 μM) of the present invention on the ratio of each cycle in the cell cycle after the action of 6 hours to 72 hours. As shown in the second to third (C), after 60 hours of KMUP-1 (100 μM), the cell cycle was clearly observed. The block is in the G0/G1 phase.

Please refer to the fourth figure, which is a cell cycle region map analyzed by flow cytometry, which is the effect of KMUP-1 (100 μM) of the present invention on cell cycle distribution after comparison with the control group and the solvent control group thereof. . In the fourth panel, after 72 hours of KMUP-1 (100 μM), the cell cycle was clearly arrested in the G0/G1 phase.

3. Performance of NOS, sGC and PKG In order to determine the expression levels of eNOS, iNOS, sGC, PKG, HIF-1a, VEGF, ROCKII, p38, Bax, Bc1-2 and the CDK inhibitory proteins p21 and p27 in H441 cells, cells are first introduced in the present invention. All proteins were extracted and analyzed by the following Western blot method.

H441 cells were cultured in culture plates of 10 cm in diameter, and when the cells were grown to half full, the cells were stopped growing and treated with KMUP-1 for different times. In some experiments, KMUP-1 was administered to the cells prior to treatment with a specific inhibitor. When the amount of iNOS expression was measured, it was treated with KMUP-1, and the amount of expression was measured at 30 minutes after TNF-a (100 ng/ml). After the cells were treated, they were washed with PBS (pH 7.4) and placed in an extraction buffer (10 mM Tris solution with a pH of 7.0, containing 140 mM sodium chloride, 2 mM phenylmethylsulfonate PMSF, 5 mM dithiothreitol DTT, 0.5% surfactant NP-40, 0.05 mM gastric protein inhibitor A and 0.2 mM calpain inhibitor leupeptin) were slowly shaken and then at 12,500 g Centrifuge at the speed for 30 minutes. Next, the cell extract was boiled in a sample solution (100 mM Tris solution having a pH of 6.8 containing 20% glycerol, 4% SDS, and 0.2% bromophenol blue) in a ratio of 1:1. 50 mg of protein was placed in each sample cell of a 10% SDS-polyacrylamide gel, and electrophoresis was carried out at 10 volts (V) and 40 mA. The electrophoretically separated protein was transferred to a polyvinylidene difluoride (PVDF) membrane at 100 volts for 90 minutes, and then the non-specific IgGs antibody was blocked with 5% skim milk powder, and the specific antibody was silenced. Set for an hour. After applying the film to the anti-mouse or anti-goat IgG antibody (1:1000) combined with alkaline phosphatase for one hour, a chemical color enhancer (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) makes the protein band visible.

Please refer to the fifth figure (A) (B), which shows that H441 cells 12, 24 are treated under normal and hypoxic conditions in the presence (Fig. 5B) or absence (Fig. 5A) of KMUP-1 (10 μM). The effect of its eNOS performance after 48 and 72 hours. Under normal conditions, KMUP-1 (10 μM) stimulates the expression of eNOS in H441 cells with a temporal correlation. The performance of eNOS reached its maximum at 48 hours. In hypoxia, the expression of eNOS protein in H441 cells showed a time-related decrease, but it was suddenly increased by KMUP-1.

Please refer to the sixth figure, which is treated with different concentrations of KMUP-1 (0.001, 0.01, 0.1, 1.0, and 10 μM) under normal oxygen content for 48 hours. The effect of eNOS performance. As shown in Figure 6, the induction of eNOS expression by KMUP-1 was also concentration-dependent within 48 hours.

Please refer to the seventh figure (A) and the seventh figure (B), in which the KMUP-1 of the present invention is normal and hypoxic after the cells are pretreated with the commercially available NOS inhibitory drug L-NAME (100 μM) for 30 minutes. The effect of the state on the amount of eNOS expression. As shown in the seventh panel (A) (B), the KMUP-1 was reduced in the amount of expression of the eNOS protein after pretreatment with the NOS inhibitor L-NAME (100 μM) in both states.

