WO2007013147A1 - Inhibiteur de l’angiogenèse et agent de régression vasculaire - Google Patents

Inhibiteur de l’angiogenèse et agent de régression vasculaire Download PDF

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Publication number
WO2007013147A1
WO2007013147A1 PCT/JP2005/013713 JP2005013713W WO2007013147A1 WO 2007013147 A1 WO2007013147 A1 WO 2007013147A1 JP 2005013713 W JP2005013713 W JP 2005013713W WO 2007013147 A1 WO2007013147 A1 WO 2007013147A1
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vascular
dihydroxyphenylalanine
hydrogen atom
angiogenesis inhibitor
angiogenesis
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PCT/JP2005/013713
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English (en)
Japanese (ja)
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Syunji Natori
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Inbiotex Inc.
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Priority to PCT/JP2005/013713 priority Critical patent/WO2007013147A1/fr
Publication of WO2007013147A1 publication Critical patent/WO2007013147A1/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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Angiogenesis inhibitors and vascular regression agents are included in angiogenesis inhibitors and vascular regression agents.
  • the present invention relates to an angiogenesis inhibitor and a vascular regression agent.
  • the present invention also relates to a method for screening an angiogenesis inhibitor.
  • vascular endogenesis refers to a phenomenon in which new blood vessels, that is, new blood vessels, are formed from existing blood vessels.
  • RTK receptor tyrosine kinase
  • Angiopoietin 1 (Ang-1) is known to act on vascular endothelial cells through binding to Tie-2 to induce differentiation of juvenile blood vessels built by VEGF into more mature blood vessels.
  • Ang-1 Angiopoietin 1
  • Angiopoietin 2 (Ang— 2) is a physiological inhibitor in the Ang-1 / Tie-2 system. It is thought that there is, it works competitively with Ang_l and controls angiogenesis, morphological maintenance, etc. (PC Maisonpierre et al., Science, 277: 55-60 (1997)).
  • Angiogenesis is observed in various physiological and pathological states in the living body.
  • Angiogenesis in the physiological state is observed during luteinization, placenta formation, and the like.
  • angiogenesis in a pathological state has been observed in inflammation, wound healing, tumor growth, etc.
  • angiogenesis associated with diabetic retinopathy, post-lens fibrosis, corneal transplantation, etc. Glaucoma, eye tumors, trachoma, etc.
  • the dermatology area for example, psoriasis, pyogenic granuloma, etc.
  • pediatric area for example, hemangioma, fibroangioma, etc.
  • Is for example, hypertrophic scars, granulations, etc., in the internal medicine region, for example, rheumatoid arthritis, edema sclerosis, etc., and for heart diseases, for example, atherosclerosis, etc.
  • a pathological increase is observed.
  • abnormal angiogenesis in diabetic retinopathy and trachoma drives many people blind, and in rheumatoid arthritis, abnormal angiogenesis in the joints destroys the cartilage in the joints. Wake up and annoy many people.
  • angiogenesis is caused by various inflammatory diseases (rheumatic arthritis, psoriasis, etc.), various diseases in ophthalmology (diabetic retinopathy, retinopathy of prematurity, senile macular degeneration, retinal vein occlusion, later Lens fibroproliferation, angiogenesis associated with corneal transplantation, glaucoma, eye tumors, etc.) and various tumors.
  • ophthalmology diabetic retinopathy, retinopathy of prematurity, senile macular degeneration, retinal vein occlusion, later Lens fibroproliferation, angiogenesis associated with corneal transplantation, glaucoma, eye tumors, etc.
  • corneal diseases Stevens-Johnson syndrome and related diseases
  • ocular acne and related diseases alkalis, acids, surfactants, various solvents, volatile gases, and other various cytotoxic substances.
  • the angiogenesis inhibitors developed so far can be classified as follows.
  • angiogenesis inhibitors have insufficient activity and have side effects such as toxicity, they are satisfactory as preventive or therapeutic agents for diseases involving angiogenesis. Development of a substance having an angiogenesis-inhibiting action superior to that of spiders is desired. In particular, in the field of ophthalmology, drugs must always be used with the goal of maintaining visual function, and the development of safe drugs that do not adversely affect other eye tissues is expected.
  • corneal neovascularization is known to be involved in allograft rejection during corneal transplant surgery (Jpn. J. Ophthalmol., 38 (3), 311-316 (1994)), it is thought that rejection of a graft can be suppressed by inhibiting angiogenesis after corneal transplantation.
  • the potential for treatment of ocular diseases with neovascularization with interferon is age-related macular degeneration (Fung, WE, J. ophthalmol., 112, 349 (1991)), neovascular glaucoma (Miller, JW et al., Ophthalmology) , 100, 9, (1992)), and diabetic retinopathy (Wakelee_Lynch, J.
  • N_ ⁇ -alanyl_5_S-glutathionyl_3,4-dihydroxyphenylalanine is a known substance (Patent Document 3), and 5-S-GAD is It is known to have antitumor action (Patent Documents 3 and 4), apoptosis-inducing action (Patent Document 5), osteoclast formation inhibitory action (Patent Document 5), and the like.
