WO2001056566A1 - Compositions inhibitrices de neovascularisation et procede correspondant - Google Patents

Compositions inhibitrices de neovascularisation et procede correspondant Download PDF

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Publication number
WO2001056566A1
WO2001056566A1 PCT/JP2001/000672 JP0100672W WO0156566A1 WO 2001056566 A1 WO2001056566 A1 WO 2001056566A1 JP 0100672 W JP0100672 W JP 0100672W WO 0156566 A1 WO0156566 A1 WO 0156566A1
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cancer
neovascularization
sba
tumor
benzylidene
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PCT/JP2001/000672
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English (en)
Japanese (ja)
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Mutsuyuki Kochi
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Mutsuyuki Kochi
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Publication of WO2001056566A1 publication Critical patent/WO2001056566A1/fr

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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Neovascularization inhibitor composition and method for inhibiting neovascularization are provided.
  • the present invention relates to a novel antiangiogenic composition. More specifically, the present invention relates to an inhibitor composition for neovascularization, which comprises 5,6-0-benzylidene-raescorbic acid monosodium salt as an active ingredient.
  • the present invention provides intravenous, subcutaneous or intraperitoneal administration of 5,6-0-benzylidene-raescorbate monosodium salt, or intraperitoneal administration, or oral administration to the peripheral cornea of the human eye, or the choroid and / or )
  • the present invention encompasses a composition that suppresses the formation of new blood vessels in the part, suppresses the growth and metastasis of cancer or tumor due to the formation of new blood vessels, and a method for suppressing the same.
  • the present invention includes a composition for suppressing enhanced formation of new blood vessels in a rheumatic diseased tissue of rheumatoid arthritis and suppressing pain caused thereby, and a method thereof.
  • the present invention relates to the use of 5,6-0-benzylidene-raescorbic acid mononatridium salt for the production of the above-mentioned various pharmaceutical compositions which are administered to suppress the formation of new blood vessels.
  • the present invention relates to the growth and migration of vascular endothelial cells containing 5,6-0-benzylidene-L-ascorbate monosodium salt as an active ingredient, particularly vascular endothelial cell growth factor and / or fibroblast growth factor. And other growth factors enhance the proliferation and migration of vascular endothelial cells and the resulting shape of new blood vessels Inhibitors that inhibit the enhancement of growth.
  • the present invention suppresses the action of vascular endothelial cell growth factor and / or the action of fibroblast growth factor, which activates the transcription factor NF-kappaB present in vascular endothelial cells, and the action of other growth factors. It relates to an inhibitor and an inhibition method.
  • angiogenesis is a process in which vascular endothelial cells migrate into living tissues that do not contain blood vessels, and the migrated endothelial cells become flattened in the tissues and form vessels. Formation and the formation of a vascular network within the tissue.
  • vascularization refers to the formation of a new vascular network by extending branch blood vessels from native blood vessels in living tissue. Angiogenesis and vascularization often occur simultaneously in both parties. Therefore, they are often referred to as "angiogenesis” or “angiogenesis” without strict distinction.
  • the terms neo-angiogenesis and neo-vascularization are also used, rather strictly, without distinction from one another.
  • angiogenesis plays a major role in cancer growth and metastasis, but it is said that vascularization plays a major role in angiogenesis in ophthalmic diseases.
  • neovascularization is used to include neoangiogenesis.
  • angiogenesis is a biological and physiological event that is necessary for the growth of tissues during embryonic development, repair of tissues after injury, and other tissue activities.
  • neovascularization including neoangiogenesis, is closely related to the pathological changes of the femur, and furthermore, it is associated with cancer growth.
  • Transmigration requires neovascularization, and a variety of ophthalmic diseases, such as proliferative diabetic retinopathy and macular degeneration, are often caused by abnormal neovascularization.
  • Such pathological enhancement of neovascularization requires a vascular endothelial cell growth factor and / or other growth factors to stimulate it, and such growth factors may be transmitted by cancer cells or locally. It is known that it is produced and released from cells of anemic tissues.
  • Neovascularization inhibitors may be used to treat the aforementioned diseases caused or exacerbated by excessive neovascularization.
