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WO2007032591A1 - Composition comprising 1-furan-2-yl-3-pyridin-2-yl-pr0pen0ne having anti-angiogenic activity and cancer growth inhibitory activity - Google Patents

Composition comprising 1-furan-2-yl-3-pyridin-2-yl-pr0pen0ne having anti-angiogenic activity and cancer growth inhibitory activity Download PDF

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Publication number
WO2007032591A1
WO2007032591A1 PCT/KR2006/001990 KR2006001990W WO2007032591A1 WO 2007032591 A1 WO2007032591 A1 WO 2007032591A1 KR 2006001990 W KR2006001990 W KR 2006001990W WO 2007032591 A1 WO2007032591 A1 WO 2007032591A1
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cancer
cells
fpp
disease
angiogenesis
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PCT/KR2006/001990
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French (fr)
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Jung Ae Kim
Eung Seok Lee
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Industry-Academic Cooperation Foundation, Yeungnam University
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Priority claimed from KR1020060046746A external-priority patent/KR100779610B1/en
Application filed by Industry-Academic Cooperation Foundation, Yeungnam University filed Critical Industry-Academic Cooperation Foundation, Yeungnam University
Publication of WO2007032591A1 publication Critical patent/WO2007032591A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a composition
  • l-furan-2-yl- 3-pyridin-2-yl-pro ⁇ enone having an anti-angiogenic activity and a cancer growth inhibitory activity.
  • Angiogenesis is a process of creating capillary blood vessels from pre ⁇ existing microvascular networks. Angiogenesis normally occurs during embryonic development, tissue regeneration, wound healing and corpus luteum development that is a change in cyclical female reproductive system; in any case, neovascularization is strictly regulated to progress (Folkman J et al., Int. Rev. Exp. Pathol., 16, pp207-248, 1976).
  • Angiogenesis is a complex process that generally includes the degradation of vascular basement membrane by proteases released by the stimuli of proangiogenic factors! the migration and proliferation of endothelial cells; the formation of lumen due to differentiation of endothelial cells! the reconstruction of blood vessels! and the generation of new capillary vessels.
  • angiogenesis there are diseases induced by angiogenesis that is not regulated autonomously but grows morbidly.
  • diseases associated with angiogenesis occurring in pathological states are exemplified by hemangioma, angiofibroma, vascular malformation and cardiovascular diseases, such as arteriosclerosis, vascular adhesion, scleroedema, etc.
  • Ocular diseases associated with angiogenesis include corneal graft angiogenesis, neovascular glaucoma, diabetic retinopathy, corneal disease induced by angiogenesis, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasia, granular conjunctivitis, etc.
  • angiogenesis-related diseases may include chronic inflammatory diseases such as arthritis, cutaneous diseases such as psoriasis, capillarectasia, pyogenic granuloma, seborrheic dermatitis, acne, Alzheimer's disease and obesity.
  • Tumor growth and metastases are dependent upon angiogenesis (D'Amato RJ et al., Ophthalmology, 102(9), ppl261-1262, 1995 ; Arbiser JL, J. Am. Acad. Dermatol., 34(3), pp486-497, 1996 ; O'Brien KD et al. Circulation, 93(4), pp672-682, 1996 ; Hanahan D et al . , Cell, 86, pp353-364, 1996).
  • angiogenesis plays an important role in the growth and metastasis of cancers.
  • Tumor is supplied with nutrition and oxygen necessary for growth and proliferation through new blood vessels and the new blood vessels infiltrating into the tumors make the cancer cells being metastasized to enter the blood circulation system, thus supporting the metastasis of cancer cells (Folkman and Tyler, Cancer Invasion and metastasis, Biologic mechanisms and Therapy (S.B. Day ed.) Raven press, New York, pp94-103, 19771 Polverini PJ, Crit. Rev. Oral. Biol. Med., 6(3), pp230-247, 1995).
  • the major cause of death in cancer patients is metastasis and the reasons why the chemotherapies or immunotherapies being used clinically at present do not contribute to the increase in the survival rate of cancer patients are directed to metastasis.
  • Typical diseases resulting from angiogenesis includes macular degeneration, diabetic retinopathy, etc. that occur commonly in old age, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by neovascularization, etc. (Adamis AP et al., Angiogenesis, 3, pp9-14, 1999). Among them, diabetic retinopathy that is one of the diabetic complications and a disease that retinal capillaries invade vitreous body to become blind.
  • Psoriasis characterized by red spots and scaly skin is a chronic proliferative disease occurring in skin and is accompanied with pain and malformation. Normally, keratinocytes proliferate once a month, however, in psoriasis patient, the keratinocytes proliferate at least once a week. For such rapid proliferation, a large quantity of blood is required, thus resulting in active angiogenesis (Folkman J, J. Invest. Dermatol., 59, pp40- 48, 1972).
  • angiogenesis inhibitors Since it is possible to apply angiogenesis inhibitors to agents for treating diseases associated with various angiogeneses, a variety of researches aimed at treating such diseases by inhibiting angiogenesis have continued to progress actively. Since such angiogenesis inhibitors should be administrated to patients for a long time, the most ideal inhibitor is one that should have low toxicity and be orally administrated. Accordingly, it is necessary to develop drugs that have low toxicity as angiogenesis inhibitors.
  • l-furan-2-yl-3-pyridin-2-yl-propenone has an excellent anti-angiogenesis activity and a cancer cell growth inhibitory effect and completed the present invention.
  • An object of the present invention is to provide a pharmaceutical composition comprising l-furan-2-yl-3-pyridin-2-yl-propenone for preventing and treating diseases, caused by angiogenesis, and cancer diseases.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising l-furan-2-yl-3- ⁇ yridin-2-yl-propenone expressed by chemistry figure 1 below as an active ingredient for preventing and treating diseases caused by angiogenesis, in combination with pharmaceutically acceptable carriers or excipients:
  • Such diseases caused by angiogenesis includes rheumatic arthritis, osteoarthritis, septic arthritis, psoriasis, corneal ulcer, senile macular degeneration, diabetic retinopathy, proliferative vitreous body retinopathy, premature retinopathy, ocular inflammation, conical cornea, Sjogren's syndrome, myopia eye tumor, cornea graft rejection, abnormal wound intention, bone disease, proteinuria, abdominal aortic aneurysm, regressive cartilage loss due to traumatic joint injury, demyelinating disease of central nervous system, hepatic cirrhosis, glomerular disease, premature rupture of embryonic membrane, inflammatory bowel disease, periodontitis, atherosclerosis, restenosis, inflammatory disease of central nervous system, Alzheimer's disease, skin aging or infiltration and metastasis of cancer.
  • Such cancer diseases include lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bone cancer, pancreatic cancer, skin cancer, cephalic or cervical cancer, skin or eye melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrium carcinoma, cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin' s disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma (PCNSL), spinal-cord tumor, brain stem glioma or pituitary adenoma.
  • CNS central nervous system
  • PCNSL primary
  • the compound of l-furan-2-yl-3-pyridin-2-yl- propenone in accordance with the present invention can be effectively used as a composition for preventing and treating diseases, caused by angiogenesis, and cancer diseases, since it inhibits the neovascularization and the growth of cancer cells in the chorioallantoic membrane models significantly, and also inhibits the growth of cancer and the metastasis of cancer cells in the athymic nude mice models.
  • Fig. 1 shows angiogenesis inhibitory effects of FPP-3 on glioblastoma cells
  • Figs. 2 to 7 show angiogenesis inhibitory effects of FPP-3 in HUVEC cells
  • Fig. 8 depicts volume changes of tumors according to FPP-3 treatments in athymic nude mice models
  • Fig. 9 illustrates cancer cell migration inhibitory effects of FPP-3 in HT-1080 cells
  • Figs. 10 to 13 illustrate cancer cell invasion inhibitory effects in HT-1080 cells of a control group and FPP-3 treated groups by concentrations
  • Figs. 14 and 15 illustrate MMP secretion inhibitory effects of FPP-3 in HT-1080 cells
  • Fig. 16 depicts VEGF secretion inhibitory effects of FPP-3 in HT-1080 cells!
  • Fig. 17 depicts a result of cancer cell toxicity test of FPP-3. [Best Mode]
  • a strong base such as potassium hydroxide, sodium hydroxide and the like, desirably, 34 mg of sodium hydroxide is dissolved in a low alcohol solvent of C 1 to C4 , desirably, ethanol at a temperature of 10 to 30°C under the presence of nitrogen
  • an aldehyde compound desirably, 54.85 mM of 2- ⁇ yridinecarboxaldehyde
  • the compound of l-furan-2-yl-3-pyridin-2-yl- propenone in accordance with the present invention obtained in such manner described above can be effectively used as a composition for inhibiting angiogeneses and for preventing and treating cancer diseases, since it inhibits the increase of neovascularization according to the treatment of neovascularization inducing materials such as vascular endothelial growth factor and fibroblast growth factor in the chicken chorioallantoic membrane (CAM) models, inhibits the neovascularization activated by grafting cancer cells into the chorioallantoic membrane, and further inhibits the volume expansion of glioblastoma in accordance with the above results.
