Pharmaceutical or cosmetic compositions

Abstract

Provided is a composition that is able to mature blood vessels or stabilize blood vessels.　This composition contains a mucopolysaccharide polysulfate and / or a salt thereof as an active ingredient and is useful in treatment and / or prevention of diseases with vascular lesions or lymphatic lesions. The above problem is solved by this composition.

Description

[Technical field]

[0001] The present invention relates to a composition, such as a pharmaceutical composition or a cosmetic composition, having a blood vessel maturation effect or a blood vessel stabilization effect. [Background technology]

[0002] Blood vessels are distributed throughout the body, and by circulating blood, they supply oxygen and nutrients to various organs, tissues, and cells, and discharge waste products, playing an important role in maintaining tissue homeostasis. Blood vessels have a two-layer structure in which vascular wall cells (vascular smooth muscle cells and pericytes) adhere to the outside of vascular endothelial cells, and have a stable structure formed through the processes of vasculogenesis, angiogenesis, and vascular maturation. During the process of vascular maturation, PDGF-BB, an isoform of platelet derived growth factor (PDGF) secreted by vascular endothelial cells, induces migration of vascular wall cells toward vascular endothelial cells through activation of PDGFRβ, a receptor expressed in vascular wall cells. Furthermore, angiopoietin-1 (hereinafter referred to as "Ang-1") secreted from vascular wall cells (pericytes) activates the Tie2 receptor expressed in vascular endothelial cells, induces adhesion between wall cells and endothelial cells, and stabilizes the vascular structure. On the other hand, angiopoietin-2 (hereinafter referred to as "Ang-2") is mainly secreted from vascular endothelial cells and acts antagonistically against the Ang-1 / Tie2 signal. In other words, it has been suggested that Ang-2 destabilizes the vascular structure by blocking the Ang-1 / Tie2 vascular stabilization signal, and is involved in the induction of angiogenesis. It is known that the decrease in PDGF-BB / PDGFRβ signaling and Ang-1 / Tie2 signaling during the vascular maturation process induces increased vascular permeability and leakage of plasma components, resulting in the formation of structurally unstable and immature blood vessels. Such structurally unstable blood vessels have been observed in various diseases accompanied by vascular lesions. Furthermore, it is known that PDGF-BB / PDGFRβ signaling and Ang-1 / Tie-2 signaling decrease with age, suggesting a relationship between vascular instability and aging. It has also been shown that Ang-1 / Tie-2 signaling contributes not only to the maturation and stabilization of blood vessels, but also to the stabilization of lymphatic vessels, promoting lymphatic flow.

[0003] Although Ang-1 / Tie-2 signaling and PDGF-BB / PDGFRβ signaling have been studied (for example, see Non-Patent Documents 1 and 2), almost no pharmaceuticals based on such mechanisms have been developed. Therefore, the current situation is that there is a strong demand for the prompt development of highly safe substances that have Tie2 activation activity, PDGF-BB production activity, and the like, and bring about vascular maturation or vascular stabilization, or lymphatic stabilization. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Journal of the Japanese Society of Cosmetic Technologists, 46(3):188-196, 2012 [Non-Patent Document 2] J Biochem, 163(1):3-9, 2018 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an objective of the present invention is to provide a composition that can activate Ang-1 / Tie2 signaling, increase PDGF-BB production, and the like by acting on vascular mural cells and endothelial cells, as well as lymphatic endothelial cells. [Means for solving the problem]

[0006] In order to solve the above-mentioned problems, the inventors have investigated various polysaccharides and found that polysulfated chondroitin sulfate and its salts, which are specific polysulfated mucopolysaccharides, have excellent effects such as activating the Ang-1 / Tie2 signal and increasing the production of PDGF-BB, thereby completing the present invention.

[0007] The present invention relates to a composition comprising the following polysulfated mucopolysaccharide and / or a salt thereof. (1) A composition comprising a polysulfated mucopolysaccharide and / or a salt thereof for treating and / or preventing a disease accompanied by vascular or lymphatic lesions, or for improving skin condition by improving vascular or lymphatic function. (2) The composition described in (1) above, wherein the polysulfated mucopolysaccharide comprises any one of polysulfated chondroitin sulfate, polysulfated dermatan sulfate, polysulfated keratan sulfate, polysulfated heparan sulfate, and salts thereof. (3) The composition described in (1) or (2) above, wherein the sulfate group content of the polysulfated mucopolysaccharide is 18% by weight or more. (4) The composition according to (3) above, wherein the polysulfated mucopolysaccharide has a sulfate group content of 20% by weight or more and 80% by weight or less. (5) The composition according to any one of (1) to (4) above, wherein the polysulfated mucopolysaccharide has a weight-average molecular weight of 8,000 to 10,000,000. (6) The composition according to any one of (1) to (5) above, wherein the polysulfated mucopolysaccharide has a weight-average molecular weight of 10,000 to 10,000,000. (7) The composition according to any one of (1) to (6) above, wherein the polysulfated mucopolysaccharide has an intrinsic viscosity of 0.05 to 0.30. (8) A Tie2 activator, a PDGF-BB production promoter, a vascular normalizer, a vascular stabilizer, or a lymphatic vessel stabilizer, comprising the composition according to any one of (1) to (7) above. (9) The pharmaceutical composition, wherein the disease accompanied by a vascular lesion is at least any one of peripheral arterial disease including atherosclerosis and subarachnoid hemorrhage, angioedema, lymphangioleiomyoma, inflammatory disease including psoriasis vulgaris and Schnitzler's syndrome, common warts, tuberous sclerosis, systemic sclerosis, Kaposi's sarcoma, cutaneous angiosarcoma, wrinkles, swelling, retinopathy, macular edema, skin ulcer, wound, nephropathy, peripheral neuropathy, diabetes, photoaging, retinopathy in premature infants and diabetic retinopathy, tumor, acute myeloid leukemia, hepatitis, and sepsis, comprising the composition according to any one of (1) to (7) above. (10) A pharmaceutical composition, wherein the disease accompanied by a vascular lesion is at least one of purpura (including drug-induced purpura) and chemotherapy-associated vascular disorder, the pharmaceutical composition comprising the composition according to any one of (1) to (7) above. (11) A cosmetic composition, the improvement of the skin condition being at least one of improvement of wrinkles, age spots, swelling, and alopecia, comprising the composition according to any one of (1) to (7) above. Effect of the Invention

[0008] The composition of the present invention has excellent effects such as Tie2 activation and increased production of PDGF-BB. Such a composition has a blood vessel maturation effect, a blood vessel stabilization effect, and a lymphatic vessel stabilization effect, and is therefore useful for the prevention and / or treatment of various diseases caused by a decrease in blood vessel function, lymphatic vessel function, etc. The composition of the present invention is also useful for the prevention and / or treatment of various diseases caused by blood leakage from blood vessels, etc. Furthermore, since the active ingredient, polysulfated mucopolysaccharide, is highly safe, it is possible to provide a pharmaceutical composition with few side effects. [Brief description of the drawings]

