KR100193983B1 - Protease inhibitors, tumor infiltrating inhibitors, tumor metastasis inhibitors, tumor growth inhibitors and angiogenesis inhibitors containing mesylic acid ibexate as active ingredients - Google Patents

Abstract

The present invention relates to a protease inhibitor, a tumor infiltrating inhibitor, a tumor metastasis inhibitor, a tumor growth inhibitor, and an angiogenesis inhibitor containing mexalic acid gabexate (Gabexate mesilate) as an active ingredient. More specifically, the present invention provides inhibitors of matrix degrading metalloproteinases (MMPs) and serine protease, such as type IV collagenase, containing mesylic acid gabexate as active ingredients, tumor infiltration, metastasis or growth inhibitors, A method of inhibiting protease, tumor infiltration, tumor metastasis, tumor growth or angiogenesis by administering an angiogenesis inhibitor and an electrical inhibitor. Using the inhibitor of the present invention, protease, tumor invasion, tumor metastasis, tumor growth and angiogenesis can be inhibited in a short amount of time.

Description

Protease inhibitors, tumor infiltrating inhibitors, tumor metastasis inhibitors, tumor growth inhibitors and angiogenesis inhibitors containing mesylic acid ibexate as active ingredients

The present invention relates to a protease inhibitor, a tumor infiltrating inhibitor, a tumor metastasis inhibitor, a tumor growth inhibitor, and an angiogenesis inhibitor containing mexalic acid gabexate (Gabexate mesilate) as an active ingredient. More specifically, the present invention provides inhibitors of matrix degrading metalloproteinases (MMPs) and serine protease, such as type IV collagenase, containing mesylic acid gabexate as active ingredients, tumor infiltration, metastasis or growth inhibitors, A method of inhibiting protease, tumor infiltration, tumor metastasis, tumor growth or angiogenesis by administering an angiogenesis inhibitor and an electrical inhibitor.

According to the 1986 National Health Service statistics, cancer mortality is the second highest after cardiac disease, but since that mortality due to cardiovascular disease has gradually decreased, the mortality rate due to malignant tumors has been on the rise. Has become a very important national challenge. Death of primary malignant tumors among cancer patients is extremely rare, and more than 90% of patients are known to be caused by metastatic tumors caused by the spread of cancer cells to distant organs other than the primary site. It is becoming a new strategy to reduce mortality in patients.

In addition, even if the primary tumor site is treated with a radical treatment method such as surgical extraction, in many cases, micrometastatic lesions already exist from the beginning and then gradually grow, and later become metastatic lesions, which later becomes impossible to treat. The prevention and effective treatment of metastatic lesions is very important.

As part of the development of anti-metastatic agents, a brief review of the growth, infiltration and metastasis of malignant tumors shows that the malignant tumors in epithelial cells grow very slowly in the epithelial layer until the diameter is about 2 mm and then grow rapidly when tumor angiogenesis occurs. Invading surrounding tissues (Follkman, J. et al., Int. Rev. Exp. Path, 16: 207 (1976)), and introducing cancer cells into structurally vulnerable neovascularization in the tumor. Subsequently, metastatic cancer cells bind to adhesion molecules such as endothelial leucocyte adhesion molecules (ELAMs) of distant endothelial vascular endothelial cells (see Philips, MD et al., Science, 250: 1130-1132 (1990); Berg; , EL et al., J. Biol. Chem., 266: 1-5 (1991)), bind to matrix of cells around blood vessels, ie, substrates of cells such as laminin and type IV collagen Yamada, KM et al., J. Cell Biochem., 28: 79-97 (1985); Dahiya, R. et al., Biochem. Int., 27: 567-577 (1992). Extracellular matrix is hydrolyzed by proteolytic enzymes such as type IV collagenase and urokinase type plasminogen activator (uPA), which are mainly secreted from cancer cells (Dahiya, R. et al., Biochem.Int., 27: 567 -577 (1992); Gottesman, M., Semin. Cancer Biol., 1: 97-160 (1990), infiltrating cancer cells using cell motility (Liotta, LA, et al, 1986, Proc. Natl. Acad. Sci. USA., 83: 3302-3312) to form metastatic lesions These metastatic lesions, like the primary lesions, are known to grow rapidly during angiogenesis, invading surrounding tissues or producing other metastatic lesions. Liotta, LA et al., Cell, 64: 327-336 (1991); Fidler, IJ et al., Cell, 79: 185-188 (1994); Liotta, LA, Scientfic American, 34-41, Feb. (1992).