  • neovascularization inducing materials such as vascular endothelial growth factor and fibroblast growth factor in the chicken chorioallantoic membrane (CAM) models
  • the compound of l-furan-2-yl-3- pyridin-2-yl-propenone in accordance with the present invention inhibits angiogenesis by human umbilical endothelial cells (HUVEC) dose-dependentIy, inhibits the volume expansion of tumors in the athymic nude mice models, into which HT-1080 human fibrosarcoma cells are grafted, inhibits metastasis of cancer cells by obstructing the migration and invasion of cancer cells in HT- 1080 human fibrosarcoma cells, and further inhibits the activation and expression of MMP and VEGF.
  • HUVEC human umbilical endothelial cells
  • the present invention provides an efficacious pharmaceutical composition
  • l-furan-2-yl-3-pyridin-2-yl-propenone prepared in the above process as an active ingredient for preventing and treating diseases, caused by angiogenesis, and cancer diseases, in combination with pharmaceutically acceptable carriers or excipients.
  • Doses and ways of application of the pharmaceutical composition comprising l-furan-2-yl-3-pyridin-2-yl-propenone expressed by chemistry figure 1 in accordance with the present invention may be varied based on the formulations and the use purposes thereof.
  • the pharmaceutical composition comprising the compound of the present invention for preventing and treating diseases, caused by angiogenesis, and cancer diseases is composed of 0.1 to 50% by weight of the compound based on the total weight of the composition.
  • composition comprising the compound of the present invention may further comprises suitable carriers, excipients or diluents used commonly in preparing pharmaceutical compositions.
  • the carriers, excipients or diluents that may be contained in the composition comprising the compound of the present invention are exemplified by lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • composition comprising the compound of the present invention may be prepared in various formulations, such as oral formulations including powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., agents for external application, suppository and sterilizing injection solutions.
  • oral formulations including powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., agents for external application, suppository and sterilizing injection solutions.
  • compositions comprising the compound of the present invention may be formulated into various dosage forms using diluents or excipients such as fillers, expanders, bonding agents, humectants, disintegrants, surfactants, etc.
  • Solid dosages for oral administration include tablets, pi 1 lets, powders, granules, capsules, etc.
  • Such solid dosages are prepared by mixing the compound of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium stearate, talc, etc. may be added.
  • Liquid dosage forms for oral administration such as suspensions, internal solutions, emulsions, syrups, etc.
  • Dosage forms for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc.
  • Non-aqueous solvents and suspensions may be prepared using propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate.
  • bases for suppositories witepsol, macrogol, Tween 61, cacao oil, laurinic acid, and glycerogelatine are useful.
  • the dosages of the compound of the present invention may be varied according to various relevant factors, such as age, sex, weight. In general, 0.1 to 100 mg/kg of the compound of the invention can be administrated once or several times a day. Moreover, the dosages of the compound may be increased and decreased according to administration path, severity of disease, sex, weight, age, etc. Accordingly, the dosages do not limit the scope of the present invention in any aspect.
  • the pharmaceutical composition can be administrated to mammals such as mice, rats, livestock, humans, etc. through various paths.
  • mammals such as mice, rats, livestock, humans, etc.
  • it can be administrated by oral and parenteral (e.g., rectal, intravenous, intramuscular, cutaneous, intrauterine or intracerebroventricular injection) administrations.
  • parenteral e.g., rectal, intravenous, intramuscular, cutaneous, intrauterine or intracerebroventricular injection
  • 2-pyridinecarboxaldehyde (5.2 ml, 54.85 mM) was added to 34 mg of solid phase sodium hydroxide solution dissolved in ethanol at 25 ° C under the presence of nitrogen. The resulting solution was mixed with 2-acetylfuran (5 ml, 49.86 mmol) and stirred at 25°C for 3 hours. Subsequently, 80 ml of water was added to the mixture and the mixture was extracted with 120 ml of dichloromethane. The organic layer was washed with 80 m#x2 of water and 80 ml of saturated sodium chloride (NaCl) solvent and dried with sodium sulfate (Na2 SO4 ).
  • Fertilized eggs were purchased from Yeungnam University affiliated stock farm (Gyeongsan, Korea).
  • Chang liver cells that are a line derived from normal human liver, A172 human glioblastoma cells, SK-N-SH human neuroblastoma cells, HT-1376 human bladder cancer cells, HT-29 human colon cancer cells, HepG2 human hepatoma cells, HT-1080 human fibrosarcoma cells were purchased from ATCC (Rockville, MD, USA).
  • human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics (San Diego, CA, USA)
  • Athymic nude mice aged 5 weeks were purchased from Orient Co., Ltd. (Seoul , Korea) .
  • 2-carboxaldehyde, 2-acetylfuran and cortisone acetate were purchased from Aldrich Chemical Co. (St. Louis, MO, USA), fibroblast growth factors (FGF) were from Invitrogen (USA), vascular endothelial growth factors (VEGF) were from R&D systems (Minneapolis, MN, USA), Tetrac and XT199 were supplied from the Pharmaceutical Research Institute of Albany and Albany College of Pharmacy (USA) and whatman filter discs were purchased from Whatman Inc. (UK).
  • TM spectrometry was used under the control of the Xcalibur software.
  • the silica gel Kieselgel 60 F254 (230 to 240 mesh) of Merck was applied thereto.
  • HT-29 human colon cancer cells HT-1376 human bladder cancer cells, HepG2 human hepatoma cells, SK-N-SH human neuroblastoma cells, A172 human glioblastoma cells, HT-1080 human fibrosarcoma cells and Chang liver cells were purchased from ATCC (Rockville, MD, USA).
  • Cells were cultured in a powdered Eagle' s minimum essential medium (MEM, Sigma Chemical Co.) containing 10% fetal bovine serum (GIBCO), 200 IU vd penicillin, 200 ⁇ g/id streptomycin, ImM sodium pyruvate in a humidifying incubator under the conditions of 37°C, 5% CO 2 and 95% air.
  • MEM powdered Eagle' s minimum essential medium
  • GEBCO fetal bovine serum
  • 200 IU vd penicillin 200 IU vd penicillin
  • 200 ⁇ g/id streptomycin ImM sodium pyruvate
  • the culture medium was replaced with a new
  • HUVEC cells were cultured in a flask coated with 0.2% gelatin. Then, the HUVEC cells were cultured in endothelial cell basal medium-2 (EBM-2, Clonetics, San Diego, CA) containing fetal bovine serum (FBS), hydrocortisone, human basic fibroblast growth factor (hFGF-B) , vascular endothelial growth factor (VEGF), human recombinant insulin-like growth factor (R3-IGF-1), ascorbic acid, human epidermal growth factor (hEGF) and heparin.
  • EBM-2 endothelial cell basal medium-2
  • FBS fetal bovine serum
  • hFGF-B human basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • R3-IGF-1 human recombinant insulin-like growth factor
  • ascorbic acid human epidermal growth factor (hEGF) and heparin.
  • HUVEC cells between the first and sixth passages were
  • chorioallantoic membrane assay was carried out (Nguyen M et al . , Microvascular Res., 47, pp31-40, 1994). Fertilized chicken eggs were cultured keeping the temperature at 37°C and the relative humidity at 55%. On the tenth day, the first small hole was made in the region of air sac and the second hole was dug in the flat region of egg, through which a window is to be made, using a hypodermic needle (Greencross Medical Science, Korea).
  • Each egg was deflated through the first hole in the region of the air sac so that the chorioallantoic membrane was split from the egg shell. Subsequently, a window was made by cutting the second hole using a grinding wheel (Multipro 395JA, Dremel, Mexico). Next, whatman filter discs #1 (Whatman Inc. USA) were treated with 3 mg/ml of cortisone acetate and dried. The filter discs were drenched with the fibroblast growth factor (FGF) in a concentration of 15 ng/CAM and the vascular endothelial growth factor (VEGF) in a concentration of 20 ng/CAM.
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • the filter disc was put on the vessels through the window previous made and the compound FPP-3 of Example 1 in accordance with the present invention was dissolved in dimethylsulfoxide (DMSO) and diluted with phosphate buffered saline (PBS) to treat by concentrations (100 ng, 1 ⁇ g, 5 ⁇ g, 10 ⁇ g and 20 ⁇ g ).
  • DMSO dimethylsulfoxide
  • PBS phosphate buffered saline
  • concentrations 100 ng, 1 ⁇ g, 5 ⁇ g, 10 ⁇ g and 20 ⁇ g
  • the CAMs, on which each filter disc was placed were separated and washed with PBS to take images using a stereomicroscope (Stemi SV6 stereomicroscope, Carl Zeiss, Germany) and Image- Pro Plus software (Media Cybernetics; Silver Spring, MD, USA).
