Use of paeonol for inhibiting angiogenesis or for enhancing radiosensitization

Abstract

The present invention relates to a composition comprising paeonol of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient for inhibiting angiogenesis, or for enhancing radiosensitization. The present invention also relates to novel use of paeonol of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a composition for inhibiting angiogenesis, or for the manufacture of a composition for enhancing radiosensitization. Also, the present invention relates to a method for inhibiting angiogenesis which comprises administrating to a subject in need thereof an effective amount of paeonol of formula (I) or a pharmaceutically acceptable salt thereof, or a method for enhancing radiosensitization which comprises administrating an effective amount of paeonol of formula (I) or pharmaceutically acceptable salts thereof to a subject in need of radiotherapy. ##STR00001##

Claims

1. A method for treating cancer comprising administering low-dose radiotherapy and an effective amount of paeonol or a pharmaceutically acceptable salt thereof to a subject in need of such treatment.

2. The method of claim 1, wherein the cancer is uterine cancer, lung cancer, or skin cancer.

3. The method of claim 1, wherein the paeonol or pharmaceutically acceptable salt thereof is comprised in a pharmaceutical composition in amount of 1-99 w/w %.

4. The method of claim 1, wherein the paeonol is administered before or during the radiotherapy.

5. The method of claim 1, further comprising administering an anti-cancer drug.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graph showing inhibitory effect of paeonol on angiogenesis in mouse Matrigel model.

(2) FIG. 2 is a graph showing inhibitory effect of paeonol on tumor growth in solid tumor induced mouse.

(3) FIG. 3 is a graph showing effect of paeonol on elongation of survival time in solid tumor induced mouse.

(4) FIG. 4 is a graph showing synergy effect of paeonol on inhibition of tumor growth in combination with other anti-cancer drugs.

(5) FIG. 5 is a graph showing synergy effect of paeonol on elongation of survival time in combination with other anti-cancer drug.

(6) FIG. 6 is a graph showing effect of paeonol on radiosensitization of human lung cancer cell.

(7) FIG. 7 is a graph showing effect of paeonol on radiosensitization of human uterine carcinoma cell.

(8) FIG. 8 is a graph showing decrease of tumor volume in lung cancer cell-injected mouse by treatment of radiation and paeonol.

(9) FIG. 9 is a graph showing increase of survival rates in lung cancer cell-injected mouse by treatment of radiation and paeonol.

(10) FIG. 10 is a graph showing decrease of tumor volume in melanoma cell-injected mouse by treatment of radiation and paeonol.

(11) FIG. 11 is a graph showing survival rates of normal and cancer cell by the treatment of paeonol.

MODE FOR INVENTION

(12) To investigate effects of the present composition comprising paeonol on angiogenesis or radiosensitization, paeonol was purified from the root of Paeonia suffruticosa and then activities of paeonol were investigated using animal experiment.

(13) Purification and Identification of Paeonol

(14) 2 kg of dried root powder of Paeonia suffruticosa was extracted in 5 liters of methanol at 70 C. for 5 hours by reflux extraction, and the extraction was repeated three times. The filtrated extract was lyophilized, and then 500 g of methanol extract was obtained. Methanol extract was suspended in 3 liters of water, and then was fractionated with 3 liters of hexane three times. Hexane-soluble fraction was lyophilized, and then 67 g of hexane fraction was obtained. Hexane fraction was applied into silica gel column chromatography using hexane:acetone solvent (60:1 to 1:1), and then 7 fractions (Fr. 17) were obtained.

(15) TLC(Thin Layer Chromatography) Data

(16) Silica gel plate 60 F254 (Merck)

(17) hexane:aceotone=3:1

(18) Rf value: 0.44 (detection at UV 254 nm)

(19) Precipitant obtained from Fr. 2 was recrystalized with ethanol, and then compound 1 (2100 mg) showing needle-shaped white crystal was obtained.