Based on the growth, infiltration and metastasis of the malignancies described above, the anti-transfer strategies can be roughly classified into the following: (1) modulation of tumor vascularization; (2) inhibition of proteases, inhibition of adhesive interactions, cell surface alteration, alteration of cell motility, and the use of monoclonal antibodies Prevent invasion of tumor cells; (3) use anticoagulation and antiplatelet agents; And (4) immunotherapy.

Among these, the most widely studied anti-transfer strategy is to inhibit protease. As mentioned above, MMPs such as type IV collagenase and uPA are deeply involved in invasion and metastasis of epithelial carcinoma. And are also known to be protease enzymes essential for angiogenesis including tumor vessels (Liotta, LA, et al., Cell, 64: 327-336 (1991); Fidler, IJ et al. , Cell, 79: 185-188 (1994)), and several studies have been reported to inhibit cancer invasion, metastasis and angiogenesis using tissue inhibitor of metalloproteinases (TIMPs), a type IV collagenase inhibitor (see: Liotta, LA, Scientific American., 34-41, Feb. (1992); Albini, A. et al., J. Natl. Cancer Inst., 83: 775-779 (1991); Alvarez, OA et al., J. Natl. Cancer Inst., 82: 589-595 (1990); DeClerck, YA et al., Cancer Res., 51: 2151-2157 (1992); Naito, K. et al., Int. J. Cancer, 58: 730-735 (1994).

However, TIMPs have different protease inhibitory effects depending on the type of cancer cell (see Naito, K. et al., Int. J. Cancer, 58: 730-735 (1994); Reich, R. et al. , Cancer Res., 48: 3307-3312 (1988); Throgeirsson, UP et al., J. Natl. Cancer Inst., 69 (5): 1049-1054 (1982); Persky, B. et al., Cancer Res., 46: 4129-4134 (1986), because of their limited clinical application, they have not been put to practical use as effective antimetastatic agents.

In addition, various types of tumor cells can secrete or utilize a wide variety of proteases such as cathepsin, cysteine protease, in addition to serine proteases such as MMPs and uPA, including type IV collagenase. (See Liotta, LA et al., Cell, 64: 327-336 (1991); Liotta, LA, Scientific American, 34-41, Feb. (1992)); Schlechte, W. et al., Cancer Communications, 2 (5): 173-179 (1990); Reich, R. et al., Cancer Res., 48: 3307-3312 (1988)), there is an urgent need for the development of new drugs that inhibit various protease enzymes and also have an inhibitory effect on angiogenesis.

On the other hand, the mesylic acid gabexate represented by the following structural formula has its common name [ethyl-p- (6-guanidino hexanoyloxy) benzoate] methyl sulfonate ([ethyl p- (6-guanidino hexanoyloxy) benzoate methyl sulfonate):

This mesylic acid babexate is known to have different levels of inhibitory effects on serine proteases such as plasma kallikrein, thrombin, plasmin, activator X, C ₁ esterase, and trypsin. See: Ohno, H. et al, Thromb. Res., 19: 579 (1980); Tamura, Y. et al., Biochem. Biophys. Acta., 484: 417 (1977). In addition, mesylic acid gabexate inhibits protease as a competitive inhibition mechanism and does not require antithrombin-III, so it lacks this material. : DIC) has a stronger anticoagulant effect than heparin (Taenaka, N. et al., Critical Care Medicine, 11: 238 (1983)). In addition, mesylic acid gabexate has been reported to have effects of inhibiting phenomena such as kinin, antibody complement, and platelet aggregation, which contribute to the pathology of DIC (Nomura, T., Blood and Vessels, 11: 136 (1980).

Under this background, the inventors have as many multispecificities as possible that act on the different types of proteases that tumor cells secrete, and are easy to develop, leading to new cancer infiltrations that can easily be used in clinical approaches. As a result of intensive research to develop a transfer inhibitor, it was the first time that a serine protease inhibitor, mesylic acid gabecate, strongly inhibited not only serine proteases such as uPA but also MMPs such as type IV collagenase. Cancer invasion and metastasis experiments using human colorectal cancer cell lines were found to have significant anti-invasion and anti-metastatic effects. In addition, the present inventors have found for the first time that mesylic acid gabexate not only inhibits cancer invasion and metastasis but also inhibits tumor growth and angiogenesis, thereby completing the present invention.

