Hydrogel comprising hyaluronic acid derivative polymer

Abstract

The present invention relates to a drug delivery and preparation method for tissue regeneration and treatment through the promotion of angiogenesis, comprising synthesizing various kinds of hyaluronic acid derivatives, and delivering the hyaluronic acid derivatives by using Pluronic F127, which is a temperature-sensitive polymer. More specifically, angiogenesis efficacy of the hyaluronic acid derivatives was analyzed by introducing ethylene diamine, hexamethylene diamine, decane diamine, and gallic acid into hyaluronic acid. As a result of analyzing the angiogenesis effect thereof through an in ovo chick chorioallantoic membrane (CAM) assay, the synthesized hyaluronic acid derivatives exhibited an enhanced angiogenic effect compared to an untreated group or a group treated with hyaluronic acid alone. It is considered that the hyaluronic acid derivatives can be utilized in various medical engineering fields requiring new blood vessel regeneration such as those of damaged tissue or organ regeneration, and cell therapy.

Description

Hydrogel containing hyaluronic acid derivative polymer

[0001] The present invention relates to a hydrogel comprising a hyaluronic acid derivative polymer having an angiogenic effect.

[0002]

[0003] Hyaluronic acid, a substance abundant in the human body, has been reported to promote tissue regeneration by inhibiting inflammation and promoting cell migration and angiogenesis during tissue regeneration and recovery. Consequently, hyaluronic acid is widely used in biomedical applications, including wound healing agents, intra-articular injections, and artificial tears. However, considering hyaluronic acid's rapid degradation rate and the tissue microenvironment, the development of new materials that can maintain and enhance its function over time is necessary.

[0004] Meanwhile, diabetic macular edema (DMO, also known as DME) and age-related macular degeneration (AMD) are diseases that cause vision loss affecting the central region of the macula and are a leading cause of blindness in high-income countries (Bressler, 2004). DMO is caused by disruption of the inner and outer blood-retinal barriers as a result of increased expression of proangiogenic isoforms of vascular endothelial growth factor (Perrin et al., 2005). As a result, new blood vessels grow, leaking fluid and proteins from the vasculature into the retina, and increasing fluid transport across the retinal pigment epithelium cells into the retina, leading to retinal edema and vision loss. Exudative AMD (also known as wet AMD or wAMD) is the most severe form of AMD, primarily arising from the choroidal circulation beneath the macula and characterized by choroidal neovascularization (CNV) (Ferris et al., 1984). CNV, the abnormal growth of new blood vessels from the choroid into the retinal pigment epithelium (RPE) (Patz et al., 1977), is thought to lead to vision loss due to leakage of blood and serous fluid under and through the RPE, ultimately leading to photoreceptor loss, retinal detachment, and dense macular scarring (Fine et al., 2000; Campochiaro et al., 2006). Vascular endothelial growth factor (VEGF), a key factor in angiogenesis and vascular leakage (Dvorak et al., 1995), is upregulated during the progression of DMO and CNV (Spilsbury et al., 2000; Anderson et al., 2002; Das et al., 2003) and has become a major therapeutic target for the treatment of exudative AMD.

[0005]

[0006] One object of the present invention is to provide a hydrogel comprising a polymer to which a hyaluronic acid derivative is bound.

[0007] Another object of the present invention is to provide a composition for promoting angiogenesis comprising the hydrogel.

[0008] Another object of the present invention is to provide a method for producing a polymer by synthesizing hyaluronic acid and diamine in the presence of a chlorinated organic solvent and N-hydroxysuccinimide (NHS).

[0009]

[0010] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0011]

[0012] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0013]

[0014] Accordingly, in the present invention, various types of hyaluronic acid derivatives were synthesized. For this purpose, a material consisting of a carbon chain and having amine groups introduced at both ends of a hyaluronic acid polymer was used, and by introducing 2-carbon ethylenediamine, 6-carbon hexamethylenediamine, and 10-carbon decanediamine, hyaluronic acid polymer derivatives were synthesized to enhance the angiogenic effect. In addition, it was thought that increasing the adhesiveness of the hyaluronic acid derivative would enhance its angiogenic effect, and thus hyaluronic acid-decanediamine-gallic acid was synthesized. Additionally, to ensure that the hyaluronic acid derivatives can be stably fixed on the egg embryo, a thermosensitive polymer, Pluronic, was used, and the hyaluronic acid derivatives were stably fixed on the egg embryo, and the release of the hyaluronic acid derivatives was controlled over time. Therefore, the hyaluronic acid derivative developed through the present invention can be said to have secured differences and superiority over the conventional technology because it has a superior angiogenic effect than existing hyaluronic acid and can be stably released at a specific location.

