KR20030038282A - Inhibitor for angiogenesis containing 6-anilinoquinoline-5,8-quinone - Google Patents

Abstract

The present invention relates to an angiogenesis inhibitor containing 6-anilinoquinoline-5,8-quinone (6-anilinoquinoline-5,8-quinone, COMR2029) as an active ingredient, the angiogenesis inhibitor of the present invention It can be usefully used for the prevention or treatment of diseases related to angiogenesis such as cancer, rheumatoid or diabetic retinopathy.

Description

Angiogenesis inhibitor containing 6-anilinoquinoline-5,8-quinone as an active ingredient {Inhibitor for angiogenesis containing 6-anilinoquinoline-5,8-quinone}

The present invention relates to an angiogenesis inhibitor containing 6-anilinoquinoline-5,8-quinone (6-anilinoquinoline-5,8-quinone, COMR2029) as an active ingredient, the angiogenesis inhibitor of the present invention It can be usefully used for the prevention and treatment of diseases related to angiogenesis such as cancer, rheumatoid or diabetic retinopathy.

Cancer is one of the incurable diseases that all human beings should overcome. Currently, various anticancer drugs have been developed and used clinically. However, since the existing anticancer drugs are non-selective, they cause serious side effects, as well as heterogeneity and genetic instability of cancer cells. Genetic instability) causes a problem in the appearance of resistant cells. In addition, most existing anticancer drugs are not suitable for treating solid cancer. Therefore, efforts to solve the above problems by using the recent advanced cancer cell biology have been actively conducted worldwide, one of which is to treat cancer through inhibition of angiogenesis.

Solid cancers can be supplied by diffusion to the growth factor, oxygen, and nutrients necessary for growth until they are about 1 to 2 mm in size, but must be supplied through new blood vessels to grow further. In other words, by blocking angiogenesis, which is essential for cancer growth and metastasis, cancer growth can be effectively prevented.

As a result of research on angiogenesis or neovascularization at various angles, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and epipithelial growth factor (EGF) have been studied. And angiogenic factors such as angiopoietin, fibroblast growth factor receptor (FGFR), Flk-1 / KDR, Flt-1, Flt-3, Tie-1, Tie-2 / Tek, Eph, etc. Receptors of angiogenic factors with tyrosine kinase activity, as well as endogenous angiogenesis inhibitors such as angiostatin or endostatin, have been found to provide angiogenesis as well as angiogenesis. The association between production and human disease is becoming increasingly clear, and several new ways to control angiogenesis are being proposed. In particular, VEGF among the angiogenesis factors has a close correlation with the progression of cancer and the treatment rate of cancer, and its receptor, Flk-1 / KDR, is highly expressed in vascular endothelial cells. Attention has been directed to the development of production inhibitors.

The relationship between cancer and angiogenesis was first reported in 1971 as a hypothesis that neovascularization is essential for the growth of cancer presented by Dr. Fokman of the United States (J. Folkman, Tumor Angiogenesis: Therapeutic Implications.New England Journal of Medicine). , 285, 1182-1186, 1971). In 1990, fumagillin from natural products was reported to have a selective inhibitory effect on angiogenesis, and angiogenesis inhibitors began to attract attention as a treatment for cancer (D. Ingber). , T. Fujita, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature, 348, 555-557, 1990). Also recently discovered angiostatin (MS O'Reilly, L. Holmgren, Y. Shing, C. Chen, RA Rosenthal, M. Moses, WS Lane, Y. Cao, EH Sago and J. Forkman. , Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis Lung Carcinoma, Cell , 79, 315-328, 1994), endostatin (MS O'Reilly, T. Boehm, C. Chen, et al, Endostatin:.. an endogeneous inhibitor of angiogenesis and tumor growth Cell, 88, 277-285, 1997) of endogenous angiogenesis inhibitors experimental anticancer agent of angiogenesis inhibitor is demonstrated to have an excellent efficacy as an anticancer agent in an animal model, such as More attention is focused on their use as.

Moreover, when an angiogenesis inhibitor is used as an anticancer agent, there is an advantage over an existing anticancer agent. Since angiogenesis is an essential phenomenon for cancer growth or metastasis, it can block the growth and metastasis of the cancer at the same time, and angiogenesis inhibitor is an aploid. Targeting vascular endothelial cells, which are normal diploids rather than human cancer cells, does not cause problems such as resistance due to heterogeneity and genetic instability of cancer cells. In addition, angiogenesis inhibitors are effective against all types of cancer in which angiogenesis is indispensable, whereas conventional anticancer agents have shown efficacy only in certain or some types of cancers. Except for healing, women's physiology, etc., it is very rare, so if angiogenesis inhibitors are used as anticancer drugs, the side effects seen with conventional anticancer drugs will be greatly reduced.

In addition, because angiogenesis is known to cause cancer growth and metastasis, as well as so-called angiogenic diseases such as rheumatoid arthritis, diabetic retinopathy, blood vessels Inhibitors of production are expected to be useful in the treatment and prevention of diseases related to other types of blood vessel proliferation in addition to cancer.

Thus, the present inventors have been trying to find a new angiogenesis inhibitor 6-anilinoquinoline-5,8-quinone (6-anilinoquinoline-5,8-quinone) has the function of inhibiting angiogenesis without cytotoxicity The present invention was completed by revealing that 6-anilinoquinoline-5,8-quinone can be usefully used for the prevention or treatment of diseases related to angiogenesis.

An object of the present invention is to provide an angiogenesis inhibitor containing 6-anilinoquinoline-5,8-quinone (6-anilinoquinoline-5,8-quinone) as an active ingredient.

1 is a photograph of the analysis of in vivo angiogenesis inhibition by COMR2029 by administering Matrigel (subcutaneous subcutaneous) of the mouse,

2 is a photograph of the apoptosis-inducing ability of COMR2029 analyzed by agarose electrophoresis,

A: B16 (F10), lane 1 and lane 8: markers,

Lane 2 and lane 9; Solvent control group (VH), lane 3: COMR2029 0.03 μg / ml,

Lane 4: COMR2029 0.1 µg / ml, lane 5: COMR2029 0.3 µg / ml,

Lane 6: COMR2029 1 µg / ml, lane 7: COMR2029 3 µg / ml,

Lane 10: camptothecin 0.1 μg / ml,

Lane 11: camptothecin 0.1 μg / ml + COMR2029 0.03 μg / ml,

Lane 12: camptothecin 0.1 μg / ml + COMR2029 0.1 μg / ml,

Lane 13: camptothecin 0.1 μg / ml + COMR2029 0.3 μg / ml,

Lane 14: camptothecin 0.1 μg / ml + COMR2029 1 μg / ml,

Lane 15: camptothecin 0.1 μg / ml + COMR2029 3 μg / ml,

B: HL60, lane 1: marker, lane 2: solvent control group (VH),

Lane 3: camptothecin 0.3 µg / ml, lane 4: COMR2029 0.1 µg / ml,

Lane 5: COMR2029 0.3 µg / ml, lane 6: COMR2029 1 µg / ml,

Lane 7: COMR2029 3 μg / ml,

Figure 3 is a graph analyzing the change in intracellular Ca ²⁺ concentration by treatment with COMR2029 using Fura-2 / AM,