  • Non-Patent Document 1 Ingber D et al., “Nature”, 1990, No. 348, No. 6301, p.555-557
  • Non-Patent Document 2 Jung HJ et al., “Bioorganic & Medicinal Chemistry”, 2003, No. 11, No. 22, ⁇ .4743-4747
  • Non-Patent Document 3 Nakamura (Nakamura) ES, etc., “Cancer Science”, 2004, No. 95, No. 1, ⁇ .25-31
  • Non-Patent Document 4 Penn JS et al., “Investigative Ophthalmology and Visual Science”, 2003, 4th 4th, p.5423-5429
  • Non-Patent Document 5 Ozaki H et al., “American Journal of Pathology”, 2000, No. 156, No. 2, ⁇ .697-707
  • Patent Document 1 JP-A-1 279828
  • Patent Document 2 Japanese Patent Laid-Open No. 2003-26696
  • Patent Document 3 Japanese Patent Laid-Open No. 8-337594
  • Patent Document 4 Japanese Patent Laid-Open No. 2001-213799
  • Patent Document 5 Japanese Patent Laid-Open No. 2001-226283
  • An object of the present invention is to provide a novel angiogenesis inhibitor and a vascular regression agent.
  • Another object of the present invention is to provide a screening method for a novel angiogenesis inhibitor.
  • the present invention provides the following angiogenesis inhibitors, vascular regression agents, and methods for screening for angiogenesis inhibitors.
  • R 1 represents a hydrogen atom or an arbitrary amino acid residue
  • R 2 represents a hydrogen atom or the following formula ( ⁇ ):
  • R 3 represents a hydrogen atom or any amino acid residue
  • R 4 represents a hydroxyl group or any amino acid residue
  • n represents 1 or 2.
  • An angiogenesis inhibitor comprising, as an active ingredient, a 3,4-dihydroxyphenyllauran derivative represented by the formula: or a pharmaceutically acceptable salt thereof.
  • the angiogenesis inhibitor according to the above (1) or (2) which is an ocular angiogenesis inhibitor.
  • R 1 represents a hydrogen atom or any amino acid residue
  • R 2 represents a hydrogen atom or the following formula ( ⁇ ):
  • R 3 represents a hydrogen atom or an arbitrary amino acid residue
  • R 4 represents a hydroxyl group or any Amino acid residue
  • n represents 1 or 2.
  • a vascular regression agent comprising, as an active ingredient, a 3,4-dihydroxyphenyllauranin derivative represented by the formula: or a pharmaceutically acceptable salt thereof.
  • the vascular regression agent according to (5) or (6) above which is an ocular vascular regression agent.
  • vascular retraction agent which is a corneal vascular retraction agent.
  • Test substance is vascular regression, vascular endothelial cell growth inhibitory, vascular endothelial cell luminal formation inhibitory, vascular endothelial cell chemotaxis inhibitory, matrix meta-mouth protease inhibitory, plasminogen activator Step of determining whether or not it has vector inhibitory activity and receptor type tyrosine kinase inhibitory activity
  • b Has vascular regression, inhibits proliferation of vascular endothelial cells, inhibits luminal formation of vascular endothelial cells, inhibits chemotaxis of vascular endothelial cells, inhibits matrix metamouth protease, plasminogen Screening for substances that have no inhibitory activity on the inhibitor and receptor type tyrosine kinase
  • the angiogenesis inhibitor and the vascular regression agent provided by the present invention can inhibit angiogenesis based on a mechanism different from conventional angiogenesis inhibitors.
  • angiogenesis inhibitors have an inhibitory effect on vascular endothelial cell proliferation, an inhibitory effect on vascular endothelial cell lumen formation, an inhibitory action on vascular endothelial cell chemotaxis, an inhibitory action on matrix metabolite protease, a plasminogen It exerts angiogenesis inhibitory action based on one or more kinds of actions such as inhibitory action of cuticle and receptor type tyrosine kinase.
  • the angiogenesis inhibitor and the vascular regression agent of the present invention have an inhibitory action on vascular endothelial cell proliferation, an inhibitory action on luminal formation of vascular endothelial cells, an inhibitory action on chemotaxis of vascular endothelial cells, and an inhibition of matrix metamouth protease
  • it does not have any of the actions, plasminogen activator beta inhibitory action and receptor type tyrosine kinase inhibitory action, it can inhibit angiogenesis based on the vascular regression action. Therefore, the angiogenesis inhibitor and the vascular regression agent of the present invention can be administered for a long time without causing the possibility of delaying wound healing caused by excessively suppressing the proliferation of vascular endothelial cells.
  • Fig. 1 shows the results of measuring the number of cells by the Coulter Counter method after culturing HUVEC in the presence of 5-S-GAD. b) shows the results of measuring the mitochondrial metabolic activity by the MTT method after culturing HUVEC in the presence of 5-S-GAD.
  • FIG. 2 is a diagram showing the results of measuring the number of cells by the Coulter counter method after culturing HUVEC in the presence of 5_S-GAD.
  • FIG. 3 is a diagram showing the results of observation of tube formation after culturing HUVEC in the presence of 5_S-GAD.