  • the branch points were counted and the result data were analyzed (See Table D. [Table 1]
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • the window was made in the same manner as Experimental Example 1 and C6 glioblastoma cells of 1X10 cells/25 ml were inoculated into the chorioallantoic membrane (CAM) through the window according to the method of Gu JW et al . (Gu JW et al . , Cancer, 103(2), pp422-431, 2000). After two hours from the inoculations, 10 ⁇ g/CAM of XT199 and Tetrac were treated, respectively, to positive control groups, and the compound FPP-3 of Example 1 was treated to the portions, into which the cancer cells were inoculated, by concentrations (5 and 10 ⁇ g/CAM) .
  • CAM portions in which tumor tissues were formed, were removed and washed with PBS to take images using a stereomicroscope (Stemi SV6 stereomicroscope, Carl Zeiss, Germany) and Image-Pro Plus software (Media Cybernetics; Silver Spring, MD, USA). Ultimately, the branch points were counted and the result data were analyzed (See Fig. 1). Moreover, the tumor tissues were weighed and kept in 10% formalin or liquid nitrogen for the purposes of RNA isolation and morphological study. [Table 2]
  • HUVEC cells were cultured in a 96-well plate.
  • the respective wells of the 96-well plate were coated with 40 ⁇ l of Matrigel (BD Bioscience, Bedford, MA) and then the 96-well plate was used after kept at 37°C for 30 minutes.
  • Matrigel BD Bioscience, Bedford, MA
  • HUVEC cells were suspended in endothelial cell basal medium-2 (EBM-2,
  • Fig. 2 shows HUVEC cells, not treated with FPP-3, as a control group
  • Fig. 3 shows HUVEC cells treated with 100 mM of FPP-3
  • Fig. 4 shows those treated with 500 mM of FPP-3
  • Fig. 5 shows those with 1 ⁇ M of FPP-3
  • Fig. 6 shows those with 5 ⁇ M of FPP-3
  • Fig. 7 shows those with 10 ⁇ M of FPP-3.
  • Athymic nude mice aged 5 weeks were allowed to take the feed and water freely under the circumstances where the temperature was kept at 21+1°C and the light was regulated such that a light-dark cycle was repeated every 12 hours.
  • Such athymic nude mice were applied to the experiment after kept under such circumstances for at least two days.
  • the animal experiment, to be described hereinafter, was carried out pursuant to the ethical provisions of the Korean National Institute of Health (KNIH).
  • V 1/2(Z,*PF 2 ) wherein V denotes a volume of tumor; L denotes a large diameter; and W denotes a small diameter.
  • the horizontal axis denotes the days after drug treatment based on the first FPP-3 treatment and the vertical axis denotes the volume increases of tumor (nun 3 ) measured in the athymic nude mice models.
  • the horizontal axis denotes the passages of time such as 0, 6, 12, and 24 hours (Hr)
  • the upper photographs denotes the cell migrations in the control group taken after treating HT-1080 cells like the above
  • the lower photographs denotes the cell migrations in the group treated with 30 ⁇ M of FPP-3 under the same conditions as the control group, the respective photographs being magnified 100 times.
  • the numerous HT-1080 cells were cultured in the upper and lower parts of the line of 1 mm in width formed by wounding the HT-1080 cells cultured in the culture plate (0 Hr). Subsequently, with the passage of time of 6 hours, 12 hours and 24 hours, the line of 1 mm in width vanished and the HT-1080 cells migrated along with the line of 1 mm in width in the control group. However, the HT-1080 cells in the group treated with FPP-3 hardly migrated along with the line of 1 mm in width even after the lapse of 24 hours. Accordingly, it could be confirmed that the compound FPP-3 inhibited the cancer cell migration remarkably.
  • the bottom side of the polycarbonate filter was coated with 20 I of type 1 collagen in a concentration of 0.5 mg/m£ and the top side thereof was coated with 20 i of matrigel (BD Bioscience, Bedford, MA) in a concentration of 1.5 mg/iM.
  • the lower region of the polycarbonate filter was filled with a medium containing 10% fetal bovine serum (FBS) and the HT-1080 cells were inoculated into the upper region of the polycarbonate filter.
  • FBS fetal bovine serum
  • the HT-1080 cells inoculated like the above were cultured at 37°C for 18 hours. Subsequently, the cells infiltrating the bottom side of the polycarbonate were fixed with methanol and dyed with hematoxylin and eosin.
  • HT-1080 cells 6 secretion in cancer cells, 1X10 HT-1080 cells were injected into the respective wells of 6-well plate.
  • the HT-1080 cells injected were cultured in MEM medium containing 10% fetal bovine serum (FBS) for 12 hours so that the HT-1080 cells adhered to the plate.
  • FBS fetal bovine serum
  • the MEM medium containing FBS was removed and the plate were washed with a MEM medium containing no serum.
  • the HT-1080 cells were cultured again in MEM medium containing no serum for 24 hours. Then, the supernatants were collected from the 6-well o
  • the supernatants collected from the 6-well plate were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing gelatin. After the electrophoresis, the SDS-PAGE gel was washed twice with buffer solutions (50 mM Tris-HCl, pH 7.5 and 100 mM NaCl, 2.5% Triton X-IOO) to remove the SDS.
  • buffer solutions 50 mM Tris-HCl, pH 7.5 and 100 mM NaCl, 2.5% Triton X-IOO
  • the SDS-PAGE gels were cultured at 37 ° C in buffer solutions (5OmM Tris-HCl, pH7.5, 15OmM NaCl, 1OmM CaCl 2 , 0.02% NaN 3 ) and then dyed with 0.25% Coomassie Brilliant Blue R-250 solution (Sigma Chemical Co., St. Louis, MO) and destained.
  • buffer solutions 5OmM Tris-HCl, pH7.5, 15OmM NaCl, 1OmM CaCl 2 , 0.02% NaN 3
  • Coomassie Brilliant Blue R-250 solution Sigma Chemical Co., St. Louis, MO
  • Fig. 14 the respective lanes shows the activities of MMP-2 and MMP- 9 based on FPP-3 concentrations ( ⁇ M) treated to the respective HT-1080 cells for 24 hours.
  • the MMP-2 and MMP-9 are gelatinases responsible for the degradation of gelatins contained in the SDS-PAGE. Accordingly, it is possible to observe bands in the area where the MMP-2 and MMP-9 exist. First, it can be observed from Fig. 14, where FPP-3 was treated in concentrations from 0 ⁇ M to 30 ⁇ M, that the bands become dimmer as much as the concentration of FPP-3 treated becomes higher. Accordingly, it can be learned that the enzyme activities of MMP-2 and MMP-9 secreted from the HT- 1080 cells become lower as much as the concentration of FPP-3 treated becomes higher.
  • the first lane depicts the results obtained by treating no TPA and FPP-3 to the HT-1080 cells!
  • the second lane denotes the results obtained by treating 12 ng/m£ of TPA and treating no FPP-3 thereto;
  • the third lane denotes the results obtained by treating 12 ng/in£ of TPA and 20 ⁇ M of FPP-3 thereto;
  • the fourth lane denotes the results obtained by treating 12 ng/mt of TPA and 30 ⁇ M of FPP-3 thereto, respectively.
  • the MMP-9 protein having a relatively great size exists in the top side of the SDS-PAGE gel, and PRO-MMP-2 protein, a precursor of MMP-2 protein, and ACTIVE-MMP-2 protein, exist in the bottom side thereof.
  • the MMP-2 and MMP-9 as gelatinases degrade the gelatins contained in the SDS-PAGE to form bands shown in Fig. 15.
  • TPA (12-0-tetradecanoylphorbol-13-acetate)
  • the expressions of such MMP-2 and MMP-9 are increased.
  • the first lane shows no bands in MMP-9 and ACTIVE-MMP-2, since the HT-1080 cells were not stimulated by TPA.
  • a band is shown in PRO-MMP-2, a precursor of MMP-2 protein in the first lane.
  • the second lane denoting the results obtained by treating only TPA, bright bands are observed in MMP-9 and MMP-2.
  • the compound FPP-3 decreases the secretion of MMP-2 and MMP-9 activated by TPA in the HT-1080 cells.
  • VEGF vascular endothelial growth factor
  • HT-1080 cells were cultured in a 24-well plate with media containing no serum under the presence of FPP-3 of different concentrations for 24 hours.
  • HT-1080 cells used in the control group were not treated with FPP-3.
  • the supernatant media in the respective wells were collected to measure the concentrations of VEGF discharged from the cells.
  • the concentrations of VEGF discharged were measured using Quantikine human VEGF ELISA kit (R&D system, Minneapolis, MN). The measurement of VEGF concentrations was carried out pursuant to the usage of the Quantikine human VEGF ELISA kit.
  • Fig. 16 depicts VEGF secretion inhibitory effects of FPP-3 in HT-1080 cells, in which the horizontal axis denotes the concentrations ( ⁇ M) of FPP-3 and the vertical axis denotes the concentrations (ng/roC) of VEGF.