4 is a photograph showing the analysis of infiltration inhibition when COMR2029 was treated at a concentration of 3 μg / ml, 1 μg / ml or 0.3 μg / ml in B16 (F10) cells,

5 is a photograph showing the analysis of infiltration inhibition when COMR2029 was treated at a concentration of 0.03 μg / ml, 0.1 μg / ml or 0.3 μg / ml in LOX-IMVI cells,

6 is a graph comparing tumor volume with solvent control group (VH) when COMR2029 was treated in nude mice transplanted with human skin cancer cell line LOX-IMVI,

7 is a graph comparing the weight of the mouse with the solvent control group (VH) when COMR2029 was treated in nude mice transplanted with human skin cancer cell line LOX-IMVI,

FIG. 8 is a graph comparing tumor volume with solvent control group (VH) when COMR2029 and positive control adriamycin were treated in nude mice transplanted with human skin cancer cell line LOX-IMVI,

Figure 9 is a graph comparing the weight of the mouse compared to the solvent control group (VH) when treated with COMR2029 and positive control adriamycin in nude mice transplanted with human skin cancer cell line LOX-IMVI.

In order to achieve the above object, the present invention provides an angiogenesis inhibitor containing 6-anilinoquinoline-5,8-quinone (6-anilinoquinoline-5,8-quinone, hereinafter abbreviated as "COMR2029") as an active ingredient to provide.

Hereinafter, the present invention will be described in detail.

The present invention provides an angiogenesis inhibitor containing COMR2029 as an active ingredient.

In the present invention, COMR2029 has the same structure as in Chemical Formula 1 .

In the present invention, in order to analyze whether COMR2029 has basic inhibitory activity of angiogenesis, the tube formation assay of COMR2029 was tested using HUVEC cells having the ability to form a tube in a matrix due to the characteristics of the cells. Inhibits. In addition, when analyzing the cytotoxicity of HUVEC of COMR2029, COMR2029 is only slightly toxic to HUVEC cells at high concentrations but not at all (see Table 1 and Table 2 ).

In the present invention, in order to measure the ability to inhibit angiogenesis of COMR2029, in vivo angiogenesis assay was performed by administering a Matrigel containing FGF to the mouse subcutaneously, and did not administer COMR2029. The non-solvent control group appears to be red due to a lot of angiogenesis, and mice to which COMR2029 is administered appear yellow because little blood vessels are formed (see FIG. 1 ). Thus, COMR2029 functions to inhibit angiogenesis.

In addition, COMR2029 shows high cytotoxicity against most cell lines, and has high anticancer activity. In particular, COMR2029 has a particularly strong cytotoxicity to skin cancer cell lines (see Table 3 ). COMR2029 shows strong cytotoxicity against skin cancer cell lines but does not affect cell cycle and apoptosis, nor does it affect caspase-3 activity and intracellular calcium changes, a mechanism involved in cell death ( FIGS. 2 and 3). Reference).

In addition, COMR2029's membrane permeation inhibition ability was performed by in vitro invasion assay, and COMR2029 has a membrane permeation inhibition function in B16 (F10) cells, which are widely used as an experimental system for testing membrane permeability inhibitors ( 4 , cell lines that are not well used for infiltration analysis, such as LOX-IMVI cells, inhibit membrane permeation in a concentration-dependent manner compared to the solvent control group (see FIG. 5 ).

In addition, in order to examine the anticancer activity of COMR2029, when a human skin cancer cell line LOX-IMVI was implanted subcutaneously and intraperitoneally administered to COMN2029, the tumor size was reduced without affecting the weight (see FIGS . 6 and 7 ). In comparison with the COMR2029-administered group, the anti-cancer adriamycin as a positive control group showed that the tumor size was significantly reduced like the positive control group, indicating that COMR2029 had high anticancer activity (see FIGS . 8 and 9 ).

Angiogenesis inhibitors containing the COMR2029 of the present invention as an active ingredient can be usefully used in diseases related to angiogenesis such as cancer, rheumatism or diabetic retinopathy.

Angiogenesis inhibitor for the prevention or treatment of diseases related to angiogenesis of the present invention contains the COMR2029 as an active ingredient. The COMR2029 may be administered orally or parenterally during clinical administration and may be used in the form of a general pharmaceutical formulation. In other words, COMR2029 can be administered in various oral and parenteral dosage forms for actual clinical administration. When formulated, COMR2029 can be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. It is prepared. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may be used in mixed herbal extracts, including at least one excipient such as starch, calcium carbonate, sucrose, lactose and gelatin, and the like. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and gelatin may be used.

The effective dose of COMR2029 of the present invention was 10 mg / kg and a single oral dose toxicity test of COMR2029 in rats showed that LD ₅₀ far exceeded 1000 mg / kg.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Tube formation assay

In order to analyze whether the tube formation of the cells by COMR2029 is inhibited, the inventors used a tube using human UVM (Human Umbilical Vein Endothelial Cell) which forms a tube shape in a matrix due to the characteristics of the cells. A tube formation assay was performed.

All experimental tools used for the test were kept refrigerated (4 ° C.) at all times throughout the test. 40 μl of the Matrigel was dispensed into 96-well plates and polymerized in a 37 ° C. CO ₂ incubator for 30 minutes. In this experiment, Matrigel provides a matrix for HUVEC cells for tube formation, ie angiogenesis. HUVEC cells, which are human placental umbilical vein endothelial cell lines grown in EGM-2 medium, were plated at 180 μl each at a concentration of 1.1 × 10 ⁵ cells / ml. COMR2029 was treated with 0.03, 0.1, 0.3, 1, 3 μg / ml, and cultured in a 37 ° C., CO ₂ incubator for 18-24 hours, and the results were determined by photographing with a microscope.

As a result, VH was formed as 10 or more tube shapes as shown in the figure as a solvent control group. On the other hand, treatment of 3 μg / ml and 1 μg / ml concentrations of COMR2029 showed no tube formation, and starting from 0.3 μg / ml, the line-like arrangement for forming the tubes began to show little by little. However, no tube was formed up to a concentration of 0.1 μg / ml, and a low degree of tube was started at 0.03 μg / ml.