  • FIG. 4 shows the results of measuring extracellular plasminogen activity after culturing HUVEC in the presence of 5-S-GAD
  • FIG. 4 (b) shows the results of measuring cell adhesion plasminogen activator activity after culturing HUVEC in the presence of 5-S-GAD.
  • FIG. 5 is a diagram showing the results of detecting MMP-2 precursor and MMP-9 precursor after culturing HUVEC in the presence of 5_S-GAD.
  • FIG. 6 is a diagram showing the results of measuring cell chemotaxis after culturing HUVEC in the presence of 5-S-GAD.
  • FIG. 7 is a diagram showing the results of detecting PKA activity, PKC activity, eE F 2K activity and PTK activity after treating a cell lysate with 5-S-GAD.
  • FIG. 8 is a graph showing the results of measuring EGF receptor tyrosine kinase activity after treating the membrane fraction with 5-S-GAD.
  • FIG. 9 is a diagram showing the results of detecting VEGF receptor tyrosine kinase activity after treating the membrane fraction with 5-S-GAD.
  • Fig. 10 shows blood vessels that are directed to the corneal epithelial cell exfoliated part from the outer edge of the cornea in the 5-S-GAD administered group or the control group after exfoliating the corneal epithelial cells of the sputum and stimulating with silver nitrate It is a figure which shows the result of having observed the newborn.
  • FIG. 11 is a diagram showing the results of measuring the number of new blood vessels after 5-S-GAD administration by the mouse dorsal subcutaneous method.
  • FIG. 12 is a view showing the state (thickness) of an existing blood vessel after administration of 5-S-GAD.
  • FIG. 13 (a) is a diagram showing the results of observing the state of blood vessels after 5-S-GAD treatment by the chicken embryo chorioallantoic membrane method, and FIG. It is a figure which shows the result of having evaluated the presence or absence of the newborn inhibitory effect.
  • Arbitrary amino acid residues represented by R 3 or R 4 include any type of amino acid residues.
  • Examples of amino acid residues represented by R 1 R 3 or R 4 include ⁇ -amino acid residues. Groups, ⁇ -amino acid residues, ⁇ -amino acid residues, neutral amino acids (monoamino mono-functional norlevonic acid such as glycine, phosphorus, leucine), acidic amino acids (glutamic acid, aspartic acid, etc.) Minodicarboxylic acid) residues, basic amino acids (diaminomonocarboxylic acids such as arginine and phenylalanine) and the like.
  • the binding mode of any amino acid residue represented by R 1 R 3 or R 4 is an amide bond.
  • the amino acid residue represented by R 1 is preferably a ⁇ -alanine residue, and the amino acid residue represented by R 3 is preferably a glutamic acid residue, represented by R 4.
  • the amino acid residue is preferably a glycine residue, and ⁇ is preferably 1.
  • the 3,4-dihydroxyphenylalanine derivative (compound (1)) represented by formula (I) is preferably ⁇ - ⁇ -alanyl-5-S glutathionyl-3,4-dihydroxy. It is phenylalanine (5-S-GAD), ⁇ -alanil-3,4-dihydroxyphenylalanine-AD) or 5-S cystinelu 3,4-dihydroxyphenylalanine (5-S-CD).
  • compound (I) is 5-S-GAD
  • R 1 is a; 3-alanine residue
  • R 2 is a group represented by the formula (II)
  • R 3 is a gnoretamine residue
  • R 4 is glycine.
  • n 1, when compound (I) is ⁇ -AD, R 1 is an alanine residue, R 2 is a hydrogen atom, and compound (I) is 5 —S—CD, R 1 is a hydrogen atom, R 2 is a group represented by the formula ( ⁇ ), R 3 is a hydrogen atom, R 4 is a hydroxyl group, and n is 1.
  • an asymmetric carbon exists in the compound (I), the configuration of the asymmetric carbon is not particularly limited, and may be either S or R configuration.
  • compound (I) exists as an isomer based on one or more asymmetric carbons, compound (I) can be any stereoisomerically pure isomer (optical isomer, diastereoisomer). Etc.), a mixture of arbitrary isomers, a racemate and the like.
  • Examples of the pharmaceutically acceptable salt of compound (I) include acid addition salts, base addition salts, amino acid addition salts, and the like.
  • acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate and phosphate, formate, acetate, oxalate, benzoate, methanesulfonate, ⁇ _Toluene sulfonate, maleate, fumarate, tartrate, citrate, succinate, organic acid salts such as lactate, etc.
  • base addition salts include sodium salt, potassium, for example Metal salts such as salts, magnesium salts, calcium salts, and amine salts such as ammonium salts, methylamine salts, and triethylamine salts.
  • amino acid addition salts include glycine, phenylalanine, aspartic acid, and glutamic acid. With Salting can be mentioned.
  • Compound (I) is produced, for example, using 3, 4-dihydroxyphenylalanine (Dopa) as a starting material and utilizing amino acid condensation synthesis method, enzyme treatment method, liquid phase peptide synthesis method, etc. This comes out.
  • Dopa 4-dihydroxyphenylalanine
  • a specific production method of the compound (I) is as follows.
  • a certain compound (I) can be produced.