(20) Compound 1 (Paeonol)

(21) mp 4850 C., IR (KBr) v.sub.max cm.sup.1: 3400, 2940, 1725, 1625, 1570, 1500; .sup.1H-NMR (400 MHz, CDCl.sub.3) : 2.55 (3H, s, CH.sub.3), 3.83 (3H, s, OCH.sub.3), 6.406.50 (2H, m, H-3, H-5), 7.60 (1H, d, J=10.0 Hz, H-6), 12.60 (1H, s, OH); .sup.13C-NMR (100 MHz, CDCl.sub.3) : 114.1 (C-1), 165.2 (C-2), 100.8 (C-3), 166.1 (C-4), 107.6 (C-5), 132.6 (C-6), 202.5 (CO), 55.4 (OCH.sub.3), 26.1 (CH.sub.3).

(22) Because the above data of compound 1 was coincided with the data of paeonol written in the literature (Kwon et al., Kor. J. Pharmacogn. 30, p 340-344, 1999), compound 1 was identified as paeonol.

Example 1

Inhibitory Effects of Paeonol on Angiogenesis Using Animal Experiment (Mouse Matrigel Model)

(23) Effects of oral administration of paeonol on angiogenesis were quantitatively investigated in mouse Matrigel model.

(24) The mixture of 0.4 ml of Matrigel, 50 ng/ml of basic fibroblast growth factor (bFGF) and 40 units/ml of heparin which are angiogenesis-inducing factor was subcutaneously injected into C57BL/6 female mice (SLC Co., Japan; 6 week-old) adapted for 1 week.

(25) 0.5 mg of the above isolated paeonol was dissolved in 0.2 ml of 20% ethanol, and then 0.5 mg per mouse of paeonol (1 mg/day) was orally administrated into the treated group (n=8) twice a day for 4 days. In the other hand, 0.2 ml of 20% ethanol was also orally administrated into the control group (n=8) in the same manner as treated group.

(26) On the fifth day of oral administration, Matrigel was separated from excised skin of each mouse, and the level of hemoglobin in the Matrigel was measured in order to determine the inhibition of angiogenesis quantitatively.

(27) As shown in FIG. 1, the hemoglobin content of treated group was reduced up to 33% as compared with that of control group, and angiogenesis was inhibited by about 67%. Therefore, it was confirmed that the composition of the present invention comprising paeonol has anti-angiogenic activity.

Example 2

Inhibitory Effect of Paeonol on the Growth of Solid Tumor

(28) Effects of administering the present composition comprising paeonol on the growth of solid tumor were investigated.

(29) For the induction of primary tumor, B16BL6 melanoma cells (110.sup.6) suspended in 0.2 ml of PBS (potassium-buffered saline) were subcutaneously injected into C57BL6 female mice (SLC Co., Japan; 6 week-old, 20-25 g of body weight) adapted for 1 week.

(30) 0.5 mg of the above isolated paeonol was dissolved in 0.2 ml of 20% ethanol, and then 0.5 mg per mouse of paeonol (1 mg/day) was orally administrated into the treated group (n=7) twice a day for 17 days after injecting cancer cells. In the other hand, 0.2 ml of 20% ethanol was also orally administrated into the control group (n=7) in the same manner as treated group.

(31) The size of tumor was measured once per 1-2 days for 17 days after injecting cancer cells and then the volume of tumor was calculated in accordance with the following equation, (width).sup.2(length)0.52.

(32) As shown in FIG. 2, oral administration of paeonol with the dosage of 1 mg per day for 17 days prevented tumor size compared with the control group, and inhibited tumor growth by 48%.

(33) Therefore, it was confirmed that the composition of the present invention comprising paeonol has inhibitory effects on tumor growth.

Example 3

Lifetime Elongation of Tumor-Induced Mice by Administration of Paeonol

(34) Effects of the present composition comprising paeonol on lifetime elongation of tumor-induced mice were investigated.

(35) For the induction of primary tumor, B16BL6 melanoma cells (110.sup.6) suspended in 0.2 ml of PBS were subcutaneously injected into C57BL6 female mice (SLC Co., Japan; 6 week-old, 20-25 g of body weight) adapted for 1 week.

(36) 0.5 mg of the above isolated paeonol was dissolved in 0.2 ml of 20% ethanol, and then 0.5 mg per mouse of paeonol (1 mg/day) was orally administrated into the treated group (n=7) twice a day for 22 days after injecting cancer cells. In the other hand, 0.2 ml of 20% ethanol was also orally administrated into the control group (n=7) in the same manner as treated group.

