WO2016018090A1 - Peptide utilisé pour promouvoir l'angiogenèse et utilisation de ce dernier - Google Patents

Peptide utilisé pour promouvoir l'angiogenèse et utilisation de ce dernier Download PDF

Info

Publication number
WO2016018090A1
WO2016018090A1 PCT/KR2015/007968 KR2015007968W WO2016018090A1 WO 2016018090 A1 WO2016018090 A1 WO 2016018090A1 KR 2015007968 W KR2015007968 W KR 2015007968W WO 2016018090 A1 WO2016018090 A1 WO 2016018090A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
angiogenesis
composition
treatment
present
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2015/007968
Other languages
English (en)
Korean (ko)
Inventor
오억수
이유미
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Industry Academic Cooperation Foundation of KNU
Original Assignee
Industry Academic Cooperation Foundation of KNU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Industry Academic Cooperation Foundation of KNU filed Critical Industry Academic Cooperation Foundation of KNU
Publication of WO2016018090A1 publication Critical patent/WO2016018090A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids

Definitions

  • the present invention provides a composition for promoting angiogenesis comprising a novel isolated peptide, a pharmaceutical composition for preventing or treating angiogenic dependent diseases comprising the peptide, a method for promoting angiogenesis comprising administering the peptide And it relates to a method of treating angiogenic dependent disease, comprising administering the pharmaceutical composition.
  • Angiogenesis refers to the process by which new blood vessels are formed, which rarely occurs in normal living conditions, but is an essential process in embryogenesis, corpus luteum formation or wound healing.
  • the angiogenesis process is generally caused by stimulation of angiogenesis factors, resulting in the formation of lumen due to the degradation of the basal membrane, proliferation, proliferation, and differentiation of vascular endothelial cells due to proteases. Consists of being generated.
  • Angiogenesis processes are known to be tightly controlled by several types of promoters and inhibitors, including growth factors, cytokines, lipid metabolites and latent fragments of hemostatic proteins.
  • angiogenesis treatment The treatment of angiogenesis using angiogenesis is called angiogenesis treatment.
  • Angiogenesis promoters such as vascular endothelial growth factor (VEGF) are used as treatments for severe ischemia, but the isolation and purification of these factors is difficult and expensive. Therefore, there is a difficulty in clinical application, and the development of new and more effective factors for the treatment of diseases in which symptoms may be improved by vascular tissue repair is continuously required.
  • VEGF vascular endothelial growth factor
  • the present inventors have made intensive efforts to develop an effective therapeutic agent for a disease requiring the generation of neovascularization, and as a result, 16 amino acid sequences separated by MMP-7 from the extracellular domain of syndecan-2 were produced.
  • the peptides increase the proliferation, mobility and angiogenic ability of vascular endothelial cells, and exhibit an angiogenic promoting effect, it was confirmed that it can be used for the treatment of diseases caused by the lack of neovascularization, and completed the present invention.
  • An object of the present invention is to provide an angiogenesis composition
  • an isolated peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of angiogenic dependent diseases comprising the peptide.
  • Another object of the present invention is to provide a method for promoting angiogenesis, comprising administering the peptide to a subject in need of angiogenesis.
  • Still another object of the present invention is to provide a method for treating angiogenic dependent disease, comprising administering the pharmaceutical composition to a subject suspected of being an angiogenic dependent disease.
  • the angiogenic composition comprising the isolated peptide of the present invention increases the ability of neovascularization by increasing the proliferation, mobility, and the like of vascular endothelial cells to treat diseases caused by a lack of neovascularization such as wound healing, ischemic diseases, infertility, and the like. It can be usefully used.
  • FIG. 1 shows a sequence of a Cindecan-2 peptide consisting of 16 amino acid sequences constructed from some amino acid sequences of a portion cleaved by MMP-7 in the extracellular domain of Cindecan-2.
  • FIG. 2 is a graph showing the increase in the growth of vascular endothelial cells in a concentration-dependent manner when treated with Sindecane-2 peptide.
  • Figure 3 shows that the movement of the vascular endothelial cells when treated with the syndecan-2 peptide by taking a picture of the degree of movement of the cells under a microscope using a transwell analysis.
