WO2007136225A1 - Procédé d'inhibition d'angiogénèse faisant intervenir dkk1 et composition comprenant dkk1 - Google Patents

Procédé d'inhibition d'angiogénèse faisant intervenir dkk1 et composition comprenant dkk1 Download PDF

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Publication number
WO2007136225A1
WO2007136225A1 PCT/KR2007/002492 KR2007002492W WO2007136225A1 WO 2007136225 A1 WO2007136225 A1 WO 2007136225A1 KR 2007002492 W KR2007002492 W KR 2007002492W WO 2007136225 A1 WO2007136225 A1 WO 2007136225A1
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dkkl
protein
angiogenesis
diseases caused
dkkl protein
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PCT/KR2007/002492
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English (en)
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Young Guen Kwon
Jeong Ki Min
Chae Ok Yun
Young Myeong Kim
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Industry-Academic Cooperation Foundation, Yonsei University
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Publication of WO2007136225A1 publication Critical patent/WO2007136225A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors

Definitions

  • the present invention relates to a method for inhibiting angiogenesis using by DKKl protein and the composition comprising the same.
  • Angiogenesis is a process by which new capillary blood vessels are formed. This process rarely occurs under normal biological condition but it always accompanied by embryogenesis, corpus luteum formation and wound healing. Particularly, angiogenesis plays an important role in tumor metastasis (Folkman J and Klagsburn M, Science, 235(4787). pp.442-447, 1987).
  • Angiogenesis procedure consists of four steps, i.e., the 1 st step is the dissociation of capillary basal lamina by the action of protease enzyme caused by the stimulation of angiogenic factors, the 2" step is the migration and proliferation of blood endothelial cells, the 3 r step is the formation of capillary tubes due to the differentiation of blood endothelial cell, and the 4 step is the reconstruction of new capillary blood vessels.
  • an- giogenesis-involved diseases such as hemangioma, hemangiofibroma, vascular malformation, and so on; cardiovascular diseases such as arteriosclerosis, vascular ad hesion, edematous sclerosis and so on; ophthalmic diseases such as corneal graft neovascularization, neovascular glaucoma, diabetic retinopathy, corneal neovascularization, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, trachoma and so on; chronological inflammatory disease such as arthritis and so on; dermatological disease such as psoriasis, telangiectasia, pyogenic granuloma, seborrhoic dermatitis, acne and so on; alzheimer's disease, obesity and tumor
  • Angiogenesis is accompanied with many genetic changes during the differentiation of vascular endothelial cell. Accordingly, recently, many researches have been focused on the regulation of angiogenesis through the control of differentiation-controlling genes and a method for inhibiting angiogenesis. However, it has been reported that already-known inhibitors of angiogenesis have various side effects such as the damage of vessel tissues, limit to obtain desired efficacy etc. Accordingly, there has been still needed to develop new method for treating angiogenesis with safe and desirable efficacy till now.
  • DKKl a repressor protein of Wnt protein is one of the Dickkopf family members and has been firstly reported that it plays an important role in forming the head of amphibian, Xenopus (Glinka A et al., Nature, 391(6665 * ). pp.357-362, 1998). It has two specific cysteine-rich domains and is divided into various lengths of connection regions. Particularly, the protein belonged to Dickkopf family highly conserves cystein-2 region between the family members as well as 10 cysteines (Krupnik VE et al., Gene, 238m. pp.301-313, 1999).
  • the present inventors of the present invention have intensively studied to find several differentiation-control genes in endothelial cell and new effective method for inhibiting angiogenesis finally, they have found that DKKl shows potent inhibiting effect on angiogenesis and thus the protein could be useful in treatment and prevention of angiogenesis-involved diseases.
  • the present invention provides a method for inhibiting angiogenesis in a mammal, comprising the step of administering to mammal an effective amount of DKKl (Dickkopf- 1) protein or DKKl protein-encoding DNA.
  • DKKl Dickkopf- 1
  • DKKl protein-encoding DNA disclosed herein is administered to mammal using by viral vectors or non- viral vectors.
  • non-viral vectors comprises the plasmid which can be expressed in animal cells.
  • viral vectors comprises adenovirus vector, adeno- associated virus vector, retrovirus vector, lentivirus vector or herpes simplex virus vector.
  • It is another object of the present invention to provide a pharmaceutical composition comprising DKKl protein or DKKl protein-encoding DNA as an active ingredient in an effective amount to treat and prevent the diseases caused by angiogenesis.