After all, the main object of the present invention is to provide a protease inhibitor, a tumor infiltrating inhibitor, a tumor metastasis inhibitor, a tumor growth inhibitor and an angiogenesis inhibitor containing mesylic acid gabexate as an active ingredient.

Another object of the invention is a method for inhibiting protease, a method for inhibiting tumor invasion, a method for inhibiting tumor metastasis, a tumor comprising administering to a patient an effective amount of mesylic acid gabexate together with a pharmaceutically acceptable carrier. It is to provide a method for inhibiting growth and a method for inhibiting angiogenesis.

Figure 1 (a) is a photograph showing the zymography results of the protease secreted from colon cancer cells HM7.

Figure 1 (b) is a photograph showing the results of the zymography on the polyacrylamide gel containing gelatin.

FIG. 1 (c) is a photograph showing the results of zymography on a polyacrylamide gel containing casein.

FIG. 2 (a) is a photograph showing the results of the electrolysis of progelatinase A from which TIMP-2 was removed on a gelatin gel, followed by treatment of the reaction solution for zymogram with mesylic acid gabexate.

FIG. 2 (b) is a photograph showing the results of treatment of 1 mM mesylic acid gabexate with a reaction solution for zymogram after electrophoresis of progelatinase A from which TIMP-2 was removed on a gelatin gel.

FIG. 2 (c) is a photograph showing the results of treatment of 2 mM mesylic acid gabexate with a reaction solution for zymogram after electrophoresis of progelatinase A from which TIMP-2 was removed on a gelatin gel.

FIG. 2 (d) is a photograph showing the results of treatment of 5 mM mesylic acid gabexate with a reaction solution for zymogram after electrophoresis of progelatinase A from which TIMP-2 was removed on a gelatin gel.

FIG. 3 (a) is a photograph showing the inhibitory effect of mesylic acid gabexate on type IV collagenase secreted from NIH3T3 cell line.

Figure 3 (b) is a photograph showing the inhibitory effect of mesylic acid gabexate on type IV collagenase secreted from HT1080 cell line.

Figure 3 (c) is a photograph showing the inhibitory effect of mesylic acid gabexate on type IV collagenase secreted from SW480 cell line.

Figure 3 (d) is a photograph showing the inhibitory effect of mesylic acid gabexate on type IV collagenase secreted from LoVo cell line.

FIG. 3 (e) is a photograph showing the inhibitory effect of mesylic acid gabexate on type IV collagenase secreted from HM7 cell line.

Figure 4 (a) is a photograph showing the results of treatment of aprotinin to the reaction solution for the zymogram after electrophoresis of the serum-free culture medium of LS 174 T, the parent cell of HM7 human colorectal cancer cells.

Figure 4 (b) is a photograph showing the results of treatment of mesylic acid gabexate in the reaction solution for the zymogram after electrophoresis of the serum-free culture medium of LS 174 T, the parent cell of HM7 human colon cancer cells.

Figure 5 (a) is a photograph showing the results of treatment of the reaction solution for zymogram alone or a mixture with aprotinin after the electrophoresis of serum-free culture medium HM7 cell line on gelatin gel.

Figure 5 (b) is a photograph showing the results of treatment of the HT1080 cell line serum-free culture solution on the gelatin gel electrophoresis and then treated with the reaction solution for the zymogram mesylic acid gabexate alone or aprotinin.

FIG. 5 (c) is a photograph showing the results of treatment of the reaction solution for zymogram with mesylic acid gabexate alone or a mixture with aprotinin after electrophoresis of the serum-free culture solution of the SW480 cell line on gelatin gel.

Figure 5 (d) is a photograph showing the result of treating the reaction solution for zymogram alone or a mixture with aprotinin after electrophoresis of serum-free culture medium of LoVo cell line on gelatin gel.

FIG. 6 (a) shows tumorigenic ability of HM7 cell lines in nude mice 4 weeks after injecting sterile saline solution for 2 weeks.

FIG. 6 (b) shows tumorigenic ability of HM7 cell line in nude mice 4 weeks after mesylic acid gabexate treatment for 2 weeks.