(37) As shown in FIG. 3, the number of dead mice was increased in proportion to the incubation time. After 22 days, one of control group survived whereas three of treated group survived. The survival rates of treated and control groups were 43% and 14%, respectively. Therefore, it was confirmed that the composition of the present invention comprising paeonol has enhancing effects on lifetime and survival rate of solid tumor-induced mice.

Example 4

Inhibitory Effects of Paeonol on Tumor Metastasis

(38) Inhibitory effects of the present composition comprising paeonol on tumor metastasis in tumor-induced mice were investigated.

(39) In order to induce tumor, B16BL6 melanoma cells (510.sup.4) suspended in 0.2 ml of PBS were intravenously injected into tails of C57BL6 female mice (SLC Co., Japan; 6 week-old) adapted for 1 week.

(40) 0.25 mg of the above isolated paeonol was dissolved in 0.2 ml of 20% ethanol, and then 0.25 mg per mouse of paeonol (0.5 mg/day) was orally administrated into the treated group (n=6) twice a day for 14 days after injecting cancer cells. In the other hand, 0.2 ml of 20% ethanol was also orally administrated into the control group (n=6) in the same manner as treated group. After 14 days, mice were dissected, and then the numbers of colonies, which metastasized into lung, were counted.

(41) Control group had 21360 colonies per mouse whereas treated group had 13280 colonies. Oral administration of paeonol inhibited metastasis into lung by 38%. Therefore, it was confirmed that the composition of the present invention comprising paeonol has inhibitory effects on tumor metastasis.

Example 5

Synergy Effect of Paeonol in Combination with Other Anti-Cancer Drug

(42) 5-1) Inhibitory Effect on Tumor Growth

(43) Synergy effect of paeonol in combination with anti-cancer drug, 5-fluorouracil (5-FU) was investigated.

(44) For the induction of primary tumor, B16BL6 melanoma cells (110.sup.6) suspended in 0.2 ml of PBS were subcutaneously injected into C57BL6 female mice (SLC Co., Japan; 6 week-old, 20-25 g of body weight) adapted for 1 week.

(45) In 5-FU & paeonol treated group (n=7), 70 mg/kg per mouse of 5-FU was intraperitoneally injected at 3 day and 6 day after injecting cancer cells, and 1 mg per mouse of paeonol dissolved in 0.2 ml of 20% ethanol was orally administrated twice a day for 21 days after injecting cancer cells. In 5-FU treated group (n=7), 70 mg/kg per mouse of 5-FU was treated in the same manner as 5-FU & paeonol treated group. In the other hand, 0.2 ml of 20% ethanol was also orally administrated into control group (n=7) and 5-FU treated group in the same manner as 5-FU & paeonol treated group. The size of tumor was measured once per 1-2 days for 21 days after injecting cancer cells and then the volume of tumor was calculated in accordance with the following equation, (width).sup.2(length)0.52.

(46) As shown in FIG. 4, tumor growths of 5-FU treated group and 5-FU & paeonol treated group were inhibited by 50% and 69% compared with that of control group, respectively. It was confirmed that the combinational treatment with anti-cancer drug and anti-angiogenic paeonol has more inhibitory effect on tumor growth than the treatment of anti-cancer drug itself.

(47) 5-2) Lifetime Elongation of Tumor-Induced Mice

(48) Effects of paeonol on lifetime elongation of tumor-induced mice were investigated.

(49) For the induction of primary tumor, B16BL6 melanoma cells (110.sup.6) suspended in 0.2 ml of PBS were subcutaneously injected into C57BL6 female mice (SLC Co., Japan; 6 week-old, 20-25 g of body weight) adapted for 1 week.

(50) In 5-FU & paeonol treated group, 70 mg/kg per mouse of 5-FU was intraperitoneally injected at 3 day and 6 day after injecting cancer cells, and 1 mg per mouse of paeonol dissolved in 0.2 ml of 20% ethanol was orally administrated twice a day for 21 days after injecting cancer cells. In 5-FU treated group, 70 mg/kg per mouse of 5-FU was treated in the same manner as 5-FU & paeonol treated group. In the other hand, 0.2 ml of 20% ethanol was also orally administrated into control group and 5-FU treated group in the same manner as 5-FU & paeonol treated group.