[0009] [Figure 1]1A) and 1B) show the Ang-1 production from parietal cells by polysulfated chondroitin sulfate, chondroitin sulfate (Chs), hyaluronic acid (HA), xylohexaose (O-XHE), and control (-: solvent only). Polysulfated chondroitin sulfate is a type of sulfated mucopolysaccharide, and is therefore hereinafter also referred to as MPS. [Diagram 2] 2A) and 2B) show graphs showing Ang-2 production from parietal cells by polysulfated chondroitin sulfate (MPS) (FIG. 2A), chondroitin sulfate (Chs), hyaluronic acid (HA), xylohexaose (O-XHE), and the control (-: solvent only) (FIG. 2B). [Diagram 3] FIG. 1 shows the effect of polysulfated chondroitin sulfate (MPS) on Tie2 phosphorylation. [Figure 4] 4A) and 4B) show the effects of polysulfated chondroitin sulfate (MPS) on PDGF-BB production from endothelial cells, and the effects of chondroitin sulfate (Chs), hyaluronic acid (HA), xylohexaose (O-XHE), and the control (-: solvent only). [Diagram 5] FIG. 1 is a graph showing the effect of MPS on VEGF-induced vascular leakage, and is a graph showing the relationship between the amount of polysulfated chondroitin sulfate (MPS) etc. administered intradermally to mice and the amount of dye (Evans blue) indicating vascular leakage induced by vascular endothelial growth factor (VEGF). [Figure 6] 6A is a graph showing the effect of MPS on the reduction in expression of Tie2 and Ang-1 due to repeated steroid administration, and shows the relationship between the presence or absence of polysulfated chondroitin sulfate (MPS) or the like administered percutaneously to mice and the amount of Tie2 measured after the reduction in expression (FIG. 6A); and the relationship between the presence or absence of polysulfated chondroitin sulfate (MPS) or the like administered percutaneously to mice and the amount of Ang-1 measured after the reduction in expression (FIG. 6B). [Figure 7]1 is a graph showing the effect of MPS on the rate of change in TEER value of human dermal microvascular endothelial cells. [Figure 8] FIG. 1 shows the effect of MPS on Claudin-5 in human dermal microvascular endothelial cells (detected protein bands). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described in detail below. The composition of the present invention includes a pharmaceutical composition and a cosmetic composition, as described in detail below. Examples of pharmaceutical compositions include compositions that fall under the category of medical drugs, prescription drugs, over-the-counter drugs, or quasi-drugs as defined in the "Law on Ensuring Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc." Examples of cosmetic compositions include cosmetics as defined in the "Law Concerning Ensuring Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc." and compositions that fall under the category of quasi-drugs, i.e. medicated cosmetics.

[0011] [Pharmaceutical composition] The pharmaceutical composition of the present invention prevents a decline in vascular or lymphatic function and is useful for the prevention and treatment of various diseases, particularly diseases relating to blood vessels and lymphatic vessels.

[0012] <Active ingredient> The pharmaceutical composition of the present invention contains a polysulfated mucopolysaccharide or a salt thereof as an active ingredient. Polysulfated mucopolysaccharides refer to long-chain polysaccharides having repeating units of disaccharides consisting of hexosamine and uronic acid, or hexosamine and galactose, and having sulfate groups. Some naturally occurring mucopolysaccharides have sulfate groups in the sugar chain, and therefore these and those that have been further chemically sulfated are also included in the category of polysulfated mucopolysaccharides.

[0013] The term "hexosamine" broadly refers to a compound in which the hydroxyl group of a hexose is substituted with an amino group, and specific examples include D-glucosamine, D-galactosamine, etc. The amino group of the hexosamine may be substituted with an acetyl group.

[0014] Furthermore, the term "uronic acid" broadly refers to a compound in which the primary alcohol of an aldose is oxidized to a carboxyl group, and specific examples thereof include naturally occurring uronic acids such as D-glucuronic acid, L-iduronic acid, D-galacturonic acid, D-mannuronic acid, and L-guluronic acid.

[0015] The polysulfated mucopolysaccharides are preferably polysulfated polysaccharides having a repeating unit of a disaccharide consisting of D-acetylgalactosamine and uronic acid, or D-acetylgalactosamine and galactose, and specific examples thereof include polysulfated chondroitin sulfate, polysulfated dermatan sulfate, polysulfated keratan sulfate, polysulfated heparan sulfate, etc. Among these specific examples of polysulfated mucopolysaccharides, polysulfated chondroitin sulfate and polysulfated dermatan sulfate are more preferred, and polysulfated chondroitin sulfate is even more preferred.

[0016] In addition, the number of sulfate groups contained in the polysulfated mucopolysaccharides is not particularly limited, but is usually 0.55 to 5.0 on average, preferably 0.60 to 2.9 on average, and more preferably 0.70 to 2.5 on average, per repeating saccharide unit. The organic sulfate group content of polysulfated mucopolysaccharides, i.e., the percentage of sulfate groups (-SO 4 The total weight ratio of the sulfate groups (including the sulfate groups) is preferably 18% by weight or more. The sulfate group content of the polysulfated mucopolysaccharide is more preferably 20% by weight or more and 80% by weight or less, further preferably 25% by weight or more and 60% by weight or less, and particularly preferably 25% by weight or more and 40% by weight or less.

[0017] The molecular weight of the polysulfated mucopolysaccharide or a salt thereof used in the present invention varies depending on the type of polysaccharide and is not limited, but the average molecular weight is, in weight average molecular weight, about 10,000 to 10,000,000, preferably about 15,000 to 1,000,000, more preferably about 20,000 to 100,000, and even more preferably about 30,000 to 50,000. The average molecular weight of the polysulfated mucopolysaccharide or a salt thereof may be, in weight average molecular weight, about 8,000 to 10,000,000, preferably about 8,000 to 1,000,000, more preferably about 10,000 to 100,000, and even more preferably about 10,000 to 50,000. In addition, the weight average molecular weight used in this specification is calculated by the following formula when Ni polymers each having a molecular weight of Mi are present: Weight average molecular weight (Mw)=Σ(Ni·Mi 2 ) / Σ(Ni·Mi) It is a value expressed as:

[0018] The polysulfated mucopolysaccharide such as polysulfated chondroitin sulfate preferably has an intrinsic viscosity of 0.05 to 0.30. The intrinsic viscosity value of the polysulfated mucopolysaccharide is more preferably 0.07 to 0.25, and further preferably 0.09 to 0.20.

[0019] The polysulfated mucopolysaccharides used in the present invention are not particularly limited in terms of origin, but more preferred examples include those that have been sulfated by artificially subjecting naturally occurring mucopolysaccharides to a sulfation treatment, as described above.

[0020] Methods for introducing sulfate groups into mucopolysaccharides such as chondroitin sulfate include known methods, such as a method in which mucopolysaccharides are reacted with a sulfating agent by heating in an appropriate solvent. The sulfating agent is not particularly limited as long as it can achieve the purpose of polysulfation, but it is preferable to use a complex of sulfuric anhydride and pyridine or triethylamine. The ratio of mucopolysaccharide to sulfating agent can be selected according to the desired sulfation rate (or sulfur content) of sulfated mucopolysaccharide and reaction conditions, but generally, the sulfating agent is used in a ratio of 2 to 10 parts by weight per part by weight of mucopolysaccharide. Examples of the solvent include protophilic solvents such as dimethylformamide. The reaction temperature and reaction time are not particularly limited as long as the desired sulfation rate can be achieved, but for example, the reaction is carried out at 40 to 90°C for about 30 minutes to 20 days.

[0021] The polysulfated mucopolysaccharides produced as described above can be purified by purification procedures commonly used for various modified polysaccharides, such as neutralization, desalting by dialysis, recovery of precipitates by addition of an organic solvent, and recovery by freeze-drying.

[0022] In the present invention, the polysulfated mucopolysaccharides may be used in the form of a physiologically acceptable salt, and examples of physiologically acceptable salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts, and magnesium salts, and also include multiple salts of these.

[0023] "Polysulfated chondroitin sulfate," a preferred example of a polysulfated mucopolysaccharide, is a polymer containing approximately 2 to 4, and preferably approximately 2 to 3, sulfate ester residues per disaccharide unit consisting of acetylgalactosamine (D-acetylgalactosamine) and glucuronic acid (D-glucuronic acid).

