WO2004098601A1 - As a pkc inhibitor, inhibitor for angiogenesis containing 6-anilinoquinoline-5,8-quinone as an effective ingredient - Google Patents

Abstract

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

Description

AS A PKC INHIBITOR, INHIBITOR FOR

ANGIOGENESIS CONTAINING 6-

ANI INOQUINOLINE-5,8-QUINONE AS AN

EFFECTIVE INGREDIENT

FIELD OF THE INVENTION

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

BACKGROUND

In order to treat cancer, one of the diseases hard to cure, various anticancer agents have been developed and used clinically. However, conventional anticancer agents do not work particularly to a specific target, causing serious side effects and the generation of resistant cells because of heterogeneity and genetic instability of cancer cells. Moreover, the conventional anticancer agents are not appropriate for the treatment of most solid cancers. Thus, efforts have been made world-widely to solve the said problems by taking advantage of advanced cancer cell biology. One of the approaches to treat cancer based on cancer cell biology is inhibiting angiogenesis .

Solid tumor is supplied with growth factors, oxygen and nutrients by diffusion until it grows 1 —2 mm³ and, from then on, it should be supplied with them by new blood vessels for further growth. Thus, the growth of cancer can be successfully inhibited by blocking angiogenesis that is necessary for tumorigenesis and metastasis. PKC inhibitor suppresses angiogenesis caused by an angiogenesis inducer, VEGF.

PKC is a kind of serine/threonine kinases and is activated most in the presence of calcium ion and DAG (Diacylglycerol) . At least 10 classes of PKC have been found so far and their formation depends specifically on intracellular expression and activation mediated by lipid and calcium. PKC is detected during the process of signal transduction by hormones, growth factors and neurotransmitters . PKC participates in phosphorylation of various proteins, meaning that PKC regulates the protein activity. On the special occasion, phosphorylation of EGF receptor caused by PKC has a function of down-regulation of the activity of tyrosine kinase of the receptor. PKC family is divided into three major subgroups. The first subgroup is classical PKC that PKC α , β I , β II and Y belongs to. The members of the classical PKC are Ca²⁺/lipid-dependent . The second group is novel PKC exemplified by PKC δ , ε , rι and θ. Even though the members of the second group are Ca²⁺- independent, they need lipid as they are activated. The third group is atypical PKC that is not Ca²⁺, lipid-dependent and PKC \ and λ belongs to. PKC μ seems similar to the members of novel PKC, but it is classified into another group since it has membrane-spanning domain. PKC is activated by various agonists, growth factors, antigens, cytokines and neurotransmitters . Especially, phorbol ester like TPA directly stimulates classical and novel PKC. PKC plays an important role in intracellular actions such as secretion, differentiation, tumorigenesis, mitogenesis, signal transduction, gene expression and cytoskeletal regulation. In addition, PKC has continuously been reported to be important for integrin-mediated adhesion and signaling event. For the first example, adhesion, spreading and cell migration in extracellular matrix increase as TPA and some of other cellular forms are treated with PKC. On the contrary, when PKC is suppressed, cell adhesion, cell spreading, cell migration, FAK phosphorylation and focal adhesion formation are all inhibited. Secondary, several PKC isoforms are proved to be included in adhesion-dependent event. PKC α and PKC δ relate to focal adhesion. PKC α and PKC ε relate to integrin activation and locate on membrane. Thirdly, as for the integrin engagement, PLC activity, diacylglycerol level and arachidonic acid production increase as PKC is activated.

PKC inhibitor inhibits the actions related to integrin and indirectly affects various diseases and pathological condition by working on integrin receptor. Integrin receptor antagonist is believed to be a good candidate for many useful medicines. Integrin receptor is a heterodimer transmembrane receptor where cells are attached and communicate with an extracellular matrix and other cells .

As a integrin, a_vβ 3 ligand can be effectively used for the treatment and/or the suppression of recurrent stenosis (recurrence of stenosis after correctional operation of valvular disease), arteriosclerosis, diabetic retinopathy, degeneration of yellow spot, angiogenesis

(forming new blood vessels) and other viral diseases. The growth of tumor relies on the proper supply of blood and the supply of blood depends on the growth of newly formed blood vessels in the tumor; Thus, the inhibition of angiogenesis might bring about regression of tumor in animal models. Therefore, a_vβ₃ antagonist inhibiting angiogenesis can be effectively used for the treatment of cancers by suppressing the growth of tumors.

It has also been disclosed that angiogenesis is a major cause for the development and the progress of arthritis and integrin a_vβ₃ is the primary target of inflammatory arthritis. Thus, a new approach to treat arthritis such as rheumatoid arthritis can be made by a_vβ₃ antagonist inhibiting angiogenesis. The antagonist is able to inhibit angiogenesis by working as an antagonist a_vβs, a integrin receptor. The monoclonal antibody against a_vβ₅ has been confirmed to inhibit VEGF-induced angiogenesis in cornea of rabbit and chorion of chick. Therefore, it is for sure that the compounds antagonizing a_vβ₅ can be successfully used for the treatment and the prevention of degeneration of yellow spot, diabetic retinopathy, viral diseases, cancers, and metastatic tumor growth .

As of today, studies on angiogenesis (or neovascularization) have actively been made, resulting in the disclosure of angiogenesis factors such as VEGF (vascular endothelial growth factor) , bFGF (basic fibroblast growth factor) , HGF (hepatocyte growth factor) , EGF (epithelial growth factor), angiopoietin, etc., receptors of angiogenesis factor having tyrosine kinase activity such as FGFR (fibroblast growth factor receptor), Flk-1/KDR, Flt-1, Flt-3, Tie-1, Tie-

2/Tek, Eph, etc., and angiogenesis inhibitors such as angiostatin and endostatin. Owing to the disclosure, the mechanism of angiogenesis and the relation between angiogenesis and human diseases were more clearly explained, and further, various novel attempts to regulate angiogenesis have been made. Among many angiogenesis factors, VEGF draws a special attention since VEGF seems to be closely related to the extent of tumor progression and treatment in most cases. Besides, its receptor Flk-1/KDR is expressed in endothelial cells of blood vessels specifically, making VEGF a useful candidate for the development of a specific angiogenesis inhibitor.

In regards to the relation between cancer and angiogenesis, the hypothesis that angiogenesis is indispensable for tumor growth was first reported by Dr. Folkman (USA) in 1971 (J. Folkman, Tumor Angiogenesis: Therapeutic Implications. New England Journal of Medi cine, 285, 1182-1186, 1971) . After the report in 1990 that fumagillin extracted from a natural substance inhibits angiogenesis, angiogenesis inhibitor began to draw an attention as an anticancer agent (D. Ingber, T. Fujita, et al : Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Na ture, 348, 555-557, 1990). In addition, angiostatin (M. S. O'Reilly, L. Holmgren, Y. Shing, C. Chen, R. A. Rosenthal, M. Moses, W. S. Lane, Y. Cao, E. H. 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) and endostatin (M. S. O'Reilly, T. Boehm, C. Chen, et al . , Endostatin: an endogeneous inhibitor of angiogenesis and tumor growth. Cell , 88, 277-285 , 1997), recently found intra angiogenesis inhibitors, were proved to be very effective anticancer agents, which was confirmed by test animal model, and contributed to the development of angiogenesis inhibitors as anticancer agents.

As an anticancer agent, an angiogenesis inhibitor has more benefits than conventional anticancer agents. Particularly, since angiogenesis is indispensable for tumorigenesis or metastasis, inhibition of angiogenesis results in the prevention of both tumorigenesis and metastasis. Besides, since angiogenesis inhibitor targets not aneuploid cancer cells but normal diploid endothelial cells of blood vessels, the inhibitor does not cause side effects such as resistance induced by heterogeneity and genetic instability of cancer cells. The conventional anticancer agents only work against specific or some kinds of cancers. On the contrary, angiogenesis inhibitors affect every kinds of cancer requiring angiogenesis. Angiogenesis is hardly occurring but some special occasions such as wound healing, menstruation, etc. Thus, using angiogenesis inhibitors as anticancer agents might be very effective to treat cancers with less side effects . Angiogenesis has also been known to induce angiogenic diseases like rheumatoid arthritis, diabetic retinopathy, etc., in addition to tumor growth and metastasis. Therefore, angiogenesis inhibitors are expected to be effectively used for the treatment and the prevention of angiogenic diseases along with cancers.