  • DKKl protein-encoding DNA for manufacture of medicines employed for treating or preventing the diseases caused by the angiogenesis.
  • a health care food composition comprising DKKl protein or DKKl protein-encoding DNA as an active ingredient in an effective amount to prevent and alleviate the diseases caused by angiogenesis.
  • the term "disease caused by angiogenesis" disclosed herein comprises hemangioma, hemangiofibroma, vascular malformation, arteriosclerosis, vascular adhesion, edematous sclerosis, corneal graft neovascularization, neovascular glaucoma, diabetic retinopathy, corneal neovascularization, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, trachoma, arthritis, psoriasis, telangiectasia, pyo genie granuloma, seborrhoic dermatitis, acne, alzheimer's disease, obesity or tumor in human or mammal.
  • DKKl protein disclosed herein comprises the amino acid represented by
  • DKKl protein-encoding DNA comprises the gene represented by SEQ ID NO: 2.
  • DKKl protein comprises the DKKl protein isolated from the tissues of mammal and recombinant DKKl proteins.
  • inventive DKKl protein and the gene encoding the same may be prepared in accordance with the following preferred embodiment.
  • RNA purified from HUVEC is reverse-transcribed to obtain complimentary DNA at the 1 st step; PCR was performed to obtain complimentary DNA as a template and DKKl primers, preferably, the DKKl primers represented by SEQ ID NO: 5 and SEQ ID NO: 6 and amplified DKKl genes at the 2 nd step; the DKKl genes prepared from 2 TM step are treated with restriction enzyme, preferably EcoRI and Xhol, cloned into protein purifying plasmid, preferably pcDNA-His vector, to obtain plasmid at the 3 r step; the plasmid is transformed with expression cell lines, preferably, human renal cell line 293 (ATCC, USA) using gene delivery reagent, preferably, Lipofectamin (Invitrogen, USA) at the 4 step; the transformed cells are selected using by selective-transforming cell isolating reagents, preferably, G418 (Sigma, USA) and mass-cultured in medium at the 5 step; and the medium is
  • the DKKl proteins prepared by the above-described step inhibit the tube formation in HUVEC and DKKl over-expressed cell lines transformed with DKKl protein- encoding DNA, the blood vessel sprouting in arterial circle tissues of transformed mice with DKKl protein encoding DNA, and the vascular development in the embryo of the mice. Therefore, it has been confirmed that the proteins of the present invention showed potent angiogenesis-inhibiting activity. Additionally, not only full-length of the DKKl but also fragment of DKKl show similar angiogenesis-inhibiting activity each other.
  • It is another object of the present invention to provide a pharmaceutical composition comprising DKKl protein or DKKl protein-encoding DNA prepared by the above- described preparation method as an active ingredient in an effective amount to treat and prevent the diseases caused by angiogenesis.
  • DKKl protein-encoding DNA prepared by the above-described preparation method for manufacture of medicines employed for treating or preventing the diseases caused by the angiogenesis.
  • the inventive composition for treating the disease caused by the angiogenesis may comprise the above-described DKKl protein or DKKl protein-encoding DNA as 0.1 ⁇ 50% by weight based on the total weight of the composition.
  • inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).
  • composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • pharmaceutically acceptable carriers e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl
  • the formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.
  • the compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient, for example, human or mammal such as mouse, rat, livestock, etc by employing any of the procedures well known in the art.
  • compositions containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).
  • oral dosage form prowder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule
  • topical preparation cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like
  • injectable preparation solution, suspension, emulsion
  • compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection.
  • suitable examples of the base or carrier in the injection include various salt mixture such as physiological saline, inorganic salt or the mixture thereof; sugar solution such as mannitol, lactose, dextran etc; amino acid such as glycine, arginine etc; polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, organic acid solution, salt solution, or the mixture thereof etc, but are not limited to them.
  • the injectable preparation of the present invention may be prepared by adding conventional additives in injection, for example, osmotic controller, pH controller, vegetable oil, lecithin, surfactant such as non-ionic surfactant to the above-described base in order to make appropriate formulation such as solution, suspension, colloidal solution etc.
  • osmotic controller for example, osmotic controller, pH controller, vegetable oil, lecithin, surfactant such as non-ionic surfactant
  • the composition is dissolved in sterilized base prior to use in genetic therapy and liquid composition of the present invention may be used directly without particular treatment.
  • composition of the present invention can be formulated in the form of ointments and creams.
  • composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active ingredients.