[0024] Polysulfated chondroitin sulfate can be easily produced by a known method of reacting a chondroitin component such as chondroitin or chondroitin sulfate (A, C, D, E) with a sulfating agent such as chlorosulfuric acid, concentrated sulfuric acid, sulfur trioxide-pyridine complex, etc. Chondroitin sulfate A has a sulfate ester residue at the 4th position of acetylgalactosamine, chondroitin sulfate C has a sulfate ester residue at the 6th position of acetylgalactosamine, chondroitin sulfate D has a sulfate ester residue at the 6th position of acetylgalactosamine and the 2nd or 3rd position of glucuronic acid, and chondroitin sulfate E has a sulfate ester residue at both the 4th and 6th positions of acetylgalactosamine.

[0025] Preferred examples of polysulfated chondroitin sulfate include heparinoids listed in the Japanese Pharmacopoeia Non-Drug Ingredients Standards. Specifically, it is a polysulfated chondroitin sulfate exhibiting the following physicochemical properties: a) Sulfate group content: 25.8~37.3% by weight b) Intrinsic viscosity: 0.09~0.18

[0026] Polysulfated chondroitin sulfate may be used in the form of a free acid derived from the sulfate residues, but is usually used in the form of a base salt.

[0027] Examples of the base salt include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium, and magnesium salts.

[0028] As described in detail below, polysulfated mucopolysaccharides and salts thereof enable the activation of Tie2 and promotion of PDGF-BB production, and contribute to the prevention of functional decline, maturation, and stabilization of blood vessels and lymphatic vessels. Therefore, polysulfated mucopolysaccharides, polysulfated mucopolysaccharide salts, and compositions containing any of them can also be used as Tie2 activators, PDGF-BB production promoters, blood vessel normalizers, blood vessel stabilizers, lymphatic vessel stabilizers, etc.

[0029] <Dosage form of pharmaceutical composition> The pharmaceutical composition of the present invention is not particularly limited by its dosage form, and may be, for example, an oral preparation, an injection, an eye drop, a nasal drop, or an external preparation for skin, and may be used as a pharmaceutical composition together with a pharma- ceutical acceptable additive. External preparations for skin include solid preparations for external use; liquid preparations for external use such as liniments and lotions; spray preparations such as external aerosols and pump sprays; ointments; creams; gels; and patches such as plasters, patches, tapes, and poultices.

[0030] <Additives in pharmaceutical compositions> When the pharmaceutical composition of the present invention is in the form of an ointment, additives used in the ointment include bases, moisturizers, thickeners, emulsifiers, etc. As bases, higher hydrocarbons, oils and fats, waxes, fatty acids, higher alcohols, esters, etc. can be used. Examples of higher hydrocarbons include squalane, synthetic paraffin, liquid paraffin, white petrolatum, microcrystalline wax, etc. Examples of waxes include beeswax, white beeswax, lanolin, ceresin wax, etc. Examples of fatty acids include stearic acid, oleic acid, etc. Examples of higher alcohols include lanolin alcohol, cetostearyl alcohol, etc. Examples of esters include isopropyl myristate, stearyl myristate, etc.

[0031] Examples of moisturizing agents include glycerin and 1,3-butylene glycol, examples of thickening agents include carboxyvinyl polymers and carboxymethylcellulose, and examples of emulsifying agents include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants.

[0032] Additives used in patches include thickeners, moisturizers, fillers, crosslinkers, solubilizers, emulsifiers, etc. Specifically, thickeners include sodium alginate, gelatin, methylcellulose, carboxyvinyl polymer, sodium polyacrylate, etc., moisturizers include glycerin, etc., fillers include kaolin, titanium dioxide, zinc oxide, etc., crosslinkers include acetaldehyde, dimethyl ketone, aluminum sulfate, etc., solubilizers include alcohols such as ethanol and isopropanol, etc., and emulsifiers include anionic surfactants, nonionic surfactants, etc. Additives used in plasters or patches can be, for example, a support such as a nonwoven fabric, an elastic body such as natural rubber or isoprene rubber, a filler such as zinc oxide or titanium oxide, a tackifier such as a terpene resin, a release agent such as vinyl acetate, a softener such as liquid paraffin, a preservative such as dibutylhydroxytoluene (BHT), and the like, which can be used in appropriate combination. Examples of additives used in the cataplasm include water-soluble polymeric compounds such as polyacrylic acid, polyvinyl alcohol, and polyvinylpyrrolidone.

[0033] Additives used in creams include oil-soluble substances, water-soluble substances, emulsifiers, etc. As the oil-soluble substances, higher hydrocarbons, oils and fats, waxes, fatty acids, higher alcohols, esters, etc. can be used. Examples of higher hydrocarbons include squalane, synthetic paraffin, liquid paraffin, white petrolatum, microcrystalline wax, etc. Examples of waxes include beeswax, white beeswax, lanolin, ceresin wax, etc. Examples of fatty acids include stearic acid, oleic acid, etc. Examples of higher alcohols include lanolin alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, cholesterol, etc. Examples of esters include isopropyl myristate, stearyl myristate, etc.

[0034] Examples of the water-soluble substance that can be used include water (purified water), thickeners, moisturizers, etc. Examples of the thickeners include carboxyvinyl polymers and carboxymethylcellulose, and examples of the moisturizers include glycerin, propylene glycol, and 1,3-butylene glycol.

[0035] The emulsifier includes cationic surfactants, anionic surfactants, amphoteric surfactants, nonionic surfactants, and the like.

[0036] The lotion may be a suspension type, an emulsion type or a solution type lotion. Additives used in lotions include oil-soluble substances, water-soluble substances, emulsifiers, etc. Examples of oil-soluble substances that can be used include hydrocarbons, oils and fats, waxes, fatty acids, higher alcohols, esters, etc. Examples of hydrocarbons include squalane, α-olefin oligomers, synthetic paraffin, liquid paraffin, white petrolatum, microcrystalline wax, etc. Examples of waxes include beeswax, white beeswax, lanolin, ceresin wax, etc. Examples of fatty acids include stearic acid, oleic acid, etc. Examples of higher alcohols include lanolin alcohol, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, etc. Examples of esters include isopropyl myristate, stearyl myristate, octyldodecyl myristate, etc.

[0037] Examples of the water-soluble substance include water (purified water), alcohols such as ethanol and isopropanol, thickeners, moisturizers, etc. Examples of the thickeners include carboxyvinyl polymers and carboxymethylcellulose, and examples of the moisturizers include glycerin, propylene glycol, and 1,3-butylene glycol. Examples of the emulsifiers include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants. Additives for suspension-type lotions include, in addition to the above, gums such as gum arabic and gum tragacanth, celluloses such as methyl cellulose and hydroxyethyl cellulose, and suspending agents such as clays such as bentonite.

[0038] Examples of additives used in liniments include vegetable oils such as olive oil, alcohols such as ethanol or isopropanol, or mixtures of these with water.

[0039] Additives to be incorporated into the gel include bases, thickeners, etc. Bases include isopropanol, propylene glycol, etc. Thickeners include, for example, carboxyvinyl polymers, carboxymethylcellulose, etc.