The present inventors have worked hard on finding a novel angiogenesis inhibitor and finally found the fact that 6-anilinoquinoline-5, 8-quinone has a function to inhibit angiogenesis without cytotoxicity . Thus, the present inventors have accomplished this invention by confirming that the said 6-anilinoquinoline-5 , 8-quinone can be effectively used for the treatment or the prevention of angiogenesis-related diseases

SUMMARY OF THE INVENTION

It is an object of this invention to provide an agent for the prevention or the treatment of angiogenesis-related diseases containing 6- anilinoquinoline-5, 8-quinone as an effective ingredient .

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

FIG. 1 is a set of photographs showing the inhibition of angiogenesis by COMR2029 after hypodermically injecting matrigel in mouse abdomen;

FIG. 2 is a set of electrophoreses photographs showing the apoptosis-inducing capability of COMR2029, which was analyzed by agarose gel electrophoresis; A : B16(F10), Lane 1 and 8 :

Marker,

Lane 2 and 9 : Solvent control (VH) ,

Lane 3 : COMR2029 0.03 βg/ml , Lane 4 : COMR2029 0.1 βg/ml,

Lane 5 : COMR2029 0.3 βg/ml,

Lane 6 : COMR2029 1 βg/ml,

Lane 7 : COMR2029 3 μg/ml,

Lane 10: Camptothecin 0.1 βg/ml, Lane 11: Camptothecin 0.1 βg/ml + COMR2029

Lane 12: Camptothecin 0.1 βg/ml + COMR2029 0.1

Lane 13: Camptothecin 0.1 βg/ml + COMR2029 0.3

Lane 14: Camptothecin 0.1 βg/ml + COMR2029 1

Lane 15: Camptothecin 0.1 βg/ml + COMR2029 3

B : HL60, Lane 1 :

Marker,

Lane 2 Solvent control (VH) , Lane 3 Camptothecin 0.3 βg/ml, Lane 4 COMR2029 0.1 μg/ml, Lane 5 COMR2029 0.3 βg/ml, Lane 6 : COMR2029 1 βg/ml,

FIG. 3 is a graph showing the changes of intracellular Ca²⁺ concentration by treating COMR2029, resulted from the analysis using Fura- 2 /AM;

FIG. 4 is a set of photographs showing the inhibition of invasion after treating Blβ (F10) cells with 3 βg/ml, 1 βg/ml or 0.3 βg/ml of COMR2029 respectively;

FIG. 5 is a set of photographs showing the inhibition of invasion after treating LOX-IMVI cells with 0.03 βg/ml , 0.1 βg/ml or 0.3 βg/ml of COMR2029 respectively;

FIG. 6 is a graph showing the tumor volumes of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029 or solvent control (VH) ;

FIG. 7 is a graph showing the body weights of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029 or solvent control (VH) ;

FIG. 8 is a graph showing the tumor volumes of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029, positive control (adriamycin) or solvent control

(VH) ;

FIG. 9 is a graph showing the body weights of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029, positive control (adriamycin) or solvent control (VH) ;

FIG. 10 is an electrophoresis photograph showing the inhibition activity of COMR2029 to phosphorylation of MAPK (ERK 1/2);

FIG. 11a - FIG. lie are electrophoresis photographs showing the inhibition activity of COMR2029 to transcription regulating elements, in which each test results were obtained using LOX- IMVI (FIG. 11a), B16 (F10) (FIG. lib) and HUVEC (FIG. lie) cells; FIG. lid is a set of electrophoresis photographs showing the result of confirmation test for FIG. lla-Fig. lie, in which the inhibition activity of COMR2029 to phosphorylation of AP-1 and NF-kB in LOX-IMVI is revealed;

FIG. 12 is a set of graphs showing the effect of COMR2029 on PKC activity;

FIG. 13 is a graph showing the effect of

COMR2029 on sGC activity;

FIG. 14 is a graph showing the effect of COMR2029 on uPA activity as time passes;

FIG. 15 is a diagram showing the mechanism of uPA protein working on cell migration and invasion,

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides an agent for the prevention or the treatment of angiogenesis- related diseases containing 6-anilinoquinoline- 5, 8-quinone (referred "COMR2029" hereinafter) as an effective ingredient. Further features of the present invention will appear hereinafter.

The present invention provides an agent for the prevention or the treatment of angiogenesis- related diseases containing COMR2029 as an effective ingredient.

The structure of COMR2029 of the present invention is presented in the below <Chemical Formula 1>.

<Chemical Formula 1>

The PKC inhibitor of the present invention that inhibits angiogenesis induced by angiogenic factors can be used for the prevention or the treatment of a disease selected from a group consisting of diabetic retinopathy, macular degeneration, retinopathy of immature infants, angiogenic glaucoma, arthritis and solid tumors, but not always limited thereto (D'Amato, R.J. and Adamis, A, P. (1995) Ophthalmol . , 102:1261-1262; Arbiser, J.L. (1996) J. Am . Acad . Derm . , 34 (3) :486-497; O'Brien, K.D. et al . (1996) Circula tion , 93 (4 ): 672-682; Hanahan, D. and Folkman, (1996) Cell , 86:353-364).

In order to investigate whether COMR2029 had the inhibiting activity to angiogenesis, the present inventors performed tube formation assay using HUVEC cells that were able to make tube form in matrix. As a result, COMR2029 inhibited tube formation by HUVEC cells even at the low concentration. As for the cytotoxicity of COMR2029 against HUVEC cells, COMR 2029 did not show cytotoxicity against HUVEC cells except when the cells were treated with high concentration of COMR2029. Even at that time, minimum cytotoxicity was observed (see Table 1 and Table 2) . In order to measure the angiogenesis inhibiting capacity of COMR2029, the present inventors hypodermically injected matrigel containing FGF in mouse abdomen and performed in vivo angiogenesis assay. As a result, solvent control group, in which COMR2029 was not injected, turned red owing to the active angiogenesis, while mice, in which COMR2029 was injected, colored yellow since angiogenesis was hardly occurring

(see FIG. 1) . Therefore, COMR2029 was confirmed to inhibit angiogenesis.

In addition, COMR2029 showed cytotoxicity against almost all the tumor cell lines and especially, very strong cytotoxicity against skin cancer cell line (see Table 3) . Even it's strong cytotoxicity against skin cancer cell line, COMR2029 did not affect cell cycle, apoptosis, caspase-3 activity that was related to apoptosis, and intracellular Ca²⁺ concentration (see FIG. 2 and FIG. 3) . In order to investigate the ability of COMR2029 to inhibit membrane transmission, the present inventors performed in vi tro invasion assay. As a result, COMR 2029 functioned to inhibit membrane transmission in B16(F10) cells that have been widely used for the experiments testing the ability to inhibit membrane transmission in general (see FIG. 4) . In LOX-IMVI cells that have not been commonly used for invasion assay, COMR2029 also inhibited membrane transmission dose-dependently (see FIG. 5). In order to investigate the anticancer activity of COMR2029, the present inventors hypodermically implanted LOX-IMVI cells, a human skin cancer cell line, in a nude mouse and then injected COMR 2029 into the peritoneal cavity of the nude mouse. Resultingly, the weight of the mouse did not change but the tumor size became smaller (see FIG. 6 and FIG. 7) . Instead of COMR2029, injected adriamycin, a sort of anticancer agent, in positive control and compared the tumor sizes between the two groups. Resultingly, the extent of diminution of tumor size was not very different. Thus, COMR2029 was confirmed to have strong anticancer activity (see FIG. 8 and FIG. 9) .

The present inventors also investigated the effect of COMR2029 on MAPK regulation. As a result, the inventors confirmed that COMR2029 dose-dependently inhibited the activity of ERKl/2 among MAPKs, intracellular transmitters (see FIG.