  • the DKKl protein-encoding DNA disclosed herein to be supplied into the affected part may be used in the inserted form of vectors, for example adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, herpes simplex virus vector or plasmid expressed in mammal cells.
  • vectors for example adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, herpes simplex virus vector or plasmid expressed in mammal cells.
  • the desirable dose of the inventive composition varies depending on the condition such as age, sex etc and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to topically administer into the affected part at the amount ranging from 0.001 to 100mg/kg, preferably, 0.1 to 100mg/kg by weight/week one or more than once a week of the inventive composition of the present invention through catheter after surgery. The dose may be administered in single or divided into several times per day or week.
  • compositions therein can be added to food, additive or beverage, wherein, the amount of the above described protein or DNA in food or beverage may generally range from about 0.01 to 95w%, preferably 1 to 80w% of total weight of food for the health care food composition.
  • the present invention provides a composition of the health care beverage comprising a DKKl protein or DKKl protein-encoding DNA for preventing and alleviating the disease caused by the angiogenesis in mammal.
  • examples of addable food comprising the above- described protein or DNA of the present invention are various food, beverage, gum, vitamin complex, health improving food and the like, and can be used as powder, granule, tablet, chewing tablet, capsule or beverage etc.
  • composition of the present invention has no toxicity and adverse effect therefore they can be used with safe.
  • the above-described composition therein can be added to food, additive or beverage, wherein, the amount of the above-described DKKl protein or DKKl protein-encoding DNA in food or beverage may generally range from about 0.01 to 80w/w%, preferably 0.01 to 15w/w% of total weight of food for the health food composition and 0.02 to 5g, preferably 0.3 to Ig on the ratio of 100ml of the health care beverage composition.
  • the health care beverage composition of present invention contains the above-described DKKl protein or DKKl protein-encoding DNA as an essential component in the indicated ratio
  • the other component can be various deodorant or natural carbohydrate etc such as conventional beverage.
  • natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc.
  • natural deodorant such as taumatin, stevia extract such as levaudioside A, gly- cyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al.
  • the amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 Cl of present beverage composition.
  • the other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al.
  • the other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination.
  • the ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition.
  • Examples of addable food comprising aforementioned protein or DNA therein are various food, beverage, gum, vitamin complex, health improving food and the like.
  • the inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural anti-oxidants, such as polyphenol, catechin, ⁇ - tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc.
  • organic acid such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid
  • phosphate such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate
  • natural anti-oxidants such as polyphenol, catechin, ⁇ - tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc.
  • the above-described inventive DKKl protein may be 20 to 90 % high concentrated liquid, power, or granule type.
  • the above-described DKKl protein or DKKl protein-encoding DNA can comprise additionally one or more than one of lactose, casein, dextrose, glucose, sucrose and sorbitol.
  • Inventive DKKl protein or DKKl protein-encoding DNA of the present invention has no toxicity and adverse effect therefore; they can be used with safe.
  • the DKKl showed potent inhibitory effect on the tube formation of HUVEC, sprouting of arterial circle tissues and vascular development of mice embryo. Therefore, it can be used as the therapeutics or functional health food for treating and preventing diseases caused by the angiogenesis.
  • Fig. 1 shows the differentiation procedure of the HUVEC (Human umbilical vein endothelial cell) on Matrigel
  • Fig. 2 represents the expression of DKKl gene on differentiation of the HUVEC according to each stage
  • Fig. 3 presents the confirmed result of the expression and purification of DKKl recombinant
  • FIG. 4 shows the inhibition of tube formation on HUVEC according to the concentration of treated DKKl recombinant
  • Fig. 5 represents the produced result of DKKl expression cell line and DKKl repression cell line using by lentiviruses
  • FIG. 6 shows the comparison result of the tube formation on DKKl expression and repression cell lines
  • Fig. 7 presents the vector diagram for producing DKKl -transgenic mouse
  • Fig. 8 represents the confirmed result of DKKl -transgenic mouse by DNA amplification
  • FIG. 9 shows the comparison results of mouse size of normal and DKKl -transgenic mouse
  • Fig. 10 presents the repression of the sprouting of endothelial cells from aorta of DKKl -transgenic mouse
  • Fig. 11 represents the comparison results of angiogenesis in embryo of normal mouse and DKKl -transgenic mouse
  • FIG. 12 shows the comparison results of angiogenesis in the head region of embryo
  • Fig .13 presents the growth of descending aorta in embryo of normal mouse and DKKl -transgenic mouse
  • Fig. 14 presents the growth of segmental vessel in embryo of normal mouse and DKKl -transgenic mouse. Best Mode for Carrying Out the Invention [106] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.