[0040] Additives to be incorporated into the spray include a base, a foaming agent, and the like. Examples of bases include water, glycerin, 1,3-butylene glycol, dipropylene glycol, propylene glycol, D-sorbitol, polyethylene glycol, 2-ethylhexanediol, isostearyl alcohol, octyldodecanol, hexyldecanol, oleyl alcohol, isostearic acid, oleic acid, oleyl oleate, isopropyl myristate, octyldodecyl myristate, glycerin triisooctanoate, cetyl 2-ethylhexanoate, isostearyl palmitate, medium-chain fatty acid triglyceride, castor oil, olive oil, triacetin, squalane, light liquid paraffin, liquid paraffin, squalene, white petrolatum, and gelling hydrocarbons. Examples of foaming agents include polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene polyoxypropylene glycol, glyceryl stearate, polyoxyethylene hydrogenated castor oil, decaglyceryl trioleate, polyoxyethylene cetyl ether, polyoxyethylene sorbit tetraoleate, sucrose stearate, polyoxyethylene polyoxypropylene cetyl ether, polyoxyethylene sorbit beeswax, polyoxyethylene glyceryl monostearate, polyoxyethylene lauryl ether, self-emulsifying glycerin monostearate, polyoxyethylene sorbitan monooleate, decaglyceryl monooleate, decaglyceryl monostearate, polyethylene glycol monostearate, polyoxyethylene behenyl ether, polyoxyethylene sorbitan laurate, sucrose laurate, polyoxyethylene sorbitan ... Examples of such an acid ester include uric acid ester, polyoxyethylene stearyl ether, sodium laurate, potassium palmitate, arginine stearate, sodium lauryl sulfate, potassium lauryl sulfate, sodium cetyl sulfate, polyoxyethylene triethanolamine lauryl sulfate, sodium lauroyl sarcosine, sodium N-stearoyl-N-methyl taurine, sodium N-myristoyl-N-methyl taurine, sodium monostearyl phosphate, sodium polyoxyethylene oleyl ether phosphate, sodium polyoxyethylene stearyl ether phosphate, sodium polyoxyethylene cetyl ether phosphate, sodium di-2-ethylhexyl sulfosuccinate, monosodium N-lauroyl glutamate, sodium N-stearoyl-L-glutamate, arginine N-stearoyl-L-glutamate, and sodium N-myristoyl-L-glutamate.

[0041] Specific examples of cationic surfactants that can be used in each formulation of the external skin preparation include cetyltrimethylammonium chloride, lauryldimethylbenzylammonium chloride, tetrabutylammonium chloride, dioctadecyldimethylammonium chloride, and the like. Examples of the anionic surfactant include sodium alkylbenzene sulfonate, sodium dodecyl sulfate, sodium coconut alcohol ethoxy sulfate, sodium α-olefin sulfonate, emulsified cetostearyl alcohol, and the like. Examples of nonionic surfactants include polyoxyethylene alkyl ethers such as polyoxyethylene oleyl ether and polyoxyethylene octyldodecyl ether, polyoxyethylene alkylphenol ethers, polyoxyethylene hydrogenated castor oil, polyoxyl stearate, glycerin monostearate, self-emulsifying glycerin monostearate, glycerin monoisostearate, glycerin palmitate, glycerin myristate, glycerin oleate, and glycerin triisooctanoate, and other glycerin fatty acid esters, diglycerin laurate, diglycerin stearate, and diglycerin oleate, and other diglycerin fatty acid esters, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan coconut oil fatty acid, sorbitan tristearate, and sorbitan trioleate, and other sorbitan fatty acid esters, and polyethylene glycol fatty acid esters such as polyethylene glycol monolaurate, polyethylene glycol monostearate, and polyethylene glycol monooleate. Examples of the amphoteric surfactant include N-alkyl-N,N-dimethylammonium betaine and imidazoline-type amphoteric surfactant. Any of the above surfactants may be used alone or in combination.

[0042] In the above-mentioned formulation of the external skin preparation, a pH regulator, a preservative, macrogols, etc. may be used as necessary.

[0043] Examples of pH adjusters include diisopropanolamine, triisopropanolamine, triethanolamine, potassium hydroxide, sodium hydroxide, sodium citrate, phosphoric acid, tartaric acid, dl-malic acid, glacial acetic acid, etc., and examples of preservatives include thymol, dibutylhydroxytoluene, sodium edetate hydrate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, etc.

[0044] In addition, when the pharmaceutical composition is in the form of an oral preparation, various additives can be selected and used as necessary to improve the taste, odor, and color, which are necessary for improving stability, ease of handling, or compliance. For example, (1) excipients such as lactose, starch, crystalline cellulose, D-mannitol, glucose, and calcium phosphate, (2) disintegrants such as starch and croscarmellose sodium, (3) sweeteners and flavoring agents such as D-mannitol, D-sorbitol, aspartame, and citric acid, (4) plasticizers such as polyethylene glycol, (5) stabilizers such as polyaminocarboxylic acid chelating agents and their salts, sodium pyrosulfite, and 1-ascorbic acid, (6) preservatives such as paraoxybenzoic acid esters and benzalkonium chloride, as well as colorants and flavoring agents can be used.

[0045] The types and amounts of these additives can be appropriately determined taking into consideration the properties of the active ingredient, such as stability, taste, etc. Furthermore, when the pharmaceutical composition requires the property of being soluble in water, the additives can be appropriately selected taking into consideration the solubility in water. Furthermore, a binder such as hydroxypropyl cellulose or polyvinylpyrrolidone may be added to the pharmaceutical composition as necessary, as long as it does not impair the rapid disintegration and solubility of the oral granules. In order to ensure rapid disintegration and solubility, it is preferable that the binder has a weak binding force, and the amount of the binder is preferably 5% by weight or less, particularly preferably 2% by weight or less, based on the weight of the composition.

[0046] Dry syrup is a useful dosage form of oral granules. Dry syrup is a syrup that is dissolved or suspended when used, and is an excellent preparation that masks the bitterness or unpleasant taste of medicines with the sweetness and viscosity of sugars, as well as various flavoring agents added, and is easy to take due to the pleasant color tone of appropriate coloring agents. In addition to water, liquids containing water such as juice or milk can be used as a solvent when dissolving or suspending dry syrup. Oral granules similar to dry syrup, that is, granules, powders and fine granules which contain a large amount of sucrose and are essentially dissolved or suspended when used, are also suitable embodiments of the pharmaceutical composition.

[0047] When the pharmaceutical composition is in the form of an injection, additives to be added include pH adjusters, buffers, stabilizers, isotonicity agents, local anesthetics, soothing agents, diluents, surfactants and the like. Examples of the pH adjuster and buffer include hydrochloric acid, citric acid, sodium citrate, sodium acetate, sodium phosphate, sodium hydroxide, etc. Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid, etc. Examples of the buffer include sodium citrate, phosphoric acid, sodium phosphate, potassium phosphate, sodium acetate, boric acid, etc. As the local anesthetic, procaine hydrochloride, lidocaine hydrochloride, etc. can be used, and as the isotonicity agent, sodium chloride, D-mannitol, D-sorbitol, glucose, glycerin, etc. can be used. As a soothing agent, procaine hydrochloride and the like can be used. Examples of diluents include water, ethyl alcohol, macrogol, and propylene glycol.

[0048] When the pharmaceutical composition is in the form of an eye drop formulation, additives to be added include a solvent, an isotonicity agent, a preservative, a viscosity agent, a pH adjuster, and the like. Examples of the solvent include sterilized purified water, physiological saline, buffer solutions, vegetable oils, propylene glycol, and liquid paraffin. Examples of the isotonicity agent include sodium chloride, boric acid, sodium nitrate, and potassium nitrate. Examples of the preservative include paraoxybenzoic acid esters, bezalkonium chloride, chlorobutanol, sodium dehydroacetate, and polymyxin B sulfate. Examples of the viscosity agent include methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, polyvinyl alcohol, and sodium chondroitin sulfate. Examples of the pH regulator include acids and bases, and examples of the acids include hydrochloric acid, phosphoric acid, citric acid, and acetic acid, and examples of the bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate.