  • Syndecane-2 peptide is indicated as SDC2 in the figures.
  • 4 is a graph showing the number of cells treated and treated with the dedecane-2 peptide.
  • Figure 5 is a photograph showing the degree of angiogenesis after treatment with the synthecan-2 peptide under a microscope. VEGF was used as a positive control.
  • FIG. 6 is a graph showing the treatment of the syndecan-2 peptide and measuring the length of the blood vessel formed. VEGF was used as a positive control.
  • Figure 7 shows by observing through the microscope the appearance of microvascularization when treated with syndecane-2 in vivo .
  • Figure 8 is a graph showing the measurement of the number of micro-vascular generated when treated with indecane-2 in vivo .
  • FIG. 9 is an image of Western blot results of analyzing expression of vascular endothelial protein.
  • Figure 9a shows the increase in tyrosine phosphorylation of the proteins when treated with the dedecane-2 peptide, Western blot
  • Figure 9b shows VEGFR, PI3K, Akt and JNK when treated with the dedecane-2 peptide Phosphorylation was increased by Western blot
  • Figure 9c shows that the expression level of ⁇ v ⁇ 4 integrin was increased by comparing the expression level of integrin after the treatment of syndecan-2 peptide.
  • FIG. 10 is an image of a western blot result of analyzing protein expression of cancer cells.
  • Figure 10a shows that the expression of HIF-1 and the phosphorylation of Akt308 increased as the treatment concentration increased when the HCT116 cell line treated with the dedecane-2
  • Figure 10b shows the HCT116 cell line
  • the increase in HIF-1 expression and phosphorylation of Akt308 when treated with 5 nM of the Sindecane-2 peptide was shown by Western blot.
  • FIG. 10C shows that HIF- was treated when 5 nM of the dec29-2 peptide line was treated with Sindecane-2 peptide.
  • 1 Expression of protein, increased phosphorylation of Akt308 and Akt473 is shown by Western blot.
  • FIG. 11 is an image of RT-PCR results confirming the increase in VEGF gene expression by synthecan-2 peptide treatment.
  • FIG. Figure 11a shows the amount of VEGF mRNA after 24 hours treatment with 5nM of the decane-2 peptide in the HCT116 cell line through RT-PCR
  • Figure 11b is 5nM of the dec29- in the HT29 cell line After 24 hours of treatment with 2 peptides, the amount of VEGF mRNA was confirmed by RT-PCR.
  • 12 is an image of the result of confirming the state of HIF-1 protein by synthecan-2 peptide treatment in a hypoxic state (5% O 2 ).
  • 12 a shows the amount of HIF-1 protein expression in the HT29 cell line through Western blot
  • FIG. 12 b shows the immunoprecipitation of ubiquitination of the HIF-1 protein in the HT29 cell line. ) Is shown through the assay.
  • angiogenesis composition comprising an isolated peptide, consisting of an amino acid sequence represented by SEQ ID NO: 1 as an aspect.
  • Amino acid sequences used in the present invention are abbreviated as follows according to the IUPAC-IUB nomenclature.
  • Protein or isolated peptide of the present invention refers to a peptide having angiogenic activity.
  • the peptide is a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 may be specifically composed of 16 amino acids, but is not limited thereto.
  • the peptide comprising the amino acid of SEQ ID NO: 1 is added or removed at the terminal portion of the sequence, as long as it exhibits angiogenic activity, or even if some amino acids of the sequence is mutated in the scope of the present invention Inclusion is self-evident.
  • the peptide comprising the amino acid sequence of SEQ ID NO: 1 is an amino acid sequence 90% or more, specifically 96%, more specifically 98% or more, even more specifically 99% or more identical to the sequence, and promote angiogenesis Peptides with activity are also included within the scope of the present invention.
  • the peptide of the present invention may be protected from protein cleavage enzymes in vivo, and its N-terminus and / or C-terminus may be chemically modified or protected with an organic terminus, or amino acids may be added to the peptide terminus. It may be in a modified form.
  • acetylation of the N-terminus and / or amidation of the C-terminus are performed to remove such charges. It may be, but is not particularly limited thereto.
  • the peptides of the present invention can be synthesized by methods well known in the art, for example by automated peptide synthesizers, depending on their length, and can also be produced by genetic engineering techniques. For example, through genetic engineering, a fusion gene encoding a fusion protein, comprising a fusion partner and a peptide of the present invention, is prepared and transformed into a host cell, and then expressed in the form of a fusion protein, a protease or compound The peptide of the present invention may be cleaved and separated from the fusion protein to produce a desired peptide.