  • the CONTROL represents the results obtained by treating no FPP-3 to HT-1080 cells.
  • VEGF was secreted most much in the control group and the concentration of VEGF secreted from the HT- 1080 cells was decreased as much as the concentration of FPP-3 was increased by 1, 10 and 20 ⁇ M. Accordingly, it can be confirmed that the compound FPP-3 decreases the secretion of VEGF secreted from the HT-1080 cancer cells.
  • DMSO dimethylsulfoxide
  • the compound FPP-3 shows toxicity for human cancer cells of various types only in a high concentration but does not show the toxicity in a low concentration.
  • the toxicity concentration for Chang cells shows more than 50 ⁇ M of 50% inhibitory concentration (IC50), thus representing the low cell toxicity.
  • the above ingredients were mixed with one another and tableted according to a conventional method of preparing tablets, thus preparing a tablet.
  • the above ingredients were mixed with one another and packed in a gelatin capsule according to a conventional method of preparing capsules, thus preparing a capsule.
  • An injection was prepared to 2m£ of an ampoule containing the above ingredients according to a conventional method of preparing injections.
  • the above ingredients were added to purified water to be dissolved and lemon fragrance of optimum dose was added thereto. Then the above ingredients were mixed with one another and purified water was added thereto to regulate the resulting solution as HXM in total.
  • the solution was filled into a brown bottle and sterilized according to a conventional method of preparing liquids, thus preparing a liquid.

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Abstract

Disclosed relates to a composition comprising l-furan-2-yl-3-pyridin-2- yl-propenone expressed by chemistry figure 1 having an ant i -angiogenic activity and a cancer growth inhibitory activity. The compound of the present invention can be effectively used as a composition for preventing and treating diseases, caused by angiogenesis, and cancer diseases, since it inhibits the neovascularization and the growth of cancer cells in the chorioallantoic membrane models significantly, and also inhibits the growth of cancer and the metastasis of cancer cells in the athymic nude mice models.

Description

[DESCRIPTION] [Invention Title]
COMPOSITION COMPRISING 1-FURAN-2-YL-3-PYRIDIN-2-YL-PR0PEN0NE HAVING ANTI-ANGIOGENIC ACTIVITY AND CANCER GROWTH INHIBITORY ACTIVITY [Technical Field]
The present invention relates to a composition comprising l-furan-2-yl- 3-pyridin-2-yl-proρenone having an anti-angiogenic activity and a cancer growth inhibitory activity. [Background Art]
Angiogenesis is a process of creating capillary blood vessels from pre¬ existing microvascular networks. Angiogenesis normally occurs during embryonic development, tissue regeneration, wound healing and corpus luteum development that is a change in cyclical female reproductive system; in any case, neovascularization is strictly regulated to progress (Folkman J et al., Int. Rev. Exp. Pathol., 16, pp207-248, 1976).
The vascular endothelial cells are growing slowly and do not divide well relatively as compared with other types of cells in adult body. Angiogenesis is a complex process that generally includes the degradation of vascular basement membrane by proteases released by the stimuli of proangiogenic factors! the migration and proliferation of endothelial cells; the formation of lumen due to differentiation of endothelial cells! the reconstruction of blood vessels! and the generation of new capillary vessels.
However, there are diseases induced by angiogenesis that is not regulated autonomously but grows morbidly. Such diseases associated with angiogenesis occurring in pathological states are exemplified by hemangioma, angiofibroma, vascular malformation and cardiovascular diseases, such as arteriosclerosis, vascular adhesion, scleroedema, etc. Ocular diseases associated with angiogenesis include corneal graft angiogenesis, neovascular glaucoma, diabetic retinopathy, corneal disease induced by angiogenesis, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasia, granular conjunctivitis, etc. Furthermore, angiogenesis-related diseases may include chronic inflammatory diseases such as arthritis, cutaneous diseases such as psoriasis, capillarectasia, pyogenic granuloma, seborrheic dermatitis, acne, Alzheimer's disease and obesity. Tumor growth and metastases are dependent upon angiogenesis (D'Amato RJ et al., Ophthalmology, 102(9), ppl261-1262, 1995 ; Arbiser JL, J. Am. Acad. Dermatol., 34(3), pp486-497, 1996 ; O'Brien KD et al. Circulation, 93(4), pp672-682, 1996 ; Hanahan D et al . , Cell, 86, pp353-364, 1996).
Especially, angiogenesis plays an important role in the growth and metastasis of cancers. Tumor is supplied with nutrition and oxygen necessary for growth and proliferation through new blood vessels and the new blood vessels infiltrating into the tumors make the cancer cells being metastasized to enter the blood circulation system, thus supporting the metastasis of cancer cells (Folkman and Tyler, Cancer Invasion and metastasis, Biologic mechanisms and Therapy (S.B. Day ed.) Raven press, New York, pp94-103, 19771 Polverini PJ, Crit. Rev. Oral. Biol. Med., 6(3), pp230-247, 1995). The major cause of death in cancer patients is metastasis and the reasons why the chemotherapies or immunotherapies being used clinically at present do not contribute to the increase in the survival rate of cancer patients are directed to metastasis.
Arthritis, a typical disease in inflammatory diseases, is initiated as an autoimmune disorder. As the progression of the disease, the chronic inflammation occurring in the synovial cavity between joints induces angiogenesis to destroy the cartilage. That is, the proliferations of synovial cell and vascular endothelial cell in the synovial cavity are activated by cytokines that induce inflammations, thus resulting in the development of angiogenesis. Finally, the articular cartilage playing a role of cushion is destroyed by articular pannus, a connective tissue layer formed in cartilaginous part (Koch AE et al., Arthritis. Rheum., 29, pp471-479, 1986; Stupack DG et al . , Braz J. Med. Biol. Res., 32(5), pp578-581, 1999; Koch AE, Atrhritis. Rheum., 41(6), pp951-962, 1998).
The ocular diseases, from which millions of people are losing their eyesight all over the world every year, result mainly from angiogenesis (Jeffrey MI et al., /. Clin. Invest., 103, ppl231-1236, 1999). Typical diseases resulting from angiogenesis includes macular degeneration, diabetic retinopathy, etc. that occur commonly in old age, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by neovascularization, etc. (Adamis AP et al., Angiogenesis, 3, pp9-14, 1999). Among them, diabetic retinopathy that is one of the diabetic complications and a disease that retinal capillaries invade vitreous body to become blind.
Psoriasis characterized by red spots and scaly skin is a chronic proliferative disease occurring in skin and is accompanied with pain and malformation. Normally, keratinocytes proliferate once a month, however, in psoriasis patient, the keratinocytes proliferate at least once a week. For such rapid proliferation, a large quantity of blood is required, thus resulting in active angiogenesis (Folkman J, J. Invest. Dermatol., 59, pp40- 48, 1972).
Since it is possible to apply angiogenesis inhibitors to agents for treating diseases associated with various angiogeneses, a variety of researches aimed at treating such diseases by inhibiting angiogenesis have continued to progress actively. Since such angiogenesis inhibitors should be administrated to patients for a long time, the most ideal inhibitor is one that should have low toxicity and be orally administrated. Accordingly, it is necessary to develop drugs that have low toxicity as angiogenesis inhibitors.
It has been reported that l-furan-2-yl-3-pyridin-2-yl-propenone inhibits NF-kB activity to prevent the generations of nitric oxide and tumor necrosis factor-* (TNF-« ) , thus resulting in an anti-inflammation effect (Lee ES et al., Biol. Pharm. Bull., 27(5), pp617~620, 2004), however, there have been no teachings or disclosures that the compound of the present invention has an anti-angiogenic activity and a cancer growth inhibitory activity.
Accordingly, the inventors of the present invention have confirmed that l-furan-2-yl-3-pyridin-2-yl-propenone has an excellent anti-angiogenesis activity and a cancer cell growth inhibitory effect and completed the present invention.
[Disclosure)
[Technical Problem]
An object of the present invention is to provide a pharmaceutical composition comprising l-furan-2-yl-3-pyridin-2-yl-propenone for preventing and treating diseases, caused by angiogenesis, and cancer diseases.
[Technical Solution]
To accomplish the above objects, the present invention provides a pharmaceutical composition comprising l-furan-2-yl-3-ρyridin-2-yl-propenone expressed by chemistry figure 1 below as an active ingredient for preventing and treating diseases caused by angiogenesis, in combination with pharmaceutically acceptable carriers or excipients:
[Chemistry Figure 1]
Figure imgf000005_0001
Such diseases caused by angiogenesis includes rheumatic arthritis, osteoarthritis, septic arthritis, psoriasis, corneal ulcer, senile macular degeneration, diabetic retinopathy, proliferative vitreous body retinopathy, premature retinopathy, ocular inflammation, conical cornea, Sjogren's syndrome, myopia eye tumor, cornea graft rejection, abnormal wound intention, bone disease, proteinuria, abdominal aortic aneurysm, regressive cartilage loss due to traumatic joint injury, demyelinating disease of central nervous system, hepatic cirrhosis, glomerular disease, premature rupture of embryonic membrane, inflammatory bowel disease, periodontitis, atherosclerosis, restenosis, inflammatory disease of central nervous system, Alzheimer's disease, skin aging or infiltration and metastasis of cancer.