[0049] When the pharmaceutical composition is in the form of a nasal drop formulation, additives to be added include solubilizers, isotonicity agents, pH adjusters, etc. In the case of a nasal drop suspension, a dispersant or stabilizer may be added as necessary to obtain a uniform state of the active ingredient. In addition, when storing in a filled container, etc., a preservative may be added as necessary.

[0050] <Method of manufacturing pharmaceutical composition> The pharmaceutical composition of the present invention can be prepared by a conventional preparation method. For example, an ointment or cream can be prepared by kneading, emulsifying or suspending the base material according to the respective dosage forms to prepare a base, adding the active ingredient and the like, and mixing in a mixer such as a screw mixer.

[0051] The lotion can be produced, for example, by adding various additive components to purified water, mixing and stirring, then adding and mixing the active ingredient and the like, and filtering as desired. The liniment can be produced, for example, by dissolving the active ingredient in an additive and, if desired, adding and mixing other ingredients thereto.

[0052] The cataplasm can be produced, for example, by mixing the active ingredient and additives, heating and then cooling.

[0053] Plasters, patches and the like can be produced by conventional methods such as the solution method and the heat-pressure method. Gels include aqueous gels, oily gels, etc. Aqueous gels can be produced by adding a polymer compound, other additives, and purified water to the active ingredient, dissolving or suspending the mixture, and then heating and cooling the mixture or adding a gelling agent to crosslink the mixture. Oily gels can be produced by adding a liquid oily base such as glycols or higher alcohols, and other additives to the active ingredient, and mixing the mixture. A spray can usually be produced by preparing a solution or suspension of an active ingredient, filtering it if necessary, and then filling it into a container.

[0054] In the step of mixing the active ingredient or additives in the above-mentioned production method, various pharmaceutical additives may also be mixed in.

[0055] The oral pharmaceutical composition can be produced according to a conventional method for producing oral granules.As the granulation method, a general granulation method such as extrusion granulation, stirring granulation, rolling granulation, fluidized bed granulation, crushing granulation, and crushing granulation can be used, but the fluidized bed granulation method is particularly suitable for obtaining granules with the following characteristics: 1) high porosity, 2) low bulk density, and 3) good dispersibility in water.

[0056] The pharmaceutical composition can also be used to manufacture oral granules such as granules, powders, and fine granules. These preparations can be taken orally directly, or can be dissolved or suspended in a solvent containing water before use. Preparations that are dissolved or suspended before use are solid when shipped, but are dissolved or suspended between the time the drug is opened and the time of application, and are usually dissolved or suspended immediately before application.

[0057] When preparing a pharmaceutical composition as an injection, it can be prepared by a conventional method, for example, by dissolving, suspending or emulsifying in water for injection or other aqueous or non-aqueous solvent to produce an intravenous, intramuscular, subcutaneous or intradermal injection. It is preferable that the injection is sterilized and isotonic with blood.

[0058] When preparing eye drops, the above-mentioned additives may be added to the active ingredient, if necessary, and the mixture may be dissolved or suspended in a solvent to produce the eye drops.

[0059] When preparing nasal drops, the active ingredient is dissolved or suspended as it is or with the addition of a solvent or other additives. If necessary, the liquid nasal drops are produced by adding the above-mentioned additives, filtering, etc. If necessary, the fine powder nasal drops can be produced by mixing the active ingredient as fine particles with additives as necessary to make it homogenous.

[0060] <Amount of each ingredient> The content of polysulfated mucopolysaccharides such as polysulfated chondroitin sulfate, which is an active ingredient in the pharmaceutical composition of the present invention, is preferably 0.0001 to 30 wt%, more preferably 0.001 to 10 wt%, and particularly preferably 0.01 to 1 wt%, based on the total weight of the pharmaceutical composition. The total content of the above-mentioned additives is, for example, 70% by weight or more, 90% by weight or more, 99% by weight or more, etc., based on the total weight of the pharmaceutical composition.

[0061] <Application of pharmaceutical compositions> The dosage and frequency of the pharmaceutical composition of the present invention may be appropriately adjusted depending on the target disease and the severity of its symptoms, the concentration of polysulfated mucopolysaccharide such as polysulfated chondroitin sulfate in the composition, age, body weight, etc. For example, in the case of systemic administration, 50 mg to 5 g, preferably 100 mg to 1 g of polysulfated mucopolysaccharide may be administered per day, for example, once a day, twice a day, or three times a day. In the case of local administration, 2 A dose of 0.03 μg to 30 mg, and preferably 0.3 μg to 3 mg, may be applied once or several times a day.

[0062] <Diseases for which the pharmaceutical composition is applicable> Diseases caused by decreased PDGF-BB / PDGFRβ signaling or Ang-1 / Tie2 signaling include tumors, rheumatoid arthritis, diabetes, hyperlipidemia, hypertension, hepatitis, sepsis, angioedema, systemic sclerosis, angiosarcoma, hemangioma, psoriasis, warts, and other diseases accompanied by vascular lesions. According to the present invention, it is possible to provide a preventive and / or therapeutic drug for diseases accompanied by lesions in blood vessels or lymphatic vessels, which contains the above-mentioned polysulfated mucopolysaccharide as an active ingredient. Furthermore, since it is known that PDGF-BB / PDGFRβ signaling and Ang-1 / Tie2 signaling are decreased with age, the above-mentioned pharmaceutical composition is also expected to serve as a preventive and / or therapeutic agent for age-related diseases.

[0063] As described above, a pharmaceutical composition containing polysulfated mucopolysaccharides activates Ang-1 / Tie2 signaling and increases the production of PDGF-BB, and therefore exhibits angiogenesis inhibitory, vascular maturation, vascular normalization, vascular stabilization, and lymphatic vessel stabilization effects, improves vascular and lymphatic vessel functions, and can be used for the treatment or prevention of the following diseases that are thought to be accompanied by lesions in blood vessels and lymphatic vessels: cardiovascular diseases, skin diseases, endocrine, nutritional, and metabolic diseases, diseases associated with aging, eye diseases, and other diseases.

[0064] Examples of circulatory system diseases include capillary leak syndrome, coronary artery disease, chronic heart failure, peripheral arterial disease (atherosclerosis, microvascular angina, myocardial infarction, stroke, subarachnoid hemorrhage), hypertension, angioedema, ischemic disease caused by side effects of surgery or organ tissue damage, lymphangioleiomyoma, disorders caused by lymph leakage from lymphatic vessels, Raynaud's disease, etc. Among these, the pharmaceutical composition is considered to be particularly effective in treating and preventing peripheral arterial disease including atherosclerosis, subarachnoid hemorrhage, etc., angioedema, lymphangioleiomyoma, etc. Examples of skin diseases include rosacea, inflammatory diseases (psoriasis vulgaris, Schnitzler's syndrome), common warts, wounds, bedsores, frostbite, tuberous sclerosis, multiple sclerosis, systemic sclerosis, Kaposi's sarcoma, cutaneous angiosarcoma, purpura, severe wrinkles, age spots, swelling, etc. Among these, the pharmaceutical composition is considered to be particularly effective in treating and preventing inflammatory diseases such as psoriasis vulgaris and Schnitzler's syndrome, common warts, tuberous sclerosis, systemic sclerosis, Kaposi's sarcoma, cutaneous angiosarcoma, severe wrinkles, swelling, etc.