[0015]

[0016] The present invention relates to the synthesis and delivery of a polymer derivative capable of promoting angiogenesis for the purpose of regenerating new blood vessels, such as for regenerating damaged tissues or organs or for cell therapy, and more specifically, to the synthesis of a hyaluronic acid polymer derivative, a method for producing a pluronic hydrogel encapsulating the same, and delivery of the hyaluronic acid polymer derivative using the same.

[0017]

[0018] In the present invention, by introducing amine groups at both ends of a hyaluronic acid polymer that significantly affects angiogenesis and carbon chains such as ethylene diamine (EDA), hexamethylene diamine (HDA), and decane diamine (DAD), a hyaluronic acid polymer derivative was synthesized in order to enhance the angiogenic effect. In addition, in order to introduce a galol group that is reported to have excellent adhesiveness, gallic acid was introduced into the synthesized hyaluronic acid-decane diamine, thereby synthesizing a hyaluronic acid derivative with a galol group introduced. Finally, the angiogenic effect of the synthesized hyaluronic acid polymer derivative was analyzed through an in ovo chick Chorioallantoic membrane (CAM) assay, thereby completing the present invention.

[0019]

[0020] The compounds or hydrogels of the present invention may be used in anti-angiogenic therapy in the eye or various tissues. Anti-angiogenic therapy preferably includes the treatment or prevention of any disease or disorder associated with abnormal angiogenesis or abnormal overproduction of pro-angiogenic VEGF isoforms (VEGFxxx). Such diseases and disorders include, for example, diabetic retinopathy, trachoma, retinal hyperplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, corneal neovascularization associated with ocular injury or infection, and proliferative diabetic retinopathy. Anti-angiogenic therapy according to the present invention may also include non-therapeutic treatments performed on healthy subjects, such as inhibiting blood vessel development for cosmetic purposes.

[0021]

[0022] 1. Hydrogel containing a polymer combined with a hyaluronic acid derivative

[0023] In one embodiment of the present invention, a hydrogel comprising a polymer to which a hyaluronic acid derivative is bound is provided.

[0024] In the present invention, the "hyaluronic acid" or "HA" is a biosynthetic natural substance found abundantly in the skin of animals and other organisms. Because it contains many hydroxyl groups (-OH), it is a hydrophilic substance and acts as a moisturizing agent in the skin of animals and other organisms. It is also found in human skin, and is known to be particularly abundant in the skin of earthworms. Because of its moisturizing effect, it is widely used in cosmetics and other products. Furthermore, hyaluronic acid possesses a three-dimensional structure in solution, resulting in extensive internal hydrogen bonding, limited polymer chain mobility, and unique secondary (helical) and tertiary (coiled coil) structures. In particular, it forms hydrophilic polymers through cross-linking and can absorb water in amounts hundreds of times its dry weight. Hydrogels, due to their excellent biocompatibility and hydrophilic properties, have diverse applications in the medical and pharmaceutical fields. It is known to regulate various physiological functions by reacting with the CD44 protein expressed in various epithelial cells, and is represented by the following chemical formula 7.

[0025] [Chemical Formula 7]

[0026]

[0027] In the present invention, examples of the “hyaluronic acid derivative” or “Hyaluronic Acid derivative” may include, but are not limited to, substances including salts such as sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, and tetrabutylammonium hyaluronate.

[0028] In the present invention, the “polymer” or “polymer” is a molecule formed into a long chain with regular repeating units through a chemical reaction of monomer molecules. It is a compound word of the Greek words polus (many) and meros (parts), and is also called macromolecule.