Such cancer diseases include lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bone cancer, pancreatic cancer, skin cancer, cephalic or cervical cancer, skin or eye melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrium carcinoma, cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin' s disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma (PCNSL), spinal-cord tumor, brain stem glioma or pituitary adenoma. [Advantageous Effects]
As described above, the compound of l-furan-2-yl-3-pyridin-2-yl- propenone in accordance with the present invention can be effectively used as a composition for preventing and treating diseases, caused by angiogenesis, and cancer diseases, since it inhibits the neovascularization and the growth of cancer cells in the chorioallantoic membrane models significantly, and also inhibits the growth of cancer and the metastasis of cancer cells in the athymic nude mice models. [Description of Drawings]
Fig. 1 shows angiogenesis inhibitory effects of FPP-3 on glioblastoma cells;
Figs. 2 to 7 show angiogenesis inhibitory effects of FPP-3 in HUVEC cells;
Fig. 8 depicts volume changes of tumors according to FPP-3 treatments in athymic nude mice models;
Fig. 9 illustrates cancer cell migration inhibitory effects of FPP-3 in HT-1080 cells;
Figs. 10 to 13 illustrate cancer cell invasion inhibitory effects in HT-1080 cells of a control group and FPP-3 treated groups by concentrations;
Figs. 14 and 15 illustrate MMP secretion inhibitory effects of FPP-3 in HT-1080 cells; Fig. 16 depicts VEGF secretion inhibitory effects of FPP-3 in HT-1080 cells! and
Fig. 17 depicts a result of cancer cell toxicity test of FPP-3. [Best Mode]
The compound of l-furan-2-yl-3-pyridin-2-yl-propenone in accordance with the present invention can be obtained as follows:
A solution, where a strong base, such as potassium hydroxide, sodium hydroxide and the like, desirably, 34 mg of sodium hydroxide is dissolved in a low alcohol solvent of C1 to C4 , desirably, ethanol at a temperature of 10 to 30°C under the presence of nitrogen, is mixed with an aldehyde compound, desirably, 54.85 mM of 2-ρyridinecarboxaldehyde and ketone compound, preferably, 49.86 mM of 2-acetylfuran, and stirred at 10 to 30°C for 2 to 5 hours. Subsequently, 5 to 10 times percent by weight of water is added to the mixture and a water-soluble fraction is extracted with dichloromethane, thus obtaining water-soluble fraction and dichloromethane- soluble fraction. The dichloromethane-soluble fraction that is an organic layer is washed with water and a saturated sodium chloride solvent and dried with sodium sulfate. Collected solvent is dried under reduced pressure and the residue is purified by silica gel column chromatography (SiU2 column chromatography) with a mixed solvent of ethyl acetate and n-hexane, desirably, with a 1:2 mixed solvent of ethyl acetate and n-hexane, thus isolating and identifying the compound expressed by chemistry figure 1.
It has been confirmed that the compound of l-furan-2-yl-3-pyridin-2-yl- propenone in accordance with the present invention obtained in such manner described above can be effectively used as a composition for inhibiting angiogeneses and for preventing and treating cancer diseases, since it inhibits the increase of neovascularization according to the treatment of neovascularization inducing materials such as vascular endothelial growth factor and fibroblast growth factor in the chicken chorioallantoic membrane (CAM) models, inhibits the neovascularization activated by grafting cancer cells into the chorioallantoic membrane, and further inhibits the volume expansion of glioblastoma in accordance with the above results.
Moreover, it has been confirmed that the compound of l-furan-2-yl-3- pyridin-2-yl-propenone in accordance with the present invention inhibits angiogenesis by human umbilical endothelial cells (HUVEC) dose-dependentIy, inhibits the volume expansion of tumors in the athymic nude mice models, into which HT-1080 human fibrosarcoma cells are grafted, inhibits metastasis of cancer cells by obstructing the migration and invasion of cancer cells in HT- 1080 human fibrosarcoma cells, and further inhibits the activation and expression of MMP and VEGF.
Accordingly, the present invention provides an efficacious pharmaceutical composition comprising l-furan-2-yl-3-pyridin-2-yl-propenone prepared in the above process as an active ingredient for preventing and treating diseases, caused by angiogenesis, and cancer diseases, in combination with pharmaceutically acceptable carriers or excipients.
Doses and ways of application of the pharmaceutical composition comprising l-furan-2-yl-3-pyridin-2-yl-propenone expressed by chemistry figure 1 in accordance with the present invention may be varied based on the formulations and the use purposes thereof.
The pharmaceutical composition comprising the compound of the present invention for preventing and treating diseases, caused by angiogenesis, and cancer diseases is composed of 0.1 to 50% by weight of the compound based on the total weight of the composition.
In addition, the composition comprising the compound of the present invention may further comprises suitable carriers, excipients or diluents used commonly in preparing pharmaceutical compositions.
The carriers, excipients or diluents that may be contained in the composition comprising the compound of the present invention are exemplified by lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
The composition comprising the compound of the present invention may be prepared in various formulations, such as oral formulations including powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., agents for external application, suppository and sterilizing injection solutions.
Moreover, the composition comprising the compound of the present invention may be formulated into various dosage forms using diluents or excipients such as fillers, expanders, bonding agents, humectants, disintegrants, surfactants, etc. Solid dosages for oral administration include tablets, pi 1 lets, powders, granules, capsules, etc. Such solid dosages are prepared by mixing the compound of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be added. Liquid dosage forms for oral administration, such as suspensions, internal solutions, emulsions, syrups, etc., may comprise simple diluents, e.g., water and liquid paraffin, as well as various excipients, e.g., humectants, sweeteners, aromatics, preservatives, etc. Dosage forms for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspensions may be prepared using propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate. As bases for suppositories, witepsol, macrogol, Tween 61, cacao oil, laurinic acid, and glycerogelatine are useful.
The dosages of the compound of the present invention may be varied according to various relevant factors, such as age, sex, weight. In general, 0.1 to 100 mg/kg of the compound of the invention can be administrated once or several times a day. Moreover, the dosages of the compound may be increased and decreased according to administration path, severity of disease, sex, weight, age, etc. Accordingly, the dosages do not limit the scope of the present invention in any aspect.
The pharmaceutical composition can be administrated to mammals such as mice, rats, livestock, humans, etc. through various paths. For example, it can be administrated by oral and parenteral (e.g., rectal, intravenous, intramuscular, cutaneous, intrauterine or intracerebroventricular injection) administrations.
Hereinafter, the present invention will now be described in detail with reference to the accompanying drawings, in which examples, formulation examples and experimental examples of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the examples, formulation examples and experimental examples set forth herein. Rather, these examples, formulation examples and experimental examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Example 1'- Preparation of l-furan-2-yl-3-pyridin-2-yl-ρropenone
2-pyridinecarboxaldehyde (5.2 ml, 54.85 mM) was added to 34 mg of solid phase sodium hydroxide solution dissolved in ethanol at 25°C under the presence of nitrogen. The resulting solution was mixed with 2-acetylfuran (5 ml, 49.86 mmol) and stirred at 25°C for 3 hours. Subsequently, 80 ml of water was added to the mixture and the mixture was extracted with 120 ml of dichloromethane. The organic layer was washed with 80 m#x2 of water and 80 ml of saturated sodium chloride (NaCl) solvent and dried with sodium sulfate (Na2 SO4 ). Solvent was dried under reduced pressure and the residue was purified by silica gel column chromatography (EtOAc:n-hexane = 1:2, v:v) to give 3.84 g of yellow crystalline l-furan-2-yl-3-pyridin-2-yl-propenone having the following material properties (yield: 38.7%, hereinafter, referred to as FPP-3).
TLC(EtOAc: n-Hexane = 1:2, v:v), Rf =0.167
1H-NMR (250 MHz, CDCl3): δ 8.70 (ddd, J = 4.8, 1.7, 0.9Hz, 1 H,
pyridine H-6), 7.97 (d, J = 15.4Hz, 1 H, -CH=CH-CO), 7.84 (d, J = 15.4Hz, 1 H, -CH=CH-CO-), 7.75 (dt , J = 7.7, 1.8Hz, 1 H, pyridine H-4) , 7.68 (dd, J = 1.7, 0.7Hz, 1 H, furan H-5) , 7.48 (dt , J= 7.8, 1.2Hz, 1 H, pyridine H-3), 7.42 (dd, J= 3.6, 0.7Hz, 1 H, furan H-3), 7.31 (ddd, J= 7.6, 4.8, 1.1Hz, 1 H, pyridine H-5), 6.61 (dd, J= 3.6, 1.7Hz, 1 H, furan H-4)
Reference Example 1: Analysis of experimental materials, reagents and equipment
Fertilized eggs were purchased from Yeungnam University affiliated stock farm (Gyeongsan, Korea). Chang liver cells that are a line derived from normal human liver, A172 human glioblastoma cells, SK-N-SH human neuroblastoma cells, HT-1376 human bladder cancer cells, HT-29 human colon cancer cells, HepG2 human hepatoma cells, HT-1080 human fibrosarcoma cells were purchased from ATCC (Rockville, MD, USA). Moreover, human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics (San Diego, CA, USA)
Athymic nude mice aged 5 weeks were purchased from Orient Co., Ltd. (Seoul , Korea) .