  • neovascularization in the retina and / or in the choroid occurs, causing fundus hemorrhage, which is a major cause of human blindness.
  • Neovascularization also occurs in the formation of hemangiomas, which occur in the skin and mucous membranes, and in the growth and metastasis of cancer.
  • Various studies have heretofore been attempted on the development of a compound having a neovascularization inhibitory activity capable of blocking nutrient supplementation as a neovascularization inhibitor. These previous studies are elucidating the cytological and molecular mechanisms of neovascularization.
  • a variety of neovascularization-inhibitors have also been identified that can block several steps in the body's signaling that regulate and regulate neovascularization.
  • Id1 and Id3 genes are expressed at very low levels in adult humans, we are developing a new type of angiogenesis inhibitor that can target and inhibit the Id gene. Is also under study.
  • the present inventor has conducted research to search for a powerful and safe new neovascularization inhibitor that can meet the above strong demands.
  • 5,6-0-benzylidene-rascorbic acid or its monosodium salt is a known compound with extremely low toxicity.
  • the enhanced formation of various vascular endothelial cells in humans cultured in culture media containing various growth factors was found to be due to the presence of 5,6-0-benzylidene-raescorbic acid mononatridium salt.
  • SBA 5,6-0-benzylidene-raescorbic acid monosodium salt
  • VEGF vascular endothelial cell growth factor
  • FGF fibroblast growth factor
  • the glioma cells When the glioma cells are transplanted into the rat brain and SBA is intraperitoneally administered daily to the rat at a dose of lOmg kg per rat, the growth of the glioma is physiologically enhanced in the second week. It was found to be significantly suppressed as compared to the control group to which saline was administered. Along with this inhibition of tumor growth, neovascularization in and around the tumor was also suppressed by SBA administration.
  • patients with cancer or tumor tissue in the body such as patients with brain tumor in brain stem (1 case), patients with gastric cancer (4 cases), patients with femoral tumor (1 case), maxilla Patients with sinus (1), or patients with malignant gonorrhea (2), patients with cervical cancer (1), patients with thyroid cancer (1), patients with esophageal cancer (1) 2), patients with colorectal cancer (3 cases), patients with primary liver cancer (2 cases), patients with breast cancer (5 cases), patients with lung cancer (5 cases), patients with mediastinal tumor (2 cases), patients with knee cancer (2 cases), patients with scrotal cancer (2 cases), patients with uterine cancer (1 case), patients with liposarcoma (1 case), or acute leukemia cancer 1 patient (1 patient) or patient with prostate tumor (1 patient), one dose less than 10 mg, especially 1 SBA was administered intravenously at a low dose of 10 mg, in which case the tumor or cancer shrank or disappeared, and the number of small neovessels in or near the shrunken tumor or cancer tissue was reduced.
  • 1 SBA was administered intraven
  • SBA 5,6-0-benzylidene-raescorbic acid monosodium salt
  • the first aspect of the present invention is characterized in that it comprises 5,6-0-benzylidene-raescorbic acid monosodium salt as an active ingredient, and further comprises a pharmaceutically acceptable carrier for the active ingredient.
  • a composition for inhibiting neovascularization is provided.
  • composition for inhibiting neovascularization of the first aspect of the present invention comprises an ophthalmologic disease induced or aggravated by excessive neovascularization in the eye and the resulting hemorrhage of the fundus oculi, for example, proliferative diabetic retinopathy, corneal margin It is useful for medical or prophylactic treatment of degeneration and age-related macular degeneration.
  • composition for inhibiting neovascularization of the first aspect of the present invention is useful for medical or prophylactic treatment of hemangiomas in skin and mucous membranes.
  • the first anti-angiogenesis composition of the present invention can be used for a medical treatment for inhibiting the growth and metastasis of various tumors or cancers dependent on neovascularization, for example, for the medical treatment of glioblastoma. It is also useful for treatment.
  • Intravenous administration of SBA at doses of 1 mg / body to 10 mg / body per adult, 1-2 times per day, preferably less than 10 mg, will result in excessive SBA.
  • the present inventors have found that neovascularization can be suppressed most efficiently.