[0029] In the present invention, the "hydrogel" or "hydrogel" is also called a hydrogel, and refers to a material that does not dissolve in an aqueous environment and can contain a significant amount of water, as a network structure in which water-soluble polymers form three-dimensional cross-links through physical (hydrogen bonds, van der Waals forces, hydrophobic interactions, or polymer crystals) or chemical (covalent bonds) bonds. Hydrogels can be made from various water-soluble polymers and therefore have various chemical compositions and properties. Furthermore, they are easy to process and can be transformed into various shapes depending on the application. As can be seen from their successful applications in the peritoneum and various other parts of the body, hydrogels have high biocompatibility due to their high water content and physicochemical similarity to the extracellular matrix. Due to these characteristics, hydrogels have attracted attention as one of the most attractive materials for medical and pharmacological applications.

[0030]

[0031] In another embodiment of the present invention, a hydrogel is provided wherein the hyaluronic acid derivative is represented by the following chemical formula 9:

[0032] [Chemical Formula 9]

[0033]

[0034] In the above chemical formula 1, R1 is selected from the group consisting of a hydroxyl group, a carbonyl group, a carboxyl group, a hydroperoxy group, a carboxamide group, a primary amine group, and a secondary amine group.

[0035]

[0036] In another embodiment of the present invention, a hydrogel is provided in which a diamine is chemically bonded to the R1 group of the hyaluronic acid derivative.

[0037] In the present invention, the above “diamine” or “diamine” means having two amine groups, and the above “amine” or “amine” refers to a compound in which the hydrogen atoms of ammonia are replaced with hydrocarbon groups, and melamine is a type of amine. In other words, it refers to an organic compound whose functional group is an amino group (-NH2). It is similar to amide in that it is an organic compound containing nitrogen, and the two compound systems have many similarities, such as being classified into 1st, 2nd, and 3rd degrees depending on the number of functional groups actually bonded.

[0038]

[0039] In another embodiment of the present invention, the diamine is a saturated C2-C 12 , a linear, branched, or cyclic diamine; wherein the amine group of the diamine is primary or secondary.

[0040] In the present invention, the above “diamine” is a saturated C2-C 12 (e.g., C4-C12, C6-C 12 , C8-C 12 , C 10 -C 12 , C2-C 10 , C4-C 10 , C6-C 10 , C8-C 10, C2-C8, C4-C8, C6-C8, C2-C6, C4-C6, or C2-C4) linear, branched, or cyclic diamines. It should be understood that the modifiers “linear,” “branched,” and “cyclic” will only apply to certain embodiments where such embodiments are chemically possible, as understood by those skilled in the art. For example, it is not possible for the diamine to be a saturated C2 cyclic diamine, and such embodiments should therefore be considered excluded from the broader embodiments described in this paragraph. Suitable non-limiting examples of diamines include ethylenediamine, diaminopropane, diaminobutane, diaminopentane, hexamethylenediamine, diaminooctane, diaminononane, diaminodecane, diaminododecane, piperazine, methylpiperazine, dimethylpiperazine, homopiperazine, and bis-(aminomethyl)-cyclohexane.

[0041]

[0042] In another embodiment of the present invention, a hydrogel is provided in which gallic acid or a gallic acid derivative is additionally chemically bonded to the amine group of the diamine.

[0043] The above “gallic acid” or “Gallic acid” or “GA” is a substance whose molecular formula is C7H6O5 and whose chemical formula is C6H2(OH)3COOH, and is represented by the following chemical formula 8.

[0044] [Chemical Formula 8]

[0045]

[0046] The above gallic acid is 3,4,5-trihydroxybenzoic acid, a type of phenolic acid found in extracts from various plants including Galla Rhois, Orostachys japonica, Damnacanthus major, and Acanthopanax koreanum.

[0047] In the present invention, the term "gallic acid derivative" or "gallic acid derivative" refers to a derivative having three hydroxyl groups as a basic structure and capable of including a functional group capable of bonding with a carboxyl group or a linker. The gallic acid derivative can exhibit the angiogenic effect provided by the present invention by having three hydroxyl groups present in the meta and para positions. Non-limiting examples of the gallic acid derivative are as follows.

[0048]

[0049] In another embodiment of the present invention, the gallic acid derivative provides a hydrogel represented by the following chemical formula 2:

[0050] [Chemical Formula 2]

[0051]

[0052] In the above chemical formula 2, R2 is selected from the group consisting of a hydroxyl group, a carbonyl group, a carboxyl group, a hydroperoxy group, a carboxamide group, a primary amine group, and a secondary amine group.