10) . Once a stimulus by a growth factor like EGF is given, Erk is activated and cell division begins. However, in aged cells, caveolin protein on cell membrane inhibits the activity of growth factor receptor, resulting in the interference of signal transduction. A signal transduction system by calcium is one of many important signal transduction systems. Calcium induces the activation of transcription factors related to gene expression, which is regulated by various signal transduction enzyme system and those enzymes are exemplified by PKC (protein kinase C) , CaM kinase (calcium/calmodulin dependent protein kinase) and ERK (extracellular signal regulated kinase) , a kind of MAP (mitogen-activated protein) kinase. Recently reported ERK has been known to be the most important enzyme for the expression of a gene relating to long-term memory. Activated ERK moves into nucleus and activates transcription factors directly or do indirectly by activating other enzyme systems. COMR2029 of the present invention inhibits the expression of ERK, meaning that one of the signal transduction systems is blocked and the activation of transcription factor and gene expression are all affected. In addition, the ERK protein is the last of signal transduction system Ras-Raf-MEK-ERK, a signal transduction system of angiogenesis inhibitor. The present inventors also investigated the effect of COMR2029 on the expressions of various transcription regulating factors with 4 transcription regulating factors in 3 cell lines. As a result, the expressions of CREB and Octamer did not change, while the expressions of AP-1 and NF-kB varied (see FIG. 11a - FIG. lie) . To reconfirm the above result, the present inventors carried out the same experiment again to detect the changes of AP-1 and NF-kB using LOX-IMVI cells.

As a result, the expression of NF-kB was not changed, but the expression of AP-1 was decreased

(FIG lid) . From the results of RT-PCR, it was also confirmed that p21 and uPA were decreased just like AP-1, suggesting that AP-1 binding sites were located in promoter region, and thus COMR2029 of the present invention related to angiogenesis inhibitors .

The present inventors further investigated the effect of COMR2029 on the intracellular activity of PKC (protein kinase C) . As a result,

COMR2029 inhibited the PKC activity (see FIG. 12) .

PKC is a kind of serine/threonine kinases and is activated most in the presence of calcium ion and DAG (Diacylglycerol) . At least 10 kinds of PKC have been known and they change their forms specifically according to the level of their expression and activation caused by lipid and calcium in tissues. PKC can be observed during the signal transduction process induced by hormones, growth factors and neurotransmitters . PKC induces phosphorylation of various proteins, resulting in the variation of protein activity. On special occasions, phosphorylation of EGF receptor induced by PKC functions to down-regulate the activity of tyrosine kinase of the receptor. As seen in FIG. 12, COMR2029, along with MAPK and transcription regulating factor, inhibits PKC activity, which is the same result as others.

The present inventors also investigated the effect of COMR2029 on the activity of sGC (soluble guanynyl cyclase) and uPA. As a result, COMR2029 inhibited the activity of sGC as LY83583 that was known as a structural analogue of sGC and sGC inhibitor did (see FIG. 13) . COMR2029 also inhibited the activity of uPA little (see FIG. 14)

UPA is an important enzyme relating to migration and invasion as shown in FIG. 15. The angiogenesis inhibitor of the present invention for the prevention or the treatment of the angiogenesis-related diseases contains the above COMR2029 as an effective ingredient. The above COMR2029 can be administered orally or parenterally and be used in general form of pharmaceutical formulation. The COMR2029 of the present invention can be prepared for oral or parenteral administration by mixing with generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients. Solid formulations for oral administration are tablets, pill, dusting powders and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the abovementioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, and suppositories. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerinated gelatin, etc.

The effective dosage of COMR2029 of the present invention is 10 mg/kg and its estimated LD50 value is much greater than 1000 mg/kg, which are confirmed by acute toxicity assay with rats tested via oral administration.

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1 : Tube formation assay

In order to investigate whether COMR2029 could inhibit tube formation in cells, the present inventors performed tube formation assay using HUVEC (Human Umbilical Vein Endothelial Cell) cells characterized by forming tube shape in matrix .

Every experimental instruments were kept in cool condition (4°C) all the time. Matrigel was distributed to 96-well plate by 40 μl and polymerized in C0₂ incubator at 37 ^°C for 30 minutes. In that experiment, matrigel was to provide matrix that was necessary for tube formation, that is, angiogenesis by HUVEC cells. HUVEC cells cultured in EGM-2 medium were distributed into wells of a plate by 180 βl with the concentration of 1.1X10⁵ cells/m^. The wells of the plate were treated with COMR2029 by 0.03, 0.1, 0.3, 1, and 3 βg/ml respectively and were cultured in C0₂ incubator at 37 ^°C for 18-24 hours. After cultivation, microphotographs were taken for detection . As a result, as a solvent control, VH formed more than 10 tubes. In the meantime, tube formation was not observed in the wells treated with 3 βg/ml and 1 βg/ml of COMR 2029 each. Just a line to form a tube was seen dimly from the concentration of 0.3 βg/ml, yet the tube was not formed until the concentration reached 0.1 βg/ml . The tube began to be formed from the concentration

From the above results, it was confirmed that COMR2029 inhibited the tube formation of HUVEC cells even with the low concentration.

Example 2: Measurement of cytotoxicity against

HUVEC cells

The present inventors investigated if COMR2029 had cytotoxicity against HUVEC cells, a human umbilical vein endothelial cell line, with cell growth inhibition test using SRB (SRB method, Skehan, et al., Proc . Am . Assoc . Cancer Res . , 30: 612, 1989) . The above cell line was cultured in EGM-2 medium. COMR2029 was dissolved in 100% dimethylsulfoxide (referred "DMSO" hereinafter) and was diluted consecutively to adjust the concentration to 0.01, 0.03, 0.1, 0.3, 1 βg/ml respectively. Cells were counted and then distributed into wells of 96-well plate by 5X10⁴, 8 10⁴, 10X10⁴ cells /ml each. On the next day, Tz (Time Zero) plate was fixed using 50% TCA to determine the cell concentration before cells were treated with reagents. The plate that would be treated with COMR2029 was divided into two groups. One group was left for 24 hours, and then treated with 5 different concentrations of COMR2029 that were adjusted by 0.1% DMSO. The other group was treated with COMR2029 right away and was transferred into a fresh medium two hours later, which was treated with COMR2029 again after 24 hours. Tz plate was washed with tap water after an hour. A plate treated with reagents was fixed by treating 50% TCA (50 β /well ) after two days, which was washed with tap water 1 hour later. The washed plate was dried at room temperature. Then, 0.4% SRB solution (dissolved in 1% acetic acid) was added by 100 μl per well. After 30 minutes since then, the plate was washed with 1% acetic acid solution, which was dried at room temperature. 10 mM tris base (pH 10.5) was added into each well by 100 μ . Finally, optical ^'density was measured at 540 nm using a microplate reader. The group treated with COMR2029 24 hours later was presented in the below Table 1 and the other group treated with COMR2029 right away, transferred into a fresh medium two hours later and treated with COMR2029 again after 24 hours, was presented in the below Table 2.

<Table 1>

<Table 2>

Concentratio 5X10⁴ 8X10⁴ 10X10⁴ n (βg/ml ) cells/m£ cells/mβ cells /ml

As a result of the cytotoxicity analysis on COMR2029 done by treating HUVEC cells with different concentrations of COMR2029, the present inventors confirmed that cell growth was not inhibited by COMR2029 with and below 0.3 βg/ml, but was remarkably inhibited from 1 βg/ml .

Example 3: Analysis on matrigel plug angiogenesis

In order to investigate the capacity of COMR2029 to inhibit angiogenesis, the present inventors performed in vivo angiogenesis assay after hypodermically injecting matrigel in mice.

Heparin was diluted in PBS containing 1 mg/ml of albumin, which was added to matrigel with the final concentration of 64 unit/m-€. and aFGF was adjusted to 1 ng/ml . The prepared matrigel was injected subcutaneously by 0.5 ml into the abdominal regions of 7 week old female C57BL/6 mice. COMR2029 was prepared as follows; COMR2029 5%, Cremophor 20% and 0.85% saline 75%. COMR2029 was administered into the abdominal cavity of the mice with the concentration of 10 mg/kg everyday from day 1 to day 8. Cremophor solution without COMR2029 was administered for the solvent control. On the 9^th day after implanting matrigel under the skin of the mice, the mice were sacrificed for examination and photographs were taken.

As a result, mice of solvent control not treated withCOMR2029 turned red by angiogenesis, while mice treated with COMR2029 colored yellow since angiogenesis was hardly occurred (FIG. 1) . Therefore, it was confirmed that COMR2029 functioned to inhibit angiogenesis.