Please refer to the eighth and ninth figures, which are the effects of KMUP-1 (10,100 μM) of the present invention on the sGC/PKG message pathway in H441 cells under normal and hypoxic conditions. The cells were divided into a control group containing no KMUP-1 and a group containing KMUP-1, which were treated under normal and anoxic conditions for 48 hours, respectively. As shown in the eighth panel, the expression of sGCa in H441 cells was increased by KMUP-1 (10,100 μM) in both states. In addition, as shown in the ninth figure, the expression of PKG in H441 cells was significantly increased by the action of KMUP-1 (10,100 μM), and increased to 146.8±13.9% and 157.9±12.1% of the control group under normal conditions, respectively. In the oxygen state, it increased to 130.1±11.7% and 166.6±18.0% of the control group, respectively.

In previous studies that when human cells are PKG activation, and effective inhibition of the phenomenon to trigger apoptosis in cell growth, cell migration can be suppressed phenomenon ^10. In the present invention, KMUP-1 (10, 100 μM) enhances the expression of sGC and PKG in H441 cells under normal or hypoxic conditions, indicating that it has the ability to stimulate apoptosis and inhibit cell growth and migration. . In general, activation of eNOS and sGC helps to increase the performance of PKG by relying on cGMP. Based on the results of the above experiments, it is speculated that the inhibitory effect of high concentration of KMUP-1 on cell growth is related to long-term increase of NO/peroxynitrite and cGMP/PKG.

4. HIF-1a, VEGF and ROCKII messages Please refer to the tenth figure, which is the effect of KMUP-1 (1 μM) of the present invention on the amount of HIF-1a expression after exposure of H441 cells to anoxic state for 3 to 72 hours. After 3 hours of exposure to hypoxia, HIF-1a performed more significantly and reached its maximum at 6 to 12 hours. However, after 18 to 24 hours, the performance of HIF-1a protein decreased rapidly. Under the action of KMUP-1, the performance of HIF-1a was inhibited at 12 hours, most obviously at 24 hours.

Please refer to the eleventh figure, which is the effect of KMUP-1 (1 μM) of the present invention on the amount of VEGF expression after exposure of H441 cells to anoxic state for 3 to 72 hours. The expression of VEGF was also enhanced under hypoxic conditions, while the inhibitory effect of KMUP-1 (1.0 μM) was 59.9±3.0% and 39.6±4.4% of the control group at 12, 18, 24, 48 and 72 hours, respectively. 37.0 ± 5.1%, 50.7 ± 3.5%, and 44.2 ± 3.3%.

Please refer to Fig. 12, which is a 24 hour inhibition of HIF-1α and VEGF expression after KMUP-1 (1 μM) of the present invention was pretreated for 30 minutes with L-NAME (100 μM). From the twelfth map It is known that HIF-1α does not show well under normal conditions, but is clearly visible in anoxic state. When pretreated with L-NAME, the effect of KMUP-1 on the expression of HIF-1a was not observed, but L-NAME was combined with KMUP-1, and HIF-1a was not observed under hypoxia. Further suppression. .

Refer to Figure 13 (A) and Figure 13 (B) for different concentrations of KMUP-1 (0.01, 0.1, 1.0, 10 and 100 μM) for HIF-1a under hypoxic conditions ( Figure 13A) and VEGF (Figure 13B) show the effect of the amount at 24 hours. As shown in Figure 13 (A) and Figure 13 (B), the expression of HIF-1a and VEGF was correlated with KMUP-1 (0.01-100 μM).

Please refer to Figure 14 (A) and Figure 14 (B) for H441 cells in the hypoxia state, control group, 1.0 μM KMUP-1, 1.0 μM YC-1, 100 μM SNP and 100 The amount of expression of HIF-1a (Fig. 14A) and VEGF (Fig. 14B) after 24 hours of different treatments of μM IBMX. According to the data in Fig. 14 (A), the HIF-1a protein was inhibited under different treatments as follows (100% of the data from the control group): 34.8 ± 2.3% (KMUP-1, 1.0 μM), 31.6 ±3.4% (YC-1, 1.0 μM), 78.7 ± 3.1% (SNP, 100 μM), 15.9 ± 5.9% (IBMX, 100 μM). The data in Figure 14 (B) shows that the VEGF protein was inhibited under different treatments as follows (100% of the data from the control group): 45.5 ± 3.1% (KMUP-1, 1.0 μM), 47.2 ±4.4% (YC-1, 1.0 μM), 80.1 ± 2.8% (SNP, 100 μM), 20.2 ± 4.7% (IBMX, 100 μM).