(51) As shown in FIG. 5, the number of dead mice was increased in proportion to the incubation time. After 22 days, each one of control group (n=7) and 5-FU treated group (n=7) survived (survival rate 14%) whereas four of 5-FU & paeonol treated group (n=7) survived (survival rate 57%). Therefore, it was confirmed that the combinational treatment with anti-cancer drug and anti-angiogenic paeonol has more enhancing effects on lifetime and survival rate of solid tumor-induced than the treatment of anti-cancer drug itself.

Example 6

Effect of Paeonol on Radiosensitization in Lung Carcinoma Cell Line, H460

(52) Effect of treatment of paeonol during radiation into lung carcinoma cell line on radiosensitization was investigated.

(53) 10.sup.2-10.sup.3 of human lung carcinoma cell line H460 was irradiated with various doses of radiation (0, 1, 2, 3 Gy) for control groups (n=3) just once, and was irradiated with 3 Gy of radiation and the above isolated paeonol (1 g/ml, 2 g/ml in 20% ethanol) for paeonol treated group (n=3) just once. At 7 day after irradiation, the clone formation (%) of each groups were measured and then the survival rates of each groups were compared with each other.

(54) Irradiation with high dose of radiation into cancer cell line prevents clone formation.

(55) As shown in Table 1 and FIG. 6, irradiation with 0, 1, 2 and 3 Gy of radiation showed 100%, 77.0%, 49.3% and 30.7% of clone formation, respectively.

(56) In the case of radiation & paeonol treated groups, treatment with 1 g/ml and 2 g/ml of paeonol showed 25.8% and 21.2% of clone formation, respectively.

(57) The clone formation of radiation & paeonol treated groups decreased by 16% (1 g/ml of paeonol) or 31% (2 g/ml of paeonol) compared with that of control group irradiated with 3 Gy of radiation.

(58) Therefore, it was confirmed that treatment of paeonol into lung carcinoma cell line during irradiation enhances the radiosensitization of lung carcinoma cell line.

(59) TABLE-US-00001 TABLE 1 Radiation (Gy) Paeonol (g/ml) Clone formation (%) 0 100 1 100 2 100 1 77 12 72.5 2 71.1 2 49.3 1 46.4 2 43.3 3 30.7 1 25.8 2 21.2

Example 7

Effect of Paeonol on Radiosensitization in Uterine Carcinoma Cell Line, HeLa

(60) Effect of treatment of paeonol during radiation into uterine carcinoma cell line on radiosensitization was investigated.

(61) 10.sup.2-10.sup.3 of human lung carcinoma cell line HeLa was irradiated with various doses of radiation (0, 1, 2, 3 Gy) for control groups (n=3) just once, and was irradiated with 3 Gy of radiation and the above isolated paeonol (1 g/ml, 2 g/ml in 20% ethanol) for paeonol treated group (n=3) just once. At 7 day after irradiation, the clone formation (%) of each groups were measured and then the survival rates of each groups were compared with each other.

(62) As shown in Table 2 and FIG. 7, irradiation with 0, 1, 2, and 3 Gy of radiation showed 100%, 73.8%, 49.4%, and 15.9% of clone formation, respectively.

(63) In the case of radiation & paeonol treated groups, treatment with 1 g/ml and 2 g/ml of paeonol showed 12.3% and 10.9% of clone formation, respectively.

(64) The clone formation of radiation & paeonol treated groups decreased by 23% (1 g/ml of paeonol) or 39% (2 g/ml of paeonol) compared with that of control group irradiated with 3 Gy of radiation.

(65) Therefore, it was confirmed that treatment of paeonol into uterine carcinoma cell line during irradiation enhances the radiosensitization of uterine carcinoma cell line.

(66) TABLE-US-00002 TABLE 2 Radiation (Gy) Paeonol (g/ml) Clone formation (%) 0 100 1 100 2 100 1 73.8 1 73.4 2 72.8 2 49.4 1 45.5 2 44.7 3 15.9 1 12.3 2 10.9

(67) In accordance with the results of Table 1 and Table 2, it was confirmed that the treatment of paeonol into lung carcinoma cell line as well as uterine carcinoma cell line during irradiation enhances the radiosensitization of carcinoma cell lines, so it has enhancing effects on radiotherapy.