  • 1N hydrochloric acid is added to the reaction mixture, and after extraction with ethyl acetate under acidic conditions, the ethyl acetate layer is concentrated under reduced pressure. This can be done by collecting the precipitated crystals and subjecting them to HPLC treatment.
  • R 1 is a hydrogen atom and R 2 is a group represented by the formula (II)
  • Compound (I) can be produced. Purification of compound (I) from the reaction solution can be performed by HPLC treatment.
  • n is as defined above.
  • S-GAD is the force to collect body fluid after wounding and rearing adult Scarlet fly, homogenized and used as a raw material, which is separated by ion column chromatography and reversed-phase HP LC. It can also be obtained by subjecting to fractionation and collecting a fraction having antibacterial activity.
  • Compound (I) has an angiogenesis inhibitory action and a vascular regression action.
  • Compound (I) can relieve blood vessels and inhibit angiogenesis based on a vascular retraction effect.
  • the angiogenesis inhibitory action of compound (I) is not limited to the action based on the vascular regression action.
  • Compound (I) inhibits the proliferation of vascular endothelial cells, inhibits the formation of lumens of vascular endothelial cells, inhibits the chemotaxis of vascular endothelial cells, inhibits matrix meta-mouth protease, and inhibits plasminogen activator. And angiogenesis can be inhibited on the basis of the vascular retraction action, although it does not have any action of inhibiting the receptor tyrosine kinase. Therefore, Compound (I) has an excessive effect on inhibiting the proliferation of vascular endothelial cells. Therefore, it can be administered for a long period of time without the risk of causing a delay in wound healing.
  • the angiogenesis inhibitor and the vascular regression agent of the present invention may be composed only of the compound (I), but usually usually together with one or more pharmaceutically acceptable carriers and Z or additives. Formulated according to law. When formulating, the compounding amount of compound (I) can be adjusted as appropriate.
  • Examples of the pharmaceutically acceptable carrier include water, pharmaceutically acceptable organic solvents, collagen, polybutyl alcohol, polybutyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran. , Sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum anolebumin, mannitol, sorbitol, ratatoose Etc.
  • additives used in the formulation include excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, buffers, antioxidants, preservatives, and isotonic agents. PH regulators, solubilizers, stabilizers, and the like. These additives can be appropriately selected according to the dosage unit form of the preparation.
  • routes of administration include oral administration and parenteral administration such as intracerebral, intraperitoneal, intraoral, intratracheal, intrarectal, subcutaneous, intramuscular and intravenous.
  • routes of administration include oral administration and parenteral administration such as intracerebral, intraperitoneal, intraoral, intratracheal, intrarectal, subcutaneous, intramuscular and intravenous.
  • examples include tablets, granules, capsules, powders, solutions, suspensions, syrups, sprays, ribosomes, emulsions, suppositories, injections, eye drops, ointments, tapes.
  • oral administration and parenteral administration such as intracerebral, intraperitoneal, intraoral, intratracheal, intrarectal, subcutaneous, intramuscular and intravenous.
  • examples include tablets, granules, capsules, powders, solutions, suspensions, syrups, sprays, ribosomes, emulsions, suppositories, injections
  • tissue in which the angiogenesis inhibitor and the vascular regression agent of the present invention can exert an angiogenesis inhibitory effect and vascular regression activity is not particularly limited, but it is excellent in the eye, particularly in the cornea. Angiogenesis inhibitory action and vascular regression action can be exerted.
  • the angiogenesis inhibitor and the vascular regression agent of the present invention are used as injections, and directly injected into a diseased tissue such as cornea or vitreous body or adjacent tissue with a thin injection needle. You can do it. It can also be administered as an intraocular perfusate using a pump or the like. Further, by pre-impregnating the angiogenesis inhibitor and the vascular regression agent of the present invention as components of contact lenses, administration to the eye including the cornea can be performed.
  • the sclera is a thin avascular layer of highly ordered collagen network surrounding most of the vertebrate eye circumference. Because the sclera is avascular, injections that do not inherently pose a risk of bleeding can be quickly lost from the eye. Thus, the sclera can be used as a natural drug storage location. In addition, by using the sclera as a natural drug storage location, it is possible to supply drugs to the underlying tissue.
  • the angiogenesis inhibitor and the vascular regression agent of the present invention are, for example, incorporated into a sustained-release polymer pellet or microcapsule to form a sustained-release agent, and the sustained-release agent is surgically inserted into the tissue to be treated. Can be transplanted.
  • sustained-release polymers include ethylene vinyl acetate, polyhydrometatalylate, polyacrylamide, polyvinyl pyrrolidone, methyl cellulose, lactic acid polymer, and lactic acid / glycolic acid copolymer.
  • biodegradable polymers Lactic acid polymers and lactic acid glycolic acid copolymers are preferred. Examples that can be referred to when using sustained-release agents include the use of inserts and implants (US Pat. No.
  • An insert is a device inserted on the eye, such as on the conjunctival layer, which generally consists of a polymer matrix containing the active compound.
  • the angiogenesis inhibitor and the vascular regression agent of the present invention can be administered to mammals including humans.