Hereinafter, the present invention will be described in detail.

The present inventors are based on the report that MMPs such as type IV collagenase and uPA are deeply involved in the invasion and metastasis of epithelial cell carcinoma, and that they are very essential proteolytic enzymes for angiogenesis including tumor vessels. In response to the urgent need for multispecific protease inhibitors to act on different types of proteases that cells secrete, numerous studies have been conducted to develop novel protease inhibitors.

As a result, it was found for the first time that mesylic acid gabexate strongly inhibits not only serine proteases such as uPA but also MMPs such as type IV collagenase. In addition, if mesylic acid gabexate effectively inhibits proteolytic enzymes such as MMPs and uPA, it is predicted that not only cancer invasion and metastasis but also tumor angiogenesis may be inhibited.

Against this background, in vitro cell motility and invasiveness studies using various human colorectal cancer cell lines, mesylic acid gabexate markedly inhibited cancer cell invasion without affecting cell motility. In addition, in tumor growth and liver metastasis experiments using nude mice, mesylic acid gabexate inhibited the growth of subcutaneous tumors and significantly reduced liver metastasis. In addition, in angiogenesis experiments of rabbit cornea, mesylic acid gabexate effectively inhibited angiogenesis.

Therefore, the mesylic acid gabexate of the present invention can be used as an active ingredient of inhibitors of serine proteases such as MMPs and uPA, type IV collagenase, tumor invasion inhibitors, tumor metastasis inhibitors, tumor growth inhibitors and angiogenesis inhibitors. Can be. In addition, the mesylic acid gabexate of the present invention can be used as angiogenesis inhibitors as well as inhibiting angiogenesis of malignant tumors, as well as various benign angiogenic diseases such as hemangiomas, keratoconjunctival diseases, keloids, and skin vascular diseases. Using the inhibitor of the present invention, protease, tumor invasion, tumor metastasis, tumor growth and angiogenesis can be inhibited in a short amount of time.

The pharmaceutical compositions of the various inhibitors described above containing mesylic acid gabexate having such inhibitory activity as an active ingredient may be administered orally or in the form of a composition containing a pharmaceutically acceptable carrier.

Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredients, are diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), suspending agents (e.g. silica, Talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols, and the tablets also contain binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpi It is preferred to contain a sealing agent (such as starch, agar, alginic acid or its sodium salt) or a boiling mixture and / or absorbents, colorants, flavors and sweeteners as the case may be. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries (eg, preservatives, stabilizers, wetting or emulsifier solution promoters, salts and / or buffers for osmotic control). In addition, they may contain other therapeutically valuable substances.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1

Characterization of Protease Secreted from Various Human Colon Cancer Cell Lines

To characterize the proteases secreted by human colorectal cancer cell lines, LoVo (ATCC CCL229), SW480 (ATCC CCL228), HM7 (see Kuan, S.-F. et al., Cancer Res., 47). (5715-5724 (1987)) Human colon cancer cell line and control group NIH3T3 (ATCC CRL1658), HT1080 (ATCC CCL121) cell line 10% fetal bovine serum (FBS), penicillin (100 µg / ml) and streptomycin (100 µg) / ml) was incubated in DMEM (Dulbeco's modified Eagle's medium) at 5% CO ₂ , 37 ℃. At this time, when the cell density was increased, the cells were subcultured so as to be in a logarithmic phase when experimented with trypsin treatment (0.05% trypsin, 0.02% EDTA in Hank's balanced salt solution without calcium and magnesium) for several minutes, and cells within 15 passages. Was used in the following experiment.

For zymography analysis of the protease secreted from the cell line, the cancer cells grown about 80% were washed with PBS, incubated for 24 hours in DMEM medium without serum, and centrifuged at 1,000 × g for 5 minutes. Cells were then removed and concentrated 10-fold with Centricon-10 concentrator (Amicon Corp., USA). Proteins in this concentrated culture were separated on 10 mg polyacrylamide gels containing 1 mg / ml gelatin (Fisher Chemical Co., USA) or casein. After electrophoresis, the gel was washed with 2.5% Triton-X 100 and allowed to react for 3 days in 50 mM Tris buffer (pH 8.0) containing 5 mM CaCl ₂ and 0.02% NaN ₃ . Then, since the protease in the concentrated culture solution dissolves gelatin at each characteristic molecular weight, the gel is decolorized when stained with Coomassie brilliant blue R250.