[0053] The above "carboxyl group" or "carboxyl group" is a functional group composed of carbon, oxygen, and hydrogen, and is represented by the functional group of carboxylic acid, -COOH. Furthermore, the name carboxyl group is derived from the combination of the two elements it contains: a carbonyl group and a hydroxyl group. The structure of a carboxyl group consists of a central carbon atom, with one oxygen atom double bonded to it, and one hydroxyl group single bonded to it. In molecular formula terms, a carboxyl group has a hydroxyl group attached to a carbonyl group in the form of -C=O-. Due to the difference in electronegativity between oxygen and carbon, the carbon atom carries a partial positive charge. Therefore, the carboxyl carbon atom is susceptible to nucleophilic attack. The hydroxyl portion of the carboxyl group is relatively acidic, and the proton is readily donated to a suitable partner. The increased acidity (acid strength) compared to alcohols is due to the resonance stabilization of the corresponding base, which is a negatively charged carboxylate anion. In the carboxylate anion (-COO-), the two oxygen atoms are equivalent, meaning that the negative charge is distributed across both oxygen atoms and both CO bonds have the characteristics of a partial double bond.

[0054] That is, the carboxyl group of the above chemical formula 2 combines with the amine group of the diamine to form a chemical bond. The chemical bond may be any chemical bond, such as a non-covalent chemical bond or a covalent chemical bond, and there is no limitation thereon.

[0055]

[0056] In another embodiment of the present invention, a hydrogel is provided wherein the polymer is hyaluronic acid-ethylenediamine represented by the following chemical formula 3:

[0057] [Chemical Formula 3]

[0058]

[0059] In the above chemical formula 3, x:y is 99:1 to 50:50, and preferably, x:y in the above chemical formula 3 is 95:5 to 70:30, but is not limited thereto.

[0060]

[0061] In another embodiment of the present invention, a hydrogel is provided wherein the polymer is hyaluronic acid-hexamethylenediamine represented by the following chemical formula 4:

[0062] [Chemical Formula 4]

[0063]

[0064] In the above chemical formula 4, x:y is 99:1 to 50:50, and preferably, x:y in the above chemical formula 4 may be 95:5 to 70:30, but is not limited thereto.

[0065]

[0066] In another embodiment of the present invention, a hydrogel is provided wherein the polymer is hyaluronic acid-decanedyneinamine represented by the following chemical formula 5:

[0067] [Chemical Formula 5]

[0068]

[0069] In the above chemical formula 5, x:y is 99:1 to 50:50, and preferably, x:y in the above chemical formula 5 may be 95:5 to 70:30, but is not limited thereto.

[0070]

[0071] In another embodiment of the present invention, the polymer provides a hydrogel of hyaluronic acid-decanediamine-gallic acid represented by the following chemical formula 6:

[0072] [Chemical Formula 6]

[0073]

[0074] In the above chemical formula 6, x:y is 99:1 to 80:20, and preferably, x:y in the above chemical formula 6 may be 99:1 to 90:10, but is not limited thereto.

[0075]

[0076] 2. A composition for promoting blood vessel formation comprising the hydrogel.

[0077] In one embodiment of the present invention, a composition for promoting angiogenesis comprising the hydrogel is provided.

[0078] In the present invention, the term "angiogenesis" or "neovascularization" may include the process of directly generating new blood vessels from vascular progenitor cells (angioblasts) (vasculogenesis), the process of forming new blood vessels from existing blood vessels (angiogenesis), and the process of forming new blood vessels by the addition of vascular muscle cells (arteriogenesis). Such angiogenesis may be involved in various physiological and pathological phenomena, such as wound repair, embryonic development, tumor formation, chronic inflammation, and obesity.

[0079] Angiogenesis is particularly essential for wound healing and tissue regeneration. For example, a deficiency in angiogenesis in the body can lead to tissue or organ dysfunction, resulting in necrosis, ulceration, and ischemia. Furthermore, impaired blood supply can also lead to cardiovascular diseases such as ischemic heart disease, arteriosclerosis, myocardial infarction, and angina. Therefore, the development of treatments that induce or promote angiogenesis has been sought to reduce tissue damage caused by angiogenesis deficiency and treat cardiovascular diseases caused by it.