Example 4 : Measurement of cytotoxicity against

cancer cell lines

In order to investigate whether COMR2029 had organ specific or cell line specific cytotoxicity to cancer cell lines originated from different organs, the present inventors detected out the anticancer activity of COMR2029 with SRB method (Skehan, et al . , Proc . Am . Assoc . Cancer Res . , 30: 612, 1989) . For the experiment, the present inventors used leukemia cell lines such as MOLT-4F, RPMI8226, HL60, P388/ADR, P388, K562/ADR, K562/VIN and K562, human melanoma cell lines such as LOX- IMVI, SK-MEL-28, SK-MEL-5, UACC62, Hs 294T, Hs 695T, SK-MEL-2, RPMI7951, A253, G361, M14 and HT1080, a mouse melanoma cell line, B16(F10), a renal cancer cell line, UO-31, colon cancer cell lines such as Colo205, HCT116, HCC2998, HCT15 and SW620, lung cancer cell lines such as A549, NC1- H522 and NC1-H23, a CNS cancer cell line, SNB19, an ovarian cancer cell line, SK-OV-3, a prostate cancer cell line, PC-3, breast cancer cell lines such as Hs 578T, MCF7/ADR and MCF7. All the cell lines used for this invention were distributed from NCI and ATCC, USA and maintained at Korea Research Institute of Bioscience and Biotechnology. The cancer cell lines were cultured in RPMI

1640 medium supplemented with 10% FBS and the culture cells were adjusted to be in exponential growing phase. Cell concentration was adjusted to 4X10⁴ - 10X10⁴ cells/m£ on RPMI 1640 medium supplemented with 5% FBS based on cell growth rate, which was then distributed into wells of 96-well plate. On the next day, Tz (Time Zero) plate was fixed by using 50% TCA to determine cell concentration before the plate would be treated with reagents.

COMR2029 was dissolved in 100% DMSO and was diluted consecutively to adjust the concentration to 0.01, 0.03, 0.1, 0.3 and 1 βg/ml respectively. The final concentration of sample in the plate that would be treated with COMR2029 was also adjusted to 5 different concentrations using 0.1% DMSO. Tz plate was washed with Tap water one hour later and two days later, the plate where COMR2029 was treated was fixed by treating 50 βl of 50% TCA per well, which was washed with Tap water after an hour had passed. The washed plate was dried at room temperature. 0.4% SRB solution (dissolved in 1% acetic acid) was added into each well by 100 βl , which was washed with 1% acetic acid solution after 30 minutes. The plate was dried at room temperature, again. 10 mM Tris-base (pH 10.5) was added into each well by 100 βl and dissolved.

Finally, optical density was measured at 540 nm using a microplate reader. And then, cytotoxicity against each cell line was presented in Table 3. In the Table 3, GI₅₀ (βg/ml ) means the concentration of a compound inhibiting cancer cell growth up to 50%.

<Table 3>

As shown in Table 3, COMR2029 responded sensitivity to K562, K562/VIN and K562/ADR among 8 kinds of blood cancer cell lines but did not to P388 and P388/ADR, showing over 1.0 βg/ml in GI₅₀ value. COMR2029 showed 0.3 βg/ml in GI₅₀ value for kidney cancer cell line UO-31 or central nervous system cancer cell line SNB19, 0.2 βg/ml for colon cancer cell lines, HCT15 and HCT116 and 0.5 βg/ml for Colo205, HCT15 and SW620. COMR2029 showed low cytotoxicity to ovarian cancer cell line SK-OV-3, while did comparatively high cytotoxicity to breast cancer cell line or prostate cancer cell line (about 0.2 βg/ml of GI₅₀) . In regard to 13 skin cancer cell lines, GI50 value was under 0.1 βg /ml in 9 of those 13 cell lines, SK-MEL-5, UACC62, Hs 294T, Hs 695T, SK-MEL-2, RPMI7951, A253 and G361, suggesting that COMR2029 had strong cell growth inhibiting activity. Especially, GI50 value in LOX-IMVI was under 0.03 βg/ml, which meant the strongest cytotoxicity was observed in LOX-IMVI. In SK-MEL-28, M14, HT1080 and B16, the cell growth inhibiting effect was not detected with all different concentrations except 1 βg/ml, the highest concentration where minor cell growth inhibiting activity was observed. GI₅₀ value in lung cancer cell lines NCI-H522 and NCI-H23 was about 0.1 μg/ml . Resultingly, COMR2029 worked selectively to skin cancer

Example 5: Experiment for measuring cell cycle

As seen in Example 4, the present inventors confirmed that COMR2029 had skin cancer cell line- specific cytotoxicity, and performed experiment for measuring cell cycle to investigate whether COMR2029 affected cell cycle.

Particularly, melanoma cell line LOX-IMVI was cultured in RPMI 1640 medium supplemented with 10%

FBS. The required COMR2029 was dissolved in 100%

DMSO, which was diluted serially to prepare five different concentrations (0.0003, 0.001, 0.003,

0.01, 0.003 βg/ml each). Cells were distributed into T-75 flask by the density of 6X10⁶ cells/mi, which was then treated with COMR2029. On the next day, the cells were collected by centrifugation

(1,000 rpm, 10 min.), and then fixed with 70% ethanol. After centrifuged again, the cells were left in a 37 ^°C water bath containing 400 μl of PBS,

1 μg/ml of RNAase and 400 μg/ml of PI for 30 minutes,

Then, the cells were measured with FACS

(Fluorescence-activated cell sorter) . After 48 hours passed, the experiment was repeated. The cell cycles on 24 hours after being treated with COMR2029, which were confirmed by FACS measurement, were represented in Table 4 and the cell cycles on 48 hours after being treated with COMR2029 were represented in Table 5.

<Table 4>

<Table 5>

As a result, it was confirmed that the

COMR2029 treatment on the skin cancer cell line did not affect the ratio of cell cycle phases,

GO/Gl phase, S phase and G2/M phase, both 24 and 48 hours after the treatment.

Example 6: Experiment for measuring apoptosis

In order to measure the capacity to induce apoptosis of COMR2029, the present inventors took advantage of several methods such as agarose gel electrophoresis, quantification of intracellular DNA, caspase-3 activity measurement and the measurement of intracellular calcium change.

A typical phenomenon of apoptosis, regular cut of DNA, can be seen as ladder type on agarose gel . Concerning diphenylamine method, diphenylamine infiltrates in-between DNA and dyes dark blue over the apoptosis region. Caspase-3 is required for the last step of apoptosis, that is, almost every cell needs this enzyme to be progressed to apoptosis. Therefore, when apoptosis is occurring, DAN is seen as ladder type on agarose gel, cells are stained dark blue by diphenylamine, resulting in the high value of optical density and the activity of caspase-3 increases .

<6-l> Agarose gel electrophoresis Skin cancer cell line B16(F10) and human blood cancer cell line HL60 were distributed into 12-well plate by 2xl0⁶ cells/m-β, which was treated with COMR2029 in different concentrations such as 0.03, 0.1, 0.3, 1 and 3 βg/ml respectively. The cells were cultured for 24 hours and then transferred in 1.5 ml tubes. Centrifugation was performed using mini-centrifuge (1300 rpm, 20 sec.) . Cell precipitates were dissolved in 500 βl of lysis buffer (10 mM Tris buffer, 1 M EDTA, 0.2% Triton X-100, pH8.0) at room temperature for 15 minutes. Lysed cells were centrifuged at 15,000 rpm for 20 minutes, resulting in the separation of DNA fragments from the precipitates. The DNA fragments in supernatants were precipitated in the mixture of isoprophyl alcohol and 0.5 M NaCl at -

20^°C, and then left thereof for a day. The samples were centrifuged, after which the DNA samples were dried. The dried DNA samples were dissolved in 40 βl of TE solution (10 mM Tris, 1 mM EDTA, pH7.4) and 20 βl of loading buffer (15 mM EDTA, 2% SDS, 50% glycerol, 0.5% bromophenol blue, 10 unit RNase) , and then heated at 65°C for 10 minutes, followed by electrophoresis on 1.5% agarose gel containing 1.0 βg/ml of ethidium bromide at 50 V for 50-60 minutes.

As a result of electrophoresis, DNA was cut on agarose gel, so that a ladder type bands were observed, but the DNA cut was not observed in B16(F10) and HL60, either (FIG. 2).