2-carboxaldehyde, 2-acetylfuran and cortisone acetate were purchased from Aldrich Chemical Co. (St. Louis, MO, USA), fibroblast growth factors (FGF) were from Invitrogen (USA), vascular endothelial growth factors (VEGF) were from R&D systems (Minneapolis, MN, USA), Tetrac and XT199 were supplied from the Pharmaceutical Research Institute of Albany and Albany College of Pharmacy (USA) and whatman filter discs were purchased from Whatman Inc. (UK).
For the structure identification of synthesized substances, H-NMR spectra were acquired using a Bruker AMX 250 MHz model. As liquid chromatography/mass spectrometry, Finnigan LCQ Advantage LC/MS/MS
TM spectrometry was used under the control of the Xcalibur software. As thin- layer chromatography and column chromatography, the silica gel Kieselgel 60 F254 (230 to 240 mesh) of Merck was applied thereto.
Reference Example 2: Cell culture
HT-29 human colon cancer cells, HT-1376 human bladder cancer cells, HepG2 human hepatoma cells, SK-N-SH human neuroblastoma cells, A172 human glioblastoma cells, HT-1080 human fibrosarcoma cells and Chang liver cells were purchased from ATCC (Rockville, MD, USA). Cells were cultured in a powdered Eagle' s minimum essential medium (MEM, Sigma Chemical Co.) containing 10% fetal bovine serum (GIBCO), 200 IU vd penicillin, 200 μg/id streptomycin, ImM sodium pyruvate in a humidifying incubator under the conditions of 37°C, 5% CO2 and 95% air. Here, the culture medium was replaced with a new one once every other day. ConfluentIy grown cells were trypsin- treated with 0.25% trypsin-EDTA solution and subjected to subculture.
Meanwhile, HUVEC cells were cultured in a flask coated with 0.2% gelatin. Then, the HUVEC cells were cultured in endothelial cell basal medium-2 (EBM-2, Clonetics, San Diego, CA) containing fetal bovine serum (FBS), hydrocortisone, human basic fibroblast growth factor (hFGF-B) , vascular endothelial growth factor (VEGF), human recombinant insulin-like growth factor (R3-IGF-1), ascorbic acid, human epidermal growth factor (hEGF) and heparin. In the present experiment, HUVEC cells between the first and sixth passages were used.
Experimental Example 1'- Angiogenesis inhibitory effects of FPP-3 via CAM assay
To identify anti-angiogenic effect in vivo, the following chorioallantoic membrane assay was carried out (Nguyen M et al . , Microvascular Res., 47, pp31-40, 1994). Fertilized chicken eggs were cultured keeping the temperature at 37°C and the relative humidity at 55%. On the tenth day, the first small hole was made in the region of air sac and the second hole was dug in the flat region of egg, through which a window is to be made, using a hypodermic needle (Greencross Medical Science, Korea).
Each egg was deflated through the first hole in the region of the air sac so that the chorioallantoic membrane was split from the egg shell. Subsequently, a window was made by cutting the second hole using a grinding wheel (Multipro 395JA, Dremel, Mexico). Next, whatman filter discs #1 (Whatman Inc. USA) were treated with 3 mg/ml of cortisone acetate and dried. The filter discs were drenched with the fibroblast growth factor (FGF) in a concentration of 15 ng/CAM and the vascular endothelial growth factor (VEGF) in a concentration of 20 ng/CAM.
The filter disc was put on the vessels through the window previous made and the compound FPP-3 of Example 1 in accordance with the present invention was dissolved in dimethylsulfoxide (DMSO) and diluted with phosphate buffered saline (PBS) to treat by concentrations (100 ng, 1 μg, 5 μg, 10 μg and 20 μg ). After 3 days from the drug treatments, the CAMs, on which each filter disc was placed, were separated and washed with PBS to take images using a stereomicroscope (Stemi SV6 stereomicroscope, Carl Zeiss, Germany) and Image- Pro Plus software (Media Cybernetics; Silver Spring, MD, USA). Ultimately, the branch points were counted and the result data were analyzed (See Table D. [Table 1]
Figure imgf000013_0001
As shown in Table 1, it could be learned that the increase rate of neovascularization according to the treatment of neovascularization inducing materials such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) was decreased owing to FPP-3 treated thereto.
Experimental Example 2'- Angiogenesis inhibitory effects of FPP-3 using chicken CAM models into which cancer cells were grafted
To each fertilized egg on the tenth day of the culture, the window was made in the same manner as Experimental Example 1 and C6 glioblastoma cells of 1X10 cells/25 ml were inoculated into the chorioallantoic membrane (CAM) through the window according to the method of Gu JW et al . (Gu JW et al . , Cancer, 103(2), pp422-431, 2000). After two hours from the inoculations, 10 μg/CAM of XT199 and Tetrac were treated, respectively, to positive control groups, and the compound FPP-3 of Example 1 was treated to the portions, into which the cancer cells were inoculated, by concentrations (5 and 10 μg/CAM) . Then, the samples were cultured further for 7 days. The CAM portions, in which tumor tissues were formed, were removed and washed with PBS to take images using a stereomicroscope (Stemi SV6 stereomicroscope, Carl Zeiss, Germany) and Image-Pro Plus software (Media Cybernetics; Silver Spring, MD, USA). Ultimately, the branch points were counted and the result data were analyzed (See Fig. 1). Moreover, the tumor tissues were weighed and kept in 10% formalin or liquid nitrogen for the purposes of RNA isolation and morphological study. [Table 2]
Treatment Branch points ± SEM % of inhibi t ion + SEM
C6 (1 X 10 cel ls/CAM) 187.0 ± 11.9
C6 + XT199 (10«g) 146.1 ± 10.3 21.8 + 5.5
C6 + Tetrac (lOrøO 181.7 + 13.1 2.9 ± 7.0
C6 + FPP-3 (5 UR) 111.5 + 12.2 40.4 + 6.5
C6 + FPP-3 (10 UK) 118.3 ± 11.7 36.8 + 6.3
[Table 3]
Figure imgf000014_0001
As shown in Table 2, the neovascularization activated in the chorioallantoic membrane, into which cancer cells were grafted, was inhibited owing to FPP-3 treated thereto. Moreover, as shown in Table 3, the volume expansion of glioblastoma was also inhibited in accordance with the above results shown in Table 2.
Experimental Example 3: Angiogenesis inhibitory effects of FPP-3 on HUVEC cells To examine angiogenesis inhibitory effects of FPP-3 on HUVEC cells, HUVEC cells were cultured in a 96-well plate. Here, the respective wells of the 96-well plate were coated with 40 μl of Matrigel (BD Bioscience, Bedford, MA) and then the 96-well plate was used after kept at 37°C for 30 minutes.
HUVEC cells were suspended in endothelial cell basal medium-2 (EBM-2,
4
Clonetics, San Diego, CA) containing 2% of fetal bovine serum (FBS). 1X10 suspended HUVEC cells were injected into the respective wells coated with the matrigel. Subsequently, FPP-3 was added to the respective wells by concentrations and the resulting wells were kept for 7 hours. Photographs of HUVEC cells treated like this were taken by a digital camera connected with an inverted microscope and the photographs were shown in Figs. 2 to 7.
Fig. 2 shows HUVEC cells, not treated with FPP-3, as a control group; Fig. 3 shows HUVEC cells treated with 100 mM of FPP-3; Fig. 4 shows those treated with 500 mM of FPP-3; Fig. 5 shows those with 1 μM of FPP-3; Fig. 6 shows those with 5 μM of FPP-3; and Fig. 7 shows those with 10 μM of FPP-3.
First, referring to Fig. 2, it could be observed that numerous vessels were formed in the control group. Then, it could be learned from the rest figures that the amount of angiogenesis was decreased as much as the concentration of FPP-3 was increased. Accordingly, it was confirmed that FPP- 3 inhibited angiogenesis of HUVEC cells.
Experimental Example 4: Angiogenesis inhibitory effects of FPP-3 using athymic nude mice models into which cancer cells were grafted
Athymic nude mice aged 5 weeks were allowed to take the feed and water freely under the circumstances where the temperature was kept at 21+1°C and the light was regulated such that a light-dark cycle was repeated every 12 hours. Such athymic nude mice were applied to the experiment after kept under such circumstances for at least two days. The animal experiment, to be described hereinafter, was carried out pursuant to the ethical provisions of the Korean National Institute of Health (KNIH).