  • a protease such as Factor Xa or enterokinase
  • a compound such as CNBr or hydroxylamine
  • the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is prepared based on the sequence of syndecane-2.
  • Syndecan-2 protein is a cell binding receptor present on the cell surface and is a transmembrane heparan-sulfate proteoglycan.
  • syndecane-2 it has been reported that it can be used for diagnosing bowel cancer by quantitatively analyzing the methylation of the syndecane-2 gene (Korean Patent No. 1,145,406), but a specific site thereof, in particular SEQ ID NO: 1 Peptide sites comprising the amino acid sequence of have not yet been associated with angiogenesis.
  • the syndecan-2 gene and protein information may be obtained from a known database such as the National Center for Biotechnology Information (NCBI), for example, NM_002998 and NP_002989 registered with the NCBI, but one mutated sequence having the activity thereof may be used. It can include without limitation.
  • NCBI National Center for Biotechnology Information
  • angiogenesis refers to any phenomenon in which blood vessels are newly formed as new blood vessels are formed.
  • cells constituting existing blood vessels proliferate and migrate to form new blood vessels. It may refer to a phenomenon, and may include a phenomenon in which blood vessels are newly generated by progenitor cells that make blood vessels.
  • angiogenesis promotion means to promote angiogenesis such that the production of blood vessels such as capillaries can be made in a faster time and / or more blood vessels.
  • blood vessels such as capillaries
  • the formation and growth of the fetus, the formation of organs, and the healing of wounds are essential for the formation of blood vessels and the circulation of blood. Can be.
  • the isolated syndecane-2 peptide for angiogenesis promotion of the present invention may be characterized by increasing the proliferative capacity of vascular endothelial cells, the ability of vascular endothelial cells to move, angiogenesis and angiogenesis.
  • vascular endothelial cells increased depending on the concentration when treated with the isolated Shindecan-2 peptide (Fig. 2), also through the transwell movement analysis of vascular endothelial cells It was confirmed that the increase (Fig. 3 and 4). From this, it can be seen that the level similar to the proliferation and migration of vascular endothelial cells induced by VEGF treated with a positive control. In addition, as shown in FIG. 5 and FIG. 6, it was confirmed that angiogenesis of vascular endothelial cells was increased by the syndecan-2 peptide.
  • the composition of the present invention comprising the syndecane-2 peptide may be a composition for promoting angiogenesis, and in particular, may be a composition for preparing blood vessels for skin plate regeneration, wound and burn treatment, artificial skin transplantation and transplantation, but is not limited thereto. It is not.
  • skin plate regeneration, wound and burn treatment, artificial skin transplantation and implantation of blood vessels to induce the formation of tissues the smooth supply of blood is essential, promoting the angiogenesis using the peptide of the present invention to increase the effect Can be.
  • compositions for the prevention or treatment of angiogenic dependent diseases comprising the isolated peptide.
  • the angiogenesis-dependent disease refers to a disease that occurs or worsens due to inability to supply blood or oxygen due to abnormal blood vessels caused by various causes, and specifically refers to a disease requiring angiogenesis, but is not limited thereto.
  • one or more days selected from the group consisting of ischemia, infertility, diabetic foot ulcer, ischemic stroke, ulcer, arteriosclerosis, myocardial infarction, angina pectoris, ischemic heart failure, pressure sores, alopecia, acute posterior limb ischemia and cerebrovascular dementia May be, but is not limited thereto.
  • Ischemia, ischemic stroke, atherosclerosis, myocardial infarction, and angina are caused by blockage or narrowing of blood vessels, which can lead to disorders of body tissues and organs if oxygen and nutrients are not properly supplied through blood vessels.
  • this vascular dysfunction occurs in the heart muscle, the heart stops and myocardial infarction, angina pectoris, etc. occurs in the hands or the tip of the foot, ischemic retardation disease, and when it occurs in the brain, ischemic stroke, cerebrovascular dementia, etc. Can be.
  • Ischemic heart failure and acute posterior limb ischemia can also be caused by lack of oxygen due to inadequate blood supply due to blockage or narrowing of the ducts.
  • Diabetic foot ulcers are the most common complications of diabetes and are caused by high blood sugar levels in the blood vessels of diabetic patients.