  • the dose of the angiogenesis inhibitor or the vascular regression agent of the present invention should be appropriately increased or decreased according to the conditions such as the age, sex, weight, symptoms, and administration route of the patient.
  • the drug of the above dosage may be administered every day or may be administered at intervals of several days, for example:!
  • the angiogenesis inhibitor or vascular regression agent of the present invention can be used in combination with other drugs, and the dosage in that case is the same as above.
  • the angiogenesis inhibitor and the vascular regression agent of the present invention may be used for diseases involving angiogenesis, such as , Diabetic retinopathy, retinopathy of prematurity, macular degeneration, neovascular glaucoma, retinal vein occlusion, retinal artery occlusion, pterygium, rubeosis, ocular diseases such as corneal neovascularization, solid tumors It can be used as a prophylactic or therapeutic agent for cancer diseases such as hemangioma, tumor growth and metastasis.
  • diseases involving angiogenesis such as , Diabetic retinopathy, retinopathy of prematurity, macular degeneration, neovascular glaucoma, retinal vein occlusion, retinal artery occlusion, pterygium, rubeosis, ocular diseases such as corneal neovascularization, solid tumors
  • cancer diseases such as hemangioma, tumor growth
  • the test substance is a vascular regression action, a vascular endothelial cell proliferation inhibitory action, a vascular endothelial cell lumen formation inhibitory action, a vascular endothelial cell chemotaxis inhibitory action, a matrix metamouth.
  • a protease inhibitory action a plasminogen activator inhibitory action and a receptor tyrosine kinase inhibitory action can be determined by known methods of in vitro or in vivo systems. In in vitro methods, for example, vascular endothelial cells derived from human, ushi, mouse, rat, etc.
  • a membrane fraction of a cell expressing a desired receptor (for example, a host cell into which an expression vector having a desired receptor gene has been introduced) can be used.
  • mammals such as ushi, hidge, goat, horse, pig, usagi, inu, cat, rat and mouse can be used as model animals.
  • test substance when the test substance can reduce, for example, the number of blood vessels, the thickness of the blood vessels, the length of the blood vessels, etc., it is possible to determine that the test substance has a vascular retraction action. it can.
  • the concentration of the test substance is usually 10 ⁇ M or less, preferably 50 ⁇ ⁇ or less, more preferably 100 a ⁇ or less, the test is conducted when the growth of vascular endothelial cells is not inhibited. It can be determined that the substance has no vascular endothelial cell growth inhibitory action.
  • the concentration of the test substance is usually 10 ⁇ or less, preferably 50 ⁇ or less, more preferably 100 ⁇ or less, the lumen formation of vascular endothelial cells is not inhibited. Sometimes it can be determined that the test substance has no inhibitory effect on the formation of vascular endothelial cells.
  • the concentration of the test substance is usually 10 ⁇ or less, preferably 50 ⁇ or less, more preferably 100 ⁇ or less, vascular endothelial cell chemotaxis is achieved. Can be discriminated as having no inhibitory effect on the chemotaxis of vascular endothelial cells.
  • the concentration of the test substance is usually 10 ⁇ M or less, preferably 50 ⁇ or less, more preferably 100 ⁇ or less, when the matrix metaprotease activity is not inhibited.
  • concentration of the test substance is usually 10 ⁇ M or less, preferably 50 ⁇ or less, more preferably 100 ⁇ or less, when the matrix metaprotease activity is not inhibited.
  • the test substance does not have a matrix meta-oral protease inhibitory action.
  • “Matrix meta-oral protease” includes various types of matrix meta-oral proteases as long as they are related to angiogenesis. For example, the gelatinase group ( ⁇ ⁇ -2, ⁇ -9) that degrades the basement membrane. ), MP-3, MMP-14, etc. that activate these are included.
  • plasminogen activator activity is inhibited when the concentration of the test substance is usually 10 ⁇ M or less, preferably 50 ⁇ or less, more preferably 100 ⁇ or less. If not, it can be determined that the test substance has no plasminogen activator inhibitory action.
  • the concentration of the test substance is usually 10 / ig / mL or less, preferably 50 ⁇ g / mL or less, more preferably 100 ⁇ g / mL or less, receptor type 1 tyrosine kinase activity. Can be discriminated as having no inhibitory effect on the receptor type thymosine kinase.
  • “Receptor-type thymosine kinase” includes various types of receptor-type thyrosin kinase as long as it is involved in angiogenesis. For example, EGF receptor, FGF receptor, VEGF receptor (Flt _ l, Flk_ l), PDGF receptor, etc.
  • the screening method of the present invention has a vascular retraction effect, inhibits the proliferation of vascular endothelial cells, inhibits luminal formation of vascular endothelial cells, inhibits chemotaxis of vascular endothelial cells, inhibits matrix meta-mouth protease N- ⁇ -alanyl-5 _S _Dartathonyl-3,4-dihydroxyphenylalanine-like angiogenesis inhibition of substances that do not have plasminogen activator inhibitory activity or receptor tyrosine kinase inhibitory action It can be screened as a substance.