<6-2> Quantification of intracellular DNA fragmentation

The present inventors analyzed DNA components in cell precipitates and supernatants using diphenylamine (referred as "DPA" hereinafter) method. Treated COMR2029 to a mouse skin cancer cell line B16(F10), a human blood cancer cell line HL60, a human skin cancer cell line LOX-IMVI and a human breast cancer cell line MCF7 for 24 hours, followed by centrifugation. The concentrations of

COMR2029 for the test were 0.03, 0.1, 0.3, 1, and

3 βg/ml respectively and the final concentration was adjusted to 0.1% using DMSO as a solvent. Dissolved the precipitates containing DNA in 250 μl of lysis buffer. Added 250 βl of 0.5 N perchloric acid and heated at 95 °C for 15 minutes. Added 500 μl of DPA reagent (88 mM DPA in 98% (v/v) glacial acetic acid, 1.5% (v/v) sulfuric acid, 0.5% (v/v) of 1.6% acetaldehyde solution). Left thereof at room temperature for overnight, after which measured DNA contents with a spectrophotometer at 595 nm. The percentages of DNA fragmentation ([supernatant OD x 2] x 100/ [pellet OD + supernatant OD x 2] ) on B16(F10) cell line were shown in Table 6 and those on LOX- IMVI, MCF7 and HL60 were shown in Table 7. At that time, OD represents optical density.

<Table 6>

<Table 7>

When COMR2029 and camptothecin, an apoptosis inducing substance, were respectively treated to B16(F10), LOX-IMVI and MCF7 cell lines, DNA fragmentation was not observed in all the experimental groups. When COMR2029 and camptothecin were respectively treated to HL60 cell line, DNA fragmentation was observed in both cases, which was because HL60 cell line was more sensitive to apoptosis or DNA fragmentation. LOX- IMVI reacted sensitively to COMR2029 showed no DNA fragmentation even under the concentration inducing severe cytotoxicity.

<6-3> Measurement of caspase-3 activity

Cultured HL60 cell line at the density of 2xl0⁶ cells/m-E for 24 hours, and then transferred into 1.5 ml tubes, followed by centrifugation with mini-centrifuge (13,000 rpm, 20 sec). Cooled lysis buffer of caspase fluorometric assay kit, which was distributed to cells by 25 μl per 1x10^s cells and then left at 4 ^°C for 10 minutes. Put 50 μl of cell lysates and 2X reaction buffer in each well of 96 well plate for measuring fluorescence. Added substrate into each well by 5 μl . Reacted at 37 ^°C for 2 hours under dark condition. Measured fluorescence at 400/505 nm using a fluorometer and the results were represented in Table 8.

<Table 8>

In case of positive control group where camptothecin was treated, the caspase-3 activity was increased, suggesting that apoptosis was occurring. In case of experimental group where COMR2029 was treated, there was no significant difference comparing to solvent control group. By the experiment using z-VAD.fmk, a caspase-3 specific inhibitor, it was also confirmed that the increase of caspase-3 activity was induced by only camptothecin .

<6-4> Measurement of intracellular Ca²⁺ change using Fura-2/AM After culturing HL60 cell line in RPMI1640 medium, transferred the cells into a 50 ml tube, which was centrifuged at 1,000 rpm for 10 minutes. Adjusted the cell density to lxlO⁷ cells/mβ with RPMI1640 medium. As Fura-2/AM gets into cells, it turns to [Fura-2] ^2~ and has both Ca²⁺ binding fluorescent wavelength and Ca²⁺ not binding fluorescent wavelength. Treated Fura-2/AM (final cone: 5 uM) , labeled at 37 ^°C under the dark condition for 30 minutes, and then washed with cold PBS three times. Adjusted the cell density to lxlO⁶ cells/m£ with serum-free RPMI1640 medium and set the excitation wavelength of fluorometer to 340 nm, the excitation wavelength of [Fura-2]^2- where Ca²⁺ was bound to 380 nm which was the excitation wavelength of [Fura-2]^2- itself. Observed the changes of fura-2 fluorescence for 200-500 seconds after treating COMR2029 by 0.03, 0.1, and 0.3 βg/ml . Investigated the changes of Ca²⁺ based on the changes of fluorescence and the ratios at both wavelengths .

In order to confirm the increase of intracellular Ca²⁺ density that is the typical phenomenon of apoptosis, the present inventors performed fluorescent assay using Fura-2. As a result, the changes of intracellular Ca⁺ density were not observed. Thus, COMR2029 seems not to be directly related with apoptosis induction (FIG. 3) .

Example 7: In vi tro invasion assay

After confirming the inhibiting activity of COMR2029 to angiogenesis and cancer cell growth, the present inventors further performed in vi tro invasion assay to investigate metastasis. One of the characteristics of cancer cells, unlike normal cells, is invasion and as the invasion increases, the malignancy of cancer cells becomes severe. Thus, in vitro invasion assay can be used to provide a prediction index of invasion that affects the formation of cancer tissues.

Before using chamber (BioCoat) , swelled it at room temperature for 120 minutes by adding serum- free DMEM. After swelling, removed medium from invasion chamber, which was transferred into 24- well plates containing HT 1080 condition medium

(600 μl/vrell ) . Loaded B16(F10) skin cancer cells

(4.5 x 10⁴ cells) into each chamber by 450 μl , and then added 0.3, 1 and 3 βg/ml of sample solutions each by 50 βl thereto. Likewise, loaded LOX-IMVI skin cancer cells (4.5 x 10⁴ cells) into each chamber by 450 βl , and then added 0.03, 0.1 and 0.3 βg/ml of sample solutions each by 50 βl thereto. Cultured the plates at 37 °C for 24 hours. After fixing the cells with MeOH, stained with 5% crystal violet. Cut off the stained filter by a knife, which was transferred into 24-well plates. Distributed 30% acetic acid into each well containing the filter by 200 βl to dissolve the stained part, which was, then, loaded into each well of a 96-well plate by 100 βl . Measured OD of the plate with a microreader at 450 nm and the results were represented in Table 9. <Table 9>

For the experiments testing the inhibitors of membrane transmission, B16 cell line is used in general. In the embodiments of the present invention, the membrane transmission was great in solvent control group that was not treated with COMR2029, so that lots of cells were stained, as seen in FIG. 4. In the COMR2029-treated group, matrix invasion was inhibited regardless of the COMR2029 concentration, so that the less cells were stained than the control group. The inhibiting activity to membrane transmission was also observed in LOX-IMVI cell line, which was sensitive to COMR2029, even with 10 times as low concentration as in B16(F10) cell line. As seen in Table 9, regarding solvent control group as 100, COMR2029-treated group showed 10-30, suggesting that COMR2029 had strong inhibiting activity to tissue invasion (FIG. 5). Example 8: LOX-IMVI anticancer test 1

The samples were prepared by adding 0.5% tween 80. The dosage of COMR2029 was determined to be 1 mg/kg. 0.5% tween 80 was used as a solvent control group. Human melanoma cell line LOX-IMVI was used as a cell line. Particularly, adjusted the cell density to 3X10⁶ cells/mouse, and implanted hypodermically at the right armpit of mice (0.2 ffll/ ouse) . Samples were administered from the next day. Administered COMR2029 abdominally once a day by the dosage of 1 mg/kg for day 1-2, and from then on, twice a day by the dosage of 3 mg/kg for day 3-4, by the dosage of 6 mg/kg for day 5, and by the dosage of 10 mg/kg for day 6-12. Measured the tumor size of each mouse, and the results were represented in Table 10. Measured the animal weight every day from day 1, and the results were represented in Table 11.

<Table 10>

<Table 11>

As a result, human melanoma cell line LOX- IMVI showed very fast growth, so that it could be detected from the 3^rd day after implantation but the administration of COMR2029 with the dosage of

1 mg/kg lowered the growth to 53.9%. The cancer cell growth inhibition rate did not changed, though, by the increase of dosage to 10 mg/kg or by the increase of administration times to twice a day. From the result of measuring the weight, significant weight change was not observed both in solvent control group and COMR2029-treated group. The increase of dosage or administration times did not induce cytotoxicity (FIG. 6 and FIG. 7)

Example 9: LOX-IMVI anticancer test 2

The samples were prepared by adding 0.5% tween 80. The dosage of COMR2029 was determined to be 10 and 30 mg/kg. 0.5% tween 80 was used as a solvent control group and adriamycin (2 mg/kg) was used as a positive control group. Human melanoma cell line LOX-IMVI was used as a cell line. Particularly, adjusted the cell density to 3X10⁶ cells/mouse, and implanted hypodermically at the right armpit of mice (0.3 ml/mouse) . Samples were administered from the next day. From day 1 through day 14, samples (0.5% tween 80 for solvent control group, COMR2029 for experimental group) were administered to abdominal cavity by 10, 30 mg /kg twice a day and adriamycin for the positive control group was administered to abdominal cavity as well by 2 mg/kg at the interval of 2 days. 10 days after sample administration, changed solvent into DMSO 5% + cremophor 20 % + 0.85% saline 75%. Measured the tumor size of each individual and the results were represented in Table 12. Measured the animal weight every day from day 1 and the results were represented in Table 13