In addition, in order to investigate the characteristics of proteases, serine proteolytic enzymes such as DTT and 1,10-phenan throline, which are neutral metalloproteinase inhibitors, The reaction was inhibited with pepstatin, an inhibitor of acidic protease, such as aprotinin and leupeptin.

Figure 1 (a) shows the zymography results of the protease secreted from colon cancer cell HM7. In FIG. 1 (a), the control group is treated with a neutral metalloproteinase inhibitor when 1 is not treated with a protease inhibitor, and 2 is treated with a serine protease inhibitor, respectively. . As shown in FIG. 1 (a), gelatin dissolution was observed in HM7 culture at 160kDa, 110kDa, 72kDa, 54kDa and 30kDa or less, and the dissolution activity of 30kDa or less was very weak. For analysis of each gelatin degrading activity, experiments using various protease inhibitors showed that 72 and 54 kDa gelatin lytic activity was completely inhibited by neutral metalloproteinase inhibitors, partially by lupetin. Inhibited and not inhibited by pepstatin, HM7 cells were estimated to secrete neutral metalloproteinases and serine proteases.

Figure 1 (b) shows the zymography results on a polyacrylamide gel containing gelatin. As shown in Figure 1 (b), the NIH3T3 fibroblast and HT1080 fibrosarcoma cell lines used as controls showed very strong 92 kDa, 72 kDa type IV collagenase activity, and 54 kDa gelatin, MMPs, in the HT1080 cell line. Dissolution activity could be observed. In addition, gelatin lytic activity was observed at 92kDa, 72kDa and 54kDa in SW480 and LoVo human colorectal cancer cells, of which 54kDa activity was particularly strong in LoVo cell lines. For the analysis of each gelatin degrading activity, experiments using various protease inhibitors showed that 92, 72 and 54 kDa gelatin lytic activity was inhibited only by neutral metalloproteinase inhibitors. LoVo cancer cells were estimated to secrete neutral metalloproteinases.

The first (c) also shows the results of the zymography on the polyacrylamide gel containing casein. As shown in FIG. 1 (c), SW480 and HM7 cell lines showed a single band of 55kDa, and LoVo cell lines showed two bands of 55kDa and 60kDa. For analysis of each casein degradation activity, experiments using various protease inhibitors showed that both 55 and 60 kDa casein lytic activity was inhibited by the uPA inhibitor 1 mM amylolide, SW480 And HM7 cell lines secreted a single chain of uPA, and LoVo cell lines were estimated to secrete a double chain of uPA.

Example 2

Inhibitory Activity of Protease Using Mesylic Acid Gabexate

To determine if mesylic acid gabexate can inhibit progelatinase A (72 kDa type IV collagenase) in tablets from which TIMP-2 has been removed, progelatinase A 0.125 μg, 0.25 μg, 0.5 Zymograms were performed in the same manner as in Example 1 using μg (see also 2 (a), 2 (b), 2 (c) and 2 (d)). FIG. 2 (a) shows a control group not treated with mesylic acid gabexate, and FIGS. 2 (b), 2 (c) and 2 (d) show a case where 1mM, 2mM and 5mM of mesylic acid gabecate were treated, respectively. . 2 (a) to 2 (d), 1 represents 0.5 μg of progelatinase A, 2 represents 0.25 μg of progelatinase A, and 3 represents 0.125 μg of progelatinase A, respectively. Indicates. As shown in Figs. 2 (a) to 2 (d), the adverse effect of progelatinase A by mesylic acid gabexate was found to be concentration-dependent and completely inhibited at 5mM concentration of mesylic acid gabexate. .