[0080] Meanwhile, for cardiovascular diseases associated with angiogenesis induction and its deficiency, therapies using vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived endothelial growth factor (PDEGF) are being studied. However, these factors have limitations in clinical application due to the difficulty in isolating and purifying them, and their therapeutic effects on cardiovascular diseases caused by angiogenesis deficiency are minimal.

[0081]

[0082] Here, according to a feature of the present invention, the composition for promoting angiogenesis according to one embodiment of the present invention may further include an oxidizing agent and a pH adjusting agent. For example, the oxidizing agent may include at least one selected from the group consisting of sodium periodate, hydrogen peroxide, horseradish peroxidase, and tyrosinase, and the pH adjusting agent may include at least one selected from the group consisting of sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide, and barium hydroxide.

[0083] The composition for promoting angiogenesis according to one embodiment of the present invention may be administered by at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intratracheal injection, and topical application to the skin.

[0084] According to another feature of the present invention, the content of HA-CA hydrogel in the composition for promoting angiogenesis according to one embodiment of the present invention may be 1% by volume to 5% by volume with respect to the total volume of the composition for promoting angiogenesis.

[0085]

[0086] Since the hydrogel, hyaluronic acid, gallic acid, etc. used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0087]

[0088] 3. A pharmaceutical composition for preventing or treating angiogenesis-dependent disease comprising the hydrogel.

[0089] The term “angiogenesis-dependent disease” may refer to a disease accompanied by symptoms of insufficient blood supply or improper angiogenesis, i.e., a disease caused by insufficient angiogenesis. The type of disease is not particularly limited as long as it can be prevented or treated by the angiogenesis-promoting effect of the fusion protein according to one aspect, but in one specific example, the angiogenesis-dependent disease may be any one selected from the group consisting of ischemic disease, wound, burn, psoriasis, chronic ulcer, cardiovascular hemorrhage, cerebral hemorrhage, bedsore, diabetes, retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, diabetic foot ulcer, and pulmonary hypertension.

[0090] The above ischemic disease may be any one selected from the group consisting of cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, angina pectoris, myocardial infarction, heart failure, cardiomegaly, retinal ischemia, ischemic colitis, ischemic acute renal failure, ischemic stroke, cerebrovascular dementia, brain trauma, and neonatal hypoxia.

[0091] The term “wound” refers to a state in which a living body is damaged, and encompasses pathological conditions in which tissues forming the internal or external surface of a living body, such as skin, muscle, nerve tissue, bone, soft tissue, internal organs, or vascular tissue, are divided or destroyed. The above wound may be any one selected from the group consisting of non-healing traumatic wounds, tissue destruction by radiation, abrasions, gangrene, lacerations, avulsions, penetrating wounds, gunshot wounds, wounds, chronic wounds, cuts, frostbite, contusions or bruises, skin ulcers, dry skin, keratosis, cracks, bursts, dermatitis, pain due to dermatophytosis, surgical wounds, wounds due to vascular diseases, corneal wounds, bedsores, ulcers, conditions related to diabetes and poor circulation such as diabetic skin erosions, chronic ulcers, suture sites after plastic surgery, spinal trauma wounds, gynecological wounds, chemical wounds, and acne, and may include damage to any part of the subject, but is not limited thereto.

[0092] The term “tissue regeneration” refers to the process of tissue recovery from damage caused by external and internal causes. Damage caused by external causes may include ultraviolet rays, external pollutants, wounds, trauma, etc., and damage caused by internal causes may include stress, etc.

[0093] The aforementioned "diabetic foot ulcer" is the most common complication of diabetes, affecting more than 50% of diabetic patients. This complication occurs when high blood sugar levels in the blood vessels of diabetic patients damage certain cellular functions, resulting in the death of microvessels and nerve cells in the legs.

[0094] The above "diabetic retinopathy" refers to a complication that occurs when capillaries narrow and eventually become occluded due to lesions in the capillary walls (thickening of the basement membrane, reduction of mural cells, excessive proliferation of endothelial cells) caused by diabetes, resulting in circulatory disorders in the retinal microvessels.