As shown in Figure 13 (A) (B), KMUP-1 (1-100 μM) with concentration Increased inhibition of hypoxia-induced VEGF and HIF-1a expression. On the other hand, as shown in Fig. 12, under normal conditions, KMUP-1 at a concentration lower than 1.0 μM did not give any significant expression of VEGF, and it also showed that it did not have the ability to promote angiogenesis. Combining the above results, KMUP-1 not only overcomes the phenomenon of VEGF production by relying on NO in the absence of oxygen, but also has the potential to inhibit inflammation and anti-angiogenesis. In the present invention, KMUP-1 inhibits VEGF and HIF-1a proteins under hypoxia, and exhibits an activity of inhibiting angiogenesis and antitumor, which is marked by a hypoxia protein marker.

Please refer to the fifteenth panel, which is the effect of KMUP-1 (10, 100 μM) of the present invention on the expression of ROCKII protein in H441 cells under normal and hypoxic conditions. The performance of ROCKII was positively correlated with KMUP-1 concentration. In the twenty-fifth panel, the effect of KMUP-1 was inhibited by the cGMP antagonist Rp-8-CPT-cGMP (10 μM).

5. Cell migration and inhibition of ROCKII H441 cells were cultured in six-well plates until the cells grew to over 90%. A wound is drawn on the cell layer by means of the tip of the micropipette in a manner that spans the culture cell, and the cells are washed four times with the culture solution to remove cell debris. After treating the cells with 10 μM of KMUP-1 and Y-27632 for 24 hours and 48 hours, the edges of the wound (Eclipse TS100, Nikon) were visualized and photographed using a microscope. In the same culture, the maximum and minimum positions of the wound width were measured and averaged.

In order to correspond to the results of ROCKII expression, the present invention demonstrates the effect of KMUP-1 on cancer cell metastatic activity using experiments of cell migration. Please refer to Figure 17 (A) (B), which shows that H441 cells are subjected to 10% fetal bovine serum, serum-free medium, KMUP-1 (1-100 μM), and ROCK inhibitor Y27632 (10 μM). The relative distance of the wound width in the normal (Fig. 17A) and hypoxic (Fig. 17B) states after 48 hours of different treatments. As shown in Fig. 17 (A) and Fig. 17 (B), KMUP-1 can inhibit the migration activity of H441 lung epithelial cells under normal or hypoxic conditions. Especially when the concentration of KMUP-1 was higher than 50 μM, the migration of H441 cells across the wound width after 48 hours of culture was more significantly reduced, and the effect was more remarkable under normal conditions than under hypoxia. The present invention also utilizes the results of the ROCKII inhibitor Y27632 in comparison with KMUP-1. Y27632 (10 μM) also inhibits ROCKII under normal conditions, but not in the absence of oxygen. Since the migration phenomenon of cancer epithelial cells increases the risk of metastasis of cancer cells, in the present invention, the inhibitory effect of KMUP-1 on ROCKII provides potential for metastasis in lung epithelial cells.

6. Performance of p21 and p27 Please refer to the eighteenth (A) and eighteenth (B) drawings, which are the KMUP-1 of the present invention in the normal and hypoxic state, for the non-cGMP antagonist, Rp-8-CPT-cGMP pre- The effect of p21 expression in H441 cells treated (Fig. 18A) and after 30 minutes of pretreatment with Rp-8-CPT-cGMP (10 μM) (Fig. 18B). The cells were cultured for 48 hours under normal and hypoxic conditions with or without KMUP-1. As shown in Fig. 18(A), after the comparison with the control group, p21 was affected by KMUP-1 (10,100 μM) and increased as follows: under normal conditions, 181.2±17.6% and 172.9±18.1%, In the hypoxic state, it was 151.7±7.7% and 135.1±14.7%. As shown in Figure 18 (B) It was shown that Rp-8-CPT-cGMPS (10 μM) did not inhibit the p21 protein induced by KMUP-1.

