TW201004624A - Xanthine-based cyclic GMP-enhancing Rho-kinase inhibitor inhibits physiological activities of lung epithelial cell line - Google Patents

Abstract

A pharmaceutical composition is provided. The pharmaceutical composition comprises a compound of 7-[2-[4-(2-Chlorobenzene)piperazinyl]ethyl]-1, 3, which is a Rho kinase inhibitor, wherein the pharmaceutical composition inhibits a physiological activity of a lung epithelial cell.

Description

201004624 IX. OBJECTS OF THE INVENTION: TECHNICAL FIELD The present invention relates to a compound having a xanthine-based structure capable of reducing physiological activity of a lung epithelial cell line, particularly with respect to an increase in cyclization Cyclic Guanine Monophosphate (cGMP) active Rho-kinase inhibitor inhibits cell migration by increasing cGMP to modulate the expression of Rho-kinase π/vascular endothelial growth factor (ROCK/VEGF) in lung epithelial cells. [Prior Art] Lung epithelial cells often play an important role in the pathogenesis of obstructive bronchial diseases such as obstructive bronchopneumonia and cancer cell metastasis. The nitric oxide synthase/cyclized ornithine (NOS/cGMP) message in epithelial cells is involved in the regulation of airway contraction and regulation of cell growth. It should be closely related to obstructive bronchial disease. Epithelial cells can release many inhibitory effects on smooth muscle. Regulatory substances, such as • Rapid release of NO via epithelial tissue can affect contraction and growth of smooth muscle cells in adjacent lungs. There are many immunological evidences that have shown that NOS/cGMP messages have characteristics in the respiratory epithelium I,2. (7- [2- KMUP-1 [4-(2-chl〇r〇benzene) piperazinyl]ethyl]- 1,3- dimethylxanthine) is a kind of bottom structure with yellow β ticket, which is Rho- with cGMP promotion. Kinase inhibitors have been found in previous studies to show that KUMP-1 can increase cGMP in cytoplasm by activating guanyl cyclase (sGC) and N〇 human enzyme (NOS) in epithelial cells. And reach the relaxation gas ^ 201004624 contraction effect. In addition, KMUP-1 can also participate in the activation of sGC and selenate by inhibiting the expression of inducible NO synthase (iNOS) induced by Tumor necrosis factor-a (TNF-a). The mechanism of inhibition of phosphodiester (PDE) leads to an increase in cyclized guanylate/protein kinase G (cGMP/PKG) in the smooth muscle of the trachea. Therefore, in the present invention, KMUP-1 is 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 the roles of these substances are described as follows: YC-1 is an SGC activator having NO-dependent cGMP enhancing activity. In different types of cells, γ (>1 appears to have an effect against cell proliferation, anti-angiogenesis, anti-cancer, and resistance to inflammation, and thus is used in the present invention as a positive control compared with KMUP4. In the inhibition of lung epithelial cell migration, r〇ckii is located downstream of the cGMP/PKG message pathway and is associated with cell migration activity. Therefore, in the present invention, the ROCK inhibitor Y27632 was also used to observe KMUP-1 for inhibiting lung epithelial cells. The effect of migration. For the inhibition of angiogenesis, VEGF is currently known to be an important pro-angiogenic factor and a necessary substance for tumor growth. The expression of VEGF is triggered by many factors, including hypoxia. When the hypoxia-activated transcription factor, hypoxic induced factor l mF1, is expressed, ΗΠΜ can regulate the VEGF gene of 201004624. Since YC-1 is a NO-dependent cGMp promoter, YC- 1 also plays an important role in the vascular system and inhibits the expression of VEGF and HIF-1 a in Hep3B cells. The cyclin-dependent kinase (CDK) inhibitory proteins p21 and p27 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, the present invention also observes another Phosphorylation of protein kinase 38. p38 plays an important role in inflammatory cells, cell proliferation of respiratory tract cells, and cell survival. 8, In addition, in the case of cell apoptosis, the cells are also analyzed in the present invention. In the present invention, a protein BaX/Bcl-2/protease caspase 3 which is accompanied by p21 and p27 is expressed in the cycle. In the present invention, a compound 'KMUP-1' which inhibits the physiological activity of lung epithelial cells is provided to affect eNOS. /sGC/PKG message, apoptosis message Bax/Bcl-2/caspase 3, and the performance of p21 and p27 in the cell cycle', especially in the presence of ROCKII/VEGF/HIF-la under hypoxia, resulting in inhibition of lung Epithelial cell proliferation, migration, and inflammation-promoting activities. SUMMARY OF THE INVENTION The present invention first proposes that KMUP-1 can affect eNOS/sGC/PKG information related to cGMP, and cell apoptosis information Bax/Bcl- 2/caspase 3 and the expression of ROCKII/VEGF/HIF-la under hypoxic state, thereby achieving an effect of inhibiting the activity of proliferation, inflammation, and migration of lung epithelial cells. Therefore, the present invention provides a pharmaceutical composition comprising a 7-[ 2- 201004624 4 Benzene Benzene) (4) yl]ethyl]+ 3, wherein