KR100888889B1 - Pharmaceutical Composition for Angiogenesis Stimulation Comprising Visfatin As an Active Ingredient - Google Patents

Abstract

The present invention relates to an angiogenesis-promoting agent containing bispatin as an active ingredient. More specifically, bispatin induces angiogenesis, migration, invasion and vascular formation of vascular endothelial cells associated with angiogenesis. It relates to a new use to promote angiogenesis by activating it.

Pharmaceutical compositions containing bispartin according to the present invention induce angiogenesis and thus are useful for the treatment of angiogenic diseases such as chronic ulcers, delayed wound healing, ischemic stroke, myocardial infarction, angina pectoris and cerebrovascular dementia.

Bispartin, Angiogenesis

Description

Pharmaceutical Composition for Angiogenesis Stimulation Comprising Visfatin As an Active Ingredient}

The present invention relates to an angiogenesis-promoting agent containing bispatin as an active ingredient. More specifically, bispatin induces angiogenesis, migration, invasion and vascular formation of vascular endothelial cells associated with angiogenesis. It relates to a new use to promote angiogenesis by activating it.

Angiogenesis is a phenomenon in which endothelial cells of existing blood vessels decompose, move, divide, and differentiate extracellular matrix to form new capillaries. Wound repair, embryonic development, tumor formation, chronic inflammation, obesity, etc. Involved in physiological and pathological phenomena.

Angiogenesis is a process of vascular endothelial cell proliferation and migration from the vessel wall to the surrounding tissue in the direction of stimulation. Subsequently, various proteolytic enzymes are activated so that vascular endothelial cells infiltrate the basement membrane and form loops, and the formed loops are differentiated to form tubes. These angiogenic processes are known to be tightly regulated by several types of promoters and inhibitors.

Recently, there have been active attempts to treat angiogenesis-dependent diseases such as cancer, rheumatoid arthritis, psoriasis, ulcers, ischemia, arteriosclerosis, myocardial infarction, angina, and cerebrovascular diseases using factors that induce or inhibit angiogenesis. (Folkman J., J. Nat . Med ., 1:27, 1995; Jackson JR, et al ., FASEB J. , 11: 457, 1997; Risau W., Nature , 386: 671, 1997; Bussolino, F., et al ., Trends Biochem . Sci ., 22: 251, 1997; Hanahan D., et al ., Cell , 86: 353, 1996).

In normal adults, the replacement time of endothelial cells that make up blood vessels generally varies from 47 days to 20,000 days and is under very strict control. In general, angiogenesis inhibitors such as thrombospondin-1, platelet factor-4, angiostatin, vascular endothelial growth factor, fibroblasts Angiogenesis-promoting factors such as basic fibroblast growth factor usually maintain quantitative equilibrium so that angiogenesis does not occur.However, in the case of wounds or cancers, angiogenesis inhibitors are necessary for regeneration of wounded tissues and cancer growth. The quantitative equilibrium of and promoters is broken and new blood vessels are formed, at which time the overexpression of angiogenic promoters is involved.

Excessive blood vessel formation may be a major cause of disease deterioration, but the formation of blood vessels is also a cause of serious disease. Angiogenesis is an essential phenomenon for wound healing or tissue regeneration.For example, the placenta that has not developed blood vessels is an important cause of miscarriage, and in the case of necrosis, ulceration and ischemia due to the formation of blood vessels, tissues or organs It may cause dysfunction or cause death. In addition, diseases such as arteriosclerosis, myocardial infarction and angina are caused by poor blood supply. Therefore, there is a need to develop a treatment to reduce tissue damage caused by hypoxic or hypotrophic conditions caused by the formation of blood vessels and to induce or promote new blood vessel formation for smooth tissue regeneration.

In particular, angiogenesis must be involved in the necessary wound healing process for the rejuvenation of wounded tissue. In the early stages of the wound, inflammatory reactions occur due to necrosis of the cells and destruction of blood vessels, which are accompanied by devascularization of blood components, activation of platelets and coagulation, and kallikrein, thrombin and plasmin. It goes through a series of processes in which biological mediators are formed.