Example 8

Effect of Paeonol on Radiosensitization in Lung Carcinoma Cell Line, LLC-Transplanted Animal

(68) Effect of treatment of paeonol during radiation into lung carcinoma cell line-transplanted animal on radiosensitization was investigated.

(69) 110.sup.6 of mouse lung carcinoma cell line, LLC (Lewis lung carcinoma) cell were suspended in 0.2 ml of PBS, and then were injected into left leg of mouse in order to induce the tumor. LLC-transplanted mice were classified into 4 groups such as control group (n=10), irradiation-treated group (n=10), paeonol-treated group (n=10), and irradiation & paeonol-treated group (n=10).

(70) After the volumes of tumor were about 100-200 mm.sup.3 (6-7 day after LLC injection), 5 Gy of .sup.60Co radiation was irradiated to irradiation-treated group and irradiation & paeonol-treated group three times at an interval of 2 or 3 days.

(71) After the existence of tumor was detected by touching with hand (4 day after LLC injection), 5 mg/kg of paeonol dissolved in 20% ethanol was orally administered into paeonol-treated group and irradiation & paeonol group once a day for 20 days. For control group and irradiation group, 20% ethanol was orally administered in the same manner as paeonol-treated group.

(72) Enhancing effects of paeonol in combination with radiation on efficacy of radiotherapy was determined by comparing both tumor growth rates and mouse survival rates of 4 groups with each other groups.

(73) The sizes (width, length) of tumor were measured for 30 days at an interval of 2 or 3 days, and then the volumes of tumor were calculated by the following equation, (width).sup.2(length)0.52. The tumor growth rates were also calculated by converting into the time which it takes for tumor volume to be 2000 mm.sup.3.

(74) As shown in Table 3 and FIG. 8, it took 16 days for tumor volume to be 2000 mm.sup.3 for control group, whereas the tumor growth rates of irradiation-treated group, paeonol-treated group, and irradiation & paeonol-treated group were 19, 19, and 22 days, respectively. The tumor growth rates of irradiation-treated group and paeonol-treated group went down 1.2-fold as compared with that of control group whereas that of irradiation & paeonol-treated group went down 1.4-fold.

(75) TABLE-US-00003 TABLE 3 Tumor growth rate (Time which it takes for tumor Group volume to be 2000 mm.sup.3) LLC-injected group 16 (control group) LLC-injected, 19 irradiation-treated group LLC-injected, 19 paeonol-treated group LLC-injected, 22 irradiation & paeonol-treated group

(76) As shown in FIG. 9, mice of irradiation & paeonol-treated group survived for 78 days whereas mice of control group, irradiation-treated group, and paeonol-treated group survived for 42, 60, and 69 days, respectively. The survival rate of irradiation & paeonol-treated group increased about 2 times compared with that of control group.

(77) In accordance with the results of tumor-transplanted animal experiment, tumor growths of irradiation-treated group and paeonol-treated group were inhibited more than that of control group, and especially the combinational treatment of irradiation and paeonol inhibited tumor growth rate and increased the survival time and survival rate by enhancing radiosensitization with paeonol.

(78) Therefore, it was confirmed that treatment of paeonol enhances the therapeutic efficacy of radiotherapy by increasing the radiosensitization.

Example 9

Effect of Paeonol on Radiosensitization in Melanoma-Transplanted Animal

(79) Effect of treatment of paeonol during radiation into melanoma-transplanted animal on radiosensitization was investigated.

(80) In order to induce primary tumor, B16BL6 melanoma cells (110.sup.6) suspended in 0.2 ml of PBS was subcutaneously injected into C57BL6 female mice (SLC Co., Japan; 6 week-old of body weight) adapted for 1 week.

(81) Melanoma-transplanted mice were classified into 6 groups: control group (n=10), irradiation group (n=10), paeonol-treated groups (0.5 or 1 mg/kg per day) (n=10), and irradiation & paeonol-treated groups (0.5 or 1 mg/kg per day) (n=10), and then it was observed whether the combinational treatment of irradiation and paeonol enhances the radiosensitization of melanoma-transplanted animal.

(82) 5 Gy of .sup.60Co radiation was irradiated to irradiation-treated group and irradiation & paeonol-treated at 4 and 6 day after tumor injection.