  • the cure of such a disease depends on the formation of new connective tissue and epithelial tissue by cell proliferation, and it is effective to promote or stimulate the proliferation and differentiation of cells.
  • pressure sores are also called pressure ulcers, and vascular ulcers are caused by dying tissues due to constant or repeated pressure applied to any part of the body. Can be.
  • infertility may be caused by poor blood circulation in the uterus and blood circulation in the capillaries, resulting in poor conception of fertilized eggs. It can be cured.
  • hair loss refers to an abnormally high number of hair dropouts, and may occur when blood supply is insufficient.
  • One of the methods of treating hair loss is a method of promoting blood circulation through the expansion of capillaries, and by promoting the angiogenesis using the peptide of the present invention can prevent or treat hair loss or promote hair growth.
  • the diseases may be alleviated or ameliorated by a method such as transplanting blood vessels or angiogenesis by bypassing blood vessels, and the disease may be caused by angiogenesis or transplantation of blood vessels through a pharmaceutical composition comprising the peptide of the present invention. Improve or heal them.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound to be injected without stimulating the organism.
  • the kind of the carrier usable in the present invention is not particularly limited, and any carrier can be used as long as it is a conventionally used and pharmaceutically acceptable carrier in the art.
  • Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more thereof.
  • the carrier may include a non-naturally occuring carrier.
  • composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose), gelatin and the like can be mixed.
  • lubricants such as magnesium stearate, talc and the like can also be used.
  • Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable esterol such as ethyl oleate, and the like can be used.
  • the pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.
  • composition of the present invention may be administered in a pharmaceutically effective amount.
  • the term "pharmaceutically effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to an individual's type and severity, age, sex, activity of the drug. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts.
  • Preventive agent for angiogenic dependent diseases such as ischemia, infertility, diabetic foot ulcer, ischemic stroke, ulcer, arteriosclerosis, myocardial infarction, angina pectoris, ischemic heart failure, pressure sores, alopecia, acute posterior limb ischemia or cerebrovascular dementia or
  • the therapeutic agent may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two or three times.
  • the compositions of the present invention can be used alone or in combination with other drug treatments for the prevention or treatment of angiogenic dependent diseases. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.
  • a method for promoting angiogenesis comprising administering the angiogenic composition to a site in need of angiogenesis.
  • the term "individual” means all animals including humans in need of angiogenesis, and the angiogenesis composition may be administered to an individual to promote angiogenesis.
  • the subject means an entire mammal including a dog, cow, horse, rabbit, mouse, rat, chicken or human, but the mammal of the present invention is not limited to the above examples. Specifically, the subject may be an individual except a human.
  • the isolated syndecane-2 peptide of the present invention exhibited an effect of promoting angiogenesis even in vivo (FIGS. 7 and 8), and promoting angiogenesis including the peptide.
  • the composition can be administered to promote angiogenesis.
  • Another aspect of the present invention provides a method for treating angiogenic dependent disease, comprising administering the pharmaceutical composition for preventing or treating angiogenic dependent disease to a subject suspected of being angiogenic dependent disease.
  • the term “individual” means all animals including humans in need of preventing or treating angiogenic dependent diseases, and the disease can be effectively prevented and treated by administering the pharmaceutical composition.
  • the subject means an entire mammal including a dog, cow, horse, rabbit, mouse, rat, chicken or human, but the mammal of the present invention is not limited to the above examples. Specifically, the subject may be an individual except a human.
  • the route of administration of the pharmaceutical composition may be administered via any general route as long as it can reach the target tissue.
  • the composition of the present invention may be administered parenterally, intraperitoneally, intravenously, subcutaneously, subcutaneously, or orally, as desired.