Please refer to the nineteenth (A) and nineteenth (B) drawings, which are the KMUP-1 of the present invention in the normal and hypoxic state, for the non-cGMP antagonist, Rp-8-CPT-cGMP pre- The effect of p27 expression in H441 cells after treatment (Fig. 19 A) and after 30 minutes of pretreatment with Rp-8-CPT-cGMP (10 μM) (Fig. 19B). As shown in Fig. 19(A), the increase in p27 by KMUP-1 (10,100 μM) is as follows: 162.5 ± 12.5% and 201.6 ± 23.5% under normal conditions, respectively, under hypoxic conditions 172.4 ± 19.5% and 242.2 ± 21.5%. As shown in Fig. 19(B), Rp-8-CPT-cGMPS (10 μM) also failed to inhibit the p27 protein induced by KMUP-1. In addition, the expression of p21 and p27 is also increased in cells lacking serum culture.

In the cell cycle, the CDK inhibitory proteins p21 and p27 are indicators of DNA replication in growing cells, usually activated by p53 when DNA is damaged. When the gene is damaged, p21 and p27 will cause the cell cycle to stop at the G0/G1 checkpoint through a divergent mechanism. KMUP-1 (10-100 μM) inhibits proliferation of H441 cells under hypoxia, but does not have this inhibition at concentrations below 1.0 μM. However, more and more evidence supports the importance of the regulation of p21 and p27 at the translation level. The KMUP-1 of the present invention increases the expression of p21 and p27 under hypoxic stress, indicating its effect on the translational level of cancer cell growth. However, the effect of KMUP-1 on the expression of p21 and p27 was not affected by Rp-8-CPT-cGMPS, indicating that it involved cell cycle progression independent of cGMP.

7. Bax/Bcl-2 and protease caspase 3 Please refer to the twentieth (A) and twentieth (B) drawings, which are the KMUP-1 (10,100 μM) of the present invention, Bax (Fig. 20A) and Bcl in H441 cells under normal and hypoxic conditions. -2 (Fig. 20B) The role of performance. KMUP-1 (10,100 μM) increased the performance of Bax in the normal state to 125.6±11.4% and 103.6±10.8% in the control group, while the Bcl-2 expression in the normal state decreased to 31.9±1.2% in the control group. The decrease in Bcl-2 expression in hypoxic conditions was 56.5 ± 1.3% in the control group. Therefore, as shown in the twenty-first figure, the ratio of Bax/Bcl-2 increases with the concentration of KMUP-1. As shown in Figures 22 and 23, L-NAME and Rp-8-CPT-cGMP inhibited the increase in the ratio of KMUP-1 to Bax/Bcl-2.

Please refer to Figure 24 for different concentrations of KMUP-1 (1.0, 10 and 100 μM) for the ratio of unactivated procaspase-3/activated caspase-3 in H441 cells under normal and hypoxic conditions. The effect, in which the proportion of unactivated procaspase-3/activated caspase-3 was increased by the concentration of KMUP-1, was more pronounced under normal conditions than under hypoxia.

According to the cell cycle experiment of the present invention, after exposure to hypoxia for 72 hours, H441 cells will have a sub-G1 peak (sub-G1 peak, also representing the peak of apoptosis), while in KMUP-1 ( 100 μM) co-culture further enhanced the peak of apoptosis. In addition, KMUP-1 (10,100 μM) also increased Bax and decreased Bcl-2 performance in the absence of oxygen, resulting in an increase in the Bax/Bcl-2 ratio. KMUP-1 also enhances the performance of the protease caspase-3, which show activity on apoptosis in H441 cells ^11.

It is worth noting that KMUP-1 can increase eNOS expression in normal or hypoxic conditions, so theoretically NO-induced angiogenesis and toxic peroxidic nitrite ions (ONOO ^- ) will also increase. Moreover, the effect of KMUP-1 on the Bax/Bc1-2 ratio was also inhibited by the NOS inhibitor L-NAME and the PKG inhibitor Rp-8-CPT-cGMPS (refer to Figure 22 and Figure 23). Therefore, the effect of KMUP-1 on apoptosis of H441 cells in the present invention may be attributed to the passage of NO-dependent Bax/Bcl-2 and cGMP pathways, triggering excessive expression of oxidized nitrite ions, and YC-1 is not in HA22T cells. The anti-proliferative activity dependent on cGMP is different ¹² .