  • ⁇ _ ⁇ -Varanyl-1 5 _ S _Dartathonyl-3, 4-dihydroxyphenylalanine-like angiogenesis inhibitor means 5-S— Like GAD, blood vessel endothelial cell growth inhibitory action, blood Inhibition of luminal formation of vascular endothelial cells, inhibition of chemotaxis of vascular endothelial cells, inhibition of matrix metalloprotease, inhibition of plasminogen activator and inhibitor of receptor type 1 tyrosine kinase Despite not having it, it is a substance that can inhibit angiogenesis based on vascular regression.
  • a 5-S-GAD-like angiogenesis inhibitor can be administered for a long period of time because there is no risk of causing delayed wound healing caused by excessively suppressing the proliferation of vascular endothelial cells.
  • Example 1 is known literature (Nakamura M, Katsuki Y, Shibutani Y, Oikawa T. Dienogest, a synthetic steroid, suppresses both embryonic and tumor-cell-induced angiogenes is.Eur J Pharmacol.386, 33-40 (1999). ).
  • HUVEC human umbilical vein endothelial cells (10 3 cells / well) was seeded on a gelatin-coated 24-well plate and cultured at 37 ° C, 5% CO for 4 hours.
  • HUVEC culture complete medium (MCDB131 (Sigma), 10% fetal bovine serum, 1% antibiotic (10,000 U / ml penicillin and 10,000 ⁇ g / ml streptomycin mixture) (Manufactured by GIBCO)), 10 ⁇ gZmL endothelial cell growth supplement (Upstate Biotechnology Sento), 10ng / mL epidermal growth factor, 10 ⁇ g / mL heparin) It was. Then, 5-S-GAD (0 xM, 0. ⁇ , 0. ⁇ , ⁇ , ⁇ , ⁇ ) was added, and further cultured at 37 ° C, 5% CO for 72 hours. After culture, trypsin HUVEC
  • Fig. 1 (a) The results are shown in Fig. 1 (a).
  • HUVE C was cultured in the same manner as described above using a complete medium with a concentration of 1% fetal bovine serum, and the number of cells was measured by a Coulter counter method. The result is shown in figure 2.
  • HUVEC (10 4 cells / well) was seeded in a gelatin-coated 96-well plate and cultured at 37 ° C and 5% CO for 4 months. Then, 5-S-GAD (0 M, 0.01 / iM, 0. ⁇ , 1 / i
  • Mitochondrial metabolic activity was measured by the MTT method. The results are shown in Fig. 1 (b). As shown in Fig. 1 and Fig. 2, 5-S-GAD did not affect HU VEC proliferation at concentrations below ⁇ ⁇ .
  • Example 2 is a known document (Oikawa T, Sasaki M, Inose M, Shimamura M, Kuboki H, Hi rano S, Kumagai H, Ishizuka M, Takeuchi T. Effects of cytogenin, a novel microbial product, on embryonic and tumor cell. -induced angiogenic responses in vivo. Anticancer Res. 17, 1881-1886 (1997)).
  • FIG. 3 A, B, C, and D show the results when the concentration of 5-S-GAD is 0 ⁇ M, 1 ⁇ , 10 ⁇ M, and 100 ⁇ , respectively.
  • 5-S-GAD did not inhibit HUVEC tube formation at concentrations below 100 ⁇ ⁇ .
  • Example 3 is a known literature (Nakamura M, Katsuki Y, Shibutani Y, Oikawa T. Dienogest, a synthetic steroid, suppresses both embryonic and tumor—cell—induced angiogenes is. Eur J Pharmacol. 386, 33-40 (1999). ).
  • Plasminogen activator activity includes ubiquitinogen (bovine plasminogen) and H—D—Val—Leu—Lys—p—nitroanilide 0 ⁇ 1 M Tris / HCl
  • the sample was added to (pH 7.4), incubated at 37 ° C, and quantified by measuring the released P-nitrotrolide based on the absorbance at 405 nm.
  • 5-S-GAD did not inhibit plasminogen activator activity at concentrations below 100 ⁇ .
  • Example 4 is known literature (Nakamura M, Katsuki Y, Shibutani Y, Oikawa T. Dienogest, a synthetic steroid, suppresses both embryonic and tumor-cell-induced angiogenes is. Eur J Pharmacol. 386, 33-40 (1999). ).
  • MCDB131 manufactured by Sigma
  • 10 ⁇ g / mL endothelial cell growth supplement manufactured by Upstate Biotechnology
  • 10 gZmL ⁇ , 5 A medium supplemented with S-GAD (0 M, 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, ⁇ , 100 ⁇ ) was added and cultured for 18 hours.
  • the culture supernatant was collected and centrifuged, and the ⁇ -2 activity and 9-9 activity of the supernatant were analyzed by a gelatin-zymography method.
  • the gelatin zymography method is a method for detecting the activity of MMP by utilizing gelatin, which is a denatured product of collagen, as a substrate for many MMPs.
  • gelatin is mixed with SDS-polyacrylamide electrophoresis gel and a sample containing MMP is separated on this gel and then subjected to an enzymatic reaction, MMP moves through the gel by a fixed distance depending on the size of the molecule. Place It stays in and breaks down the gelatin. Therefore, after the reaction, when the gel is dyed with a protein stain, only the portion where MMP is present is dyed and becomes a transparent gel, and the activity of MMP is detected. The results are shown in FIG.