From the above results, COMR2029 was shown to inhibit the tube forming ability of HUVEC cells with the ability to form tubes at low concentrations.

Example 2 Cytotoxicity Measurement on HUVEC Cells

The present inventors have carried out the cytotoxicity against HUVEC, a umbilical vein endothelial cell line of human placenta, using COMR2029 (SRB method, Skehan, et al., Proc. Am. Assoc. Cancer Res. , 30: 612, 1989). The cancer cell line was cultured using EGM-2 medium. The COMR2029 used in the above method was dissolved in 100% dimethylsulfoxide (hereinafter abbreviated as "DMSO"), and diluted in steps to adjust the concentration of the COMR2029 to 0.01, 0.03, 0.1, 0.3, 1 µg / ml. The number of cells was measured and dispensed into 96-well plates at 5 × 10 ⁴ , 8 × 10 ⁴ , 10 × 10 ⁴ cells / mL, and Tz (to determine the concentration of cells after one day and before treatment with the drug). Time Zero) Plates were fixed using 50% TCA. The plate to be treated with COMR2029 was divided into two groups, one group was treated for 24 hours, and then the final concentration of COMR2029 was adjusted to 5 concentrations with 0.1% DMSO, and the other group was treated immediately with COMR2029 and after 2 hours, fresh media was added. After 24 hours, COMR2029 was processed again. Tz plate was washed with tap water after 1 hour, and the plate treated with sample was fixed with 50 μl of 50% TCA per well after 2 days, and also with tap water after 1 hour. Washed. The washed plate was dried at room temperature, and then 100 μl of 0.4% SRB solution (solution dissolved in 1% acetic acid) was added per well, and then washed with 1% acetic acid solution after 30 minutes and dried again at room temperature. Next, 100 mM of 10 mM Tris base (pH 10.5) was added to dissolve the wells again, and the absorbance was measured at 540 nm using a microplate reader. The group treated with COMR2029 after 24 hours was shown in Table 1 below. , The group treated immediately with COMR2029 and replaced with a fresh medium after 2 hours and then treated with COMR2029 again after 24 hours is shown in Table 2 below.

Concentration (µg / ml) 5 × 10 ⁴ cells / ml 8 × 10 ⁴ cells / ml 10 × 10 ⁴ cells / ml 0.01 104.7 ± 6.8 113.2 ± 4.8 100.5 ± 4.3 0.03 102.5 ± 5.8 109.2 ± 8.0 102.0 ± 3.6 0.1 100.5 ± 1.3 106.8 ± 9.0 100.1 ± 3.3 0.3 102.0 ± 9.4 107.2 ± 4.8 99.6 ± 7.5 One -6.0 ± 2.7 -24.0 ± 8.9 -28.1 ± 14.8

Concentration (µg / ml) 5 × 10 ⁴ cells / ml 8 × 10 ⁴ cells / ml 10 × 10 ⁴ cells / ml 0.01 103.7 ± 8.7 99.6 ± 10.0 105.1 ± 7.0 0.03 106.0 ± 4.6 103.7 ± 11.5 107.1 ± 13.5 0.1 102.2 ± 6.8 97.5 ± 9.5 104.3 ± 2.5 0.3 96.8 ± 4.6 91.0 ± 6.6 94.9 ± 6.6 One -7.9 ± 3.9 -19.3 ± 9.1 -38.9 ± 6.0

From the above results, when the toxicity was analyzed by treating HUVEC cells with various concentrations of COMR2029, cell growth was not inhibited at concentrations of 0.3 µg / ml and below, but cytotoxicity was observed with rapid growth inhibition from 1 µg / ml. It could be confirmed.

Example 3 Matrigel plug angiogenesis analysis

In order to measure the angiogenesis inhibitory ability of COMR2029, the present inventors performed an in vivo angiogenesis assay by administering Matrigel subcutaneously.

Heparin was diluted in PBS containing 1 mg / ml of albumin and added to matrigel at a final 64 unit / ml and aFGF was prepared to be 1 ng / ml. 0.5 ml of s.c (Subcutaneous) was administered to the abdomen of 7 week old females of C57BL / 6 mice. COMR2029 was prepared with 5%, Cremophor 20% and 0.85% saline 75%. COMR2029 was intraperitoneally administered daily from day 1 to day 8 at a dose of 10 mg / kg. In the case of the solvent control group, a cremophor solution containing no drug was administered in the same manner. Animals were autopsied and photographed on day 9 after Matrigel was implanted subcutaneously in mice.

As a result, the solvent control group that was not administered COMR2029 appeared red due to a lot of angiogenesis, but mice that were administered COMR2029 showed yellow because little blood vessels were formed ( FIG. 1 ). Therefore, it can be confirmed that COMR2029 has a function of inhibiting angiogenesis.

Example 4 Cytotoxicity Measurement on Cancer Cells

In order to determine whether COMR2029 has organ-specific or cell line-specific cytotoxicity against cancer cell lines derived from different organs, the present inventors have investigated the anti-cancer activity of cancer cells in the SALB method (SRB method, Skehan, et al., Proc . Am. Assoc. Cancer Res., 30: 612, 1989). At this time, the blood cancer cell lines MOLT-4F, RPMI8226, HL60, P388 / ADR, P388, K562 / ADR, K562 / VIN and K562, human skin cancer (Melanoma) cell lines LOX-IMVI, SK-MEL-28, SK-MEL-5, UACC62, Hs 294T, Hs 695T, SK-MEL-2, RPMI7951, A253, G361, M14 and HT1080, mouse skin cancer (Melanoma) cell line B16 (F10), renal cancer cell line UO -31, Colon cancer cell lines Colo205, HCT116, HCC2998, HCT15 and SW620, lung cancer cell lines A549, NCl-H522 and NCl-H23, carcinoma of the nervous system (CNS) cell line SNB19, ovarian cancer (Ovary) Cell lines SK-OV-3, prostate cancer cell line PC-3, breast cancer cell lines Hs 578T, MCF7 / ADR and MCF7 were used. A cell line distributed by NCI and ATCC in the US and maintained by Korea Research Institute of Bioscience and Biotechnology was used.

The cancer cell lines were cultured using RPMI 1640 medium containing 10% FBS, and the cultured cells were adjusted and maintained to be in an exponential growing phase before the experiment. The cell concentration used to measure anticancer activity was adjusted to 4 × 10 ⁴ -10 × 10 ⁴ cells / ml with RPMI 1640 medium containing 5% FBS according to the growth rate of the cells and dispensed into 96 well plates. Tz (Time Zero) plates were fixed using 50% TCA to determine the concentration of cells one day after the cells were dispensed and before drug treatment.