  • HUVECs Human umbilical vein endothelial cell
  • the collected human umbilical vein endothelial cells was washed and poured to the coated T75 flask used for tissue culture with 0.1% of gelatin.
  • the cells were cultured in EGMTM-2 complete medium (Cambrex, MD, USA) in 5% CO 2 culture incubator at 37°C and when the cells became to confluent phase, the cells were separated from trypsin-EDTA solution.
  • the cells of 3-4 passage obtained from the above process were used in the experiment.
  • PCR cycles was performed 30 times using by polymerase (Stratagen, USA) as follows; pre- denaturation at 94°C for 5min; denaturation at 94°C for 30sec; annealing at 50 0 C for 30sec using by DKKl primers represented by SEQ ID NO: 5 and 6; extension at 72°C for 30sec.
  • the cloned plasmid was transformed into human embryonic kidney cell line 293 (ATCC, USA) using by Lipofectamin (Invitrogen, USA).
  • the transformed cells were selected using by G418 reagent (Sigma, USA), a selective transformation cell isolating agent and selected cells were cultured in medium on a large scale.
  • the secreted DKKl proteins in the medium were purified with nickel column (Ni-NTA agarose, Qiagen, USA) and the expression of recombinant DKKl protein was confirmed ( See, Fig. 3).
  • RNA from the cells was performed to reverse-transcription and polymerization to confirm the expression phase of DKKl mRNA as follows: total RNA was isolated using by TRIzol reagent (Invitrogen, USA), performed to reverse transcription using by oligo (dT) primer and following PCR cycles were repeated 30 times using by reverse transcriptase (Stratagen, USA); pre-denaturation at 94°C for 5min using by polymerase (Stratagen, USA), denaturation at 94°C for 30sec, annealing at 50°C for 30sec using by primers and extension at 72°C for 30sec.
  • the DKKl over expression mouse was prepared using the Tie2 transcription regulatory region activated in only vascular endothelial cells to determine the effect of DKKl gene on angiogenesis in vivo (Schlaeger TM et al., Proc. Natl. Acad. ScL USA, 94(7). pp.3058-3063, 1997).
  • mouse DKKl gene represented by SEQ ID NO: 4 was treated with Hindm and NotI(NEB, England) and cloned into Psp vector (Clontech, USA).
  • the cloned plasmid was treated with SaII(NEB, England) to prepare DNA fragments and the prepared DNA fragments were injected into the ovules isolated from the mouse (C57BL6, Orient Inc, Korea) of which ovulation had been stimulated by gonadotropin releasing-hormone (Sigma, USA) to induce transduction, and then, the DKKl-transgenic ovules were implanted on surrogate mother mouse after fertilization.
  • the tail of the mouse born after the 21 st fertilization was cut, treated with proteinase K (Sigma, USA) to isolate DNA, and the isolated DNA was amplified by PCR [(pre-denaturation at 94°C for 5min, denaturation at 94°C for 30sec, annealing at 55°C for 30sec and extension at 72°C for 30sec)x30 cycles and post-extension at 72°C for lOmin].
  • differentiation consists of 3 steps; the 1 st step is the beginning of differentiation used as control group, the 2 nd step is the formation of blood vessel-like structure due to cell transfer and the 3 step is the completion of blood vessel-like structure formation.
  • the HUVECs prepared in Reference Example 1 were cultured in M 199 growth medium (Invitrogen, USA) containing 20 % (v/v) fetal bovine serum (FBS, Hyclone, USA), 100 units/D of penicillin (Invitrogen, USA), 10D/D of streptomycin (Invitrogen, USA), 3ng/D of bFGF (basic fibroblast growth factor; Upstate Biotechnology, USA) and 5 units/D of heparin (Sigma, USA) and trypsin was added thereto to obtain cultured cells.
  • M 199 growth medium Invitrogen, USA
  • FBS % (v/v) fetal bovine serum
  • FBS fetal bovine serum
  • penicillin Invitrogen, USA
  • the cells were suspended in the growth medium and spread onto Matrigel layer in the concentration of 2x10 cells/well to induce the differentiation of cells ( See, Fig. 4). 50ng/D and lOOng/D of DKKl was treated thereto and then the cells were cultured for 20 hours. The rate of tube formation was measured by an optical microscopy (ZEISS, Germany) and the group which was not treated with DKKl was regarded as a negative control group.