  • SBA is administered for the purpose of suppressing the enhanced formation of new blood vessels in the eye and the resulting ocular fundus bleeding, the SBA should be administered at a rate of 1 mg to 2 times per day per adult per day.
  • Intravenous administration at a dose of 10 mg / body was found to give good results in suppressing neovascularization.
  • SBA was administered once daily at a dose of 3 to 4 ms / body to a cancer-bearing patient with rheumatoid arthritis. It was also found that the formation of new blood vessels in the tissues of the rheumatic lesions was suppressed, and that rheumatic pain was reduced or ameliorated. SBA can be administered not only intravenously but also by intraperitoneal or subcutaneous injection or orally. Therefore, in the second invention, the age-related retinal macular degeneration having neovascularization of the peripheral portion of the cornea of the eye, or the neovascularization of the choroid and / or the retina, or the proliferative blood having the retinal neovascularization of the eye.
  • a composition used to be administered characterized in that it contains 5,6-0-benzylidene-L-ascorbic acid monosodium salt as an active ingredient, and has an abnormal ocular neovascularity.
  • Inhibitor composition for suppressing formation and resulting fundus hemorrhage Things are provided.
  • 1 mg / body to 10 mg which is effective for suppressing the formation of new blood vessels in and / or around cancer or tumor tissue generated in a human body.
  • 5,6-0-benzylidene-reascorbic acid monosodium salt at a dose of 1 mg / body to 4 mg / body, especially at a dose of 1 to 2 times a day
  • the present invention provides an inhibitor composition for suppressing the formation of new blood vessels in a cancer or tumor or its peripheral region.
  • intravenous, subcutaneous, or intraperitoneal administration of SBA inhibits or prevents neovascularization in and around cancer or tumor tissue.
  • the cancer or tumor in the above subject that is inhibited is a brain tumor, including neuroglioma, bladder cancer, maxillary sinus cancer, cervical cancer, goiter, esophagus cancer, stomach cancer, colon cancer, primary liver cancer, breast cancer It can be a lung, mediastinal, Teng, ovarian, uterine, acute leukemia, malignant lymphoma, liposarcoma, or femoral head or prostate tumor.
  • the lmg / body to 10 mg / body effective for suppressing the enhanced formation of neovascularization and the resulting pain in rheumatoid diseased tissues of rheumatoid arthritis.
  • 5,6-0-benzylidene-raescorbate monosodium salt at a dose of lmg / body to 4mg / body, especially once or twice a day in patients with rheumatoid arthritis
  • a composition used for intradermal, subcutaneous or intraperitoneal administration characterized in that it contains 5,6-0-benzylidene-L-ascorbic acid monosodium salt as an active ingredient.
  • the present invention provides an inhibitor composition for enhancing neovascularization in rheumatic lesion tissue and suppressing pain caused thereby.
  • the first to fourth neovascularization inhibitor compositions according to the present invention preferably have a dosage unit for intravenous administration of 1 mg / body to: 10 mg / body, preferably in a dosage of 10 mg / body. Is preferably in a form containing a monosodium salt of 5,6-0-benzylidene-L-ascorbinate at a dose of 1 mg / body to 4 mg / body.
  • first to fourth neovascularization inhibitor compositions according to the present invention are used for medical treatment or prophylactic treatment for the general purpose of inhibiting neovascularization, they are generally effective.
  • SBA as a component is appropriately mixed with pharmaceutically acceptable additives, for example, pharmaceutically necessary additives such as carriers, excipients, diluents, etc., and powders, granules, tablets, capsules
  • pharmaceutically acceptable additives for example, pharmaceutically necessary additives such as carriers, excipients, diluents, etc., and powders, granules, tablets, capsules
  • the composition can be formulated in the form of syrups, injections, topical creams, nasal drops, eye drops and the like.
  • the present composition can be administered to humans both non-peripherally and orally.
  • SBA as an active ingredient used in the present invention is incorporated in an effective amount to achieve a predetermined purpose of administration.
  • the dose of SBA as an active ingredient varies depending on the route of administration, symptoms, patient weight, sex, age, etc., but for the purpose of suppressing neovascularization, for example, once a day for adult patients SBA is administered at a dose of ⁇ 10 mg body.