COMR2029 was dissolved in 100% DMSO and diluted in steps to adjust the concentration of COMR2029 to 0.01, 0.03, 0.1, 0.3, 1 μg / ml. The plate to be treated with COMR2029 was treated by adjusting the final concentration of the sample to 5 concentrations with 0.1% DMSO. Tz plate was washed with tap water after 1 hour, and the plate treated with COMR2029 was fixed with 50 μl of 50% TCA per well after 2 days, and then with tap water after 1 hour. Washed. The washed plates were dried at room temperature, then 100 μl of 0.4% SRB solution (solution dissolved in 1% acetic acid) was added per well, and then washed with 1% acetic acid solution after 30 minutes and dried again at room temperature. . Next, 10 mM Tris base (pH 10.5) was added to 100 μl per well to dissolve again, and the absorbance was measured at 540 nm using a microplate reader, and the cytotoxicity for each cell line is shown in Table 3 below. At this time, GI ₅₀ (μg / ml) indicates the concentration of the compound that inhibits the growth of cancer cells by 50%.

Tumor cell line GI ₅₀ : μg / ml Tumor cell line GI ₅₀ : μg / ml Tumor cell line GI ₅₀ : μg / ml MOLT-4F 0.817 Colo205 0.575 LOX-IMVI 0.030 RPMI8226 0.363 HCT116 0.155 SK-MEL-28 0.369 HL60 0.843 HCC2998 0.951 SK-MEL-5 0.022 P388 / ADR > 1.000 A549 1.068 UACC62 0.091 P388 > 1.000 NCl-H522 0.136 Hs 294T 0.133 K562 / VIN 0.277 NCl-H23 0.104 Hs 695T 0.085 K562 / ADR 0.163 SNB19 0.284 SK-MEL-2 0.088 K562 0.357 SK-OV-3 1.076 RPMI7951 0.095 B16 (F10) 0.776 Hs 578T 0.225 A253 0.141 UO-31 0.309 MCF7 / ADR 0.151 G361 0.045 HT1080 0.733 MCF7 0.234 M14 0.353 SW620 0.561 HCT15 0.138 PC-3 0.250

From the above results, COMR2029 showed a very sensitive response to K562, K562 / VIN, and K562 / ADR, while P388 and P388 / ADR showed low reactivity over 1.0 μg / ml. In the case of renal cancer UO-31 and central nervous system SNB19, the activity was about 0.3 μg / ml. In five rectal cancers, HCT15 and HCT116 showed GI50 of 0.2 µg / ml and Colo205, HCT15, and SW620 showed 0.5 ㎍ / ml of GI50. Ovarian cancer, SK-OV-3, had low activity, while breast and prostate carcinomas showed a GI ₅₀ of about 0.2 μg / ml. 13 In respect to the skin cancer cell line nine cells SK-MEL-5, UACC62, Hs 294T, Hs 695T, SK-MEL-2, RPMI7951, A253, a GI ₅₀ value in G361 suppressed robust cell growth to less than 0.1 ㎍ / ㎖ In particular, LOX-IMVI showed the strongest cytotoxicity in all the cells whose GI ₅₀ was observed to be 0.03 μg / ml or less. SK-MEL-28, M14, HT1080 and B16 showed weak cell growth inhibition at the highest concentration of 1 ㎍ / ml, but no inhibitory effect was observed at all experimental concentrations. Lung cancer recorded about 0.1 μg / ml of GI ₅₀ in NCI-H522 and NCI-H23. In conclusion, COMR2029 has a selective effect on skin cancer.

Example 5 Cell Cycle Measurement Test

In Example 4, it was confirmed that COMR2029 exhibited specific cytotoxicity to skin cancer cell lines, and cell cycle measurement tests were performed to determine whether COMR2029 affects the cell cycle.

Human skin cancer (Melanoma) cell line LOX-IMVI was cultured using RPMI 1640 medium containing 10% FBS. The COMR2029 used in the method was dissolved in 100% DMSO and diluted in steps to adjust the concentration of COMR2029 to 0.0003, 0.001, 0.003, 0.01, 0.003 μg / ml. Measure the number of cells and dispense into 6 × 10 ⁶ cells / ml into T 75 flask, treat COMR2029, and then collect the cells by centrifugation (1,000 rpm, 10 minutes) and fix with 70% ethanol. It was. After centrifugation (1,200 rpm, 10 minutes), PBS 400 μl, RNAase 1 μg / ml and PI 400 μg / ml were placed in a thermostat heated at 37 ° C. for 30 minutes, and the cells were FACS (Fluorecence-activated cell sorter). After 48 hours, the experiment was repeated.

The FACS measurement results were shown in Table COMR2029 treatment after a 24-hour period after the cells 4 are shown COMR2029 cell cycle 48 hours after post-treatment are shown in Table 5.

Concentration (µg / ml) G0 / G1 phase S step G2 / M stage VH 39.4 16.9 14.4 0.0003 39.9 16.9 16.4 0.001 36.0 15.8 15.7 0.003 40.1 15.6 16.7 0.01 34.9 16.2 16.2 0.03 42.2 16.8 22.5

Concentration (µg / ml) G0 / G1 phase S step G2 / M stage VH 31.9 15.6 14.8 0.0003 34.8 18.7 17.3 0.001 33.3 18.9 15.5 0.003 34.9 17.1 15.0 0.01 32.8 16.9 13.9 0.03 35.8 17.8 16.4

From the above results, when COMR2029 was treated in the skin cancer cell line, it was confirmed that there was no change in the ratio of the cell cycle stages G0 / G1 stage, S stage, G2 / M stage for 24 hours and 48 hours.

Example 6 Apoptosis Test

In order to measure apoptosis-induced ability by COMR2029, the present inventors used agarose gel electrophoresis, intracellular digested DNA quantification method, caspase-3 activity measurement and intracellular calcium change to measure cell death. The inducing ability was examined.

Regular DNA cleavage, a typical phenomenon of cell death, can be seen on the agarose gel, which shows a ladder type. In the diphenylamine method, diphenylamine is inserted into DNA so that cells with many cells are dyed and dark blue. Caspase-3 is the last enzyme in which cell death occurs, and almost all cells undergo this enzyme before cell death occurs. Thus, if cell death occurs, the DNA becomes a ladder in the agarose gel, the diphenylamine becomes dark blue, the absorbance is high, and the caspase-3 activity increases.