  • V wf von Willebrand Factor
  • Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2D ample and sterilizing by conventional injection preparation method.
  • Powder preparation was prepared by mixing above components and filling sealed package.
  • Tablet preparation was prepared by mixing above components and entabletting.
  • Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [197]
  • Liquid preparation was prepared by dissolving active component, and then filling all the components in IOOOD ample and sterilizing by conventional liquid preparation method.
  • Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 0 C for 1 hour, filtered and then filling all the components in IOOOD ample and sterilizing by conventional health beverage preparation method.
  • the DKKl showed potent inhibitory effect on the tube formation of HUVEC, sprouting of arterial circle tissues and vascular development of mice embryo. Therefore, it can be used as the therapeutics or functional health food for treating and preventing diseases caused by the angiogenesis.
  • SEQ ID NO: 1 is amino acid sequence of human DKKl
  • SEQ ID NO:2 is DNA sequence of human DKKl
  • SEQ ID NO:3 is amino acid sequence of mouse DKKl
  • SEQ ID NO:4 is DNA sequence of mouse DKKl
  • SEQ ID NO:5 is forward primer sequence of human DKKl
  • SEQ ID NO:6 is reverse primer sequence of human DKKl
  • SEQ ID NO:7 is forward primer sequence of mouse DKKl
  • SEQ ID NO:8 is reverse primer sequence of mouse DKKl.

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Abstract

L'invention concerne un procédé d'inhibition d'angiogénèse faisant intervenir la protéine DKK1 et une composition comprenant DKK1. La protéine DKK1 et l'ADN codant la protéine DKK1 de l'invention présentent un effet inhibiteur sur la formation de tubes dans des cellules endothéliales de veine ombilicale humaine (HUVEC), le bourgeonnement de tissus circulaires artériels et le développement vasculaire dans des embryons de souris. Par conséquent, l'invention peut être utilisée en tant que thérapeutique ou qu'aliments diététiques pour traiter et pour prévenir des maladies provoquées par l'angiogénèse.
PCT/KR2007/002492 2006-05-24 2007-05-23 Procédé d'inhibition d'angiogénèse faisant intervenir dkk1 et composition comprenant dkk1 WO2007136225A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2009036338A2 (fr) * 2007-09-14 2009-03-19 Farjo Rafal A Procédés pour traiter des maladies oculaires par interférence avec la voie de signalisation wnt
EP2098244A1 (fr) 2008-03-04 2009-09-09 Medizinische Hochschule Hannover Composition pharmaceutique pour le traitement de l'infarctus du myocarde
EP2626073A1 (fr) * 2012-02-13 2013-08-14 Harmonic Pharma Composé destiné à être utilisé dans la prévention et/ou le traitement d'une maladie neurodégénérative ou d'une maladie impliquant une activation de phosphodiesterase-4 (PDE4)
CN110393792A (zh) * 2019-08-15 2019-11-01 安济盛生物医药技术(广州)有限公司 Dickkopf-1蛋白在制备用于治疗X-连锁低磷血症佝偻病的药剂中的用途

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WO2009036338A2 (fr) * 2007-09-14 2009-03-19 Farjo Rafal A Procédés pour traiter des maladies oculaires par interférence avec la voie de signalisation wnt
WO2009036338A3 (fr) * 2007-09-14 2010-08-12 Farjo Rafal A Procédés pour traiter des maladies oculaires par interférence avec la voie de signalisation wnt
EP2098244A1 (fr) 2008-03-04 2009-09-09 Medizinische Hochschule Hannover Composition pharmaceutique pour le traitement de l'infarctus du myocarde
EP2626073A1 (fr) * 2012-02-13 2013-08-14 Harmonic Pharma Composé destiné à être utilisé dans la prévention et/ou le traitement d'une maladie neurodégénérative ou d'une maladie impliquant une activation de phosphodiesterase-4 (PDE4)
WO2013120896A1 (fr) * 2012-02-13 2013-08-22 Harmonic Pharma Composé destiné à être utilisé dans la prévention et/ou le traitement d'une maladie neurodégénérative ou d'une maladie comprenant une activation de la phosphodiestérase-4 (pde4)
CN110393792A (zh) * 2019-08-15 2019-11-01 安济盛生物医药技术(广州)有限公司 Dickkopf-1蛋白在制备用于治疗X-连锁低磷血症佝偻病的药剂中的用途

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