  • the dose of SBA can be continuously or intermittently administered as long as the SBA concentration in the blood due to SBA administration does not exceed a certain amount.
  • the appropriate dosage and frequency of SBA administration under certain conditions must be determined by a specialist based on the above guidelines.
  • the content of the active ingredient SBA in the pharmaceutical composition of the neovascularization inhibitor composition according to the present invention varies depending on the preparation type, but is usually 0 :! to 99% by weight, preferably 1 to 90% by weight. is there.
  • the injection usually contains 0.1 to 5% by weight of SBA.
  • oral administration it is used in the form of tablets, capsules, powders, granules, dry syrups, oral preparations, liquids, syrups and the like together with the above-mentioned solid carriers or liquid carriers.
  • the content of SBA is generally 3 to 99% by weight, preferably 5 to 90% by weight, and the balance is a carrier.
  • the excipients and carriers to be incorporated in the first to fourth compositions according to the present invention can be pharmaceutically acceptable, and the type thereof depends on the route and method of administration.
  • a liquid carrier a buffer solution of about pH 7.4, physiological saline, ethanol or animal and vegetable oils such as soybean oil, sesame oil, mineral oil, and synthetic oils are used.
  • Maltose styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, styrene, stylcellulose, and cellulose derivatives such as hydroxypropylcellulose, polysaccharides such as cyclodextrin, and organic acid salts such as magnesium stearate.
  • glycerol amino acids
  • cellulose derivatives such as hydroxypropylcellulose
  • polysaccharides such as cyclodextrin
  • organic acid salts such as magnesium stearate.
  • the liquid carrier is generally physiological saline, various buffers having a pH of about 7.4, glucose, inositol, a saccharide solution such as mannitol, or the like. Glycols such as ethylene glycol and polyethylene glycol are desirable. Also, SBA is dissolved in physiological saline together with excipients such as sugars such as inositol, mannitol, glucose, mannose, maltose, and sucrose, and amino acids such as phenylalanine. Thereafter, a freeze-dried preparation can be obtained.
  • an appropriate solvent for injection for example, sterile water, physiological saline, a buffer solution of pH 7.4, a glucose solution, an electrolyte solution, an amino acid aqueous solution or the like for intravenous administration.
  • SBA used as an active ingredient in the first to fourth compositions according to the present invention was stimulated by human vascular endothelial cell proliferation and migration, particularly various growth factors, as shown in the test examples described below. It has the biological activity of inhibiting the proliferation and migration of vascular endothelial cells, thereby inhibiting neovascularization. Therefore, in the fifth invention, abnormal proliferation and migration of endothelial cells of neovascular vessels, particularly vascular endothelium, comprising 5,6-0-benzylidene-raescorbic acid monosodium salt as an active ingredient Provided are inhibitors of the abnormal growth and migration of vascular endothelial cells and the resulting formation of new blood vessels caused by cell growth factors and / or fibroblast growth factors and other growth factors.
  • the present invention provides a method for producing an ocular peripheral cornea neovascular or choroidal neovascular and 5) Monosodium 5,6-0-benzylidene-ascorbate is used in the manufacture of a neovascularization inhibitor composition that is administered to control the abnormal formation of retinal neovascularization and the resulting hemorrhage of the fundus. Encompasses the uses used.
  • the present invention is intended to suppress the formation of new blood vessels in and / or around cancer or tumor tissue generated in the body of a human, thereby suppressing the growth and metastasis of cancer or tumor.
  • the use of 5,6-0-benzylidene-reascorbic acid mononatridium salt for the preparation of a neovascularization inhibitor composition to be administered to a patient is included.
  • cancers or tumors of tissues targeted for suppressing neovascularization include, for example, maxillary sinus cancer, cervical cancer, goiter, esophageal cancer, stomach cancer, colorectal cancer, primary liver cancer, breast cancer, lung cancer, mediastinal tumor , Cervical cancer, ovarian cancer, uterine cancer, acute leukemia, malignant lymphoma, liposarcoma, femoral head tumor, bladder cancer or brain tumor, especially neuroglioblastoma, or prostate tumor.