the compound is a compound of the base 具有 具有 具有 肺 肺 肺 肺 肺 肺 肺 肺 肺 肺 肺 肺It is contemplated that the pharmaceutical composition further comprises - pharmaceutically acceptable according to the above concept, wherein the physiologically active ~ migration activity and - proinflammatory activity are at least one of - proliferative activity, activity according to the reliance 'where the migration activity is - heart Cell-transfer material pain and inhibition of Si plus lung epithelial fines The present invention further provides a method for inhibiting a 2, which comprises administering a dose of a sputum and a physioactive thiol thiophene 7-[2·[4-(2-gas stupid) brigade I-based J-ethyl]-1,3-dimethylxanthine; melon-mammal, wherein the compound is required to be administered by phantom-1 kinase Agent. And, according to the above concept, wherein the compound further comprises a pharmaceutically effective according to the above concept, wherein the physiological activity and the inflammatory activity are at least one. 3 Activity, activity According to the above concept, one of the two cells of the feed material is transferred to the mu' in the blister, wherein the compound is caused by increasing the physiological activity of the lung epithelial cells. In addition, the present invention provides a method of preparing a pharmaceutical composition, wherein the pharmaceutical composition comprises a 7-[2-[4-(2-cepbenzene)piperazine A compound of ethylethyl]-1,3-dimethylxanthine. 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, wherein 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. [Embodiment] 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 1. Cell viability 9 201004624 NCI-H441 cells obtained from the American Type Culture Collection (ATCC) were cultured in RPMI 1640 medium and added with 2 mM glutamic acid, penicillin/streptococcus And 10% fetal bovine serum. 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 test of cell survival and proliferation, H441 cells were cultured in a 24-well plate at a density of 1〇5 cells per cell, and treated with different concentrations of KMUP-1 for various times, and cells were obtained by MTT assay. Survival rate. All experimental data are expressed as mean ± standard error, and the number of samples is n=4. For unpaired and paired samples, their 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 performed • Dunnett’s or Tukey analysis, where corpse < 0.05 was considered significant. The experimental values were analyzed via SigmaStat: Version 2.03, continued 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 μΜ) of KMUP-1 in the present invention after 24, 48 and 72 hours of normal (Fig. 1A) and anoxic (Fig. 1B) conditions. Inhibition of 441 cell survival rate. KMUP-1 (1〇, 1〇, 1〇〇 μΜ) inhibited the survival rate of Η441 cells under normal and hypoxic conditions. As shown in the first figure (Α) and the first figure (Β), 201004624 shows that the 'local concentration of KMUP-1 0 //Μ) significantly inhibited the survival rate of H441 cells. 2. Cell cycle distribution The cells after the protease were collected. After washing with PBS, 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 〇.〇5 mg/ml propidium iodide, 〇mM ethylenediaminetetraacetic acid (EDTA), 0.1% in PBS. The surfactant TritonX-i〇〇 and the 1 mg/mi rnA decomposing enzyme RNase A were stained 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 1〇〇μΜ) on the cell cycle distribution ratio (%). Under normal conditions, the results of flow cytometry analysis showed the effect of KMUP-1 on the progression of the cell cycle, where the cell cycle distribution was affected by KMUP-1 concentration (0.01, 〇·1, 1.0, 10, 100 μΜ). Impact, showing concentration correlation. The results in the second graph show that the ratio of G0/G1 phase increases with the concentration of KMUP-1. The ratio of S phase to G2/M phase decreases with the concentration of KMUP-1. Please refer to the third figure (Α) to the third figure (〇, which shows the effect of KMUP-1 (1.〇μΜ) of the present invention on the proportion of each cycle in the cell cycle after 6 hours to 72 hours. As shown in the second to third (C), after 72 hours of KMUP-1 (100 μΜ), the cell cycle 11 201004624 was significantly blocked in G0/G1 phase. Please refer to the fourth figure The cell cycle region map analyzed by flow cytometry is the effect of KMUP-1 (1 〇〇μΜ) of the present invention on the cell cycle distribution after comparison with the control group and the 1 solvent control group. In the fourth figure After KHUP-1 (100 μΜ) for 72 hours, the cell cycle was obviously arrested in G0/G1 phase. 