(83) 0.5 mg/kg or 1.0 mg/kg of paeonol dissolved in 20% ethanol was orally administered once a day after tumor injection.

(84) For control group and irradiation group, 20% ethanol was orally administered in the same manner as paeonol-treated group.

(85) The sizes (width, length) of tumor were measured for 30 days at an interval of 2 or 3 days, and then the volumes of tumor were calculated by the following equation, (width).sup.2(length)0.52.

(86) During the experiment, any side effects and special features of tested animal were not found except for tumor.

(87) As shown in FIG. 10, tumor volumes of paeonol-treated groups (0.5 mg/kg or 1.0 mg/kg of paeonol) and irradiation-treated group decreased by 6.4% (0.5 mg/kg of paeonol), 9.3% (1.0 mg/kg of paeonol, and 33.1% compared with that of control group, respectively. Irradiation & paeonol-treated groups decreased by 58.9% of the maximum, so it was confirmed that the combinational treatment of irradiation and paeonol is more effective on inhibition of tumor growth by enhancing the radiosensitization of tumor.

(88) In the case of irradiation & paeonol-treated group (1.0 mg/kg of paeonol), tumor growth was more inhibited compared as irradiation & paeonol-treated group (0.5 mg/kg of paeonol), and it means that paeonol has concentration-dependent inhibitory effects on tumor growth.

(89) Therefore, it was confirmed that treatment of paeonol enhances the therapeutic efficacy of radiotherapy by increasing the radiosensitization

Example 10

Toxicity of Paeonol on Cell and Animal

(90) 10-1) Cytotoxicity Test on Cell

(91) In order to examine the cytotoxicity of paeonol, 210.sup.5 of normal human lung cell (BEAS-2B), human lung carcinoma cell (NCI-H460), human uterine carcinoma cell (HeLa) were treated with various concentrations (0.25, 0.5, 1, 2, 10 g/ml) of paeonol for 24 hours, and then viabilities of each cells were measured by MTT assay.

(92) As shown in FIG. 11, all viabilities were decreased by paeonol in the concentration-dependent manner, and normal cell, NCI-H460, and HeLa showed 6%, 18%, and 15% of cytotoxicity in the treatment of 10 g/ml of paeonol, respectively. Also, paeonol did not show enough toxicity to calculate LD.sub.50 for both normal cell and cancer cell.

(93) Therefore, it was confirmed that paeonol has hardly cytotoxicity against normal cell and show a little cytotoxicity against cancer cell.

(94) 10-2) Acute Toxicity Test in Animal

(95) Paeonol of the present invention showed no side effects during administration at the dosage of 100 mg/kg, 50 times as much as 1-5 mg/kg of paeonol which was in vitro effective on radiosensitization, so it seems that paeonol is safe enough for treatment of diseases.

INDUSTRIAL APPLICABILITY

(96) The composition of the present invention has inhibitory effects on angiogenesis, growth of solid tumor, and metastasis of tumor, and it enhances inhibition of tumor growth and survival time of patients in combination with other anti-cancer drugs. Also, the composition of the present invention can be used for the prevention and treatment of angiogenesis-related diseases such as cancer, angioma, angiofibroma, diabetic retinopathy, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involution macula, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis, acne, arthritis, atherosclerosis, obesity and Alzheimer's disease.

(97) Also, the composition of the present invention comprising paeonol for radiosensitization has superior effects on enhancing radiosensitization of various cancers in combination with radiotherapy. The combination of paeonol and irradiation can decrease viability of cancer cell, can inhibit the viability of cancer cell, and can increase survival rates and survival time of cancer patients. The combination of paeonol and lower therapeutic dosage of irradiation can show therapeutic effects of radiotherapy like using high dosage of radiation. So, the combinational treatment of irradiation and paeonol on cancer patients can decrease side effects of radiotherapy using high dosage of radiation such as damage of normal tissue, and can maximize therapeutic efficacy of radiotherapy.

(98) As shown above, paeonol has radiosensitization-enhancing activity as well as anti-angiogenic activity, and so the composition of the present invention comprising paeonol as an active ingredient can be used, especially for treatment of angiogenesis-related disease using radiotherapy.