  • the present invention provides a method of treating a patient with rheumatoid arthritis, wherein the neovascularization is administered to suppress the enhanced formation of neovascularization in the tissue of a lesion of rheumatic disease and the resulting pain.
  • Use of 5,6-0-benzylidene-raescorbic acid monosodium salt in the manufacture of a composition for inhibiting formation is provided.
  • the present invention relates to a patient having abnormal formation of peripheral corneal neovascularization of the eye, or abnormal formation of choroidal neovascularization and / or retinal neovascularization, and retinal hemorrhage thereby.
  • the present invention provides a method for treating a patient having a new blood vessel which grows in and around a cancer or tumor tissue generated in a human body.
  • the method comprising inhibiting the formation of new blood vessels in and around the cancer or tumor tissue, and thereby suppressing the growth and metastasis of the cancer or tumor.
  • the present invention is directed to a patient with rheumatoid arthritis who has an increase in the formation of new blood vessels in the lesions and tissues of rheumatic diseases and has a pain caused by it.
  • Benzylidene-raescorbic acid monosodium salt at a dose of 1 mg / body to 10 mg body, especially at a dose of 1 mg / body to 4 mg / body, twice a day, intravenously, subcutaneously or It includes a method of enhancing neovascularization in rheumatoid arthritis lesion tissue of rheumatoid arthritis and suppressing pain caused by the administration intraperitoneally.
  • the SBA may be administered at a dose of 1 mg / body to 10 mg / body per dose, preferably 1 mg / body to 4 mg / body. It has also been found that intravenous or subcutaneous injections at dosages also have the effect of inhibiting neovascularization.
  • NFSB transcription factor NF-kappaB
  • VEGF vascular endothelial cell growth factor
  • bFGF fibroblast growth factor
  • SBA was found to act as a potent NB activity inhibitor.
  • SBA is considered to have a function of suppressing neovascular growth by suppressing the action of vascular endothelial cell growth factor that causes NB activation.
  • Test Example 7 the present inventor described this using human microvascular endothelial cells using the NFSB p50 Electrophoretic Mobility Shift Assay (Muller, Rupee and Baeuerle, Method, Vol. 1, pp. 301-312). (1997)).
  • vascular endothelial cell growth factor and / or the action of fibroblast growth factor that activates the transcription factor NF-kappaB present in vascular endothelial cells, and other growth factors A vascular endothelial cell growth factor and a vascular endothelial cell growth factor, comprising an active ingredient comprising 5,6-0-benzylidene-ascorbate mononatridium salt as an active ingredient.
  • A) fibroblast growth factor as well as other growth factor inhibitor compositions are provided.
  • a vascular endothelial cell growth factor that activates a transcription factor NF-kappaB present in vascular endothelial cells and / or the action of a fibroblast growth factor and other growth factors Treating vascular endothelial cells with an effective amount of 5,6-0-benzylidene-ascorbate monosodium salt to inhibit the effect of the factor, comprising vascular endothelial cell growth factor, fibroblast growth factor and (Or)
  • a method for inhibiting the activation of NF-kappaB by other growth factors is provided.
  • a vascular endothelial cell growth factor and / or fibroblast growth factor and other In order to achieve the suppression of neovascularization by the growth factor of SBA by SBA treatment, SBA is injected intravenously, subcutaneously or intraperitoneally at a dose effective to suppress the growth factor.
  • the SBA can be supplied to the bloodstream, which allows the SBA to be taken up by vascular endothelial cells from the bloodstream containing the SBA.
  • Examples 1 to 3 show examples of preparations of the first to fourth neovascularization inhibitors according to the present invention.
  • the first to fourth compositions of the present invention are not limited to Examples 1 to 3 below.
  • Mono sodium salt 50 g Magnesium aluminate metasilicate 14 g Maize starch 21 g Lactose 35 g Crystalline cellulose 60 g Carboxymethyl cellulose (CMC) calcium salt 18 g
  • CMC Carboxymethyl cellulose
  • SBA lmg Sterile water 50ml
  • the above SBA lg was dissolved in 50ml sterile water.
  • the obtained solution was filtered through a micropore membrane for sterilization.