  • the composition may also be administered by any device in which the active agent may migrate to the target cell. Frequency of administration is as described above.
  • the angiogenic dependent disease is a group consisting of ischemia, infertility, diabetic foot ulcer, ischemic stroke, ulcer, arteriosclerosis, myocardial infarction, angina pectoris, ischemic heart failure, pressure sores, hair loss, acute posterior limb ischemia and cerebrovascular dementia It may be at least one selected from.
  • Residues 79-94 (named hS2LQ: LTSAAPKVETTTLNIQ; SEQ ID NO: 1) of syndecan-2, a peptide consisting of 16 amino acid sequences in the syndecan-2 extracellular domain, were constructed.
  • scrambled peptides for hS2LQ (Scr: IPNTSVKTLTAQLAET; SEQ ID NO: 2) as a control were synthesized using an improved version of the solid-phase method using Fmoc chemistry (Anygen Inc., Gwangju, Korea) (FIG. 1). .
  • Human umbilical vein endothelial cells isolated from human umbilical cord (HUVEC) in 1% gelatin coated dish 20% FBS, 1% penicillin / streptomycin, 5 unit / Incubated in a 37 ° C. thermostat with 5% carbon dioxide using M199 (Hyclone, Logan) medium containing ml Heparin and 2 ng / ml basic-fibroblast growth factor (bFGF).
  • M199 Hyclone, Logan
  • bFGF basic-fibroblast growth factor
  • Colon cancer cell lines HCT116 and HT29 cells were cultured in a 37 ° C thermostat with 5% carbon dioxide using McCoy 5A medium (Welgene, Seoul, Korea) containing 10% FBS, 1% penicillin / streptomycin.
  • vascular endothelial cells were dispensed in a 96-well plate coated with 1% gelatin, 20% FBS, 1 After culturing for about 24 hours in M199 medium containing% penicillin / streptomycin, 5 unit / ml heparin, and 2 ng / ml bFGF, it was changed to low serum medium (M199 medium containing 0.5% FBS) and incubated for 16 hours. The effect of growth factors was removed.
  • VEGF vascular endothelial growth factor
  • the proliferative capacity of vascular endothelial cells was increased when treated with the dedecane-2 peptide, and the proliferative capacity was increased to 5 nM or more.
  • the effect of syndecan-2 peptide on the motility of vascular endothelial cells was observed using a transwell migration assay.
  • a 24-well transwell (Costar, 8 ⁇ m pore size) was coated with type I collagen (0.5 mg / ml) and used after drying for 1 hour at room temperature.
  • a 10 nM synthecan-2 peptide-treated medium was placed and the chamber was incubated for 24 hours with the same number of vascular endothelial cells along with a medium without FBS, and the number of cells passing through the membrane was counted.
  • 20 ng / ml VEGF was treated as a positive control.
  • the separated cells were fixed with methanol for 1 minute, stained with hematoxylin for 10 minutes, and eosin for 4 minutes, and the cells on the unmoved membrane were removed with a cotton swab and balsam on slide glass.
  • the membranes were fixed by staining and the stained cells were checked under a microscope. And counting the number of cells moved to the graph.
  • the effect of the syndecan-2 peptide on the vascular endothelial ability of vascular endothelial cells was observed using a tube formation assay. Specifically, 60 ⁇ l of Matrigel (Matrigel, BD) was laid on a 96-well plate, and then gelled at 37 ° C. for 30 minutes. After culturing the vascular endothelial cells in a medium treated with 10 nM syndecane-2 peptide 8 hours later, tube formation was confirmed under a microscope. Medium treated with 20 ng / ml VEGF was used as a positive control. In addition, the length of the formed tube was measured and graphed.
  • Matrigel Matrigel
  • CAM deposit chorioallantoic membrane
  • the purchased fertilized egg was raised for 3 days while maintaining a saturation humidity of 90% in an incubator at 37 °C, after removing about 3 ml of albumin using a syringe, the egg shell was broken and a window of a certain size was identified, and confirmed as a fertilized egg Only one was sealed with glass tape.
  • 10 nM of syndecan-2 peptide was applied to the Thermanox cover slip, dried, placed on the CAM site, sealed with tape, and incubated for 3 days.
  • PMA phorobol 12-myristate 13-acetate
  • the protein changes in vascular endothelial cells were analyzed by Western blot after treatment with synthecan-2 peptide. Specifically, after treatment with 10 nM syndecan-2 peptide to vascular endothelial cells, cells were recovered after 24 hours, lysed cells using RIPA buffer, and protein phosphorylation and expression changes were confirmed by Western blot. The negative control was observed by treating the scrambled peptide (Scr).
  • VEGF receptors VAGFR
  • PI3K phosphoinositide 3-kinase
  • AKT phosphoinositide 3-kinase
  • JNK c-JUN N-terminal kinase
  • Primary antibodies include anti-phosphorylated VEGF receptor (hosphor-VEGFR) antibodies, anti-phosphorylated phosphoinositide 3-kinase (hosphor-PI3K) antibodies, anti-phosphorylated hosphor-AKT (AKT) antibodies, anti-phosphorylated Erk (hosphor-Erk) , Anti-phosphorylated p38 (hosphor-p38) and anti-phosphorylated c-JUN N-terminal kinase (hosphor-JNK) antibodies were reacted for 16 hours.