<Table 12>

<Table 13>

As the first anticancer test, the second anticancer test resulted in the confirmation of cancer cell growth inhibition by COMR2029. When

10 and 30 mg/kg of samples were treated, 52.3% and

53.5% of mice showed tumor formation after implantation of cancer cells, and about 60% of mice showed tumor formation after the 8^th day. 10 days later, the solvent was changed and the number of animals showing tumor formation dropped to 50% after day 12. As for the weight changes, mice of solvent control group and COMR2029-treated group showed no change in weights but mice of positive control group lost about 4 g, suggesting that the adriamycin used in the positive control group had a cytotoxicity (FIG. 8 and FIG. 9) . Example 10 : Measurement of immune cell

proliferation

After dislocating cervical vertebra of a BDFl mouse having 20-25 g weight, sterilized the abdomen with 70% alcohol. Dissected the abdomen and extracted the spleen, which was put on a petri dish containing EBSS buffer solution. Isolated spleen cells using a syringe plunger. Prepared cell suspension containing the isolated spleen cells and transferred thereof into tubes, which was, then, left on ice for 5-10 minutes. Obtained supernatants and centrifuged (1,200 rpm, lOminutes). Removed supernatants and resuspended the precipitates in RPMI1640 medium [10% FCS

(fetal calf serum) , 2-mercaptoethanol (2-ME) ] , after which counted the number of cells. Loaded the cell suspension (1X10⁶ cells/mi) into each well of a 96-well plate (200 C/well) . In order to activate T cells, added Con A into the wells (5 μg/ml/πell ) . Dissolved COMR2029 in 100% DMSO, and then diluted serially to make the concentration to 0.01, 0.03, 0.1, 0.3 and 1 βg/ml, which were treated to the plate. Cultured the plate at 37 °C for 3 days in a C0 incubator. 18 hours before finishing the culture, added 1 uCi of [³H]- thymidine into each well. Collected the cells using an automatic cell collector and measured the level of [³H] -thymidine absorption, leading to the measurement of the inhibition of immune cell proliferation. The results were represented in Table 14 and Table 15.

<Table 14>

<Table 15>

As a result, when the experimental group was treated with COMR2029, there were no changes in the number of immune cells in every group each treated with different concentrations of COMR2029. But, there was an exception. When the group was treated with high concentration of COMR 2029, the number of immune cells was decreased, suggesting that the group was affected by cytotoxicity induced by COMR2029.

Example 5: Measurement of cytotoxicity of pre-

treated COMR2029 to LOX-IMVI

Investigated the anticancer activity of COMR2029 to human melanoma cell line LOX-IMVI with SRB method (Skehan, et al . , Proc . Am . Assoc . Cancer Res . , 30: 612, 1989). Cultured the said cancer cell line in RPMI1640 medium supplemented with 10% fetal bovine serum. Maintained the cells by passing once or twice a week. Set the cell density to 3,000-6,000 cells m£ for the experiment to measure the anticancer activity. All the reagents used for the experiment were dissolved in 100% DMSO, and then diluted serially to make the concentrations 0.01, 0.03, 0.1, 0.3 and 1 μg/ml . Counted the number of cells and loaded the required number of cells into wells of a 96-well plate. On the next day, fixed another plate having the same cell density as a sample treating plate with 50% TCA, which would be used as Tz

(Time Zero) plate necessary for measuring the basic OD of cells. Divided the sample treating plate into two groups . Treated group 1 with samples after 24 hours, for which the final concentration of sample was adjusted to 5 different concentrations by 0.1% DMSO. Treated group 2 with samples right away and then transferred to fresh medium two hours later.

Treated the group 2 with samples again after 24 hours. Washed Tz plate with Tap water 1 hour later. Two days later, fixed the sample treating plate with 50% TCA (50 C/well) and washed with Tap water after 1 hour. Dried the plates at room temperature. Added 0.4% SRB solution (dissolved in 1% acetic acid) into wells of the plates (100 μ /vi&l± ) and 30 minutes later, washed the plates with 1% acetic acid solution, which were, then, dried at room temperature, again. Dissolved again by adding 10 mM tris base (100 μl /well, pH 10.5) and measured OD₅₄o by enzyme-linked immunosorbent assay (ELISA) using ELISA reader. The results were represented in Table 16. In the Table 16, GI₅₀ (μg/ml) means the concentration of compound that could inhibit cancer cell growth to 50%.

<Table 16>

The experiment was to investigate the sensitivity of cells to drugs that went through the steps of exposing on drugs, retrieving the drugs and re-exposing on the drugs. As a result, the sensitivity of cells was not affected by the above treatment.

Example 12: Measurement of MMP-2 activity

<12-1> Drawing a standard curve

Standard material and substrate having fluorescence to MMP-2 were purchased from SIGMA (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) . Used a fluorescent spectrophotometer (Perkin-Elmer LS- 50B) for the experiment. Operated the machine using a computer software (Fluorescence Data Manage, Perkin-Elmer) . Set excitation and emission wavelength to 325 nm (slit 5.0 mm) and 420 nm(slit 3.0 mm) respectively, and used 20 mM Tris-HCl, 5 mM CaCl₂ and 0.15 M NaCl (reaction buffer, pH 7.5) for all the experiments.

Dissolved a standard material Mca-Pro-Leu-Gly

(1 mM) in DMSO, which was diluted with reaction buffer in the range of 15.6-250.0 pmoles on microplates, resulting in the making a standard curve .

<12-2> Measurement of MMP-2 activity

Measured the MMP-2 activity using the method of Night et al . with a slight modification. Particularly, cultured 1 nM proMMP-2 without TIMP- 2 at 37 °C for 15 minutes along with 0.5 mM APMA. Added test samples and substrate (Mca-Pro-Leu-Gly- Leu-Dpa-Ala-Arg-NH2; 10 uM) to reaction mixture. Cultured 30 more minutes. Stopped the reaction by adding 0.1 N sodium acetate buffer (pH 4.0) . Measured the hydrolysis of the substrate with a fluorescent spectrophotometer (Perkin-Elmer LS- 50B) . Set excitation and emission wavelength to 325 nm (slit 5.0 mm) and 420 nm(slit 3.0 mm) respectively. Obtained IC₅₀ value by floating the remaining enzyme activity and sample concentration, The results were represented in Table 17.

<Table 17>

MMP (Matrix MetalloProteinase) is a metastasis related enzyme, whose most sensitive and representative one is MMP-2. The experiment was to measure the MMP-2 activity. Precisely, the substrate of MMP-2 was loaded and a standard curve was made, which was compared with that of experimental group to measure the activity. As a result, when COMR2029 was treated with the concentration of 0.03, 0.1, 0.3, 1, 3 and 10 uM respectively, the enzyme activity did not change. The MMP-2 activity seemed to be decreased with the concentration of over 3 uM, which was probably because of color quenching. Thus, COMR2029 was confirmed not to inhibit the MMP-2 activity. Example 13: The effect of COMR2029 on the

regulation of MARK (ERK 1/2)

<13-1> Cell culture and sample treatment

Used human melanoma cell line LOX-IMVI as a cell line and RPMI1640 medium supplemented with 10% FBS as a medium. Adjusted cell density to 5 X 10⁶ cells/m*. Pre-treated COMR2029 (30 minutes earlier) . Added 10 ng/ml of TNF- α and reacted for 2 hours. The concentration of COMR2029 for the treatment was 0.01, 0.1 and 1 μg/ml respectively.

<13-2> Cytosolic extract preparation

After washing the cell line twice with PBS stored at 4 ^°C , collected the cells using a scraper. Added PBS by 0.5 ml and centrifuged to precipitate the cells. Added 200 μl of lysis buffer to suspend the cells. The lysis buffer was composed of 70 mM b-glycerophosphate (pH 7.2), 0.1 mM meta/ortho- vanadate, 2 mM MgCl₂, 1 mM EGTA, 1 mM DTT (dithiothreitol) , 0.5% triton-X-100 , 0.2 mM PMSF

(phenyl-methylsulfonyl fluoride) , 5 mg/ml pepstatin

A, 5 mg/ml chymostatin, 5 mg/ml leupeptin and 5 mg/ ml peptin. Left thereof at 4^°C for 15 minutes and then centrifuged (4^°C, 12,000 rpm, 10 min.). Obtained supernatants, which was distributed by 20 μl , resulting in the preparation of samples (stored at -80°C) . Quantified the amount of protein with a protein assay kit (Bio-Rad Laboratories, Richmond, CA, USA) .