  • Example 5 is a known literature (Oikawa T, Murakami K, Sano M, Shibata J, Wierzba K, Yamada Y. A potential use of a synthetic retinoid TAC-101 as an orally active agent t hat blocks angiogenesis in liver metastases of Human stomach cancer cells. Jpn J Cancer Res., 92, 1225-1234 (2001)).
  • HUVEC (4.5 X 10 5 cells / mL, 2 mU) was seeded on a 35 mm culture dish (IWAKI) and cultured for 24 hours, and then 0.1% sushi serum was added to MCDB131 (Sigma).
  • Culture medium supplemented with albumin, 10 ig / mL endothelial cell growth supplement, 10 ig / mL heparin and 5-S-GA ⁇ (0 ⁇ , 0. ⁇ , 0.
  • Example 6 is a known document (Fukazawa H, Li PM, Mizuno S, Uehara Y. Method for simu Itaneous detection of protein kinase A, protein kinase C, protein tyrosine kinase, an d calmodulin-dependent protein kinase activities.Analy Biochem., 212, 106-110 (199
  • Mouse NIH3T3 cells expressing the v_src oncogene were treated with hypotonic buffer (ImM Hepes-NaOH (pH7.4), 5 mM MgCl, 25 ⁇ g / mL antipain, leupe).
  • hypotonic buffer ImM Hepes-NaOH (pH7.4), 5 mM MgCl, 25 ⁇ g / mL antipain, leupe).
  • the cell lysate was prepared by stirring for 1 minute with a vortex mixer. The cell lysate was centrifuged at 500 ⁇ g for 5 minutes at 4 ° C., and the centrifuged supernatant was recovered as the enucleated fraction. Add an appropriate amount of reagent to the enucleated fraction, protein concentration lmg / mL, 20 mM Hepes—NaOH (pH 7.
  • PKA protein kinase 8
  • PLC protein kinase ⁇
  • eEF _ 2KJ eEF-2 kinase (eukaryotic elongation factor 2 kinase; calmodulin-dependent protein kinase ⁇ )
  • ⁇ indicates the position of tyrosine kinase.
  • the disappearance of the band represents inhibition of activity.
  • Example 7 is a known document (Hanai N, Nores G, Torres-Mendez C, Hakomori S. Modifie d ganglioside as a possiole modulator of transmembrane signaling mechanism through growth factor receptors: a preliminary note. Biochem Biophys Res Commun, 147, 1 27-134 (1987); Bertics PJ, Gill GN. Self-phosphoylation enhances the protein-tyr osine kinase activity of the epidermal growth factor receptor. J Biol Chem, 260, 146 42-14647 (1985)) It was.
  • a membrane fraction was prepared from A431 (human epidermoid carcinoma) cells. 15 ⁇ L reaction
  • EGF EGF receptor tyrosine kinase inhibitor AG1 478 (0. 001, 0. l ig / mL), 25.
  • 5 ⁇ L [ ⁇ - 32 P]
  • a TP 5 iCi / assay; l / iM
  • the reaction was stopped and the supernatant was spotted on P81 cellulose paper (Whatman) After washing, the radioactivity was measured with a scintillation counter 1. The results are shown in FIG.
  • 5-S-GAD did not inhibit EGF receptor tyrosin kinase activity at concentrations below ⁇ .
  • Example 8 is from Koyaguchi (Sawano A, Takahashi T, Yamagucni S, Shibuya M. Tne pho sphorylated 1169-tyrosine containing region of flt-l kinase (VEGFR-l) is a major binding site for PL and gamma. Biochem Biophys Res Commun., 238, 487-491 (1997) was used as a reference.
  • kinase assay buffer (5 OmM HEPES (pH7.4)) supplemented with 5_S_GAD (10xgZmL, 100 ⁇ gZmU or VEGF receptor tyrosine kinase inhibitor SU4984 (1 ⁇ g / mL, 10 ⁇ g / mL) , 0.1% Triton X-100, lOmM MnCl, 2mM MgCl,
  • the disappearance of the band represents inhibition of VEGF receptor tyrosine kinase activity.
  • the corneal epithelial cells in the silver nitrate application part were peeled off to expose the corneal surface, and silver nitrate filter paper was applied to the corneal surface for 1 minute 30 seconds. Immediately after application, it was washed with 10 mL of physiological saline. Immediately after that, 5 _S _GAD (5 _S _GAD administration group) or physiological saline (control group) was applied to each cornea using 50 ⁇ L of both corneas using an Eppendorf micropipette. An ointment (manufactured by Santen Pharmaceutical) was administered. Two weeks from the next day, 50 ⁇ L each of 5_S-GAD or physiological saline was instilled into the corneas of each group 4 times a day.
  • neovascular model hemorrhoids 7 days before, 5 _S _GAD or physiological saline was applied to both corneas 50 times a day for 4 times a day, and neovascular model hemorrhoids were prepared by the method described above. Used to observe angiogenesis over time. At that time, the number of new blood vessels in the sample eye of each group was counted and scored as follows.