  • the primary antibodies were purchased from Cell Signaling (Cell Signaling, Danvers, MA, USA) and reacted at a concentration of 0.2 ⁇ g / ml.
  • integrin one of the adhesion receptors known to increase expression when angiogenesis was increased through Western blot.
  • the primary antibody was reacted for 16 hours with anti- ⁇ v antibody, anti- ⁇ 5 antibody, anti- ⁇ 6 antibody, anti- ⁇ 1 antibody, anti- ⁇ 3 antibody and anti- ⁇ 4 antibody.
  • the primary antibodies were purchased from Cell Signaling (Cell Signaling, Danvers, MA, USA) and reacted at a concentration of 0.2 ⁇ g / ml. Then, the solution was washed with TBST solution and reacted for 16 hours using HRP-coupled goat anti-rabbit IgG antibody (AbClon, Seoul, Korea / 0.1 ⁇ g / ml) as a secondary antibody.
  • neovascularization increases the expression of various genes by increasing the transcription factor of HIF (hypoxia inducible factor) due to the hypoxic state in the tissue by cancer cells and binding to the HRE (hypoxia response element), the promoter region of the VEGF gene. Stabilized. The resulting VEGF promotes cell growth and migration specific to vascular endothelial cells and increases vascular permeability.
  • HIF hypooxia inducible factor
  • HCT116 cells were treated at various concentrations and time periods in HCT116 cells, which are colorectal cancer cell lines, and HIF-1 expression levels were compared.
  • HT29 cells which are other colorectal cancer cell lines, were treated with 5 nM of Sindecan-2 peptide for 24 hours, and then Western blot was used to analyze protein expression changes.
  • anti-HIF-1 antibodies (BD science, San Jose, California, USA / 0.2 ⁇ g / ml), anti-phosphorylated Akt 308 antibody and anti-phosphorylated Akt 473 antibody (Cell Signaling (Danvers, MA, USA) / 0.2 ⁇ g / ml) after 16 hours, washed with TBST solution, 16 hours using goat anti-mouse IgG antibody (AbClon, Seoul, Korea / 0.1 ⁇ g / ml) bound to HRP as a secondary antibody Reacted for a while.
  • Anti-HIF-1 antibodies BD science, San Jose, California, USA / 0.2 ⁇ g / ml
  • anti-phosphorylated Akt 308 antibody and anti-phosphorylated Akt 473 antibody Cell Signaling (Danvers, MA, USA) / 0.2 ⁇ g / ml) after 16 hours, washed with TBST solution, 16 hours using goat anti-mouse IgG antibody (AbClon, Seoul, Korea
  • HIF-1 expression and phosphorylation of Akt 308 at 6, 15 and 24 hours after treatment with 5 nM of the Sindecan-2 peptide were compared to the case of treatment with scrambled peptide, a negative control. Expression of -1 protein was significantly increased, and it was confirmed that phosphorylation of Akt 308 was significantly increased (FIG. 10B).
  • RT-PCR confirmed the amount of VEGF mRNA in cancer cells.
  • a PCR composition comprising a reaction buffer (ELPiS, Korea), 1 mM dNTP (ELPiS, Korea), 1 Unit Taq polymerase (ELPiS, Korea), 0.5 pmole primer (Cosmogenetech, Korea) and 1.5 ⁇ g cDNA
  • reaction buffer ELPiS, Korea
  • 1 mM dNTP ELPiS, Korea
  • Unit Taq polymerase ELPiS, Korea
  • 0.5 pmole primer Cosmogenetech, Korea
  • 1.5 ⁇ g cDNA After the reaction for 5 minutes at 94 °C (initial denaturation), 30 seconds of denaturation at 94 °C, annealing at 54 °C 30 seconds, extension 30 seconds at 72 °C was 25 cycles.
  • primer sequences used are summarized in Table 1 below.
  • VEGF mRNA As described above, the amount of VEGF mRNA was confirmed after 24 hours treatment of 5 nM syndecan-2 peptide to HCT116 and HT29 colorectal cancer cell lines, indicating that the amount of VEGF mRNA was increased in both cell lines (FIG. 11). b) of a and 11;
  • the colon cancer cell line HT-29 was incubated in a serum-free medium for 12 hours, and then treated with 5% O 2 to establish a hypoxic condition.
  • the HIF-1 stabilizing effect of the syndecan-2 peptide was confirmed by Western blot, and the effect of the syndecane-2 peptide on the HIF-1 ubiquitination was confirmed by an immunoprecipitation assay.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne: une composition utilisée pour promouvoir l'angiogenèse, contenant un nouveau peptide isolé; une composition pharmaceutique pour la prévention ou le traitement de maladies dépendantes de l'angiogenèse, contenant le peptide; une méthode pour promouvoir l'angiogenèse, comprenant une étape consistant à administrer le peptide; et une méthode de traitement de maladies dépendant de l'angiogenèse, comprenant une étape consistant à administrer la composition pharmaceutique.
PCT/KR2015/007968 2014-07-30 2015-07-30 Peptide utilisé pour promouvoir l'angiogenèse et utilisation de ce dernier Ceased WO2016018090A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020140097265A KR101721733B1 (ko) 2014-07-30 2014-07-30 혈관신생촉진용 펩타이드 및 이의 용도
KR10-2014-0097265 2014-07-30