<13-3> Western blot analysis

Used ^■ samples containing about 20 μg of protein for blotting. Particularly, mixed 10 μl of sample and 10 βl of 2X tris-glycine buffer, and then heated the mixture at 95 ^°C for 5 minutes, followed by loading on 10% SDS-PAGE. The running time was set about 2 hours in order to let staining dye all out. Set the voltage to 100 V. The proteins that went through SDS-PAGE were transferred onto nitrocellulose membrane, which was left in blocking solution (20 mM Tris-Cl containing 5% nonfat dried milk, pH 7.5, 150 mM NaCl, 0.05% Tween 20 (TBST) solution) at room temperature for 1 hour. Then, washed with TBST solution three times for 20 minutes each. Put the primary antibody diluted at the ratio of 1000:1

(added 10 μl of primary antibody to 10 ml of culture solution) in the solution containing TBST and 5% BSA, which was left at 4 °C for overnight. On the next day, shook the solution at room temperature for 1 hour and then, washed thereof with TBST three times for 20 minutes each. Put the secondary antibody diluted at the ratio of 2000:1 (added 6 g of secondary antibody to 12 ml of culture solution) in the solution containing TBST and 5% nonfat dried milk, which was left at room temperature for 1 hour. Then, washed the solution with TBST three times for 20 minutes each. After washing, developed the blot in a dark room using Amersham ECL kit (Amersham International, Buckinghamshire, UK) .

As a result, it was confirmed that COMR2029 inhibited the activity of ERK1/2 among MARK, intracellular signal transmitters, dose- dependently (FIG. 10) .

Example 14: The effect of COMR2029 on the

regulation of the expression of transcription

regulating elements

<14-1> Cell culture and sample treatment

Used human melanoma cell line LOX-IMVI as a cell line and RPMI1640 medium supplemented with 10% FBS as a medium. Adjusted cell density to IX 10⁶ cells/m£. Set the concentration of COMR2029 for the experiment to 0.01, 0.1 and 1 μg/ml respectively and the treating time was 2 hours.

Distributed the cells onto culture dishes. Cultured the cells for 3 hours and then treated COMR2029 thereto.

<14-2> Nuclear extract preparation

After washing the cell line twice with 4 °C PBS, collected cells with a scraper using PBS stored at 4 ^°C . Centrifuged (1,200 rpm, 10 minutes, 4 °C ) to precipitate the cells, which were washed with PBS. After removing PBS completely, suspended the cells in 100 μl of cell lysis buffer

(10 mM Hepes (pH 7.9), 1.5 mM MgCl₂, 1 mM DTT, 1 M PMSF, 1 q/ml of aprotinin and 1 q/ml of leupeptin). Centrifuged (10,000 rpm, 5 minutes, 4 ^°C ) after leaving at 4 ^°C for 15 minutes. Removed supernatants and then suspended cell pellet in 100 μl of nucleus lysis buffer (K buffer; 30 mM Hepes,

1.5 mM MgCl₂, 450 mM NaCl, 0.3 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 q/ml of aprotinin and 1 q/ l of leupeptin) . Left at 4 °C for 30 minutes, then centrifuged at 4 ^°C , 15,000 rpm for 20 minutes. Obtained supernatants, which was distributed by 20 μl for the use as samples (stored at -80 ^°C ) . Quantified the amount of proteins with a protein assay kit (Bio-Rad Laboratories, Richmond, CA, USA) .

<14-3> EMSA (Electrophoretic Mobility Shift Assay) Cultured ³²P-labeled DNA probe (wherein oligomers corresponding to NF-kB, Octamer, AP-1 and CREB were mixed with T4 kinase and radioisotope was added) at 37 °C for 1 hour. Put 5 μg of nuclear protein and 1 μg of poly(dl-dC) in binding buffer (100 mM NaCl, 30 mM Hepes, 0.3 mM EDTA, 1.5 mM MgCl₂, 10% glycerol, 1 mM DTT, 1 mM PMSF, and lg/ml each of aprotinin and leupeptin) , which was left at room temperature for 10 minutes. Added probe and then left at 4 °C for 10-20 minutes, Pre-ran 5% SDS-PAGE without samples over 90 minutes. After loading samples on 5% SDS-PAGE, ran it again for 120 minutes with 120 V. After finishing running, dried in a dry-oven for 30 minutes, resulting in 95% dryness. After drying, put X-ray film in a cassette in a darkroom and left at -70 ^°C for a while. Then, developed the film in the darkroom. Every antibody used in this invention was provided by Santa Cruz Biotechnology

(Santa Cruz, CA) . Double stranded DNA oligomer was prepared by synthesizing NF-kB (5-GGGGACTTTCC- 3) (Pierce et al . , 1988) and AP-1 (5-TGACTCA-3)

(Angel et al . , 1987), consensus recognition site, which was used after labeling with radioisotope using [-³²P] dATP.

As a result of the investigation on 4 transcription-regulating elements with 3 different cell lines, the expressions of CREB and Octamer did not increase or decrease, either, while the expressions of AP-1 and NF-kB were changed (FIG. 11a - FIG. lie) . In order to re-confirm the above result, the investigation on the expressions of AP-1 and NF-kB was done again using a cell line LOX-IMVI. As a result, there was no change in the expression of NF-kB, while the expression of AP-1 was decreased (FIG. lid) , which was corresponding to the result of RT-PCR confirming the decrease of p21 and uPA, suggesting that they had AP-1 binding site. Thus, COMR2029 of the present invention was also confirmed to be involved in angiogenesis inhibition. Example 15: Analysis of the effect of COMR2029 on

the PKC (Protein Kinase C) activity

<15-1> Preparation of samples for measuring PKC activity

After culturing cells in a 100 mm dish, washed the cells with PBS (5X10⁶-1X10⁷ cells/5 ml ) . Suspended the washed cells in 500 μl of cooled 2X extraction buffer (25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM mercaptoethanol (Stock 14.4 M) , 1 μg/ml leupeptin, 1 βg/ml aprotinin) , followed by homogenization with a cooled homogenizer. Centrifuged the homogenized lysates at 4 ^°C , 14,000 g for 5 minutes, which were used as samples for measuring PKC activity. Had the above extraction buffer pass through DEAE cellulose column (Whatman DE52) for pre- equilibration, followed by making the prepared samples pass through again. Washed with 1 ml of extraction buffer twice and then eluted fractions including PKC by 1 ml of extraction buffer containing 200 mM NaCl. <15-2> Preparation of reaction buffer solution for measuring PKC activity

Prepared the reaction buffer solution by dissolving 10X ATP solution, 10X biotynylated PKC pseudosubstrate solution, 10X activator solution and 10X PKC together, which was pre-treated for 30 minutes. Diluted Y -³ P-ATP containing solution from the concentration of 10 μCi// to 0.2 μCi/ £ using de-ionized water, resulting in the preparation of working solution (Table 18) .

<Table 18>

In the above table, N represents the sample number.

<15-3> PKC reaction Put 20 μl of PKC reaction mixture in a microcentrifuge tube. Added 5 βl of sample prepared in the above Example <15-1> thereto. In the meantime, added 5 βl of the same sample to 20 βl of control mixture without peptide substrate. Added 5 βl of de-ionized water instead of PKC sample to 20 βl of PKC reaction mixture, which would be used as a background control. Reacted each mixture at 25 ^°C for 15 minutes. Terminated the reaction by adding 10 βl of stop solution. When the radioactive count of control mixture was higher than that of background control, it could not be used since it reflected that nonspecific phosphorylation of the PKC sample was too high. In order to solve the problem, amended the preparation method as follows. Terminated PKC reaction by adding 3 βl of. 50% TCA and 2 βl of 1% BSA, left each reaction tube on ice for about 10 minutes and centrifuged at 14,000 g for 5 minutes. After centrifugation, transferred 25 βl of supernatants into new tubes and then added 5 βl of neutralization solution into each tube, resulting in the complete termination of reaction by neutralizing the sample.