Furthermore, to determine whether mesylic acid gabexate can inhibit type IV collagenase secreted from various colorectal cancer cell lines, a zymogram was performed in the same manner as in Example 1. 3 (a), 3 (b), 3 (c), 3 (d) and 3 (e) degrees for type IV collagenase secreted from NIH3T3 cell line, HT1080 cell line, SW480 cell line, LoVo cell line and HM7 cell line It is an electrophoresis photograph showing the inhibitory effect of mesylic acid gabexate. In Figs. 3 (a) to 3 (e), the control group shows the case where the mesylic acid gabexate is not treated, and the GM represents mesylic acid gabexate. From Figures 3 (a) to 3 (e), the mesylic acid Gabexate IC ₅₀ value for type IV collagenase secreted from control NIH3T3 and HT1080 cell lines was 0.1-1 mM and colon cancer cell lines SW480, LoVo, HM7. Since the mesylic acid Gabexate IC ₅₀ on the type IV collagenase secreted from the cell line was about 100 μM, 10 μM, and 100 μM, respectively, the inhibitory effect of mesylic acid Gabexate on human colorectal cancer cell lines was significantly greater than that of the control. It became.

Example 3

Analysis of Inhibitory Mechanism of Type IV Collagenase of Mesylic Acid Gabexate

MT-MMP and serine protease uPA, plasmin, and thrombin are involved in the activation of type IV collagenase, and uPA activity is increased in various metastatic cancers. It is reported that it is inhibited in laboratory conditions and that uPA is involved in infiltration by activating latent type IV collagenase (Sato, H. et al., Nature, 370: 61-65 (1994); Schlechte , W. et al., Cancer Communications, 2 (5): 173-179 (1990); Reich, R. et al., Cancer Res., 48: 3307-3312 (1988); Chengshi He et al., Proc Natl.Acad. Sci., USA, 86: 2632-2636 (1989).

Based on the above report, since mesylic acid gabexate, an inhibitor of serine protease, may inhibit thrombin, plasminogen activator and the like, which may interfere with the yellowing of latent type IV collagenase, To determine this, a zymogram was performed using aprotinin that inhibits serine protease as a competitive inhibitory mechanism.

4 (a) and 4 (b) show a case where aprotinin and mesylic acid gabexate were respectively treated with serum-free culture medium of LS 174 T (ATCC CCL188), which is a parent cell of HM7 human colon cancer cells. As shown in Figures 4 (a) and 4 (b), the 110 kDa serine protease of LS 174 T was completely inhibited in 1.7 μg / ml and 17 μg / ml aprotinin, while 72 kDa type IV collagenase was the same. Although there was no change in the concentration of aprotinin, it could be seen that the concentration was dependently inhibited at the concentrations of 100 μM, 1 mM, and 2 mM of mesylic acid vaccinate, and its IC ₅₀ was estimated to be about 100 μM or less.

On the other hand, to investigate the mechanism of inhibiting the type IV collagenase of mesylic acid Gabexate, 5 µg / ml aprotinin and 1 mM ap alone or mixed were tested for the type IV collagenase inhibitory ability (see, Example 5 (a ), 5 (b), 5 (c) and 5 (d) degrees, HM7, HT1080, SW480, and LoVo colorectal cancer cell lines, respectively, type IV collagenase when mixed with mesylic acid gabexate alone and aprotinin Since the degree of activity inhibition was the same, it was thought that mesylic acid gabexate inhibited plasminogen activator, plasmin, and the like, so that there was no possibility of inhibiting the activation of type IV collagenase. Was estimated by.

In FIGS. 5 (a) to 5 (d), C is not treated with a protease inhibitor, A is treated with 5 μg / ml aprotinin, and GM is treated with 1 mM mesylic acid vabeccete. A + GM represents a case where 5 µg / ml aprotinin and 1 mM mesylic acid gabecate were mixed and treated.

Example 4

Effect of Mesylic Acid Gabexate on Growth and Laboratory Infiltration of Human Colon Cancer Cell Lines

As a basic experiment for the inhibition of in vitro cancer cell motility and invasiveness using mesylic acid Gabecxate, we examined the effect of mesylic acid Gabecxate on the growth of several colorectal cancer cell lines. In other words, SW480, LoVo or HM7 colorectal cancer cell lines were dispensed at 10 ⁵ cells / well in a 24-well plate, and mesylic acid Gabexate was measured by adding mesylic acid Gabexate at concentrations of 10 μM, 100 μM and 1 mM, and measuring the number of cells daily for 3 days Was examined the effect on cell proliferation (see Table 1).

As shown in Table 1, SW480, LoVo, HM7 colorectal cancer cell lines were not affected by cell proliferation at 10 μM, 100 μM concentration of mesylic acid Gabexate (GM), but showed a significant inhibition of proliferation at 1 mM concentration.