<6-1> Agarose Gel Electrophoresis

Skin cancer cells B16 (F10) and human hematologic cancer HL60 were dispensed into 12-well plates at a concentration of 2x10 ⁶ cells / ml, and then COMR2029 was treated with 0.03, 0.1, 0.3, 1, 3 µg / ml. After incubation for 24 hours, transfer to a 1.5 ml tube and centrifuged with a minicentrifuge (1300 rpm, 20 seconds), and then precipitate the cell precipitate for 15 minutes at room temperature in 500 µl lysis buffer (10 mM Tris buffer, 1 M EDTA, 0.2% Triton X-100, pH8.0). The lysed cells are centrifuged at 15,000 rpm for 20 minutes to separate the DNA fragments from the precipitate. DNA fragments of the supernatants were allowed to stand for one day by precipitation at -20 ° C with an equal amount of isopropyl alcohol and 0.5 M NaCl. After centrifugation of the standing samples, the dry DNA samples were prepared with 40 μl TE solution (10 mM Tris, 1 mM EDTA, pH7.4) and 20 μl loading buffer (15 mM EDTA, 2% SDS, 50% glycerol, 0.5% Bromophenol Blue, 10 unit RNase). After heat treatment at 65 ° C. for 10 minutes, electrophoresis was performed at 50 V for 50-60 minutes on 1.5% agarose gel containing 1.0 μg / ml ethidium bromide.

As a result, DNA was cut on the agarose gel to see a ladder-shaped band when cells were generated, but DNA cutting was not observed in B16 (F10) and HL60 ( FIG. 2 ).

<6-2> Intracellular Degradation DNA Quantitation

The cell precipitate and the DNA components in the supernatant were all analyzed by diphenylamine (hereinafter abbreviated as "DPA") method. The cells used were treated with mouse skin cancer B16 (F10), human blood cancer HL60, human skin cancer LOX-IMVI, human breast cancer MCF7 cells for 24 hours and then centrifuged. The concentrations of COMR2029 used in the experiments were 0.03, 0.1, 0.3, 1, 3 ㎍ / mL and the final concentration used in the experiments was 0.1% using DMSO as a solvent. Precipitates containing DNA were released well with 250 μl lysis buffer, 0.5 N perchloric acid was added in the same amount as 250 μl of the supernatant of cells, and then heated at 95 ° C. for 15 minutes. 500 μl of a double dose of DPA reagent (88 mM DPA in 98% (v / v) glacial acetic acid, 1.5% (v / v) sulfuric acid, 0.5% (v / v) 1.6% acetaldehyde solution) was added. . After standing overnight at room temperature, DNA was quantified at 595 nm by absorbance. Percentage of DNA fragmentation is expressed as (ODX2) X100 / (ODX2 of OD + supernatant of pellet) in B6 (F10) cells and is shown in Table 6 below for LOX-IMVI, MCF7 and HL- 60 is shown in Table 7 below. OD is the optical density of the sample.

density % of DNA fragmentation VH 9.43 ± 2.23 Camptothecin 0.1 μg / ml 8.39 ± 0.57 0.03 μg / ml 7.62 ± 0.07 0.1 μg / ml 7.92 ± 0.79 0.3 μg / ml 5.57 ± 2.15 1 μg / ml 4.75 ± 0.69 3 μg / ml 5.81 ± 3.61

density LOX-IMVI MCF7 HL-60 VH 5.15 ± 0.43 3.31 ± 0.74 7.46 ± 0.17 Camptothecin 0.3 μg / ml 9.59 ± 0.79 3.86 ± 1.13 82.98 ± 1.22 0.1 μg / ml 5.64 ± 0.63 4.96 ± 0.62 41.58 ± 11.42 0.3 μg / ml 6.79 ± 1.92 3.72 ± 0.86 43.59 ± 11.10 1 μg / ml 5.67 ± 0.75 3.63 ± 0.48 - 3 μg / ml 7.09 ± 0.31 5.14 ± 0.57 22.44 ± 1.94

Treatment of B16 (F10), LOX-IMVI, and MCF7 cells with COMR2029 and apoptosis-inducing camptothecin did not result in DNA cleavage in all experimental groups, but in HL60, DNA cleavage was observed in both sample treatment groups. This is because HL60 is more sensitive to cell death or DNA cutting than other cells. In conclusion, LOX-IMVI, which is sensitive to COMR2029, does not have DNA cleavage at concentrations that show strong cytotoxicity.

<6-3> Caspase-3 activity measurement

HL60 cells were incubated for 24 hours at a concentration of 2 × 10 ⁶ / ml, then transferred to 1.5 ml tubes and centrifuged in a minicentrifuge (13,000 rpm, 20 seconds). The lysis buffer contained in the Caspase fluorometric assay kit (R & D) was cooled and 25 μl per 1 × 10 ⁶ cells were placed in a refrigerated state (4 ° C.) for 10 minutes. 50 μl of cell lysate and 2 × reaction buffer were placed in a compartment of a fluorescence 96 well plate, 5 μl of the substrate was added per well, and the light was blocked, and the reaction was performed at 37 ° C. for 2 hours. Fluorescence was measured at 400/505 nm using a fluorometer, and is shown in Table 8 below.

density HL-60 + z-VAD. fmk 50 uM HL-60 VH 86.0 ± 12.9 VH 8.6 ± 2.3 Camptothecin 0.3 μg / ml 909.4 ± 96.7 Camptothecin 0.3 μg / ml 9.0 ± 0.8 0.3 μg / ml COMR2029 115.1 ± 6.7 0.3 μg / ml COMR2029 16.8 ± 3.7 0.1 μg / ml 115.5 ± 13.5 0.1 μg / ml 22.8 ± 6.6 0.03 μg / ml 90.4 ± 10.3 0.03 μg / ml 28.1 ± 8.4

Although the cell death occurred due to an increase in the activity of camptothecin caspase-3, which was used as a positive control, COMR2029 showed no significant difference compared to the solvent control and z-, a selective inhibitor of caspase-3. It was also confirmed that only camptothecin increased caspase-3 by VAD.fmk.

<6-4> Intracellular Ca Using Fura-2 / AM ²⁺ Change measurement test

The HL60 cells cultured in RPMI1640 medium were transferred to a 50 ml tube, centrifuged at 1,000 rpm for 10 minutes, and the cell concentration was adjusted to 1 × 10 ⁷ / ml with culture medium RPMI1640. When Fura-2 / AM enters the cell, it becomes [Fura-2] ^{2- and} has fluorescence wavelength that binds Ca ²⁺ and fluorescence wavelength that Ca ²⁺ does not bind. Fura-2 / AM was treated at a final concentration of 5 uM, labeled at 37 ° C. for 30 minutes with light blocking, and washed three times with cold PBS. The excitation wavelength of the fluorometer was adjusted to 1x10 ⁶ / ml using RPMI1640 without serum, and the excitation wavelength of [Fura-2] ^2- attached to Ca ²⁺ and [Fura-2] ^2- Match only two excitation wavelengths of 380 nm. 0.03, 0.1,0.3 μg / mL of the COMR2029 sample was added to observe the fluorescence change of fura-2 for 200 to 500 seconds. Fluorescence at the two wavelengths and their ratios were examined to determine the extent of Ca ²⁺ change.