8. PDE-5A triggered by U46619 Please refer to the twenty-fifth figure, which is the effect of KMUP-1 of the present invention on PDE5A expression in H441 cells under normal and hypoxic conditions. Further reference is made to Figure 26, which is the effect of different concentrations of KMUP-1 (1.0, 10 and 50 μM) on the amount of PDE5A present in cells after induction with the PDE5A promoter U46619. As shown in Figure 26, the synergist U46619 (5 μM) mimicking the inflammatory factor TXA clearly caused an increase in PDE5A expression under normal conditions. However, after pretreatment with KMUP-1 (1, 10, 50 μM), U46619 caused PDE5A expression in epithelial cells, presenting a reduction associated with KMUP-1 concentration.

9. Phosphorylation of p38 and TNF-a-induced iNOS Please refer to the twenty-seventh figure, which is the effect of KMUP-1 (10, 100 μM) of the present invention on the ratio of phosphorylated p38/all p38 in H441 cells under normal and hypoxic conditions. As shown in Figure 27, the relative optical density ratio represents the ratio of phosphorylation of p38/all p38, and in the serum-free group, the ratio of expression decreases under normal conditions (number of samples n=4, p< 0.05), but there is an insignificant increase in hypoxia. KMUP-1 (10,100 μM) reduced the performance ratio to 58.9±7.1% and 50.9±9.1% under normal conditions, and reduced the performance ratio to 70.6±6.6% and 67.1±9.4% under hypoxic conditions (sample number) n=4, p<0.05). Phosphorylated p38 is affected by KMUP-1 and exhibits a decrease in performance, indicating that KMUP-1 has the ability to inhibit the proinflammatory pathway.

P38 is a protein associated with inflammation-promoting proteins and is thought to be involved in the regulation of inflammatory responses. Many factors, including hypoxia stress, activate p38. Once p38 is activated, p38 phosphorylates downstream receptors to initiate a cascade of messages that regulate the synthesis of many pro-inflammatory factors. The Rho/ROCK message responsible for regulating p38 stimuli is associated with migration of vascular smooth muscle cells and is also susceptible to the ROCK inhibitor Y27632. In the present invention, KMUP-1 attenuates the expression of ROCKII/p38 and inhibits cell migration activity under normal and hypoxic conditions, and both demonstrate that KMUP-1 can provide anti-inflammatory and anti-metastatic activity of lung epithelial cells.

Please refer to Figure 28 for the effect of different concentrations of KMUP-1 (1-100 μM) on TNF-a-induced iNOS expression under normal and hypoxic conditions. As shown in Figure 28, TNF-α (100 ng/ml) The expression of iNOS was increased, while KMUP-1 was able to inhibit iNOS induced by TNF-α in lung epithelial cells. The phenomenon of iNOS induced by TNF-α was attenuated by the action of KMUP-1. When the concentration of KMUP-1 was higher than 50 μM, it was more severely damaged under normal or hypoxic conditions, indicating that KMUP-1 is in two types. In the state, it has the ability to inhibit the inflammatory effect.

In summary, KMUP-1 has been shown to inhibit p38, ROCKII and VEGF, has been shown to be non-toxic, and has the ability to inhibit lung epithelial cell proliferation, inflammation and migration, and is more suitable for preventing inflammation. Obstructive diseases, pro-inflammatory diseases and cancer cell metastasis.

The above is only the preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Equivalent changes or modifications to the shapes, structures, features, and spirits of the present invention should be included in the scope of the present invention.

The first graph (A) and the first graph (B) are graphs showing the different concentrations (1.0, 10 and 100 μM) of KMUP-1 in the present invention in the normal (Fig. 1A) and hypoxic (Fig. 1B) states. Inhibition of H441 cell viability, where the circle represents the control group and the triangle represents 1.0 μM KMUP-1, hollow The triangle represents 10 μM KMUP-1 and the square represents 100 μM KMUP-1.