3. The expression of NOS, sGC and PKG was determined in order to determine eNOS, iNOS, sGC, PKG, HIF_la, VEGF, ROCKII, The expression levels of p38, Bax, Bcl-2 and other proteins and CDK inhibitory proteins p21 and p27 in H441 cells, in the present invention, first extract all the proteins in the cells' and analyze them by the following Western blot method. The cells are cultured in a culture dish of 1 〇 cm in diameter, and when the cells grow to half full, Growth was stopped and WKMUPd was treated for different times. In some experiments, KMUP-1 was administered in the cells with a special inhibitor. When measuring iNOS expression, it was treated with αΚΜυΐΜ and then with TNF-a (100 ng). /ml) The amount of performance was measured at 30 minutes. After the cells were processed, they were washed with PBS (pH 7.4) and placed in extraction buffer (10 mM Tris solution with pH 7.0, containing 14 mM) Sodium chloride, 2 mM phenylsulfonyl fluorocarbon PMSF, 5 mM dithiothreitol DTT, 0, 5% surfactant NP_4 〇, 〇〇 5 mM gastric protein inhibitor a, and 〇 2 mM mom protease inhibitor ieupeptin Slowly shake, then centrifuge for 30 minutes at the speed of η 5〇〇名12 201004624. Next, the cell extract is placed in the sample solution at a ratio of 1:1 (100 mM Tris solution with a pH of 6.8, containing 20%) Glycerin, 4% SDS, and 0.2% bromophenol blue) were boiled. 50 mg of protein was placed in each sample tank of a 10% SDS-polyacrylamide gel and electrophoresed at 10 volts (V) and 40 mA. The electrophoretically separated protein was spun at 100 volts for 90 minutes to polyvinylidene fluoride (p〇ly On the vinylidene difluoride (PVDF) membrane, the non-specific IgGs antibody was blocked with 5% skim milk powder and allowed to stand for one hour with the specific antibody. The membrane was combined with the phosphatase (alkaline phosphatase). One hour after anti-mouse or anti-goat IgG antibody (1:1000), the protein band was visualized by the addition of a chemical color enhancer (GE Healthcare Bio-Sciences Corp) Piscataway, NJ. Please refer to the fifth figure (A) (B), which shows that in the presence of KMUP-1 (1〇μΜ) (Fig. 5Β) or in the absence (Fig. 5Α), Η 441 cells are treated under normal and hypoxic conditions. The effect of its eNOS performance after 24, 48 and 72 hours. Under normal conditions, ΚΜυρ_1(1〇 μΜ) stimulates performance in H441 cells with a temporal correlation. The performance of eN〇s reached its maximum at 48 hours. In the hypoxic state, the expression of eN〇S protein in H441 cells showed a time-dependent decrease', but it was suddenly increased by KMUP-丨.凊 Refer to the sixth graph, which is the effect of different concentrations of iKMUPq 〇.〇1, 0.1, 1.0, and 10 μΜ) under normal oxygen content for 48 hours on the expression of eNOS in 13 201004624. As shown in the sixth panel, the induction of the expression of eN〇S by KMUP-1 was also concentration-dependent within 48 hours. Please refer to the seventh (A) and seventh (B) sections of the present invention. The KMUP-1 of the present invention is normal and hypoxic after 30 minutes of treatment with the commercially available NOS inhibitory drug L-NAME (100 μM). The effect of the state on the amount of eNOS expression. As shown in Figure 7 (A) (B), KMUP-1 was reduced in the increase in eNOS egg white matter performance after pretreatment with the NOS inhibitor L-NAME (100 μΜ) in both states. . Please refer to the eighth and ninth figures, which are the effects of KMUP-1 (10, 100 μΜ) 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 μΜ) in both states. In addition, as shown in the ninth figure, the performance of PKG in Η441 cells was significantly increased by the action of KMUP-1 (1〇, 1〇〇μΜ), which increased to 146.8±13.9% and 157.9 in the control group under normal conditions. ± 12.1%, and increased to 13〇.1±11.7% and 166.6±18.0% of the control group in the hypoxic state. It has been known in previous studies that when PKG is activated in human cells, it can effectively induce inhibition of cell growth and apoptosis, and also inhibit cell migration. In the present invention, ΚΜυΡ-i (1〇, 201004624 100 μΜ) enhances the performance of sGC and PKG in H441 cells under normal or hypoxic conditions, indicating that it stimulates apoptosis, inhibits cell growth and migration. Ability. In general, the activation of 6\〇8 and 8(}(:; helps to rely on cGMP to increase the performance of PKG. Based on the results of the above experiments, it can be speculated that the inhibitory effect of KMUP-1 on cell growth is long. Time-increased NO/peroxynitrite and eGMP/PKG are involved. 4. HIF-la, VEGF and ROCKII information Please refer to the tenth figure, which is the KMUP-1 (1 μΜ) of the invention, which is exposed to Η441 cells. The effect of HIF_la on the amount of HIF_la after 3 to 72 hours of hypoxia. After 3 hours of exposure to hypoxia, HIF_la performed more significantly and reached its maximum at 6 to 12 hours. However, after 18 to 24 hours, HIF The performance of -U protein decreased rapidly. Under the action of kmupj, the performance of HIF-la was inhibited at 12 hours, and it was most obvious at 24 hours. Φ Please refer to the eleventh figure, which is the KMUp_l of the invention (丨The effect of VEGF on the expression of VEGF after exposure to hypoxia for 3 to 72 hours. The performance of VEGF was also enhanced in hypoxic conditions, while the inhibition of ΚΜϋΡ-1 (1.0 μΜ) was at 12, 18, 24 At 48 and 72 hours, they were 59.9±3.0% and % n〇± of the control group. Il %, 50.7 ± 3.5 % and 44.2 ± 3.3%. Please refer to the twelfth figure 'KMUP-1 (1 μΜ) which is the invention, after pretreatment for 30 minutes with L-NAME (100 μΜ), for mF_la And the inhibitory effect of VEGF expression in 24 hours. It can be seen from the twelfth figure 15 201004624 that HIF-la is not obvious under normal conditions, but is obviously visible in the state of hypoxia. When pretreatment with L-NAME, The effect of kmuh on the amount of HIF-la expression was not observed' and the combination of L-NAME and KMUP-1 did not show further inhibition of HIF-la under hypoxia. See thirteenth Figure (A) and Figure 13 (B) 'These are different concentrations of KMUP-1 (0.01, 〇.1,1.