  • the filtrate was dispensed under aseptic conditions into 5 ml / 0.25 ml aliquots into 10 ml brown vials, lyophilized aseptically, and sealed with nitrogen gas.
  • a freeze-dried SBA preparation for intravenous injection was obtained.
  • the formulation in the vial was immediately and completely dissolved when added to 5 ml of sterile saline under aseptic conditions immediately before use for intravenous injection.
  • the resulting aqueous solution contained SBA at a concentration of lmg / ml and was suitable for intravenous administration.
  • SBA aqueous solution of SBA dissolved in sterile distilled water was injected intravenously at a dose of 4 m ⁇ kg from the tail vein of Wistar rats.
  • the SBA dose to the rat should be adjusted to 500 mg / kg, 1000 mg / kg ⁇ 1500 mg / kg and 2000 mg / kg.
  • Intravenous injection was performed.
  • the mortality of the treated groups of rats receiving these different SBA doses was evaluated. Therefore, SBA the LD 50 values when the given intravenously throw of SBA in Wistar rats was found to be 1693mg / kg (confidence interval is 1306 ⁇ 2194mg / kg).
  • an aqueous solution of SBA was intravenously injected into the tail vein of an NMRI mouse.
  • SBA the LD 50 values when the intravenous administration of SBA in NMR1 mice was observed to be 1815mg / kg (confidence interval is Ru 1399 ⁇ 2858mg / kg der).
  • HMVEC microvascular endothelial cells
  • HMVEC endothelial cells
  • the culture was replaced with a new culture as described below.
  • This new culture solution is obtained by removing the calf brain extract from the above-mentioned endothelial cell nutrient solution and adding the SBA of the present invention at a concentration of 0, 15, 30, 60, 125, 250 or 500 / g / ml. It becomes. Culture of endothelial cells (HMVEC) was continued for 48 hours in the replaced new culture medium.
  • This example is a test illustrating the inhibitory effect of SBA on the proliferation of another endothelial cell, human pulmonary artery endothelial cell (HPAEC).
  • HPAEC human pulmonary artery endothelial cell
  • the experimental conditions are the same as in Test Example 2. This experiment was repeated six times in the same manner (Experiment a to S. In these HPAEC cells, SBA also significantly inhibited endothelial cell (HPAEC) proliferation at a low concentration of 25 g / ml.
  • vascular endothelial cells The migration of vascular endothelial cells is an important step in conjunction with cell proliferation for neovascularization. It is known that endothelial cell migration is stimulated by various growth factors. Among them, vascular endothelial cell growth factor (VEGF) is important. Under a variety of pathological conditions, such as cancer, age-related macular degeneration of the retina, and proliferative diabetic retinopathy, the production and release of VEGF by the cells of the diseased tissue is increased, increased and released. VEGF is said to promote neovascularization. In this example, the method of Werner et al.
  • VEGF vascular endothelial cell growth factor
  • HPAECs human pulmonary artery endothelial cells
  • endothelial cells HPAEC were cultured in an EGM-2MV culture solution (Clonetic). After growing the HPAEC until the cells became confluent, the cells were dispersed by treatment with tribcine, and the cells were collected, washed, and transferred again into the EGM-2MV medium.
  • the cell concentration was adjusted to 80 ⁇ 10 4 cells / ml.
  • the cell suspension 160/1, as the culture liquid was added in a polypropylene centrifuge tube a 15 ml of EGM-2 MV broth saturated beforehand C0 2 (addition of 2% Fatal Bovine Serum (FBS) ).
  • the HPAEC was cultured for 2 hours while rotating the centrifuge tube slowly at 37 ° C.
  • the above-mentioned culture solution placed in each of these centrifuge tubes contains the SBA of the present invention at a concentration of 0, 0.78, 3.13, 12.5, 50, or 100 zg / ml. Thereafter, the cells were spun down by rotating the centrifuge tube quickly (1000 rpm) to precipitate the cells.
  • Each of the membranes in the upper chamber contains a sample of the HPAEC cell suspension (ie, 0 g / ml of SBA or 0.78 ⁇ : 0.1% BSA in EBM culture containing SBA).