The second panel is the effect of different concentrations of KMUP-1 (0.01, 0.1, 1.0, 10 and 100 μM) on the cell cycle distribution ratio (%), where FCS represents cell culture medium containing fetal bovine serum and vehicle represents culture medium. Only the solvent of KMUP-1 was added, while Go/G1, S and G2/M represent the dormant phase/growth phase, synthesis phase, and mitosis preparation/mitosis phase in the cell cycle, respectively.

The third panel is the effect of KMUP-1 (1.0 μM) of the present invention on the ratio of each cycle in the cell cycle after 6 hours to 72 hours, wherein SF and 10% FCS represent cell culture in serum-free, respectively. And in the culture medium containing 10% fetal calf serum.

The fourth panel is a cell cycle region map analyzed by flow cytometry, which is the effect of KMUP-1 (100 μM) of the present invention on cell cycle distribution after comparison with a control group and a solvent control group thereof.

Figure 5 (A) (B) shows treatment of H441 cells 12, 24, 48 and 72 in normal and hypoxic conditions in the presence (Figure 5B) or absence (Figure 5A) of KMUP-1 (10 μM) The effect of eNOS on the performance of eNOS after an hour, As a result of the normalization of the expression of -actin, the oblique line indicates the amount of eNOS at 0 hours, while the cross line strip and the white strip indicate the amount of eNOS expression at normal time and under hypoxia, respectively. ^* and ^** represent the symbol 0 hours comparison p <0.05 and p <0.01, while in the illustrated and # ## symbols are represented by the following normal and hypoxic state a certain time after comparison p <0.05 and p <0.01.

The sixth figure is the effect of different concentrations of KMUP-1 (0.001, 0.01, 0.1, 1.0, and 10 μM) under normal oxygen for 48 hours, the effect of eNOS on the performance of eNOS. As a result of the normalization of the amount of -actin, the diagonal bars represent the control group, while the white bars represent the experimental group of KMUP-1.

The seventh panel shows the effect of KMUP-1 of the present invention on the expression of eNOS under normal and hypoxic conditions after the cells were previously treated with the commercially available NOS inhibitory drug L-NAME (100 μM) for 30 minutes, wherein the oblique line represents The untreated group, while the white bars represent the groups pre-processed with L-NAME, and the seventh (A) and seventh (B) represent the results of 0.1 μM and 1.0 μM KMUP-1, respectively.

Figure 8 is the effect of KMUP-1 (10,100 μM) of the present invention on sGC expression in H441 cells under normal and hypoxic conditions, wherein SF represents cell culture in serum-free medium, and oblique lines and The white strips represent normal and hypoxic conditions, respectively.

The ninth figure is the effect of KMUP-1 of the present invention on the expression of PKG in H441 cells under normal and hypoxic conditions, wherein SF represents fine The cells were cultured in serum-free medium, while the diagonal strips and white strips represent normal and hypoxic conditions, respectively.

The tenth figure is the effect of KMUP-1 (1 μM) of the present invention on the expression of HIF-1a after exposure of H441 cells to anoxic state for 3 to 72 hours, wherein the oblique strips and the white strips respectively represent the control group. And the experimental group of KMUP-1.

The eleventh figure is the effect of KMUP-1 (1 μM) of the present invention on the expression of VEGF after exposure of H441 cells to anoxic state for 3 to 72 hours, wherein the oblique white strips and strips respectively represent the control group and Experimental group of KMUP-1.

The twelfth figure is the inhibition effect of KMUP-1 (1 μM) of the present invention on HIF-1α and VEGF expression for 24 hours after pretreatment with L-NAME (100 μM) for 30 minutes, wherein N represents normal State, H represents anoxic state.

Figure 13 (A) and (B) are different concentrations of KMUP-1 (0.01, 0.1, 1.0, 10 and 100 μM) for HIF-1a (Fig. 13A) and VEGF under hypoxic conditions (Fig. 13B). The effect of the performance on the 24 hours, in which the diagonal strips and the white strips represent the control group and the experimental group of KMUP-1, respectively.

Figure 14 (A) and (B) are the H441 cells in the hypoxia state, the control group, 1.0 μM KMUP-1, 1.0 μM YC-1, 100 μM SNP and The amount of expression of HIF-1a (Fig. 14A) and VEGF (Fig. 14B) after 24 hours of different treatments of 100 μM IBMX.