〇, 1〇 and 100 μΜ), for HIF_la under hypoxia (Figure 13Α And the expression of VEGF (Fig. 13Β) at 24 hours. As shown in Fig. 13 (A) and Fig. 13 (B), the expression of HIF-la and VEGF is correlated with KMUP-1 concentration. (〇.〇1~100 μΜ). Please refer to Figure 14 (Α) and Figure 14 (Β), which is in the hypoxic state of 'H441 cells in the control group, 1. 〇μΜ KMUP-1 , 1.0 μΜ YCM, 100 'Expression levels of HIF-la (FIG. 14 [alpha]) and VEGF (FIG. 14 [beta) after 24 hours treatment Μ SNP 100 μΜ ΙΒΜΧ and the like are different. According to the data in Fig. 14(A), the HIF-la protein was subjected to inhibition under different treatments as follows (100% of the data from the control group): 34.8 Soil 2.3% (KMUP-1, 1.0 μΜ), 31.6 ± 3.4% (YC-1, 1.0 μΜ), 78.7 ± 3.1% (SNP, 1〇〇μΜ), 15.9 ± 5.9% (ΙΒΜΧ, 100 μΜ) ° and the data in Figure 14 (Β) shows 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 μΜ), 47.2 ± 4.4% (YC-1, 1.0 μΜ), 80.1 ± 2.8% (SNP, 100 μΜ), 20.2 ± 4.7% (ΙΒΜΧ, 100 μΜ). As shown in Fig. 13 (Α) (Β), KMUP-1 (1·100 μΜ) inhibited hypoxia-induced VEGF and HIF_la expression with increasing concentration of 16 201004624. On the other hand, as shown in Fig. 12, under normal conditions, KMUP-1 at a concentration of less than 1 μμ does not give any significant expression of VEGF, and it also shows that it does not have the ability to promote angiogenesis. Combining the above results, KMUP-1 not only overcomes the phenomenon of VEGF dependent on N〇 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 la protein under hypoxia, and exhibits an activity of inhibiting angiogenesis and antitumor activity marked by an anoxic protein marker. The δ month refers to the fifteenth diagram, which is the effect of ΚΜυρ ι(1〇,1〇〇 μΜ)' of the present invention on protein expression in H441 cells under normal and hypoxic conditions. The performance of ROCKII was positively correlated with the concentration. In the twenty-fifth figure, the effect of KMUpq was inhibited by the cGMP antagonist Rp winter CPT_cGMP 〇〇 _). 5. Cell migration and inhibition of ROCKII • Culture Η441 cells in a six-well plate until the cells grow to over 90%. The π is drawn on the cell layer by the tip of the micropipette in a manner across the culture cell, and the cell debris is removed by washing the cells with the culture solution. The cells were treated with 10 μΜ of KMUIM and Υ-27632 for μ hours and after an hour, and the edges of the wound (Eclipse TS100, Nikon) were visualized and photographed using a microscope. In the same culture, measure the maximum and minimum position of the wound width and take the average. 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 an experiment of cell migration. Please refer to the 17th figure (A)(B) of the 201004624 test. The system shows that H441 cells pass through 1% fetal calf serum, serum-free medium, KMUP-1 (U1〇〇μΜ) and R〇CK inhibitor. Y27632 (1 〇μΜ) The relative distance of the wound width under normal (Fig. 17Α) and hypoxia (Fig. 17Β) conditions after 48 hours of different treatments. As shown in Fig. 17 (Α) and Fig. 17 (Β), ΚΜυρ] can inhibit the migration activity of Η441 lung epithelial cells under normal or hypoxic conditions. Especially when the concentration of KMUP-1 is higher than 50 μΜ, the migration of Η441 cells across the wound width after 48 _ hours of culture is more significantly reduced, and the effect is more significant under normal conditions than under hypoxia. The present invention also utilizes the results of the R〇CKII inhibitor Y27632 and KMUP-1, γ27632 (1〇 also inhibits ROCKII under normal conditions, but not in hypoxia. Since the migration of cancer epithelial cells increases The risk of metastasis of cancer cells, therefore, in the present invention, the inhibitory effect of KMUP-1 on R0CKII provides potential for metastasis in lung epithelial cells. 6·Performance of p21 and p27 • Please refer to Fig. 18(A) And the eighteenth (B), which is the KMUP-1 of the present invention, under normal and hypoxic conditions, for the pretreatment of Rp-8-CPT-cGMP without a cGMp antagonist (Fig. 18A) and by Rp -8-CPT-cGMP (10 μΜ) The effect of P21 expression on Η441 cells after 30 minutes of pretreatment (Fig. 18b). Under normal and hypoxic conditions, cells were added with KMUP-1 or without KMUP- The culture was carried out for 48 hours under the conditions of 1. As shown in Fig. 18, after the comparison with the control group, the mouth 21 was affected by KMUP-1 (1〇, 1〇〇μΜ) and the increase was as follows: under normal conditions It was 181.2 ± 17.6 % and 172.9 ± 18.1%, which was 151.7 ± 7.7 % and 135.1 ± 14.7% under hypoxic bears. Figure 18 (β), 201004624 shows that 'Rp-8-CPT-cGMPS (10 μΜ) does not inhibit the p21 protein induced by KMUP-1. Please refer to Figure 19 (A) and Figure 19 (B). ), which is the KMUP-1 of the present invention under normal and hypoxic conditions, for the pretreatment of Rp-8-CPT-cGMP without a cGMp antagonist (Fig. 19A) and