Therefore, the concentration of mesylic acid gabexate for cell motility and infiltration concentration to be described later was propagated to 10 μM and up to 100 μM, and the infiltration and motility under laboratory conditions were analyzed. In other words, using a Boyden chamber equipped with a polycarbonate filter having a diameter of 6.5 mm and having a diameter of 8 μm, 160 μg of Matrigel was applied above the filter, and 2 × 10 ⁵ cells were dispensed above the filter. Then, put the culture solution containing 10μM and 100μM mesylic acid gabexate under the filter, and incubate for 3 days, remove the cells above the filter with a cotton swab, move to the bottom of the filter, and attach the attached cells to hematoxylin. Invasion was determined by staining and measuring the number of cells under a 200-fold microscope (see Table 2). In addition, the mobility test was performed under the same conditions without the Matrigel applied (see Table 2).

As shown in Table 2, the inhibitory effect of mesylic acid vaccinate on the infiltrating concentrations of SW480, LoVo, and HM7 cell lines was 59.1%, 73.2%, 61.9% at 10 μM concentrations, and 79.3%, 91.1%, 83.6 at 100 μM concentrations, respectively. Inhibition of cellular motility by mesylic acid gabexate was not observed, whereas it showed a very strong invasion inhibition rate (P0.01).

Example 5

Inhibitory Effect of Mesylic Acid Gabexate on Tumor Formation in HM7 Colon Cancer Cells

In order to examine whether the mesylate gabek glyphosate inhibits tumorigenesis of the large intestine cancer, age 5 primary about 20g and the suction BALB / C nude mouse body weight with diethyl ether anesthesia, HM7 cells 10 to ⁷ / 200㎕ flank subcutaneous tissue injection in the following, periodically measuring the maximum and minimum diameters of the tumor, the tumor volume ^{(mm 3) = L × W} 2/2 (L is the maximum diameter, W represents the minimum diameter) was calculated (see Tables 3, 6 (a) and 6 (b) degrees). At this time, mesylic acid gabexate was injected intraperitoneally twice a day in an amount of 0.2 mg / 200 μl from the day before cancer cell injection to 2 weeks after injection, and the control group was injected with the same amount of sterile saline solution.

As shown in Table 3, the tumor volume was reduced by 63.6% compared to the control group at 2 weeks of mesylic acid vaccinate treatment, 36.7% at 4 weeks, 33.9% at 6 weeks (P0.05).

Figure 6 (a) shows a nude mouse 4 weeks after the injection of sterile physiological saline for 2 weeks as a control, Figure 6 (b) shows a nude mouse after 4 weeks of treatment with mesylic acid gabexate for 2 weeks. . As shown in Figures 6 (a) and 6 (b), mesylic acid gabexate was found to strongly inhibit tumor formation in vivo.

Example 6

Inhibitory Effects of Mesylic Acid Gabexate on Liver Metastasis of HM7 Colon Cancer Cells

In order to examine the transfer function between the colorectal cancer cells, were anesthetized BALB / C nude mice of about 20g body weight, one ml of incision of about 1cm in the left flank to expose the spleen injected with HM7 cells 10 ⁶ / 100㎕ then After about 1 minute, the spleen was removed and raised in a sterile thermostat, followed by laps after 4 weeks, and liver metastasis was evaluated. At this time, intraperitoneally inject 0.2 mg of mesylic acid vaccinate twice a day, 2 hours before and 10 days after injecting HM7 cells through the spleen, and then open at the fourth week (No. of tumor nodules). And liver weights were measured (see Table 4).

As shown in Table 4, the control group injected with HM7 cells alone had liver liver weight of 5.05 ± 2.16g due to severe liver metastasis, 4.9 times that of mesylic acid Gabexate treatment, and liver weight of mesylic acid Gabexate treatment 1.03 ± 0.15g. It was equal to the weight of normal liver. In addition, the number of liver metastases was 425 ± 129.9, whereas the mesylic acid vaccinate treatment was 3.5 ± 2.9, and mesylic acid vaccinate inhibited 99.2% of liver metastatic lesions. Significant hepatic metastasis inhibition was shown.

Since the dose of mesylic acid gabexate used in the liver metastasis suppression test described above is 10 mg / kg body weight, it is a CT developed by Batimastat (30 mg / kg body weight) developed by British biotechnology (United Kingdom), Celltech. (USA). It can be seen that the dosage is very small compared to 1746 (100 mg / kg).