[0065] Endocrine, nutritional and metabolic diseases include diabetes mellitus, diabetes-related retinopathy, macular edema, skin ulcers, wounds, nephropathy, peripheral neuropathy, hyperlipidemia, gout, etc. Among these, the pharmaceutical composition is considered to be particularly effective in treating and preventing diabetes, including retinopathy, macular edema, skin ulcers, wounds, nephropathy, peripheral neuropathy, etc. Age-related diseases include alopecia, osteoporosis, dermatoporosis, dementia, photoaging, age-related macular degeneration, Alzheimer's disease, vascular dementia, moyamoya disease, etc. Among these, the pharmaceutical composition is considered to be particularly effective in treating and preventing photoaging, etc.

[0066] Examples of eye diseases include age-related macular degeneration, retinopathy (premature infants, diabetes), choroidal neovascularization, retinal vein occlusion, uveitis, xerophthalmia, ocular ischemia, etc. Among these, the pharmaceutical composition is considered to be particularly effective in treating and preventing retinopathy (retinopathy in premature infants, retinopathy due to diabetes). Other diseases include lower back pain, tumors, acute myeloid leukemia, chronic rheumatoid arthritis, Sjogren's syndrome, hepatitis, sepsis, renal failure, allergies such as hay fever, etc. Other diseases include drug-induced purpura, vascular disorders associated with chemotherapy, etc. Among these, the pharmaceutical composition is considered to be particularly effective in treating and preventing tumors, acute myeloid leukemia, hepatitis, and sepsis.

[0067] [Cosmetic composition] The composition of the present invention is also useful as a cosmetic composition, and the cosmetic composition also contains at least a polysulfated mucopolysaccharide or a salt thereof. Such a cosmetic composition can also activate the Ang-1 / Tie2 signal and increase the production of PDGF-BB. Therefore, in addition to its use as a cosmetic, the cosmetic composition can also be used to improve deterioration of skin condition associated with decreased function of blood vessels or lymphatic vessels, prevent diseases associated with vascular lesions, and the like. Specifically, the cosmetic composition is useful for improving skin conditions by improving blood vessel or lymph vessel function, although the symptoms are not as severe as those of the above-mentioned diseases. Examples of skin conditions that can be improved include wrinkles, age spots, swelling, and alopecia. The cosmetic composition is useful as a cosmetic with medicinal properties because it contains the above-mentioned active ingredients, and may also include quasi-drugs (quasi-drugs as defined in the "Law Concerning the Ensuring of Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc.").

[0068] The cosmetic composition is used as a component of, for example, face washes, cosmetics for the skin, lotions, milky lotions, creams, gels, sunscreens, ointments, lotions, packs, cosmetics for wrinkles and sagging, foundations, makeup bases, beauty essences, whitening agents, deodorants, cleansers, hair care products, shampoos, rinses, hair tonics and other hair cosmetics, lipsticks, blushers and other make-up cosmetics, deodorants, aerosols, liquids, and the like.

[0069] The cosmetic composition may contain the following additives in addition to or in place of the components of the pharmaceutical composition described above. For example, surfactants (emulsifiers), moisturizing agents, antioxidants, thickeners, chelating agents, oily components, resins, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powdery components, coloring materials, pH adjusters, fragrances, acidulants, sweeteners, seasonings, coloring agents, various medicinal components, chelating agents, preservatives, moisture-proofing agents, excipients, bactericides, deodorants, blood circulation promoters, salts, refreshing agents, whitening agents, animal or plant-derived extracts, astringents, anti-inflammatory agents, active oxygen scavengers, antiseborrheic agents, cell activators, enzymes, hormones, vitamins, aqueous components, preferably purified water, etc. may be appropriately blended in the cosmetic composition. These additives may be used alone or in combination.

[0070] The content of the polysulfated mucopolysaccharide in the cosmetic composition of the present invention is preferably 0.0001 to 30 wt %, more preferably 0.001 to 10 wt %, and particularly preferably 0.01 to 1 wt %, based on the total weight of the composition. The total content of the above-mentioned additives is, for example, 70% by weight or more, 90% by weight or more, 99% by weight or more, etc., based on the total weight of the cosmetic composition.

[0071] The components of the cosmetic composition, i.e., the types of active ingredients and additives, dosage form, method of use, dosage, use, etc., may be common to those of the pharmaceutical composition. Therefore, the components, dosage form, method of use, dosage, use, etc. described above for the pharmaceutical composition can also be applied to the cosmetic composition.

[0072] The present invention will be described in detail below by showing examples of the present invention, but the present invention is not limited to the following examples. EXAMPLES

[0073] As polysulfated chondroitin sulfate or its salt (abbreviation: MPS), a heparinoid substance listed in the Japanese Pharmacopoeia (organic sulfate group: 25.8-37.3% w / w, glucuronic acid content 19.0-24.0% w / w, Maruho Co., Ltd.) was used. As control substances for comparison, hyaluronic acid (abbreviation: HA, Tokyo Chemical Industry Co., Ltd.), chondroitin sulfate (abbreviation: Chs, organic sulfate group: 15-17% w / w, Maruho Co., Ltd.), and xylohexaose (abbreviation: O-XHE, Megazyme Co., Ltd.) were used.

[0074] [Example 1] Effects of various polysaccharides on Ang-1 production from human placenta parietal cells Human placenta-derived parietal cells (PromoCell) (1×10 5 Cells) were seeded in medium (Pericyte Growth Medium + Pericyte Growth Medium Supplement Mix (PromoCell)) and incubated at 37°C, 5% CO 2 After culturing for 4 days in 95% air, the medium was replaced with one containing MPS, HA, Chs, or O-XHE at various concentrations (Pericyte Growth medium), and incubated at 37°C, 5% CO 2 The cells were then cultured for an additional 48 hours in 95% air. After the culture was completed, the Ang-1 concentration in the culture supernatant was measured using an ELISA kit (Human Angiopoietin-1 Quantikine ELISA Kit (R&D Systems)). Each evaluation substance group and the solvent group consisted of three cases (n=3).

[0075] The effect of MPS on Ang-1 production is shown in Figure 1A, and the effects of HA, Chs, and O-XHE are shown in Figure 1B. The ** in the figure indicates a significant difference (P<0.01) compared to the vehicle group (absence of drug: see (-) in Figure 1A). From the results in Figure 1A, it was confirmed that the amount of Ang-1 in the MPS group increased in a concentration-dependent manner, and a significant difference was observed in the MPS group compared to the vehicle group at concentrations of 1 μg / mL or more. On the other hand, the results in FIG. 1B show that the Ang-1 levels in the HA, Chs, and O-XHE groups were not significantly different from those in the vehicle group.

[0076] [Example 2] Effects of various polysaccharides on Ang-2 production from human dermal microvascular endothelial cells Human dermal microvascular endothelial cells (PromoCell) (1 × 10 5 Cells) were seeded in medium (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)) and incubated at 37°C, 5% CO 2 After culturing for 4 days in 95% air, the medium was replaced with one containing MPS, HA, Chs, or O-XHE at each concentration (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)), and incubated at 37°C, 5% CO 2 The cells were then cultured for an additional 48 hours in 95% air. After the culture was completed, the Ang-2 concentration in the culture supernatant was measured using an ELISA kit (Human Angiopoietin-2 Quantikine ELISA Kit (R&D Systems)). Each evaluation substance group and the solvent group consisted of three cases (n=3).

[0077] The effect of MPS on Ang-2 production is shown in Figure 2A, and the effects of HA, Chs, and O-XHE are shown in Figure 2B. From the results in Figure 2A, it was found that the amount of Ang-2 in the MPS group was reduced in a concentration-dependent manner. On the other hand, the results in Figure 2B show that the Ang-2 levels in the HA, Chs, and O-XHE groups were not significantly different from those in the vehicle group.