Rp-8-CPT-cGMP (1〇 μΜ) was used for 30 minutes (Fig. 19B) for the effect of Ρ27 expression in Η441 cells. As shown in the nineteenth figure (Α), the increase of Ρ27 by KMUP-1 (1〇, 100 μΜ) is as follows. Under normal conditions, it is 162.5±12.5 〇/〇 and 201.6±23.5 %, in the absence of The oxygen state was 172.4 ± 19.5 % and 242.2 ± 21.5 %, respectively. As shown in Fig. 19 (Β), Rp_8_CPT_cGMps (丨〇 μΜ) also failed to inhibit the ρ27 protein induced by KMUP-1. In addition, in cells lacking serum culture, the performance of P21 and ρ27 also increased. In the cell cycle, the CDK inhibitory proteins p21 and ρ27 are indicators of DNA replication in growth cells, usually activated by ρ53 when DNA is damaged. In the case of gene damage, Ρ21 and ρ27 may cause cell cycle arrest at the G〇/G1 checkpoint through the mechanism of divergence. ΚΜυΐΜ (9) μΜ) can inhibit the proliferation of H441 cells under hypoxia, but at concentrations below 1.0 μΜ There is no such inhibition. However, more and more evidence supports the importance of the adjustment of p21 and Ρ27 at the translation level. The KMUP-1 of the present invention increased the expression of p21 and ρ27 under hypoxic pressure, indicating no effect on the translational level of cancer cell growth. However, the effect of KMUi>4 on the expression of p21 and p27 was not affected by Rp_8-CPT-eGMPS, indicating that it involved cell cycle progression independent of cGMp. 19 201004624 7. Bax/Bcl-2 and protease caspase 3 Please refer to the twentieth (A) and twentieth (b) drawings, which are the normal KMUP-1 (10, 100 //M) of the present invention. And the effect of Bax (Fig. 20A) and Bcl-2 (Fig. 20B) on H44i cells in hypoxia. KMUP-1 (10, 100 μΜ) can increase the performance of Bax under normal conditions to 125.6±11.4% and 103.6±1〇.8% in the control group, while the Bcl-2 expression in the normal state is lower than that in the control group. 31.9±1.2%, the performance of reducing Bcl-2 in hypoxia was 56.5±1.3% of the control group. Therefore, as shown in Fig. 21, the ratio of 'Bax/Bcl_2 will increase with the concentration of KMup-1. As shown in Fig. 22 and Fig. 23, L-NΑΜΕ and Rp-8-CPT-cGMP inhibited the increase in the ratio of KMUP-1 to Bax/Bcl-2. Please refer to Figure 24 for the effect of different concentrations of KMUH (1.0, 10 and 100 μΜ) on the ratio of unactivated procaspase-3/activated caspase_3 in H441 cells under normal and hypoxic conditions. The expression ratio of activated procaspase-3/activated caspase_3 was increased by the concentration of KMUP-1, and it was more obvious under hypoxic state under normal conditions. According to the cell cycle experiment of the present invention, after exposure of H441 cells to 72 hours under hypoxic conditions, a sub_Gi peak (sub-Gl peak ' also represents the peak of apoptosis) appears, and i〇〇μΜ) co-cultured to further enhance the peak of this apoptosis. In addition, KMUP-1 (10,1〇〇 μΜ) also increased Bax and reduced the performance of Bcl-2 20 201004624 in the absence of oxygen, resulting in an increase in the proportion of Bax/Bcl-2. KMUP-1 enhanced the expression of protease caspase-3 and showed its activity against apoptosis in H441 cells11. It is worth noting that 'KMUP-1 can increase the amount of eNOS expression in normal or hypoxic conditions', so theoretically NO-induced angiogenesis and toxic peroxidic nitrite ions (ONOO·) will also increase. And ΚΜυρ_ι for