  • HPAEC suspension suspended endothelial cells
  • a certain wel at the bottom contains a sample of a liquid without VEGF (negative control).
  • a sample of a liquid (positive control) to which VEGF was added but SBA was not added was added to a certain lower well. Thereafter, the Boyden chamber in the C0 2 incubator, and incubated for 4 hours at 37 ° C.
  • Test Examples 2 to 4 above show that SBA inhibits vascular endothelial cell proliferation and vascular endothelial cell migration, which are the first processes required for neovascularization, in vitro. Furthermore, whether or not neovascularization was inhibited by SBA in vivo was tested in this example by the method of the mouse corneal micropocket assay 3 ⁇ 4 ⁇ (Nature Med, vol. 3, 1203-1208, 1997).
  • VEGF or fibroblast growth factor basic fibroblast growth iactor (bFGF) and sclaralate were mixed well with a hydrone, and a pellet containing 2 ng of VEGF or bFGF (1.51) was prepared from the mixture.
  • bFGF basic fibroblast growth iactor
  • Neovascularization is also required for cancer growth and metastasis.
  • human glioblastoma This is a malignant brain tumor and there is currently no cure for it.
  • the rat glioma is used as an experimental model because it resembles the human god glioblastoma.
  • rat glioma C6 cells / rat brain (Caudate pu unen) were examined in rat brain (Caudate pu unen). 20 // 1 was embedded using a stereotaxic instrument. From the following day, SBA was injected once daily intraperitoneally with 10 mg / kg for 2 weeks. The control group was injected only with a phosphate buffer (PBS, pH 7.4) supplemented with 0.9% NaCl, an injection solvent for SBA. Under anesthesia at 2 weeks, rat brain was perfused with tissue fixative The brain was removed, fixed, and embedded in paraffin.
  • PBS phosphate buffer
  • Muller et al.'S method (Methods, Volume 1, 301) showed that SBA has an activity to suppress the action of vascular endothelial cell growth factor VEGF, which activates the transcription factor NF-kappaB (abbreviated as NB).
  • VEGF vascular endothelial cell growth factor
  • NF-kappaB transcription factor NF-kappaB
  • Pp. 312 (1997), see Muller et al., “Factory Studv or gene regulation bv NFkappa B and AP-1 in response to reactive oxygen intermediatesj” (this is tested using the equivalent NFSB p50 Electrophoretic Mobility Shift Assay).
  • HMVEC human microvascular endothelial cells
  • the electrophoretic spots of the NFeBp50 Electrophoretic Mobility Shift Assay showed that NB activated by the addition of VEGF was rapidly and significantly suppressed by SBA during the culture of human microvascular endothelial cells. VEGF at 30 minutes, 60 minutes, and 6 hours of cell culture It was found that the size of activated NB spots was significantly reduced in the culture in the presence of 12.5 / g / ml SBA.
  • SBA is a potent NB inhibitor that suppresses NFSB activation by growth factors.
  • SBA inhibits vascular endothelial cell proliferation and migration without toxicity, and is especially stimulated by VEGF or bFGF to enhance Inhibit the proliferation and migration of vascular endothelial cells, and consequently suppress neovascularization.
  • VEGF vascular endothelial growth factor
  • bFGF vascular endothelial growth factor
  • Fig. 4 shows a test in which tube formation was suppressed and rheumatic pain was reduced.
  • a 61-year-old woman (named Initial T.H.) developed a brain tumor (mixed malignant gonorrheoma of the T and B types). He was treated with existing anticancer drugs, but a CT scan of the brain revealed a 6 cm X 5 cm tumor spanning the right cerebral brainstem. The patient's consciousness had completely disappeared and was in a state of terminal symptoms of cancer. After consent of the family, intravenous infusion of 1000 mg SBA twice a day for 25 days was performed. Brain CT showed that the brain tumor had disappeared and the consciousness was restored. The following day, with consent and craniotomy surgery, the tumor in the right brain had disappeared, and the number of blood vessels in the tissue after the tumor had disappeared was reduced compared to the healthy part of the left brain. However, it was found that the existence of new blood vessels was hardly recognized.