100 μl of MEM medium containing 5XlQ6 cultured HT-1080 cells was hypodermically injected to the right side of the mouse, respectively. After ten days, the volumes of tumors generated were measured. Subsequently, the tumors were measured twice a week. Small diameters and large diameters of tumor were measured, respectively, using calipers and the volumes of tumors were calculated based on math figure 1 below: [Math Figure 1]
V= 1/2(Z,*PF2) wherein V denotes a volume of tumor; L denotes a large diameter; and W denotes a small diameter.
From when the volume of tumor generated in the athymic nude mice became 200 mm3 calculated based on mathematical formula 1 like above, the compound FPP-3 of Example 1 in accordance with the present invention was orally administrated to the mice every day and the volume changes of tumors were measured. Here, the dosage of FPP-3 was set at 1 mg for 1 kg and the drug treatments were made for 20 days. Next, further description will now be made with reference to Fig. 8, a graph depicting volume changes of tumors according to FPP-3 treatments in athymic nude mice models.
In the figure, the horizontal axis denotes the days after drug treatment based on the first FPP-3 treatment and the vertical axis denotes the volume increases of tumor (nun3) measured in the athymic nude mice models.
The experimental results of the athymic nude mice, to which FPP-3 was treated every day, were represented by " FPP-3" and those, to which FPP-3 was not treated, were expressed by " CONTROL" in the figure. Here, to decrease the experimental error, the experiment was carried out with the respective groups including five athymic nude mice models.
As a result, it could be observed as depicted in Fig. 8 that the volume increase rate of the tumors in the athymic nude mice, to which FPP-3 was administrated every day, was noticeable decreased as compared with those in the control group.
Experimental Example 5: Migration inhibitory effects of FPP-3 on cancer cells 25 μg/mC of mitomycin C was pretreated to HT-1080 cancer cells, cultured 90% confluentIy, for 30 minutes. Then, the cancer cells pretreated were wounded using a tip in the culture plate so that a line having a width of 1 mm was generated (Mi-Sung Kim et al . , Cancer Research, 63, 5454-5461, 2003). Subsequently, HT-1080 cells in the culture plate were washed with hanks balanced salt solution (HBSS) and the migrations of cancer cells were observed with the passage of time.
The results were depicted in Fig. 9, photographs illustrating the cancer cell migration inhibitory effects of FPP-3 in HT-1080 cells.
In the figure, the horizontal axis denotes the passages of time such as 0, 6, 12, and 24 hours (Hr), the upper photographs denotes the cell migrations in the control group taken after treating HT-1080 cells like the above and the lower photographs denotes the cell migrations in the group treated with 30 μM of FPP-3 under the same conditions as the control group, the respective photographs being magnified 100 times.
It was seen that the numerous HT-1080 cells were cultured in the upper and lower parts of the line of 1 mm in width formed by wounding the HT-1080 cells cultured in the culture plate (0 Hr). Subsequently, with the passage of time of 6 hours, 12 hours and 24 hours, the line of 1 mm in width vanished and the HT-1080 cells migrated along with the line of 1 mm in width in the control group. However, the HT-1080 cells in the group treated with FPP-3 hardly migrated along with the line of 1 mm in width even after the lapse of 24 hours. Accordingly, it could be confirmed that the compound FPP-3 inhibited the cancer cell migration remarkably.
Experimental Example 6- Metastasis inhibitory effects of FPP-3 on cancer cells
To examine the metastasis inhibitory effects of FPP-3, the following in vitro experiment was carried out using HT-1080 cells (Mi-Sung Kim et al., Cancer Research, 63, 5454-5461, 2003; Sang-0h Yoon et al . , The Journal of Biological Chemistry, 276, 20085-20092, 2001; Sonia Zorzet et al . , The Journal of Pharmacology and Experimental Therapeutics, 295, 927-933, 2000). The culture plate applied hereto was a 24-well plate (Corning Costar, Cambridge, MA) containing a polycarbonate filter having a plurality of pores of 8mm in size. The bottom side of the polycarbonate filter was coated with 20 I of type 1 collagen in a concentration of 0.5 mg/m£ and the top side thereof was coated with 20 i of matrigel (BD Bioscience, Bedford, MA) in a concentration of 1.5 mg/iM. Here, the lower region of the polycarbonate filter was filled with a medium containing 10% fetal bovine serum (FBS) and the HT-1080 cells were inoculated into the upper region of the polycarbonate filter. The HT-1080 cells inoculated like the above were cultured at 37°C for 18 hours. Subsequently, the cells infiltrating the bottom side of the polycarbonate were fixed with methanol and dyed with hematoxylin and eosin. The results were depicted in Figs. 10 to 13, microscopic photographs of the bottom sides of polycarbonate filters taken at 400 magnifications, the photographs illustrating the cancer cell penetrating powers in the control group, 10 μM FPP-3 treated group, 20 μM FPP-3 treated group and 30 μM FPP- 3 treated group, respectively.
With reference to those figures, it could be learned that the cancer cells infiltrating into the bottom sides of the polycarbonate filters in the FPP-3 treated groups were decreased sharply as compared with those in the control group. Accordingly, it could be confirmed that the compound FPP-3 inhibited the cancer cell metastasis dose-dependentIy.
Experimental Example 7' MMP secretion inhibitory effects of FPP-3 on cancer cells
To examine the effects of FPP-3 on matrix metal loproteinase (MMP)
6 secretion in cancer cells, 1X10 HT-1080 cells were injected into the respective wells of 6-well plate. The HT-1080 cells injected were cultured in MEM medium containing 10% fetal bovine serum (FBS) for 12 hours so that the HT-1080 cells adhered to the plate. Next, the MEM medium containing FBS was removed and the plate were washed with a MEM medium containing no serum. Subsequently, the HT-1080 cells were cultured again in MEM medium containing no serum for 24 hours. Then, the supernatants were collected from the 6-well o
plate and subjected to gelatin zyraography (Herron et al . , J. Biol. Chem., 261, 2814-2818, 1986).
Briefly explaining the process of the gelatin zymography, the supernatants collected from the 6-well plate were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing gelatin. After the electrophoresis, the SDS-PAGE gel was washed twice with buffer solutions (50 mM Tris-HCl, pH 7.5 and 100 mM NaCl, 2.5% Triton X-IOO) to remove the SDS. Subsequently, the SDS-PAGE gels were cultured at 37°C in buffer solutions (5OmM Tris-HCl, pH7.5, 15OmM NaCl, 1OmM CaCl2 , 0.02% NaN3 ) and then dyed with 0.25% Coomassie Brilliant Blue R-250 solution (Sigma Chemical Co., St. Louis, MO) and destained.
Moreover, to investigate the effects of FPP-3 on MMP activity induced by TPA, 12 ng/m-β of TPA was treated also when treating FPP-3 like above.
The experimental results were depicted in Figs. 14 and 15 illustrating MMP secretion inhibitory effects of FPP-3 in HT-1080 cells.
In Fig. 14, the respective lanes shows the activities of MMP-2 and MMP- 9 based on FPP-3 concentrations (μM) treated to the respective HT-1080 cells for 24 hours.
Here, the MMP-2 and MMP-9 are gelatinases responsible for the degradation of gelatins contained in the SDS-PAGE. Accordingly, it is possible to observe bands in the area where the MMP-2 and MMP-9 exist. First, it can be observed from Fig. 14, where FPP-3 was treated in concentrations from 0 μM to 30 μM, that the bands become dimmer as much as the concentration of FPP-3 treated becomes higher. Accordingly, it can be learned that the enzyme activities of MMP-2 and MMP-9 secreted from the HT- 1080 cells become lower as much as the concentration of FPP-3 treated becomes higher.
Next, referring to Fig. 15, the first lane depicts the results obtained by treating no TPA and FPP-3 to the HT-1080 cells! the second lane denotes the results obtained by treating 12 ng/m£ of TPA and treating no FPP-3 thereto; the third lane denotes the results obtained by treating 12 ng/in£ of TPA and 20 μM of FPP-3 thereto; and the fourth lane denotes the results obtained by treating 12 ng/mt of TPA and 30 μM of FPP-3 thereto, respectively. Here, the MMP-9 protein having a relatively great size exists in the top side of the SDS-PAGE gel, and PRO-MMP-2 protein, a precursor of MMP-2 protein, and ACTIVE-MMP-2 protein, exist in the bottom side thereof.
Here, the MMP-2 and MMP-9 as gelatinases degrade the gelatins contained in the SDS-PAGE to form bands shown in Fig. 15. Under the presence of TPA (12-0-tetradecanoylphorbol-13-acetate) , the expressions of such MMP-2 and MMP-9 are increased. Accordingly, the first lane shows no bands in MMP-9 and ACTIVE-MMP-2, since the HT-1080 cells were not stimulated by TPA. However, a band is shown in PRO-MMP-2, a precursor of MMP-2 protein in the first lane. In the second lane denoting the results obtained by treating only TPA, bright bands are observed in MMP-9 and MMP-2. Moreover, it can be learned from the third and fourth lanes denoting the results obtained by treating 20 μM of FPP-3 and 30 μM of FPP-3, respectively, that the band brightness of MMP-2 and MMP-9 become dimmer as much as the concentration of FPP-3 treated becomes higher.