The effect of the Bax/Bcl-2 ratio was also inhibited by the NOS inhibitor l-NAME and the PKG inhibitor Rp_8_cPT-cGMPS (see Figures 22 and 23). Therefore, the effect of KMUP-1 on apoptosis of H441 cells in the present invention may be attributed to the excessive expression of peroxidic nitrite ions by NO through the path of Bax/Bcl-2 and cGMP, and YC_1 is independent of cGMP in HA22T cells. The anti-proliferative activity differs from I2.

8. U46619 Initiated PDE-5A 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 μΜ) on the amount of PDE5A in cells induced by the PDE5A promoter U46619. As shown in Figure 26, the synergist U46619 (5 μΜ), which mimics the inflammatory factor 明显, apparently caused an increase in PDE5A expression under normal conditions. However, after pretreatment with KMUP-1 (1, 10, 50 μΜ), U46619 caused the expression of ρ〇Ε5Α in epithelial cells, exhibiting a decrease associated with KMUP-1 concentration.

9. Disc acidification of ρ38 and TNF-a-induced iNOS 21 201004624 Please refer to the twenty-seventh figure, which is a KMUp l (1〇, 100 μΜ) of the present invention in H441 cells under normal and hypoxic conditions. The role of phosphorylation of p38/proportion of all P38. 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 performance ratio is reduced under normal conditions (sample number n=4, p< 0.05) 'But there is an insignificant increase in hypoxia. KMUP-1 (10,100 μΜ) reduced the performance ratio to 58.9±7.1% and 50.9±9.1% under normal conditions. In the anoxic state, the performance ratio was reduced to 70.6 soil 6.6% and 67. ^9.4% (sample acidified p38 is affected by KMUP-1 and shows a decrease in performance, which means that KMUP-1 has the ability to inhibit the path of inflammation. P38 is a protein related to inflammation, which is thought to be regulated. The major members of the inflammatory response are involved, and many factors, including hypoxia stress, activate the expression of 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 Rh〇/R〇CK message of p38 kinase is associated with the migration of vascular smooth muscle cells and is also susceptible to the ROCK inhibitor Y27632. In the present invention, KMUIM attenuates the expression of ROCKII/p38 and is in normal and hypoxic state. The activity of inhibiting cell migration has proved that KMUP-1 can provide anti-inflammatory and anti-metastatic activity of lung epithelial cells. Please refer to Figure 28 for different concentrations of KMUP4.

The effect of (1-100 μΜ) on TNF-a-induced iNOS expression under normal and hypoxic conditions. As shown in Fig. 28, ΤΝΚα (1〇〇 ng/mi) 22 201004624 increased the amount of mos expression while kmup_i was able to inhibit iNOS induced by TNF-α in lung epithelial cells. The phenomenon of TNF-α-induced iNOS is attenuated by the action of KMUP-1. When the ΚΜυρ_^ degree is higher than 5〇, it is more severely damaged under normal or hypoxic conditions, indicating that KMUP-1 is in two states. Both have the ability to inhibit the effects of promoting inflammation. _ 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. Causing obstructive diseases, inflammatory diseases and cancer cells

However, the above description is only intended to limit the scope of the present invention to the preferred embodiments of the present invention. Therefore, the application according to the present invention is not inhibited by the normal (Fig. 1A) and the triangle-deficient cell survival rate of the present invention.

The first graph (Α) and the first graph (Β) are graphs, the same concentration (1.0, 10 and 1〇〇μΜ) KMUP-1 gas (Fig. 1Β) state for Η441 cells, $ in the circle represents the control group The second tour master 23 201004624 The triangle represents 10 μΜ KMUP-l 'square represents 1〇〇μΜ KMUP-1. The second graph is the effect of different concentrations of KMUP-1 (〇.〇1,〇丨,j 〇, 1〇 and ΙΟΟμΜ) on cell cycle distribution ratio (%), where FCS represents cell culture medium containing boat bovine serum. 'The vehicle represents only the solvent of KMUP-1 added to the poetry solution, while Go/Gl, S and G2/M represent the dormant phase/growth phase, synthesis phase and mitosis® fission preparation/mitosis in the cell cycle, respectively. period. The second graph is the effect of the KMUP-1 (1.0 from 訄) 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 respectively represent cell culture in the absence of Serum and medium containing 10% fetal calf jk clear. The fourth panel is a cell cycle region map analyzed by flow cytometry, which is the effect of KMUIM _ _ in the invention of the cell cycle distribution after comparison with the dragon group and its solvent control group. Figure 5 (A) (B) shows that H44l cells are treated under normal and hypoxic conditions when KMUp] (1〇 μΜ) is present (Figure 5B) or absent (Figure 5A)! 2. The effect of eN〇s on the performance of 2, 24, 48 and 72 hours, in which the expression of eNOS is normalized by the expression of fi_actin❼, and the oblique line indicates the expression of eNOS in the hour, while the cross line strip and The white strips indicate the amount of eNOS in the normal and hypoxic conditions, respectively, between 24 201004624. The * and ** symbols in the graph represent the comparison with 〇 hours /? < 0.05 and Ρ < 0.01, respectively. The # and ## symbols in the indication represent the ρ < 0 05 and the reed < 〇〇 1 after comparing the normal and the anoxic state at a specific time. The sixth figure is the effect of different concentrations of KMUP-1 (o.oono^o"j 〇 and 10 μΜ) under normal oxygen demand for 48 hours, and the effect of eNOS is The results of standardization of B_actin's performance amount 'slashed bars represent the control group, while white bars represent the experimental group of Lu KMUP-1. The seventh panel shows the effect of Kmuim of the present invention on eNOS expression in normal and hypoxic conditions after 30 minutes of treatment with commercially available NOS inhibitory drug l_NAME (100 μΜ), wherein the diagonal strips represent no pretreatment. The group, while the white bar represents the group pre-processed with L-NAME, and the seventh (A) and seventh (B) represent the results of 〇1μΜ and 1·〇μΜ KMUP-1, respectively.