Treatment of biological diseases using angiogenesis is called angiogenesis treatment, and angiogenesis promoters such as vascular endothelial growth factor have already been used as therapeutic agents for severe ischemia. In addition, angiogenic factors such as fibroblast growth factor, epidermal growth factor and platelet-derived epidermal growth factor have been studied for clinical treatment. However, these factors are difficult to isolate and purify as proteins and are expensive and have difficulty in clinical application.

Adipose tissue is a well-developed blood vessel, and angiogenesis is required for the growth of fat mass. Bispartin is a secretory protein secreted from adipose tissue, and has been reported to have a function similar to that of insulin. Recently, bispartin is known to be involved in the functional maturation of vascular smooth muscle cells. However, the effect of bispartin on angiogenesis of vascular endothelial cells is unknown.

Accordingly, the present inventors have made efforts to develop angiogenesis promoters, and have confirmed that bispartin induces angiogenesis and promotes angiogenesis by activating angiogenesis, invasion and vascular formation associated with angiogenesis. The invention was completed.

It is a main object of the present invention to provide an angiogenesis-promoting agent containing bispatin as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the treatment of angiogenic dependent diseases containing bispatin (visfatin) as an active ingredient.

In order to achieve the above object, the present invention provides an angiogenesis promoter containing bispatin (visfatin) as an active ingredient.

The present invention also provides a pharmaceutical composition for the treatment of angiogenic dependent diseases containing bispartin (visfatin) as an active ingredient.

In the present invention, the angiogenic disease may be selected from the group consisting of chronic ulcers, delayed wound healing, ischemic stroke, myocardial infarction, angina pectoris and cerebrovascular dementia.

The present invention has the effect of providing angiogenesis promoter containing bispatin as an active ingredient.

According to the present invention, bispartin may be used to induce angiogenesis, and thus it may be used for the treatment of angiogenic diseases such as chronic ulcer, delayed wound healing, ischemic stroke, myocardial infarction, angina pectoris and cerebrovascular dementia.

One aspect of the present invention relates to an angiogenic promoter containing bispartin as an active ingredient and a pharmaceutical composition for treating angiogenic dependent diseases containing bispartin as an active ingredient.

In an embodiment of the present invention, the bispartin is the only protein that is secreted from adipose tissue, which is constantly active in angiogenesis in adult tissues, it was confirmed whether bispatin can induce angiogenesis. To investigate the angiogenic activity of bispartin, we investigated the effects of bispartin on the migration and invasion of human umbilical vein endothelial cells (HUVECs) and tube formation of cells. Measured. As a result, it was found that bispartin increases the migration and invasion of HUVEC cells and promotes tube formation of HUVEC cells.

Sprouting assays using rat aortic rings to determine the angiogenesis of bispartin showed that bispartin promotes sprouting formation in rat aortic rings. I could confirm it. In addition, as a result of confirming angiogenesis in vivo using chorioallantoic membrane (CAM) and matrigel plug, it was found that bispartin promotes angiogenesis.

The pharmaceutical composition of the present invention can be formulated or used in combination with drugs such as antihistamines, anti-inflammatory drugs, anticancer agents and antibiotics that are already in use.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The pharmaceutical compositions according to the invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

The composition according to the present invention may be administered in the form of an injection to a site to be mixed with an injectable composition to induce angiogenesis in a mammal, and the injectable composition is preferably an isotonic aqueous solution or suspension, and the pharmaceutical composition may be Adjuvants such as preservatives, stabilizers, wetting agents, emulsifier solution accelerators, salts for regulating osmotic pressure and / or buffers or other therapeutically useful substances.

Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention may be administered at 0.5 mg / kg to 10 mg / kg per day, and may be administered several times to once a week depending on the half-life. The dosage does not limit the scope of the invention in any aspect.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example  1: recombination Bispatin  Produce

After performing the PCR using the primers of SEQ ID NO: 2 and SEQ ID NO: 3 of the entire cDNA of the human visfatin gene, the PCR product was cloned into the expression vector pET plasmid, and The recombinant vector was introduced into E. coli to prepare a transformant. The transformant was cultured to obtain and purify bispatin protein.

SEQ ID NO: 5'-atgaatcctg cggcagaagc-3 '

SEQ ID NO: 5'-atgatgtgctgcttccagttc-3 '

Example  2: Bispatin  Effect on cell migration

Human umbilical vein endothelial cells (HUVEC, Clonetics) were grown on growth media (M199; 20% FBS, 100unit / ml penicillin, 10µg / ml striptomycin, 3ng / ml fibroblast growth factor (basic fibroblast) growth factor, bFGF), and 5 units / ml heparin) at 37 ° C., 95% (v / v) CO ₂ , and 5% (v / v) air. After culturing the cultured cells in gelatin-coated 60mm plate until the cell is about 90% filled, the razor blades are vertically placed so that the centerline is displayed, and then the blades are laid down lightly about 5mm. I drew it and wounded it. After washing the cells with medium without serum, the cells were incubated for 24 hours by adding 1 mM thymidine and bispatin (10 ng / ml, 100 ng / ml) obtained in Example 1 to the medium. Next, fixation and Giemsa staining were observed and counted on an optical microscope (FIG. 1). As a positive control, vascular endothelial growth factor (VEGF) was previously known as angiogenesis factor. As a result, it was found that as the concentration of bispatin increases, the number of moving cells increases.

Example  3: Bispatin  Effect on Cell Infiltration

The effect of bispartin on cell infiltration was measured using a transwell insertion chamber with a polycarbonate filter with a pore size of 8 μm.

The bottom of the chamber filter was coated with 5 μg of type IV collagen, and the top of the filter was coated with 25 μg of Matrigel. 600 μl of medium was added to the main chamber, 400 μl of medium containing HUVEC (2 × 10 ⁵ cells / well) cells and bistatin (100 ng / ml) were added to the insertion chamber, and the insertion chamber was placed in the main chamber. Incubated for 16 hours at 37 ℃ incubator. After removing and fixing the polycarbonate filter of the incubation chamber, the matrigel was decomposed by staining with hematoxylin-eosin, and the number of cells passing through the filter was counted on an optical microscope (FIG. 2). ). As a result, it was found that the number of HUVEC cells passing through the filter by bispartin increases.

Example  3: Bispatin  Impact on tube formation

300 μl of Matrigel at a concentration of 10 mg / ml was dropped into a 24 well plate and left at 37 ° C. for 30 minutes to gel. After the gel was formed, obtained by trypsinizing the HUVEC cells cultured to the bottom, and then cultured into growth medium (M199) to 4 × 10 ⁵ cells / well, bispatin (100ng / ㎖ and 1000ng / ㎖ ) Was cultured in 1 ml of medium, and then observed by light microscopy to see if cells penetrated into the gel to form blood vessel-like shapes (FIG. 3). As a result, it was found that the addition of bispartin promotes tube formation of HUVEC cells.

Example  4: in rat aortic ring tissue Bispatin In sprouting  Impact

Aortic ring tissues obtained from 6-week-old Sprague Dawley rats were aliquoted into 48 well plates coated with 110 μl of Matrigel, and then coated with 50 μl of Matrigel, and then 100 μg / ml bispartin, each with a final volume of 200 μl. The serum removal medium added as much as possible was put in each well, and after 5 days, the degree of spouting from each ring was photographed by optical imaging technique (FIG. 4). As a result, it was confirmed that the sprouting increased by bispatin in the aortic ring tissue of the rat.