FIG fifteenth lines of the present invention KMUP-1 (10,100 μ M) , under normal and hypoxic state for protein H441 cells ROCKII performance effect, wherein the representative SF cells were cultured in serum-free culture medium, The diagonal strips and the white strips represent the normal and hypoxic states, respectively.

The sixteenth figure is that after the cells were previously treated with commercially available Rp-8-CPT-cGMP (10 μM) for 30 minutes, after treatment with KMUP-1 (10 μM) of the present invention for 48 hours under normal and anoxic conditions, For the role of Rho's stimulating performance, the oblique strips and white strips represent normal and hypoxic states, respectively.

Figure 17 (A) and (B) show that H441 cells are treated with 10% fetal bovine serum, serum-free medium, KMUP-1 (1-100 μM) and ROCK inhibitor Y27632 (10 μM). After 48 hours, the relative distance of the wound width was normal (Fig. 17A) and hypoxia (Fig. 17B).

Figure 18 (A) and (B) are the KMUP-1 of the present invention under normal and hypoxic conditions, for the pretreatment of Rp-8-CPT-cGMP without cGMP antagonist (Fig. 18A) and after The effect of p21 expression in H441 cells after Rp-8-CPT-cGMP (10 μM) pretreatment for 30 minutes (Fig. 18B), wherein CTL represents the control group and SF represents the cells cultured in the serum-free medium. The diagonal strips and the white strips represent the normal and hypoxic states, respectively.

Figure 19 (A) and (B) are the KMUP-1 of the present invention under normal and hypoxic conditions, for the pretreatment of Rp-8-CPT-cGMP without cGMP antagonist (Fig. 19 A) and after The effect of p27 expression in H441 cells after Rp-8-CPT-cGMP (10 μM) pretreatment for 30 minutes (Fig. 19B), wherein CTL represents the control group and SF represents the cells cultured in serum-free medium. The diagonal strips and the white strips represent the normal and hypoxic states, respectively.

FIG twenty (A) and (B) may in normal and hypoxic state, (FIG. 20B) of the present invention exhibit KMUP-1 (10,100 μ M) in respect of Bax H441 cells (FIG. 20A) and Bc1-2 The role of SF is that the cells are cultured in serum-free medium, while the diagonal strips and white strips represent normal and hypoxic conditions, respectively.

The twenty-first figure is the effect of KMUP-1 (10,100 μ M) of the present invention on the ratio in H441 cells under normal and hypoxic conditions, wherein SF represents cell culture in serum-free medium, and The diagonal strips and the white strips represent the normal and hypoxic states, respectively.

The twenty-second picture shows that the cells were treated with the commercially available NOS inhibitory drug L-NAME (100 μM) for 30 minutes, and then treated with KMUP-1 (100 μM) of the present invention for 48 hours under normal and anoxic conditions. For the effect of the Bax/Bcl-2 ratio, the oblique strips and the white strips represent the normal and hypoxic states, respectively.

The twenty-third figure shows that the cells were pretreated with a commercially available cGMP antagonist, Rp-8-CPT-cGMP (10 μM) for 30 minutes, and the KMUP-1 (100 μM) of the present invention was in a normal and hypoxic state. After 48 hours of treatment, the effect on the ratio of Bax/Bcl-2, wherein the diagonal strips and the white strips represent the normal and hypoxic states, respectively.

The twenty-fourth figure is the effect of different concentrations of KMUP-1 (1.0, 10 and 100 μM) on the ratio of unactivated procaspase-3/activated caspase-3 in H441 cells under normal and hypoxic conditions, Long strips and white strips represent the control group and the experimental group of KMUP-1, respectively.

The twenty-fifth diagram is the effect of KMUP-1 of the present invention on the expression of PDE5A in H441 cells under normal and hypoxic conditions, wherein SF represents cell culture in serum-free medium, and oblique lines and white lengths. The bars represent normal and hypoxic conditions, respectively.

The twenty-fifth graph shows the effects of different concentrations of KMUP-1 (1.0, 10 and 50 μM) on the expression of PDE5A in cells induced by PDE5A promoter U46619. The oblique strips and white strips represent normal and absent, respectively. Oxygen state.