The eighth figure is the effect of KMUP-1 (1〇, 100 μΜ) of the present invention on sGC expression in Η441 cells under normal and hypoxic conditions, wherein SF represents cell culture in serum-free medium, and oblique lines. Long strips and white strips represent normal and hypoxic conditions, respectively. The ninth graph is the effect of KMUP-1 of the present invention on the amount of PKG expression in H441 cells under normal and hypoxic conditions, wherein sf represents fine 25 201004624 cell culture in serum-free medium, and oblique strips and The white strips represent normal and hypoxic conditions, respectively. The tenth figure is the effect of KMUP-1 (1 μΜ) of the present invention on the expression of HIF-la after exposure of H441 cells to hypoxia for 3 to 72 hours, wherein the oblique strips and white strips respectively represent the control group. And the experimental group of KMUP-1. The eleventh figure is the effect of KMUP-1 (1 μΜ) of the present invention on the expression of VEGF after exposure of H441 cells to hypoxia for 3 to 72 hours. The oblique white strips and strips respectively represent the control group and Experimental group of KMUP4. The twelfth figure is the inhibition effect of the KMUPd (i μΜ) of the present invention on the expression of mF_la and VEgf for 24 hours after pretreatment with L-NAME (100 μΜ) for 30 minutes, wherein n represents a normal state, and Η represents Anoxic state. Figure 13 (Α) and (Β) are different concentrations of KMUP-1 (0.01, 0.1, 1.0, 10 and 100 μΜ) for mF_la (Fig. 13A) and VEGF (Fig. 13B) under hypoxic conditions. The effect of the amount in 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 different treatments of H441 cells in the control group, 1.0 μΜ KMUP-1, 1.〇μΜ YCM, 100 μΜ SNP and 26 201004624 100 μΜ IBMX under hypoxia. The amount of HIF ia (Fig. 14A) and VEGF (Fig. 14B) after hours. The fifteenth figure is the effect of KMUP-1 (10, l00/WM) of the present invention on the expression of ROCKII protein in H441 cells under positive and hypoxic conditions, wherein SF represents cell culture in serum-free medium. Medium, and the diagonal strips and white strips represent normal and hypoxic states, respectively. The sixteenth figure is the effect on the Rho stimulating performance after the cells were treated with the commercially available Rp_8_CPT_cGMp (1 〇μΜ) for 30 minutes, and treated with the (1 〇μΜ) of the present invention for 48 hours under normal and hypoxic conditions. , wherein the diagonal strips and the white strips represent normal and hypoxic states, respectively. Figure 17 (A) and (B) show that H441 cells are treated with 1% fetal calf serum, serum-free medium, KMUP-1 (1-100 μΜ), and rock inhibitor Y27632 (10 μΜ). After 48 hours of treatment, the relative distance of the wound width was normal (Fig. 17A) and hypoxia (Fig. 17A). Figure 18 (Α) and (Β) 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. 18 A) and after The effect of p21 expression in 441 cells after Rp-8-CPT-cGMP (1〇μΜ) pretreatment for 30 minutes (Fig. 18 ,), wherein CTL represents the control group 'SF stands for cell culture in serum-free medium And the diagonal strips and the white strips represent the normal and hypoxic states, respectively. 27 201004624 The nineteenth (A) and (B) are the KMUP-1 of the present invention under normal and hypoxic conditions for Rp-8-CPT-cGMP pretreatment without cGMP antagonist (Fig. 19 A) And the effect of Ρ27 expression in Η441 cells after pretreatment with Rp_8-CPT-cGMP (10 μΜ) for 30 minutes (Fig. 19 ,), wherein CTL represents the control group, and SF represents the cell culture in the serum-free medium. The diagonal strips and the white strips represent the normal and hypoxic states, respectively. _ Figure 20 (Α) and (Β) are the KMUP-1 (10,100 yM) of the present invention in normal and hypoxic conditions for Η441 cells (Fig. 2〇α) and Bcl-2 (Fig. 20Β) The role of performance 'where SF stands for cell culture in serum-free medium' and the diagonal strips and white strips represent normal and hypoxic conditions, respectively. The twentieth-picture is the ratio of KMUIM (1, M) in the normal and hypoxic state of the invention to the H441 cell towel, wherein SF represents the cell culture medium containing the blood phase culture solution, and Slanted strips and white strips represent normal and hypoxic, respectively. The twenty-second picture shows that the cells were pretreated with the commercially available NOS inhibitory drug L-NAME (100 μΜ) for 3 minutes, and then treated with the leg of the present invention (1 μμΜ) for 48 hours under normal and anoxic conditions. After that, for the W2 ratio, the oblique line and the white strip represent the normal and anoxic states, respectively. 28 201004624 The twenty-third figure shows that the cells are pre-treated with commercially available cGMP antagonist, Rp-8-CPT-cGMP (10 μΜ) for 30 minutes, and the KMUP-1 (100 μΜ) of the present invention is normal and absent. After 48 hours of treatment under oxygen conditions, for the Bax/Bcl-2 ratio, the oblique strips and the white strips represent normal and hypoxic states, respectively. The twenty-fourth figure is the effect of different concentrations of KMUP-1 (1.0, 1〇 and 1〇〇μΜ) on the ratio of unactivated procaspase-3/activated caspase_3 in H441 cells under normal and hypoxic conditions. The diagonal strips and the white strips represent the control group and the experimental group of KMUP-1, respectively. The twenty-fifth diagram is the effect of KMUP4 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 strips and white strips are respectively Represents normal and hypoxic conditions.