Accordingly, it can be confirmed that the compound FPP-3 decreases the secretion of MMP-2 and MMP-9 activated by TPA in the HT-1080 cells.
Experimental Example 8". VEGF secretion inhibitory effects of FPP-3 on cancer cells
To investigate the effects of FPP-3 on vascular endothelial growth factor (VEGF) secretion, the following experiment was carried out with HT- 1080 cells. The VEGF is one of the cytokines secreted from cancer cells of various types and playing a role in developing angiogenesis.
HT-1080 cells were cultured in a 24-well plate with media containing no serum under the presence of FPP-3 of different concentrations for 24 hours. HT-1080 cells used in the control group were not treated with FPP-3. After the culture for 24 hours, the supernatant media in the respective wells were collected to measure the concentrations of VEGF discharged from the cells. The concentrations of VEGF discharged were measured using Quantikine human VEGF ELISA kit (R&D system, Minneapolis, MN). The measurement of VEGF concentrations was carried out pursuant to the usage of the Quantikine human VEGF ELISA kit.
Fig. 16 depicts VEGF secretion inhibitory effects of FPP-3 in HT-1080 cells, in which the horizontal axis denotes the concentrations (μM) of FPP-3 and the vertical axis denotes the concentrations (ng/roC) of VEGF. Here, the CONTROL represents the results obtained by treating no FPP-3 to HT-1080 cells.
Referring to the figure, it can be seen that VEGF was secreted most much in the control group and the concentration of VEGF secreted from the HT- 1080 cells was decreased as much as the concentration of FPP-3 was increased by 1, 10 and 20 μM. Accordingly, it can be confirmed that the compound FPP-3 decreases the secretion of VEGF secreted from the HT-1080 cancer cells.
Experimental Example 9: Cell toxicity assessment of FPP-3
To assess the cell survival rate, the respective cells of Reference Example 2 were cultured for 4 to 5 days and injected into 24-well plate in a
4 density of 5X10 cells/well and the media of the respective wells were set at
Im-C. Subsequently, FPP-3 was treated thereto in concentrations of 2, 10, 25,
50, 100 and 250 μM, respectively and then cultured for 48 hours. 100 μl of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide! 5 g MTT/
I in H2O) was added thereto and further cultured for 4 hours. Then, 200 μl of dimethylsulfoxide (DMSO) was added to the respective wells containing the corresponding cells and mixed with pipet to resolve the MTT crystals reduced. Relative cell survival rates were assessed by scanning with a microplate reader (Molecular Devices, Menlo Park, CA) having 540 nm filter (See Table 4).
As illustrated in Fig. 17, the compound FPP-3 shows toxicity for human cancer cells of various types only in a high concentration but does not show the toxicity in a low concentration. Moreover, as shown in Table 4, the toxicity concentration for Chang cells, a normal cell, shows more than 50 μM of 50% inhibitory concentration (IC50), thus representing the low cell toxicity. [Table 4]
Figure imgf000022_0001
Hereinafter, the formulation examples for the pharmaceutical composition comprising the compound of FPP-3 of the present invention will now be exemplified, however, which should not be limited thereto. Rather, these examples are provided no more than to explain the present invention in more detail .
Formulation Example 1: Preparation of Powders
FPP-3300 mg
Lactose 100 mg
Talc 10 mg
The above ingredients were mixed with one another and packed in an airtight bag, thus preparing a powder.
Formulation Example 2: Preparation of Tablets
FPP-350 mg
Corn starch 100 mg
Lactose 100 mg
Magnesium stearate 2 mg
The above ingredients were mixed with one another and tableted according to a conventional method of preparing tablets, thus preparing a tablet.
Formulation Example 3: Preparation of Capsules
FPP-350 mg
Corn starch 100 mg ΔΔ
Lactose 100 mg
Magnesium stearate 2 mg
The above ingredients were mixed with one another and packed in a gelatin capsule according to a conventional method of preparing capsules, thus preparing a capsule.
Formulation Example 4: Preparation of Injections
FPP-350 mg
Sterile distilled water for injection Proper amount pH regulator Proper amount
An injection was prepared to 2m£ of an ampoule containing the above ingredients according to a conventional method of preparing injections.
Formulation Example 5: Preparation of Liquids
FPP-3100 mg
Isomerized sugar 1Og
Mannitol 5 g
Purified water Proper amount
The above ingredients were added to purified water to be dissolved and lemon fragrance of optimum dose was added thereto. Then the above ingredients were mixed with one another and purified water was added thereto to regulate the resulting solution as HXM in total. The solution was filled into a brown bottle and sterilized according to a conventional method of preparing liquids, thus preparing a liquid.

Claims

[CLAIMS] [Claim 1]
A pharmaceutical composition comprising l-furan-2-yl-3-pyridin-2-yl- propenone expressed by chemistry figure 1 below as an active ingredient for preventing and treating diseases caused by angiogenesis, in combination with a pharmaceutically acceptable carrier or excipient: [Chemistry Figure 1]
Figure imgf000024_0001
[Claim 2]
The pharmaceutical composition for preventing and treating diseases caused by angiogenesis as recited in claim 1, wherein the disease caused by angiogenesis is at least one selected from the group consisting of rheumatic arthritis, osteoarthritis, septic arthritis, psoriasis, corneal ulcer, senile macular degeneration, diabetic retinopathy, proliferative vitreous body retinopathy, premature retinopathy, ocular inflammation, conical cornea, Sjogren's syndrome, myopia eye tumor, cornea graft rejection, abnormal wound intention, bone disease, proteinuria, abdominal aortic aneurysm, regressive cartilage loss due to traumatic joint injury, demyelinating disease of central nervous system, hepatic cirrhosis, glomerular disease, premature rupture of embryonic membrane, inflammatory bowel disease, periodontitis, atherosclerosis, restenosis, inflammatory disease of central nervous system, Alzheimer's disease, skin aging and infiltration metastasis of cancer.
[Claim 3]
A pharmaceutical composition comprising l-furan-2-yl~3-pyridin-2-yl- propenone as an active ingredient for preventing and treating cancer diseases, in combination with a pharmaceutically acceptable carrier or excipient . [Claim 4]
The pharmaceutical composition for preventing and treating caner diseases as recited in claim 3, wherein the cancer disease is at least one selected from the group consisting of lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bone cancer, pancreatic cancer, skin cancer, cephalic or cervical cancer, skin or eye melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrium carcinoma, cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin' s disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma (PCNSL), spinal-cord tumor, brain stem glioma and pituitary adenoma.
PCT/KR2006/001990 2005-09-13 2006-05-25 Composition comprising 1-furan-2-yl-3-pyridin-2-yl-pr0pen0ne having anti-angiogenic activity and cancer growth inhibitory activity WO2007032591A1 (en)

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WO2011106599A1 (en) * 2010-02-25 2011-09-01 Alcon Research, Ltd. Method of accelerating corneal wound healing
WO2014160940A3 (en) * 2013-03-29 2014-12-31 Avoscience, Llc Lipidic furan, pyrrole, and thiophene compounds for treatment of cancer, neurological disorders, and fibrotic disorders
US9371302B2 (en) 2013-03-29 2016-06-21 Avoscience, Llc Lipidic furan, pyrrole, and thiophene compounds for treatment of cancer
US9814694B2 (en) 2013-03-29 2017-11-14 Avioscience, LLC Lipidic furan, pyrrole, and thiophene compounds for treatment of cancer, neurological disorders, and fibrotic disorders
US10085962B2 (en) 2013-03-29 2018-10-02 Avoscience, Llc Lipidic furan, pyrrole, and thiophene compounds for treatment of cancer, neurological disorders, and fibrotic disorders
US10525031B2 (en) 2013-03-29 2020-01-07 Avoscience, Llc Lipid furan, pyrrole, and thiophene compounds for treatment of cancer, neurological disorders, and fibrotic disorders
US11058663B2 (en) 2013-03-29 2021-07-13 Avoscience, Llc Lipidic furan, pyrrole, and thiophene compounds for treatment of cancer, neurological disorders, and fibrotic disorders
US11833129B2 (en) 2013-03-29 2023-12-05 Avoscience, Llc Thiophene compound for treatment of exfoliating glaucoma
US10905673B2 (en) 2016-04-27 2021-02-02 Avoscience, Llc Lipidic furan, pyrrole, and thiophene compounds for use in the treatment of atrophic vaginitis
US11602518B2 (en) 2016-04-27 2023-03-14 Avoscience, Llc Lipidic furan, pyrrole, and thiophene compounds for use in the treatment of atrophic vaginitis

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