[0078] [Example 3] Effect of MPS on Tie2 phosphorylation in human dermal microvascular endothelial cells Human dermal microvascular endothelial cells (PromoCell) (1 × 10 6 Cells) were seeded in medium (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)) and incubated at 37°C, 5% CO 2After culturing for 2 days in 95% air, the cells were cultured in serum-free medium (Endothelial Cell Growth Medium MV2) for 3 hours. After that, the medium was replaced with MPS at various concentrations or Ang-1 (R&D Systems, 923-AN)-containing medium (Endothelial Cell Growth Medium MV2) as a positive control, and incubated at 37°C, 5% CO 2 The plate was then incubated for an additional 10 minutes in 95% air. After the incubation, the cells were washed with cold TBS (Tris-Buffered Saline), RIPA Buffer (Radio Immunoprecipitation Assay Buffer) containing phosphatase inhibitor (Thermo Scientific) was added, and the cells were sonicated. After the concentration of each protein was adjusted to a constant level, Sample buffer was added, and SDS-PAGE was performed at 100V for 60 minutes using a 5-20% polyacrylamide gel. After transfer to a PVDF membrane using Transfer-Blot Turbo Transfer Pack (BIO-RAD), blocking was performed for 20 minutes with Blocking One-P. Then, Tie2 phosphorylation antibody (Cell Signaling, #4221) was added and left to stand overnight at 4°C. Next, Anti-rabbit IgG HRP-linked Antibody (Cell Signaling, #7074S) was added and left to stand at room temperature for 1 hour. Protein bands were detected by chemiluminescence detection (Thermo Scientific).

[0079] The effect of MPS on Tie2 activation is shown in Figure 3. The intensity of the detected band is proportional to the amount of phosphorylated Tie2 protein. The results in Figure 3 show that Tie2 phosphorylation in the Ang-1 group (500 ng / mL) was increased compared to the solvent group (absence of drug: see (-) in Figure 3). Similarly, Tie2 phosphorylation in the MPS group was significantly increased compared to the solvent group at all concentrations.

[0080] [Example 4] Effects of various polysaccharides on PDGF-BB production from human dermal microvascular endothelial cells Human dermal microvascular endothelial cells (PromoCell) (1 × 10 5 Cells) were seeded in medium (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)) and incubated at 37°C, 5% CO 2 After culturing for 4 days in 95% air, the medium was replaced with one containing MPS, HA, Chs, or O-XHE at each concentration (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)), and incubated at 37°C, 5% CO 2 The cells were then cultured for an additional 48 hours at 95% air. After the culture was completed, the PDGF-BB concentration in the culture supernatant was measured using a Bio-Plex kit (human cytokine assay kit (BIO-RAD)). Each evaluation substance addition group and the solvent group had three cases (n=3).

[0081] The effect of MPS on PDGF-BB production is shown in Figure 4A, and the effects of HA, Chs, and O-XHE are shown in Figure 4B. * and ** in the figure indicate significant differences compared to the vehicle group (absence of drug: see (-) in Figure 4A) (*P<0.05, **P<0.01). From the results in Figure 4A, it was confirmed that the amount of PDGF-BB in the MPS group increased in a concentration-dependent manner, and for MPS, a significant difference was observed compared to the vehicle group at concentrations of 10 μg / mL or more. On the other hand, as shown in Figure 4B, the PDGF-BB levels in the HA and Chs groups were not significantly different from those in the vehicle group. In addition, the PDGF-BB levels in the O-XHE group were significantly decreased at 0.1, 10, and 100 μg / mL compared to the vehicle group, but no concentration dependency was observed.

[0082] [Example 5] Effect of MPS on vascular endothelial growth factor (VEGF)-induced vascular leakage To evaluate vascular permeability, 100 μL of 1% w / v Evans blue (Nacalai Tesque, Inc.) solution was administered intravenously to mice (Hos:HR-1, male, 7 weeks old) via the tail vein. Thirty minutes later, 20 μL of vehicle (Dulbecco's phosphate buffered saline (abbreviated as PBS, Sigma-Aldrich)) (PBS group), a mixture of mouse-derived vascular endothelial growth factor (50 ng / 20 μL / site, abbreviated as VEGF, Sigma-Aldrich) and vehicle (VEGF group), or a mixture of VEGF, MPS (2.5, 5, 10, 20 μg / 20 μL / site) and vehicle (MPS group) were administered intradermally to the back of the mice, one on each side (two sites per animal) around the midline. Twenty minutes after intradermal administration, the animals were immediately euthanized and skin samples were taken using a biopsy trephine with a diameter of 8.0 mm (Kai Industries). There were 16 samples in each of the PBS, VEGF, and MPS groups (1 sample per administration site, 8 samples from each group). 250μL of deionized formamide (The Gel Company) was added to the collected skin pieces and left to stand overnight at 55°C. After centrifugation at 10,000g for 40 minutes, the supernatant was collected and its absorbance at 620nm was measured. The amount of dye leakage (μg / site) in the skin of each individual was calculated from a calibration curve created by treating the skin in the same way with deionized formamide containing Evans blue (0, 1.25, 2.5, 5, 10, 20, 40, 80μg).

[0083] The effect of MPS on VEGF-induced vascular leakage is shown in Figure 5. In the figure, ** indicates a significant difference from the PBS group (**P<0.01), † and †† indicate a significant difference from the VEGF group (†P<0.05, ††P<0.01), respectively. From the results in Figure 5, it can be said that the amount of dye leakage was reduced concentration-dependently by the use of the MPS group, and a significant difference was observed in the MPS group compared to the VEGF group at concentrations of 10 μg / site or more. In other words, it was confirmed that MPS suppressed dye leakage from blood vessels caused by the administration of VEGF.

[0084] [Example 6] Effect of MPS on the downregulation of Tie2 and Ang-1 expression by repeated steroid administration The effect of MPS on Tie2 and Ang-1, whose expression is decreased by repeated administration of steroids, was evaluated. Dermovate Cream 0.05% (containing 0.05% clobetasol propionate, GlaxoSmithKline K.K., hereafter referred to as Dermovate Cream) was used as the steroid, and Hirudoid Soft Ointment 0.3% (containing 0.3% heparinoid, Maruho Co., Ltd., hereafter referred to as Hirudoid Soft Ointment) was used as the MPS. The back skin of mice (C57BL / 6, male, 8 weeks old) was depilated, and Dermovate cream was administered percutaneously once a day for 10 days. From the day after the final administration, the mice were divided into a non-treatment group (steroid administration + non-treatment group) in which no drug was applied, and a group administered Hirudoid Soft ointment (steroid administration + Hirudoid Soft ointment group). The steroid administration + Hirudoid Soft ointment group was administered percutaneously once a day for 7 days. The group administered neither Dermovate cream nor Hirudoid Soft ointment was set as the non-treatment group. On the day after the final administration, the animals in each group were euthanized, and the skin was collected using a biopsy trephine φ8.0 mm (Kai Industries). To the collected skin pieces, 350μL of homogenate buffer (a mixture of 10mL PBS (Thermo Fisher Scientific), 20μL Tween20 (Nacalai Tesque), and 200μL Protease Inhibitor Cocktail Set3 (Merck)) was added, tungsten beads (Biomedical Science) were added, and the skin pieces were disrupted using Shake Master NEO (Biomedical Science). The entire amount of the extracted solution was collected and centrifuged at 1870 g for 5 minutes, and the supernatant was collected. The amounts of Tie2 and Ang-1 in the collected supernatant were measured using the respective ELISA kits (Tie2: Mouse Angiopoietin-1 receptor ELISA Kit (EIAab Science Inc.), Ang-1: Mouse Angiopoietin-1 ELISA Kit (EIAab Science Inc.)). The total protein amount was measured using a CBQCA Protein Quantitation Kit (Invitrogen), and the concentrations of Tie2 and Ang-1 were calculated by correcting for the total protein amount. Each group consisted of 5 cases (n=5): the untreated group, the steroid-administered + untreated group, and the steroid-administered + Hirudoid Soft Ointment group.