<15-4> Measurement of the activity of PKC reaction Considering the case of precipitation generation when stored the washing solution at 4 ^°C , dissolved the solution at lukewarm temperature for 10-15 minutes. Added 8 Λ of avidin solution to the solution of Example <15-3> whose reaction was terminated. Left the sample at room temperature for about 5 minutes, after which put 20 βl of this reaction solution and 50 βl of washing solution together in the sample reservoirs of centrifugal ultrafiltration units. Covered the sample reservoirs tightly and centrifuged at 14,000 g for 5 minutes to remove liquid. Added 100 μl of washing solution into the sample reservoirs and washed three times, resulting in the preparation of reaction samples. In order to obtain total cpm

(counts per min) of the reaction sample, put 4 βl of reaction sample in the blank sample reservoir of centrifugal ultrafiltration units, which was used as a reference sample. Transferred the remaining reaction sample and reference sample in sample reservoirs to scintillation vials. After adding liquid scintillation cocktail solution into the vials with the required amount, set the channel to ³²P and measured the radioactivity.

As a result, COMR2029 was confirmed to inhibit PKC activity (FIG. 12) . PKC, a kind of serine/threonine kinase, is activated most in the presence of calcium ion and DAG (Diacylglycerol) .

There are at least 10 kinds of PKC and these enzymes are specialized in their intracellular expression and activation by lipid and calcium.

PKC is observed during the signal transduction by being induced by hormones, growth factors and neurotransmitters . PKC induces protein phosphorylation, causing the changes in protein activity. In some special cases, the phosphorylation of EGF receptor induced by PKC down-regulates tyrosine kinase activity. As seen in FIG. 12, COMR2029 plays a role in inhibiting

PKC activity, which was supported by other test results with MARK and transcription regulating elements

Example 16: Measurement of sGC (Soluble Guanynyl

Cyclase) activity

Cultured LOX-IMVI cells in a 24 well plate at the density of 5X10⁵ cells/mfe. Used SNP (Sodium NitroPruside) as a sGC activator and measured cGMP by using cGMP enzyme immunoassay kit (Direct cGMP Enzyme immunoassay kit, BIOMOL) . Treated with COMR2029 by 0.01, 0.1 and 1 βg/ml each. Compared 100 μM SNP-treated group and non-treated group.

As a result, COMR2029 was confirmed to inhibit sGC activity as LY83583, which was a structural analogue of COMR2029 and known as a sGC inhibitor, did (FIG. 13) .

Example 17: Measurement of uPA (Urokinase-type

Plasminigen Activator) activity

In order to measure the PA activity, performed chromogenic assay by modifying the quantifying method for cell-associated PA activity, Particularly, loaded LOX-IMVI cells to a 96 well plate (200 μβ/well) . After 24 hours, pre-treated with COMR2029. Treated with TNF- α by 10 nq/ml after 30 minutes. Two hours later, discarded supernatants and washed with PBS containing 2 mg/ ml of fatty acid-free BSA twice. Added 20 βl of mixture prepared by mixing 2XPBS and substrate at the ratio of 1:1 to 180 βl of cell solution, after which measured OD₄₀₅ on 0, 10^th, 30^th and 40^th minutes . As a result, COMR2029 was confirmed to inhibit uPA activity slightly, which was corresponding to the result of RT-PCR (FIG. 14). As shown in FIG. 15, uPA is an enzyme that plays an important role in mobility and invasion.

Example 18: Acute toxicity test in rats via oral

administration

The following experiments were performed to see if COMR2029 of the present invention has acute toxicity in rats.

6-week old SPF (specific pathogen-free) SD line rats were used in the tests for acute toxicity. COMR2029 was suspended in 0.5% methylcellulose solution and orally administered once to 2 rats per group at the dosage of 1 g/kg/ ml . Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked with eyes during autopsy.

The results showed that the COMR2029 did not cause any specific clinical symptoms, weight change, or death in rats. No change was observed in hematological tests, biochemical tests of blood, and autopsy. Conclusively, COMR2029 used in this experiment is evaluated to be a safe substance since it dose not cause any toxic change in rats up to the level of 1,000 mg/kg.

Applicable Example 1: Ophthalmic diseases such as

retinopathy, glaucoma, etc.

The examples of ophthalmic disease induced by angiogenesis are senile macular degeneration, diabetic retinopathy, retinopathy of precocious child, angiogenic glaucoma, etc. Millions of people over the world are losing their sights because of such diseases every year (D'Amato, R.J. and Adamis, A, P. (1995) Ophthalmol . , 102:1261- 1262; Ozaki, H. et al . (1996) Ophthalmi c Res . , 28 (6) :356-60; Ciulla, T.A. et al . (1999) Expert

Opin . Inves tig. Drugs . , 8 (12) : 2173-2182 ; Ambrus, J.L. et al . (2000) J. Med. , 31:278-282; Skopinski, P. et al . (2001) Int. J^". Clin . Pharmacol . Res . , 2(2):73-78; Funatsu, H. et al . (2001) Jpn . J. Ophthalmol., 45 (6) : 577-584 ; Razavi, S. et al . (2002) J. Fr. Ophthalmol., 25 (7) : 747-752 ) .

As gets older, the visual cells and the pigment epithelium on the retina lose their functions and as such degeneration exceeds, senile yellow spot degeneration is developed, leading to senile macular degeneration. As the disease progresses, the pigment epithelium on the retina peels off or angiogenesis is induced in the choroid which functions to supply blood to the retina, causing hemorrhage underneath nervous membrane of the retina, finally resulting in the loss of sight.

And diabetic retinopathy, a kind of complication of diabetes, is a disease developed by invasion of capillary vessels produced by angiogenesis on the retina into corporis vitrei, causing a mal-function in circulation of capillary vessels, resulting in the loss of sight. Precisely, as oxygen shortage area on the retina becomes wider, angiogenesis is induced to supply oxygen smoothly. But the newly generated blood vessels are not normal blood vessels, so that they burst often and new fibrous materials are growing around newly generated blood vessels, which pulls the blood vessels to cause hemorrhage, leading to the loss of sight.

Retinopathy of precocious child also induces sight trouble. The blood vessels in the retina of a fetus are formed in the last month of pregnancy. Therefore, in precocious child, abnormal blood vessels are developed by angiogenesis, so that scars are formed in the retina, followed by the sight trouble.

As for angiogenic glaucoma, when scars are formed around newly generated blood vessels by diabetic retinopathy or other diseases, retina exfoliation follows. And as the newly generated blood vessels spread to the frontal iris, angiogenic glaucoma is developed. As explained hereinbefore, the above diseases are related to angiogenesis. Thus, the compound of the present invention having function of inhibiting angiogenesis can be effectively used for the prevention and the treatment of such diseases.

Applicable Example 2: Arthritis

Arthritis is basically caused by the abnormal autoimmune response. As the disease progresses, chronic inflammation in synovial cavity induces angiogenesis and newly generated capillary vessels infiltrate through joints, resulting in the destruction of cartilage (Stupack, D.G., et al .

(1999) Bra z . J. Med . Res . , 32 (5) : 573-81 ; Wash, D.A. (1999) Rheuma tology, 38 (2) : 103-112 ; Paleolog, E.M. (2002) Arthritis Res., 3:S81-90; Bodolay, E. et al . (2002) J. Cell . Mol . Med., 6(3):357-76; Lutton, A. et al . (2002) Na t . Med. , 8 (8 ) : 831-40) .

By arthritis, the changes caused by human defense mechanism leads the proliferation of new blood vessels and the formation of osteophytes around the target place. Joint movement is limited by the osteophytes, making the disease worse. Thus, the formation of osteophytes by angiogenesis is the major cause of worsening arthritis. Therefore, the compound of the present invention having function of inhibiting angiogenesis can be successfully used for the prevention and the treatment of the mentioned disease .

INDUSTRIAL APPLICABILITY

As explained hereinbefore, an angiogenesis inhibitor of the present invention containing 6- anilinoquinoline-5, 8-quinone (COMR2029) as an effective ingredient can be effectively used for the treatment of angiogenesis-related diseases such as cancer, rheumatoid arthritis or diabetic retinopathy.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

What: is claimed is

1. A preventive or therapeutic agent for the angiogenesis-related diseases containing 6- anilinoquinoline-5, 8-quinone as an effective ingredient and having the structure of <Chemical Formula 1>.

<Chemical Formula 1>

2. The preventive or therapeutic agent for the angiogenesis-related diseases as set forth in claim 1, wherein the angiogenesis-related disease is cancer.

3. The preventive or therapeutic agent for the angiogenesis-related diseases as set forth in claim 1, wherein the angiogenesis-related disease is rheumatoid arthritis or diabetic retinopathy.