The twenty-seventh figure is the effect of KMUP-1 (10,100 μM) of the present invention on the ratio of phosphorylated p38/all p38 in H441 cells under normal and hypoxic conditions, wherein SF represents cell culture in serum-free In the culture medium, the diagonal strips and the white strips represent the normal and hypoxic states, respectively.

The twenty-eighth image shows the effect of different concentrations of KMUP-1 (1-100 μM) on TNF-a-induced iNOS expression under normal and hypoxic conditions, in which the oblique strips and white strips represent normal and absent, respectively. Oxygen state.

Citation

1. Zhan X., Li D., and Johns RA (1999) J. Histochem. Cytochem . 47, 1369-1374

2. Nijkamp FP, van der Linde HJ, Folkerts G (1993) Am Rev Respir Dis. 148 , 727-734

3. Wu, BN, Chen, CW, Liou, SF, Yeh, JL, Chung, HH, and Chen, IJ (2006) Molecular pharmacology 70 (3), 977-985.

4. Thompson, HJ, Jiang, C., Lu, J., Mehta, RG, Piazza, GA, Paranka, NS, Pamukcu, R., and Ahnen, DJ (1997) Cancer research 57 (2), 267-271 .

5. Soh, JW, Mao, Y., Kim, MG, Pamukcu, R., Li, H., Piazza, GA, Thompson, WJ, and Weinstein, IB (2000) Clin Cancer Res 6(10), 4136- 4141.

6. Yeo, EJ, Chun, YS, and Park, JW (2004) Biochemical pharmacology 68 (6), 1061-1069.

7. Chun, YS, Yeo, EJ, Choi, E., Teng, CM, Bae, JM, Kim, Ms, and Park, JW (2001) Biochemical pharmacology 61 (8), 947-954.

8. Nath, P., Leung, sY, Williams, A., Noble, A., Chakravarty, sD, Luedtke, GR, Medicherla, S., Higgins, Ls, Protter, A., and Chung, KF (2006) European journal of pharmacology 54 4 (1-3), 160-167.

9. Yoshioka, Y., Yamamuro, A., and Maeda, S. (2006) Journal of pharmacological sciences 101 (2), 126-134.

10. Deguchi, A., Thompson, WJ, and Weinstein, IB (2004) Cancer Res. 64, 3966-3973

11. Hakuma, N., Kinoshita, I., Shimizu, Y., Yamazaki, k., Yoshida, K., Nishimura, M., and Dosaka-Akita, H. (2005) Cancer Res. 65, 10776-10782

12. Zhu, B., Vemavarapu, L., Thompson, WJ, and Strada, SJ (2005) J. Cell. Biochem. 94, 336-350

Claims (10)

A pharmaceutical composition for preparing a Rho-kinase inhibitor for treating cancer cell migration or metastasis-related diseases, comprising: a 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1 A compound of 3 dimethylxanthine, wherein the pharmaceutical composition has an effect of inhibiting a physiological activity of a lung epithelial cell. The pharmaceutical composition of claim 1 further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 1, wherein the physiological activity is selected from at least one of a proliferative activity, a migration activity, and a pro-inflammatory activity. The pharmaceutical composition of claim 3, wherein the migration activity is one of a cancer cell metastatic activity. The pharmaceutical composition according to claim 1, wherein the physiological activity is achieved by increasing cGMP in the lung epithelial cells and inhibiting the activity of Rho-kinase. A compound 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine is used to prepare Rho-kinase inhibitors for the treatment of cancer cell migration or metastasis-related diseases The use of a medicament wherein the compound is synthesized from xanthine and is a Rho-kinase inhibitor. The use of claim 6 wherein the compound further comprises a pharmaceutically effective carrier. For example, the application of the scope of the patent application, wherein the physiological activity is selected At least one of a proliferative activity, a migration activity, and a pro-inflammatory activity. The use of the invention of claim 8, wherein the migration activity is one of a cancer cell transfer activity. The use of the sixth aspect of the invention, wherein the compound inhibits physiological activity of the lung epithelial cells by increasing cGMP and inhibiting Rho-kinase activity in the lung epithelial cells.