From the above examples, mesylic acid gabexate has been fully demonstrated that inhibition of MMPs such as type IV collagenase, inhibition of cancer cell infiltration, inhibition of tumorigenicity, and inhibition of liver metastasis by mesylic acid gabexate have been demonstrated. It is thought to be available.

Example 7

Inhibitory Effects of Mesylic Acid Gabexate on Rabbit Cornea

To determine if mesylic acid gabexate inhibits angiogenesis, anesthesia was injected by intramuscular injection of 30 mg / kg of ketamine in a New Zealand-produced white rabbit weighing about 3 kg, and 2% lidocaine It was injected into the back and the eye was protruded and fixed with the thumb and forefinger. Then, using the 11th operation diagram in the area slightly away from the center of the cornea, a 3 mm long partial incision is made to deepen the incision in the cornea so as not to perforate the cornea, and the 15th operation diagram is inserted into the pocket in the cornea parenchyma. Made. A polymer disc with a diameter of about 2 mm was inserted into the corneal pocket, and the control group was physiological saline, and the treatment group was instilled into the eye 4 days a day for 2 to 10 days after the operation. New blood vessels around the disc were read.

As a result, the polymer disk of the control group was unclear by the newly formed blood vessels, and many blood vessels were generated in the surrounding area, whereas the treatment group inhibited angiogenesis and the inserted polymer disk was clearly distinguished and the number of blood vessels in the surrounding area was markedly reduced. It was confirmed.

[Acute Toxicity Test]

Considering that the LD ₅₀ value of mesylic acid ibexate in mice is 8000 to 8190 mg / kg in oral administration, 4550 to 4900 mg / kg in subcutaneous injection and 248 to 260 mg / kg in intravenous injection (see Fujita, T et al., 應用 藥理, 9 (5): 743-760 (1975)), various inhibitors of the present invention containing mesylic acid vaccinate are sufficiently safe within the range of effective amounts for oral and background administration described below. It can be seen that it can be used.

[Administration method and effective amount]

In the case of an adult (based on 60 kg body weight), the amount of active ingredient administered daily is preferably 40 mg / kg body weight to 80 mg / kg body weight, and 20 mg / kg body weight to 40 mg / kg body weight by intravenous injection.

As described and demonstrated in detail above, the present invention is an MMPs and serine protease inhibitors, such as type IV collagenase containing mesylic acid vaccinate as an active ingredient, tumor infiltration, metastasis or growth inhibitory, angiogenesis A method of inhibiting protease, tumor infiltration, tumor metastasis, tumor growth or angiogenesis is provided by administering an inhibitor and an electrical inhibitor. Using the inhibitor of the present invention, protease, tumor invasion, tumor metastasis, tumor growth and angiogenesis can be inhibited in a short amount of time.

Claims (8)

An MMP (matrix degrading metalloproteinase) inhibitor comprising mesylic acid gabexate (Gabexate mesilate) as an active ingredient and a pharmaceutically acceptable carrier. A colorectal cancer metastasis inhibitor comprising mesylic acid Gabexate (Gabexate mesilate) as an active ingredient and a pharmaceutically acceptable carrier. A colorectal cancer cell growth inhibitor comprising mesylic acid Gabexate (Gabexate mesilate) as an active ingredient and a pharmaceutically acceptable carrier. Angiogenesis inhibitor comprising mesylic acid Gabexate (Gabexate mesilate) as an active ingredient and a pharmaceutically acceptable carrier. A method of inhibiting matrix degrading metalloproteinase (MMP) in mammals other than humans, the method comprising administering to a patient an effective amount of Gabexate mesilate with a pharmaceutically acceptable carrier. A method of inhibiting colorectal cancer metastasis in a mammal, except humans, comprising administering to a patient an effective amount of Gabexate mesilate with a pharmaceutically acceptable carrier. A method of inhibiting colon cancer cell growth in mammals other than humans, comprising administering to a patient an effective amount of Gabexate mesilate in association with a pharmaceutically acceptable carrier. A method of inhibiting angiogenesis in a mammal, except humans, comprising administering to a patient an effective amount of Gabexate mesilate with a pharmaceutically acceptable carrier.