  • ⁇ The number of blood vessels that do not reach the corneal epithelial cell exfoliation part from the outer edge of the cornea (however, blood vessels having a length of 1/4 or more of the distance from the outer edge of the cornea to the center of the cornea were targeted).
  • Table 1 shows the scores after 1 week and 3 weeks in each group.
  • Tumor cells S180; Sarcoma 180 tumor cell line) 0.1 5 mL (2 X 10 7 ) with a Millipore filter (pore size: 0.45 xm, Mil lipore) on both sides of the Millipore ring (Millipore) Cell Z chamber 1) was injected. Air was injected into the dorsum of the anaesthetized mouse (15-week-old female ICR mouse) subcutaneously from the dorsum. The skin was incised and a chamber was inserted into the head of the air injection part, and the insertion site was closed. One implant 1, 2, 3, 4 day chamber, site genin (C yt 0 g en i n ) (100mg / kg body weight) or 5- S- GAD (12.
  • site genin C yt 0 g en i n
  • the ip was administered.
  • the back of an anesthetized mouse was incised around the chamber, and the skin was peeled off.
  • a rubber packing of the same type as the ring was placed on the skin-contacting part of the chamber.
  • the number of new blood vessels having a length of 3 mm or more was measured with a stereomicroscope. The results are shown in FIG.
  • FIG. 12 administration of 5-S-GAD made existing blood vessels thinner.
  • Figure 12 (a) is PBS treatment, (b) is cytogenin treatment, (c) is 5-S-GAD (200mg / kg) treatment, (d) is 5-S-GAD (50mg / kg). Represents a process.
  • the chicken embryo chorioallantoic membrane (CAM) method was performed as follows.
  • 5-S-GAD inhibited angiogenesis in chicken embryos. Further, as shown in FIG. 13 (b), the inhibition of angiogenesis by 5-S-GAD was dose-dependent.

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Abstract

La présente invention concerne un nouvel inhibiteur de l’angiogenèse et un nouvel agent de régression vasculaire. L’inhibiteur de l’angiogenèse et l’agent de régression vasculaire comprennent chacun un dérivé de la 3,4-dihydroxyphénylalanine tel que N-β-alanyl-5-S-glutathionyl-3,4-dihydroxyphénylalanine, β-alanyl-3,4-dihydroxyphénylalanine et 5-S-cystéinyl-3,4-dihydroxyphénylalanine ou un sel pharmaceutiquement acceptable de celui-ci en tant que principe actif.
PCT/JP2005/013713 2005-07-27 2005-07-27 Inhibiteur de l’angiogenèse et agent de régression vasculaire WO2007013147A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01279828A (ja) * 1988-01-19 1989-11-10 Takeda Chem Ind Ltd 脈管形成阻止剤
JPH08337594A (ja) * 1995-06-13 1996-12-24 Sanwa Kagaku Kenkyusho Co Ltd 新規化合物及びその用途
JP2001213799A (ja) * 2000-01-28 2001-08-07 Inst Of Physical & Chemical Res 抗腫瘍剤
JP2001226283A (ja) * 2000-02-17 2001-08-21 Inst Of Physical & Chemical Res ５−ｓ−ｇａｄを有効成分とするアポトーシス誘導剤
JP2001278785A (ja) * 2000-03-30 2001-10-10 Inst Of Physical & Chemical Res 骨疾患の治療剤
WO2003070269A1 (fr) * 2002-02-18 2003-08-28 Cevec Pharmaceuticals Gmbh Traitement de maladies de l'oeil, de l'oreille interne et du systeme nerveux central
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01279828A (ja) * 1988-01-19 1989-11-10 Takeda Chem Ind Ltd 脈管形成阻止剤
JPH08337594A (ja) * 1995-06-13 1996-12-24 Sanwa Kagaku Kenkyusho Co Ltd 新規化合物及びその用途
JP2001213799A (ja) * 2000-01-28 2001-08-07 Inst Of Physical & Chemical Res 抗腫瘍剤
JP2001226283A (ja) * 2000-02-17 2001-08-21 Inst Of Physical & Chemical Res ５−ｓ−ｇａｄを有効成分とするアポトーシス誘導剤
JP2001278785A (ja) * 2000-03-30 2001-10-10 Inst Of Physical & Chemical Res 骨疾患の治療剤
WO2003070269A1 (fr) * 2002-02-18 2003-08-28 Cevec Pharmaceuticals Gmbh Traitement de maladies de l'oeil, de l'oreille interne et du systeme nerveux central
JP2005213159A (ja) * 2004-01-27 2005-08-11 Inbiotex:Kk 血管新生阻害剤及び血管退縮剤

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KILARSKI W. ET AL.: "Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for mechanism involving organization of the actin cytoskeleton.", EXPERIMENTAL CELL RESEARCH, vol. 291, pages 70 - 82, XP001183269 *
ZHENG Z. ET AL.: "Selective Inhibition of Src Protein Tyrosine Kinase by Analogues if 5-S-Glutathionyl-beta-alanyl-L-dopa.", CHEM.PHARM.BULL., vol. 46, no. 12, pages 1950 - 1951, XP002991712 *

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