In order to confirm the increase of intracellular Ca ²⁺ concentration which is a typical initial phenomenon of cell death, fluorescence assay using Fura-2 showed no significant change in free Ca ²⁺ concentration in cytoplasm at all concentrations of COMR2029. Did. As a result, it is determined that COMR2029 is not directly related to cell death-inducing ability ( FIG. 3 ).

Example 7 In Vitro Infiltration Analysis in vitr o invasion assay

After confirming selective inhibition of angiogenesis and cancer cell growth of COMR2029, the inventors performed an in vitro invasion assay as an experiment for metastasis of cancer cells. One of the characteristics that cancer cells differ from normal cells is that they are invasive and malignancy is generally proportional to the increase in invasiveness. Thus, in vitro invasion analysis can be used as a predictor of invasiveness that affects tumor tissue formation.

Swelling was performed by adding serum-free DMEM at room temperature for 120 minutes before using the chamber (BioCoat). The infiltration chamber from which the medium was removed after expansion was transferred to a 24-well plate dispensed with 600 μl / well of HT 1080 condition medium. 450 μl of B16 (F10) skin cancer cells (4.5 × 10 ⁴ cells) were dispensed into each chamber, and 50 μl of the sample solution was added at a concentration of 0.3, 1, 3 μg / ml. 450 μl of LOX-IMVI skin cancer cells (4.5 × 10 ⁴ cells) were dispensed and 50 μl of the sample solution was added at a concentration of 0.03, 0.1, 0.3 μg / ml. Plates were incubated at 37 ° C. for 24 hours. Infiltrated cells were fixed with MeOH and stained with 5% crystal violet. The stained filter portion was removed using a knife and transferred to a 24-well plate. Was filter rust 30% ahseteusanreul dispensed stained by 200 ㎕ part to the wells that contain, the solution exhibited to put in a 96-well by 100 ㎕ to measure each group OD value at 450 ㎚ micro reader Table 9 .

Concentration (μg / ml) LOX-IMVI Ratio (%) Concentration (μg / ml) B16 ratio (%) 0.03 22.9 ± 6.6 0.3 30.8 ± 20.0 0.1 33.0 ± 13.9 One 14.4 ± 3.3 0.3 18.0 ± 10.7 3 23.5 ± 9.0

From the above results, a large number of B16 cells are used as an experimental system to test the inhibitory ability to penetrate the membrane. The solvent control group not treated with COMR2029 has a large membrane permeation, and as a result, many cells are stained as shown in FIG. 4 . Drug treatment group treated with COMR2029 inhibited matrix permeation at all experimental concentrations, resulting in fewer cells compared to the control group. In the case of LOX-IMVI, which was sensitive to COMR2029, strong inhibitory activity was observed at concentrations 10 times lower than B16 (F10). As shown in Table 9, when the solvent control group was set to 100, the treatment group showed activity of about 10 to 30, and it was confirmed that COMR2029 had a strong tissue penetration inhibitory activity ( FIG. 5 ).

Example 8 LOX-IMVI Anticancer Test 1

Samples were prepared by adding 0.5% Tween 80 and the dose of COMR2029 sample was set to 1 mg / kg. As a solvent control group, 0.5% Tween 80 was used. The cell line was a human skin cancer (Melanoma) cell line LOX-IMVI and the cell concentration was adjusted to 3 × 10 ⁶ cells / mouse and implanted subcutaneously into the right armpit area of 0.2 ml per mouse. Sample administration started the next day. The solvent control group and COMR2029 were administered once a day for 1 to 2 days at a dose of 1 mg / kg, then 3 mg / kg for 3-4 days, 6 mg / kg for 5 days, and 10 mg / kg for 6-12 days. Intraperitoneal administration twice a day. Tumor size was measured by individuals and is shown in Table 10 , and the weight change of the animals was measured in the day intervals from the first day and is shown in Table 11 below.

3 days 4 days 5 days 6 days 7 days VH 38.0 ± 12.5 95.5 ± 16.1 154.7 ± 31.0 270.7 ± 59.4 550.9 ± 105.3 2029 20.5 ± 8.9 54.1 ± 27.1 86.8 ± 37.5 134.7 ± 42.9 287.5 ± 114.6

8 days 9 days 10 days 11 days 12 days VH 912.1 ± 206.8 1541.3 ± 426.8 1950.1 ± 364.8 2193.7 ± 506.3 2528.1 ± 649.5 2029 506.8 ± 217.8 784.7 ± 324.1 1142.8 ± 497.8 1548.8 ± 559.8 1733.0 ± 553.7

1 day 3 days 5 days 6 days 7 days VH 18.2 ± 1.18 17.9 ± 1.21 18.9 ± 1.52 19.2 ± 19.2 20.0 ± 1.66 2029 18.7 ± 1.24 18.3 ± 1.2 18.9 ± 1.52 19.2 ± 1.52 19.9 ± 1.40

8 days 9 days 10 days 11 days VH 20.8 ± 1.71 21.5 ± 1.66 21.7 ± 1.67 21.6 ± 1.75 2029 20.7 ± 1.71 21.2 ± 1.66 21.6 ± 1.67 21.8 ± 1.75

From the above results, human skin cancer, LOX-IMVI, showed very rapid growth, which could be measured from 3 days after transplantation, and showed a low growth of 53.9% when administered at 1 mg / kg. Thereafter, even if the dose was increased to 10 mg / kg or the frequency of administration was expanded twice a day, the cancer growth inhibition rate did not change. When the change in body weight was measured daily, no significant weight loss was observed in comparison with the solvent control group, and the dose increase or the frequency of administration did not cause toxicity ( FIGS. 6 and 7 ).

Example 9 LOX-IMVI Anticancer Test 2

Samples were prepared by adding 0.5% Tween 80 and the dosages of the COMR2029 samples were set to 10, 30 mg / kg. 0.5% Tween80 was used as the solvent control group and adriamycin (2 mg / kg) was used as the positive control group. The cell line was a human skin cancer (Melanoma) cell line LOX-IMVI and the cell concentration was adjusted to 3 × 10 ⁶ cells / mouse and implanted subcutaneously into the right armpit area of 0.3 ml per mouse. Sample administration started the next day. Samples of solvent control and COMR2029 were intraperitoneally administered twice a day for 1 to 14 days at doses of 10 and 30 mg / kg, and adriamycin, a positive control group, was administered intraperitoneally to 1 to 14 at doses of 2 mg / kg. It was. After 10 days of sample administration, the solvent was changed to DMSO 5% + Cremophor 20% + 0.85% Saline 75%. Tumor size was measured by individuals and is shown in Table 12 , and the weight change of the animals was measured in the day interval from the first day and is shown in Table 13 below.