[0085] The concentrations of Tie2 and Ang-1 are shown in FIG. In the figure, * indicates a significant difference compared to the untreated group (*P<0.05), and † indicates a significant difference compared to the steroid-administered + untreated group (†P<0.05). From the results in Figure 6, it can be said that the Tie2 and Ang-1 concentrations in the steroid-administered + untreated group were significantly lower than the untreated group, and the Tie2 and Ang-1 concentrations in the steroid-administered + Hirudoid Soft Ointment-administered group were significantly higher than the steroid-administered + untreated group. As described above, MPS significantly increased the expression of Tie2 and Ang-1, which were decreased by repeated steroid administration.

[0086] [Example 7] Effects of MPS on transendothelial electrical resistance (TEER) in human dermal microvascular endothelial cells The TEER value, which will be described in detail later, was measured using a real-time cell analyzer (ECIS-Zθ, Applied BioPhysics) and an electrode station 96W (ECIS-96WAS, Applied BioPhysics). A 10 mM L-cysteine ​​solution was added to a 96-well plate (96W20idf PET, Applied BioPhysics) with electrodes coated with 1 g / mL collagen I (Corning). Then, a suspension of human dermal microvascular endothelial cells (PromoCell) (4 × 10 3 Cells and medium (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)) were seeded into each well and incubated at 37°C, 5% CO 2 Measurements were started using ECIS-Zθ at ​​95% air. 24 hours after cell seeding, the medium (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)) was replaced once, and after 72 hours, each concentration of MPS or a positive control medium containing 500 ng / mL Ang-1 (R&D Systems, 923-AN) (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)) was added, and TEER values ​​were measured over time. The rate of change in TEER value at each measurement point was calculated assuming the TEER value at 72 hours after cell seeding as 100%.

[0087] The effect of MPS on the rate of change in TEER value of human dermal microvascular endothelial cells is shown in Figure 7. In the figure, * and ** indicate significant differences from the solvent group (absence of drug: see (-) in Figures 4A and B) (*P<0.05, **P<0.01). The rate of change in TEER value in the Ang-1-added group was significantly increased compared to the growth medium alone from 108 and 120 hours after seeding. The rate of change in TEER value in the MPS-added group increased in a concentration-dependent manner, and a significant increase was observed in the 1 and 10 μg / mL MPS-added groups compared to the growth medium alone from 108 and 120 hours after seeding (Figure 7).

[0088] [Example 8] Effect of MPS on Claudin-5 expression in human dermal microvascular endothelial cells Human dermal microvascular endothelial cell (PromoCell) suspension (1 × 10 5 Cells and medium (Endothelial Cell Growth Medium MV2 + Endothelial Cell Growth Medium MV2 Supplement Mix (PromoCell)) were seeded into wells and incubated at 37°C, 5% CO 2 After culturing for 3 days in 95% air, the medium was replaced with MPS-containing medium (Endothelial Cell Growth Medium MV2) of each concentration and incubated at 37°C, 5% CO 2 The cells were then cultured for an additional 2 days in 95% air. After the incubation, the cells were washed with cold TBS (Tris-Buffered Saline), RIPA Buffer (Radio Immunoprecipitation Assay Buffer, Nacalai Tesque, Inc.) was added, and the cells were sonicated. After each protein concentration was adjusted to a constant level, Sample buffer was added, and SDS-PAGE was performed at 100V for 60 minutes using a 5-20% polyacrylamide gel. After transfer to a PVDF membrane using Transfer-Blot Turbo Transfer Pack (BIO-RAD), blocking was performed for 20 minutes using 5% skim milk solution. Then, anti-Claudin-5 antibody (Invitrogen) was added and left to stand overnight at 4°C. Next, Anti-mouse IgG HRP-linked Antibody (Cell Signaling, #7076) was added and left to stand at room temperature for 1 hour. Protein bands were detected by chemiluminescence detection (Thermo scientific).

[0089] The effect of MPS on Claudin-5 in human dermal microvascular endothelial cells (detected protein bands) is shown in Figure 8. From these results, it was confirmed that MPS increased the expression level of Claudin-5 in a concentration-dependent manner, and in particular, when added at 1 and 10 μg / mL, MPS significantly increased the expression level of Claudin-5.

Claims

1. A composition for treating and / or preventing a disease accompanied by a vascular or lymphatic lesion, or for improving a skin condition by improving a vascular or lymphatic function, comprising a polysulfated mucopolysaccharide and / or a salt thereof, the polysulfated mucopolysaccharide contains at least polysulfated chondroitin sulfate, The polysulfated chondroitin sulfate has a sulfate group content of 18% by weight or more and 40% by weight or less, the disease involving vascular lesions or lymphatic lesions is at least any one of peripheral arterial diseases including atherosclerosis and subarachnoid hemorrhage, angioedema, lymphangioleiomyoma, inflammatory diseases including psoriasis vulgaris and Schnitzler's syndrome, verruca vulgaris, tuberous sclerosis, systemic sclerosis, Kaposi's sarcoma, cutaneous angiosarcoma, wrinkles, swelling, retinopathy, macular edema, skin ulcers, wounds, nephropathy, peripheral neuropathy, diabetes, photoaging, retinopathy in premature infants and diabetic retinopathy, tumors, acute myeloid leukemia, hepatitis, sepsis, purpura (including drug-induced purpura), and vascular disorders associated with chemotherapy; The composition, wherein the improvement of the skin condition is improvement of at least one of wrinkles, age spots, puffiness, and alopecia.

2. The composition of claim 1 , wherein the polysulfated mucopolysaccharide further comprises polysulfated dermatan sulfate, polysulfated keratan sulfate, polysulfated heparan sulfate, or a salt thereof.

3. The composition of claim 2 , wherein the polysulfated mucopolysaccharide comprises polysulfated dermatan sulfate and any of its salts.

4. The composition according to claim 3, wherein the sulfate group content of the polysulfated chondroitin sulfate is 25% by weight or more and 40% by weight or less.

5. The composition according to any one of claims 1 to 4, wherein the polysulfated mucopolysaccharide has a weight average molecular weight of 8,000 to 10,000,000.

6. The composition according to any one of claims 1 to 5, wherein the polysulfated mucopolysaccharide has a weight average molecular weight of 10,000 to 10,000,000.

7. The composition according to any one of claims 1 to 6, wherein the polysulfated mucopolysaccharide has an intrinsic viscosity of 0.05 to 0.

30.

8. A composition for external use used for suppressing, preventing, prophylaxis, or ameliorating a skin disease, comprising: (A) a heparinoid; (B) one or more selected from the group consisting of allantoin, pantothenic acid, vitamin A, pharma- ceutically acceptable salts thereof, and pharma-ceutically acceptable derivatives thereof; (C) a nonionic surfactant; Excluding compositions for external use, The composition according to any one of claims 1 to 7.

9. A Tie2 activator, a PDGF-BB production promoter, a blood vessel normalizer, a blood vessel stabilizer, or a lymphatic vessel stabilizer, comprising the composition according to any one of claims 1 to 8.

10. A pharmaceutical composition comprising the composition according to any one of claims 1 to 8.

11. A cosmetic composition comprising the composition according to any one of claims 1 to 8.

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