The twenty-fifth image shows the effect of different concentrations of KMUP-1 (1.0, 1〇 and 5〇μΜ) on the expression of pDE5A in cells induced by PDE5A promoter U46619, where the oblique strips and white strips represent normal And anoxic state. The seventeenth figure is the effect of KMUP-1 (10, 1〇〇μΜ) of the present invention on the ratio of acidification ρ38/all ρ38 in Η441 cells (4) under normal and hypoxic conditions. In the serum-containing culture medium, the diagonal line and the self-color strip represent the normal and hypoxic state, respectively. 29 201004624 The twenty-eighth figure is the effect of different concentrations of KMUP-1 (1-100 μΜ) on TNF-a-induced iNOS expression under normal and hypoxic conditions, in which the oblique strips and white strips represent normal And anoxic state. [Main component symbol description] None •❿

30 201004624 Citations 1. Zhan X., Li D., and Johns R.A. (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, B. N., Chen, C. W., Liou, S. F., Yeh, J. L., Chung, Η. H., and Chen, I. J. (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 Reference 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 544(1-3), 160-167. 31 201004624 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, W. J., and Strada, S. J. (2005) J. Cell. Biochem. 94, 336-350

32

Claims (1)

201004624 X. Patent Application Range: 1. A pharmaceutical composition comprising: a 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-didecyl-xanthine A compound wherein the compound is a Rho-kinase inhibitor, the pharmaceutical composition having an effect of inhibiting a physiological activity of a lung epithelial cell. 2. The pharmaceutical composition of claim 1 further comprising a pharmaceutically acceptable carrier. 3. The pharmaceutical composition according to claim 1, wherein the physiological activity is at least one selected from the group consisting of a proliferative activity, a migration activity, and an inflammatory activity. 4. The pharmaceutical composition of claim 3, wherein the migration activity is one of a cancer cell metastatic activity. 5. 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. 6. A method of inhibiting physiological activity of a lung epithelial cell comprising: administering a pharmaceutically effective amount of 7-[2-[4-(2-cephenyl)piperazinyl]ethyl]-1,3 - a compound of diterpene xanthine, which is administered to a mammal in need thereof, wherein the compound is synthesized from yellow 11-nose and is a Rho-kinase inhibitor. 7. The method of claim 6, wherein the compound further comprises a pharmaceutically effective carrier. 8. The method of claim 6, wherein the physiological activity is selected from the group consisting of at least one of a proliferative activity, a migration activity, and an inflammatory activity. 8. The method of claim 8, wherein the migration activity is one of a cancer cell transfer activity. 10. The method of claim 6, wherein the compound inhibits physiological activity of the lung epithelial cells by increasing cGMP and inhibiting Rh0-kinase activity in the lung epithelial cells. A method of preparing a pharmaceutical composition, wherein the pharmaceutical composition comprises a compound of 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl], 3-didecylxanthine. 12. The method of claim 5, wherein the pharmaceutical composition has an effect of inhibiting physiological activity of one of the lung epithelial cells, and the physiological activity is selected from the group consisting of - proliferative activity, a migration activity, and at least an inflammatory activity. one of them. The migration activity of the towel is a transfer activity of one of the cancer cells, as in the method of claim 12 of the patent application. The method of claim U, wherein the pharmaceutical composition further comprises a pharmaceutically effective carrier. 34