  • SBA was intravenously injected at 4 mg / body once a day for 16 days, and then inspected with an endoscope.
  • the blood vessels in the tumor part of the stomach completely disappeared, and the stomach wall became healthy mucous membrane.
  • Biopsies showed that the cancer cells became normal after necrosis.
  • CT findings showed disappearance of carcinomatous peritonitis, ascites and liver metastases. Treatment was discontinued after a month and a half. Six months after the start of scabbing by the SBA, there were no abnormalities.
  • mmrn 1 0 When an 82-year-old woman (initial name TM) was hospitalized for a fracture due to a tumor in the right apex of the femur, a tumor shadow of 10 cm or more centered on the right apex of the femur was observed. The apex of the femur fractured and separated, causing severe pain. Tumor biopsies were performed to identify undifferentiated adenocarcinoma. During the biopsy, fresh blood erupted from the biopsy tube. Intravenous injection of 2 mg / body of SBA was started. In the next few days, the severe pain subsided, and a giant tumor disappeared by radiographic diagnosis two weeks later.
  • the dose of SBA was increased to 4 mg / body IV of SBA.
  • a retraction was performed.
  • the tumor had completely disappeared, and it was confirmed that several ml of necrotic material was present.
  • the number of blood vessels was small as in the above case, and suppression of neovascularization was observed.
  • a 53-year-old man (named Initial K.S.) had a prostate tumor, and the tumor had spread extensively to the thoracic spine, lumbar spine, pubis, and bladder. This patient was intravenously administered 3 mg / body of SBA once a day for 2 weeks. All prostate and other metastatic tumors shrank in size and improved.
  • vascular endothelial cell growth factor and / or fibroblast a known low-toxicity compound, 5,6-0-benzylidene-raescorbic acid monosodium salt (SBA) is used for vascular endothelial cell growth factor and / or fibroblast.
  • SBA 5,6-0-benzylidene-raescorbic acid monosodium salt
  • vascular endothelial cells can suppress the growth and migration of vascular endothelial cells, which are enhanced by growth factors and other growth factors, and have an effect of inhibiting neovascularization.
  • SBA has the effect of suppressing abnormally enhanced neovascularization in the eye and abnormal neovascularization in cancer tissue and its surroundings, and also enhances neovascularization in rheumatoid arthritis lesion tissue. It has the effect of inhibiting the formation and reducing the pain caused by rheumatic diseases.
  • the above-mentioned SBA has an excellent neovascularization inhibitory action and is very useful as a new anti-angiogenesis agent having extremely low toxicity.

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Abstract

Il s'avère que le monosodium 5,6-O-benzylidène-L-ascorbate (SBA), qui est un composé connu à faible toxicité, est capable d'inhiber la prolifération et la migration des cellules endothéliales vasculaires accrue par des facteurs de croissance tels que le facteur de croissance des cellules endothéliales vasculaires et/ou le facteur de croissance du fibroblaste, et inhibe ainsi la néovascularisation. SBA inhibe la néovascularisation accrue par l'anormalité de l'oeil et, à son tour, supprime la lésion résultante de l'oeil. De plus, il inhibe la néovascularisation anormale dans un tissu cancéreux et autour du tissu, et inhibe, par conséquent, la prolifération et la métastase du cancer. SBA inhibe, de plus, la néovascularisation accrue dans une lésion d'un rhumatisme articulaire et soulage ainsi la douleur induite par ce rhumatisme. SBA est utile comme nouvel inhibiteur de la néovascularisation et présente une excellente performance d'inhibition et une très faible toxicité.
PCT/JP2001/000672 2000-02-01 2001-01-31 Compositions inhibitrices de neovascularisation et procede correspondant WO2001056566A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2114571A (en) * 1982-01-15 1983-08-24 Lilly Co Eli Ascorbic acid ethers and related compounds

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2114571A (en) * 1982-01-15 1983-08-24 Lilly Co Eli Ascorbic acid ethers and related compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARUMO TAKESHI ET AL.: "Vascular endothelial growth factor activates nuclear factor-kappa.B and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells", DIABETES, vol. 48, no. 5, 1999, pages 1131 - 1137, XP002938989 *

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