Example  5: CAM Using Bispatin  Measurement of the effects on the promotion of angiogenesis

Chorioallantoic membrane (CAM) was used to determine whether bispartin induces angiogenesis in vivo.

Egg yolk was removed from the egg albumin of the fertilized egg of the 3 day old chicken whose blood vessel formation was terminated, and egg yolk was separated from the shell film and sealed with a transparent tape so that CAM was observed. After 1 day, the cover slides were dried with negative control, 0.1 μg / ml PMA (positive control) and various concentrations of bispartin and treated with CAM. After 8 days, a 10% fat emulsion was injected into the CAM membrane to observe whether angiogenesis was promoted by anatomical microscopy (8x magnification). As a result, it was found that angiogenesis was significantly promoted when PMA or bispartin was treated as compared to the negative control (FIG. 5).

Example  6: Matrigel  Used Bispatin  Measurement of the effects on the promotion of angiogenesis

500 μl of Matrigel containing heparin (40 units / ml) and bispartin (10 μg / ml) were mixed at 4 ° C., and then injected between the abdominal membrane and the skin of 6-7 week old mice (C57BL / 6). As a positive control, vascular endothelial growth factor (VEGF) was treated. After 5-7 days, the skin of the mouse was peeled off and the formed matrigel plugs were removed from the mice, and hemoglobin analysis was performed to measure the overall degree of blood vessel formation (FIG. 6). As a result, new blood vessels were hardly formed in the negative control group, but new blood vessels were formed in the VEGF-treated group and the bispartin-treated group, and it was confirmed that the concentration increased significantly in a concentration-dependent manner. Therefore, it was found that the use of bispartin can effectively induce angiogenesis.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows the vascular endothelial cells migrated according to the treatment concentration of bispatin (visfatin).

Figure 2 shows the effect of promoting the transfer of vascular endothelial growth factor (VEGF) and bispartin vascular endothelial cells using a transwell.

Figure 3 is a culture of vascular endothelial cells on Matrigel to determine whether the formation of blood vessels when treated with bispartin.

Figure 4a shows the change in the degree of spouting by bispatin (A: photograph of the change in the degree of spouting by bispatin in the aortic ring of the rat; B: bispartin in the aortic ring of the rat Graph quantifying the effect of promoting blood vessels spouting.

Figure 5 shows the angiogenic action of bispartin. (A: micrograph showing the angiogenic action of bispartin in the chorionic villus; B: graph quantifying the number of CAMs representing angiogenesis versus the total number of CAMs in the chorionic villus).

Figure 6 is a measurement of the effect on the promotion of angiogenesis of bispartin using Matrigel (A: negative control group, positive control group (VEGF) and bispatin according to the Matrigel plug test method using a mouse B: quantitative analysis of blood vessels generated from negative control group, positive control group (VEGF), and bispartin according to the Matrigel plug test method using mice.

<110> Technology LicensingCenter Pusan University <120> PHARMACEUTICAL COMPOSITION FOR ANGIOGENESIS STIMULATION          COMPRISING VISFATIN AS AN ACTIVE INGREDIENT <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1476 <212> DNA <213> Homo sapiens <400> 1 atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60 actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120 gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180 gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240 caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300 tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360 gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420 tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480 gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540 ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600 caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660 gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720 tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780 gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840 tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900 tcgagaagta cacaggcacc actaataatc agacctgatt ctggaaaccc tcttgacact 960 gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020 tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080 ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140 ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200 tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agttgctgat 1260 cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320 gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380 gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440 cagctgaata ttgaactgga agcagcacat cattag 1476 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atgaatcctg cggcagaagc 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgatgtgct gcttccagtt c 21  

Claims (3)

Angiogenesis-promoting agent containing visfatin as an active ingredient. A pharmaceutical composition for the treatment of angiogenic dependent diseases selected from the group consisting of chronic ulcers, delayed wound healing, ischemic stroke, myocardial infarction, angina pectoris and cerebrovascular dementia containing visfatin as an active ingredient. delete

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