5 days 6 days 7 days 8 days 9 days VH 50.3 ± 11.2 136.3 ± 43.1 277.9 ± 114.5 508.1 ± 155.1 901.6 ± 216.9 2029 10mg / kg 26.3 ± 6.4 73.7 ± 32.1 126.7 ± 40.9 308.7 ± 110.1 595.6 ± 196.4 30 mg / kg 26.9 ± 9.3 70.4 ± 31.4 156.6 ± 70.0 333.5 ± 107.7 575.4 ± 180.4 ADR 2mg / kg 21.8 ± 10.2 57.2 ± 58.8 108.5 ± 76.7 294.8 ± 129.8 558.3 ± 256.6

10 days 11 days 12 days 13 days 14 days VH 1491.9 ± 378.8 1894.1 ± 283.8 2821.6 ± 518.6 3459.4 ± 808.0 4107.8 ± 756.2 2029 10mg / kg 981.6 ± 336.3 1307.5 ± 374.3 1646.3 ± 350.0 1942.0 ± 309.8 2164.1 ± 355.2 30 mg / kg 903.4 ± 349.6 1236.7 ± 371.7 1434.7 ± 393.4 1859.5 ± 390.2 2213.6 ± 393.0 ADR 2mg / kg 814.3 ± 319.5 1126.3 ± 441.7 1329.3 ± 369.9 1491.6 ± 395.2 1737.6 ± 491.5

0 days 1 day 2 days 3 days 4 days VH 17.5 ± 0.93 17.5 ± 0.85 17.7 ± 0.89 18.7 ± 0.83 18.5 ± 0.98 202910 mg / kg 17.5 ± 0.94 17.1 ± 1.07 17.2 ± 0.95 18.3 ± 1.07 18.5 ± 1.1 30 mg / kg 17.5 ± 0.84 17.5 ± 0.91 17.3 ± 0.93 18.3 ± 0.97 18.4 ± 0.99 ADR 2mg / kg 17.5 ± 0.93 17.5 ± 0.88 17.3 ± 0.82 17.9 ± 0.92 18.2 ± 0.95

5 days 6 days 7 days 8 days 9 days VH 18.9 ± 0.82 19.6 ± 0.79 20.3 ± 1.26 20.5 ± 1.15 20.8 ± 1.13 2029 10mg / kg 18.7 ± 0.99 19.4 ± 0.98 20.2 ± 0.91 20.2 ± 1.10 21.1 ± 1.38 30 mg / kg 18.6 ± 1.06 19.1 ± 1.12 19.9 ± 1.27 20.0 ± 1.08 20.5 ± 1.18 ADR 2mg / kg 17.9 ± 0.88 18.5 ± 0.99 19.0 ± 0.89 19.1 ± 0.96 19.2 ± 0.98

10 days 11 days 12 days 13 days 14 days VH 21.3 ± 1.14 21.6 ± 1.43 22.6 ± 1.37 24.0 ± 1.60 25.5 ± 1.67 202910 mg / kg 21.7 ± 1.47 22.4 ± 1.44 23.1 ± 1.58 23.6 ± 1.89 25.1 ± 2.16 30 mg / kg 21.1 ± 1.40 21.3 ± 1.86 22.1 ± 1.54 23.3 ± 2.36 25.0 ± 2.36 ADR 2mg / kg 19.3 ± 1.00 19.6 ± 1.05 19.5 ± 1.16 19.7 ± 1.21 19.0 ± 1.33

From the above results, tumor growth inhibition was shown in the second experiment as in the first anticancer experiment. Treatment of samples 10 and 30 mg / kg showed tumor formation of 52.3% and 53.5%, respectively, after cancer cell transplantation and about 60% of tumor growth after 8 days. After 10 days, the effect was increased by changing the solvent, and after 12 days, the tumor formation was 50%. In terms of weight change, the solvent control group and the COMR2029-treated group showed no weight loss, but the positive control group showed a decrease of about 4 g. ( FIGS. 8 and 9 ).

Example 10 Immune Cell Proliferation Capacity Test

BDF1 mice of 20-25 g body weight were dislocated and the abdomen was disinfected with 70% alcohol. In the sterile state, the abdomen was dissected, the spleen was removed, placed in a Petri dish containing buffer (EBSS), and the splenocytes were released using the plunge of the syringe. The suspension containing the splenocytes thus obtained was transferred to a tube, left on ice for about 5-10 minutes, and the supernatant was collected and centrifuged (1,200 rpm, 10 minutes). Discard the supernatant and resuspend the precipitate in RPMI1640 culture solution [10% FCS (fetal calf serum), 2-mercatoethanol (2-ME)], measure the cell number and measure 96- at a concentration of 1 × 10 ⁶ cells / ml. Put 200 μl into the well plate. Con A was added at a concentration of 5 μg / ml for each well for the activity of T cells. Here, COMR2029 was dissolved in 100% DMSO and diluted in steps to adjust the concentration of the reagent to 0.01, 0.03, 0.1, 0.3, 1 μg / ml, and then incubated for 3 days at 37 ° C. in a CO ₂ incubator. 1 uCi of [ ³ H] thymidine was added per well 18 hours before the end of the incubation. Cells were collected using an automatic cell harvester and the degree of inhibition of immune cell proliferation was measured by measuring the degree of absorption of [ ³ H] thymidine, and is shown in Tables 14 and 15 below.

CPM No treatment group 1547 ± 234 Positive control group 155842 ± 15492 Solvent control 148121 ± 7566 Concentration (μg / ml) CPM 0.0001 142161 ± 8633 0.001 139485 ± 13542 0.01 158398 ± 6554 0.1 176731 ± 6280 One 53270 ± 19094

CPM No treatment group 251 ± 31 Positive control group 32223 ± 1105 Solvent control 32124 ± 2197 Concentration (μg / ml) CPM 0.01 24902 ± 1203 0.03 26099 ± 2209 0.1 25650 ± 2469 0.3 25984 ± 1448 One 32072 ± 2181 3 237 ± 21

Compared with the positive control and the solvent control, there is almost no change at other concentrations, but there is a change at high concentrations, which is a decrease due to cytotoxicity.