Publications (1)

Publication Number Publication Date
WO2016018090A1 true WO2016018090A1 (fr) 2016-02-04

Family

ID=55217872

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/007968 Ceased WO2016018090A1 (fr) 2014-07-30 2015-07-30 Peptide utilisé pour promouvoir l'angiogenèse et utilisation de ce dernier

Country Status (2)

Country Link
KR (1) KR101721733B1 (fr)
WO (1) WO2016018090A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018172031A1 (fr) 2017-03-23 2018-09-27 Raimund Beck Nageltechnik Gmbh Clou en particulier dans une cloueuse

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220140176A (ko) 2021-04-09 2022-10-18 경북대학교 산학협력단 혈관신생 촉진용 약학적 조성물

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080020979A1 (en) * 2006-06-09 2008-01-24 Rapraeger Alan C Peptides of Syndecan-1 For Inhibiting Angiogenesis
US20090005295A1 (en) * 2002-01-18 2009-01-01 Ekker Stephen C Syndecans and angiogenesis

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5486599A (en) * 1989-03-29 1996-01-23 The Board Of Trustees Of The Leland Stanford Junior University Construction and use of synthetic constructs encoding syndecan
WO2014157952A1 (fr) * 2013-03-26 2014-10-02 이화여자대학교 산학협력단 Nouveau fragment de syndécan-2 et son utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090005295A1 (en) * 2002-01-18 2009-01-01 Ekker Stephen C Syndecans and angiogenesis
US20080020979A1 (en) * 2006-06-09 2008-01-24 Rapraeger Alan C Peptides of Syndecan-1 For Inhibiting Angiogenesis

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI. 3 February 2014 (2014-02-03), Database accession no. XP_005251077 .1 *
FEARS ET AL.: "Syndecan-2 is expressed tin the microvasculature of gliomas and regulates angiogenic processes in microvascular endothelial cells", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, no. 21, 2006, pages 14533 - 14536 *
NOGUER ET AL.: "Is syndecan-2 a key angiogenic element?", THE SCIENTIFIC WORLD JOURNAL, vol. 9, 2009, pages 729 - 73 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018172031A1 (fr) 2017-03-23 2018-09-27 Raimund Beck Nageltechnik Gmbh Clou en particulier dans une cloueuse