Example 11 Cytotoxicity Test after COMR2029 Pretreatment to LOX-IMVI

Anti-cancer activity against LOX-IMVI, a human skin cancer cell line, was investigated by COMR2029 by SRB method (SRB method, Skehan, et al., Proc. Am. Assoc. Cancer Res., 30: 612, 1989) It was. The cancer cell line was cultured using RPMI 1640 medium containing 10% fetal bovine serum, and the cultured cells were maintained by dispensing once or twice a week. Cell concentrations used for measuring anticancer activity were 3,000-6,000 cells / ml, and all reagents used in the above method were dissolved in 100% DMSO and diluted in steps to reduce the concentration of the reagents to 0.01, 0.03, 0.1, 0.3, 1 μg /. Adjusted to ml. A time zero (Tz) plate required to measure the number of cells, dispense them into 96-well plates at a constant concentration, and determine the basic absorbance of the cells after one day, another plate with the same cell concentration as the plate to be sampled. Was fixed using 50% TCA. The plate to be treated was divided into two groups, one group was treated for five sample concentrations after 24 hours, and the final concentration of the sample was adjusted to 0.1% DMSO. After 24 hours, the sample was processed again. Tz plate was washed with tap water after 1 hour, and the plate treated with sample was fixed with 50 μl of 50% TCA per well after 2 days, and also with tap water after 1 hour. Washed. The washed plates were dried at room temperature, then 100 μl of 0.4% SRB solution (solution dissolved in 1% acetic acid) was added per well, and then washed with 1% acetic acid solution after 30 minutes and dried again at room temperature. . Next 10 mM Tris base (pH 10.5) the redissolved added by 100 ㎕ per well, in the following by using the ELISA decoder measured at 540 ㎚ ELISA (enzyme-linked immunosorbent assay, ELISA) are shown in Table 16 It was. At this time, GI ₅₀ (μg / ml) indicates the concentration of the compound that inhibits the growth of cancer cells by 50%.

Concentration (μg / ml) Concurrent processing Pretreatment COMR 2029 ADR COMR 2029 ADR 0.01 95.8 ± 3.4 83.7 ± 2.0 94.1 ± 2.5 80.0 ± 2.0 0.03 77.2 ± 3.1 65.7 ± 1.8 72.4 ± 1.4 53.4 ± 0.7 0.1 13.0 ± 0.5 34.0 ± 2.2 8.9 ± 2.1 19.1 ± 1.5 0.3 -0.2 ± 0.9 0.3 ± 1.5 -5.1 ± 2.1 -12.4 ± 1.0 One -4.4 ± 1.0 6.1 ± 0.4 -13.1 ± 0.5 -24.4 ± 0.9 GI ₅₀ 0.0968 0.0704 0.0786 0.0398

There was little change in the experiment to see if the drug was removed after the initial exposure of the drug and if the re-exposure affected the drug sensitivity to the cells.

Example 12 Measurement of MMP-2 Activity

<12-1> Preparation of Standard Curves

The standard was purchased from Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH ₂ with a fluorescence substrate for MMP-2. Experiments were performed using a fluorescence absorbance spectrometer (Perkin-Elmer LS-50B). The computer controlled the machine using software (Fluorescence Data Manage, Perkin-Elmer). The excitation and emission wavelengths were 325 nm (slit 5.0 mm) and 420 nm (slit 3.0 mm), respectively. All experiments were performed with 20 mM Tris-HCl, 5 mM CaCl ₂ and 0.15 M NaCl (reaction buffer, pH 7.5). It was done by.

Mca-Pro-Leu-Gly (1 mM), a standard, was dissolved in DMSO to obtain a standard curve. This solution was diluted directly on microplates in the range of 15.6 to 250.0 pmoles using reaction buffer. Using the above results, we created a standard curve.

<12-2> Measurement of MMP-2 Activity

The measurement of MMP-2 activity was measured by several modifications using a method such as knight. Briefly, 1 nM proMMP-2 without TIMP-2 was incubated with 0.5 mM APMA at 37 ° C. for 15 minutes. Test samples and substrates (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH ₂ ; 10 uM) were added to the reaction mixture. 30 minutes were further performed after the incubation. The reaction was stopped by adding 0.1 N sodium acetate buffer (pH 4.0). Hydrolysis of the substrate was performed using a fluorescence absorbance spectrometer (Perkin-Elmer LS-50B). The excitation wavelengths were 325 nm (slit 5.0 mm) and 420 nm (slit 3.0 mm), respectively. IC ₅₀ values were determined by plotting the remaining enzyme activity and sample concentration. The results are shown in Table 17 below.

uM F0 F1 F1-F0 ratio(%) Solvent control 36.9 295.5 258.6 250.9 36.1 283.4 247.3 33.2 281.0 247.8 34.8 284.5 249.7 0.03 35.8 278.3 242.5 96.7 0.1 35.9 276.2 240.3 95.8 0.3 35.1 269.5 234.4 93.4 One 35.0 266.6 231.6 92.3 3 32.1 257.2 225.1 89.7 10 32.3 240.2 207.9 82.9 30 25.9 195.2 169.3 67.5 100 16.6 94.6 78.0 31.1

Matrix MetalloProteinase (MMP) is an enzyme that is involved in the metastasis of cancer cells. It is an experiment that measures the activity of MMP-2, the most representative and sensitive enzyme of which is the principle. In comparison, the activity was measured. When COMR2029 samples were added at 0.03, 0.1, 0.3, 1, 3, 10 uM, no change in the enzyme activity of MMP-2 was observed and the enzyme activity was slightly inhibited from 3 uM. This is believed to be due to color quenching because the drug shows color. The results suggest that COMR2029 does not affect the inhibitory activity of histolytic enzymes such as MMP-2.

Example 13 Oral Administration of Rats Acute Toxicity Test

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals per group were suspended orally administered once with a dose of 1 g / kg / ml in each of the COMR2029 of the present invention suspended in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, all of COMR2029 of the present invention did not show toxicity change even up to 1,000 mg / kg in rats.

As described above, the angiogenesis inhibitor containing 6-anilinoquinoline-5,8-quinone (6-anilinoquinoline-5,8-quinone, COMR2029) of the present invention as an active ingredient is cancer, rheumatism or diabetic retinopathy It can be useful for diseases related to angiogenesis such as diabetic retinopathy.

Claims (2)

An angiogenesis inhibitor containing 6-anilinoquinoline-5,8-quinone (6-anilinoquinoline-5,8-quinone) having a structure of Formula 1 as an active ingredient. <Formula 1> 2. The angiogenesis inhibitor according to claim 1, wherein said angiogenesis inhibitor is used for the prevention or treatment of cancer, rheumatoid or diabetic retinopathy.