Also Published As

Publication number Publication date
KR101721733B1 (ko) 2017-03-31
KR20160015462A (ko) 2016-02-15

Similar Documents

Publication Publication Date Title
Klöting et al. Adipocyte dysfunction, inflammation and metabolic syndrome
US8158589B2 (en) Peptides with the capacity to bind to transforming growth factor β1 (TGF-β1)
Li et al. A frog peptide provides new strategies for the intervention against skin wound healing
WO2018074682A1 (fr) Peptide à efficacité anti-obésité et anti-diabétique et son utilisation
WO2015156649A1 (fr) Peptide présentant une activité inhibitrice contre la fibrose, et composition le contenant
Barbon et al. In vitro assessment of TAT—Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration
WO2014178653A1 (fr) Utilisation d'une petite protéine humaine à fermeture éclair à leucines lors d'une procédure de différenciation d'adipocytes
WO2016018090A1 (fr) Peptide utilisé pour promouvoir l'angiogenèse et utilisation de ce dernier
WO2021256751A1 (fr) Composition comprenant une vésicule extracellulaire dérivée des fibroblastes en tant que principe actif permettant le traitement de plaies cutanées et muqueuses
JP4548335B2 (ja) 腸管細胞のインスリン産生細胞への変換誘導剤、及び糖尿病治療剤
WO2020091463A1 (fr) Composition pharmaceutique comportant des mitochondries isolées pour prévenir ou traiter la ténopathie
WO2012161519A1 (fr) Lignée de cellules souches adultes introduite avec un gène du facteur de croissance hépatocytaire et un gène du facteur de transcription neurogène ayant un motif basique hélice-boucle-hélice et ses utilisations
Zhang et al. Novel pro-myogenic factor neoruscogenin induces muscle fiber hypertrophy by inhibiting MSTN maturation and activating the Akt/mTOR pathway
WO2022220632A1 (fr) Peptide de pénétration cellulaire, peptide anti-cancéreux, et composition pharmaceutique pour prévenir ou traiter le cancer le comprenant
WO2018021771A1 (fr) COMPOSITION DE PRÉVENTION OU DE TRAITEMENT DE LA FIBROSE OU DE LA CIRRHOSE HÉPATIQUE, COMPRENANT UN ACTIVATEUR D'EXPRESSION OU D'ACTIVITÉ DE TIF1γ COMME INGRÉDIENT ACTIF
WO2019143145A1 (fr) Composition pour le traitement d'une inflammation cutanée, comprenant de la résolvine d2 en tant que principe actif
WO2023153763A1 (fr) Vaccin contre le cancer comprenant un épitope de c-met et un épitope de hif1alpha, et son utilisation
WO2023224431A1 (fr) Composition pharmaceutique pour la prévention et le traitement de maladies gériatriques comprenant des cellules souches neurales dérivées d'un organoïde d'hypothalamus tridimensionnel et son utilisation
Hampel et al. Somatostatin supports corneal wound healing in vivo
WO2014178649A1 (fr) Utilisation d'une petite protéine humaine à fermeture éclair à leucine dans un processus d'ostéogenèse
WO2014109417A1 (fr) Nouveau peptide présentant une capacité de synthétisation du collagène et utilisation correspondante
KR101337767B1 (ko) Ｖｅｇｆ 발현을 증가시키는 신규한 펩타이드 및 이를 함유하는 약학적 조성물
WO2022250413A1 (fr) Anticorps destiné à identifier des réactions spécifiques de phosphorylation de la thréonine sur la position 1010 du ncapg2 et son utilisation
WO2023033534A1 (fr) Composition pharmaceutique comprenant un polypeptide kai1 destiné à l'inhibition de la fibrose hépatique et son utilisation
WO2015111971A1 (fr) Composition pharmaceutique contenant un ligand gpr119 comme principe actif pour prévenir ou traiter une stéatose hépatique non alcoolique

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15828008

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15828008

Country of ref document: EP

Kind code of ref document: A1