[0095] The term "prevention" in the present invention refers to a reduction in the occurrence of pathological cells in an animal, or in the degree of cell damage or loss. Prevention may be complete or partial. In this case, it may refer to a reduction in the occurrence of pathological cells or abnormal immune function within the animal, compared to when the composition for preventing and treating angiogenesis-dependent diseases is not used.

[0096] The term "treatment" or "improvement" in the present invention refers to any act of clinical intervention to alter the natural process of a target or cell to be treated, and may be performed during the progression of a clinical pathological condition or to prevent it. The desired therapeutic effect may include preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, alleviating or temporarily alleviating the disease condition, or improving the prognosis. In other words, the treatment may be interpreted as encompassing all acts by which the symptoms of an angiogenesis-dependent disease are improved or completely cured by the composition.

[0097]

[0098] The compositions of the present invention can be used as pharmaceutical compositions, food compositions or cosmetic compositions.

[0099] The pharmaceutical composition of the present invention may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being intended for humans.

[0100] The pharmaceutical composition of the present invention is not limited thereto, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as external preparations, suppositories, and sterile injection solutions, each according to a conventional method. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used for injections, and bases, excipients, lubricants, preservatives, etc. may be used for topical administration. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0101] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0102] Routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0103] The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0104] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, dosage form, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, form of medicine, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0105] The food composition of the present invention can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc.

[0106] When the above-mentioned effective ingredient of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight, but is not limited thereto.

[0107] When the food composition of the present invention is manufactured in the form of a beverage, there are no special limitations other than including the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be contained as additional ingredients like in a typical beverage. Specifically, the natural carbohydrates may include monosaccharides such as glucose, disaccharides such as fructose, sucrose, and other polysaccharides, dextrin, cyclodextrin, and other typical sugars, and sugar alcohols such as xylitol, sorbitol, and erythritol. The flavoring agents may include natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0108] The food composition of the present invention may further include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0109] The above components of the present invention may be used independently or in combination. The proportion of the above additives is not a key element of the present invention, but may be selected within the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

[0110] The cosmetic composition of the present invention can be manufactured in the form of toner, nutritional lotion, nutritional essence, massage cream, cosmetic bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, suntan cream, skin lotion, skin cream, UV protection cosmetics, cleansing milk, depilatory cosmetics, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath soap, liquid soap, beauty soap, shampoo, hand cleanser (hand cleaner), medicated soap (non-medical), cream soap, facial wash, body cleanser, scalp cleanser, hair rinse, cosmetic soap, tooth whitening gel, toothpaste, etc. The composition of the present invention may further include a solvent, appropriate carrier, excipient, or diluent commonly used in the manufacture of cosmetic compositions.

[0111] The type of solvent that can be further added to the cosmetic composition of the present invention is not particularly limited, but for example, water, saline solution, DMSO, or a combination thereof can be used. In addition, carriers, excipients, or diluents include, but are not limited to, purified water, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like. In addition, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents can be included as needed.

[0112] As the oil of the present invention, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil can be used, and as the wax, beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin can be used.

[0113] The fatty acids of the present invention may include stearic acid, linoleic acid, linolenic acid, and oleic acid, and the fatty acid alcohols may include cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol, and the fatty acid esters may include isopropyl myristate, isopropyl palmitate, and butyl stearate. As the surfactant, cationic surfactants, anionic surfactants, and non-ionic surfactants known in the art may be used, and surfactants derived from natural products are preferable if possible. In addition, the composition may include absorbents, thickeners, and antioxidants widely known in the cosmetic field, and the types and amounts thereof are as known in the art.

[0114] In the above treatment method of the present invention, matters related to the complex, administration method, number of administrations, dosage, cancer, immune-related disease, neurodegenerative disease or neuroinflammatory disease, prevention, improvement and treatment are the same as described above, and thus are omitted to avoid excessive complexity of the specification.

[0115] In the present invention, the "subject" refers to a subject suspected of having a disease, and the subject suspected of having a disease refers to a mammal including a human, a rat, a livestock, etc. that has developed or can develop the disease, but an subject that can be treated with the complex of the present invention or the composition containing the same is included without limitation.

[0116] The method of the present invention may include administering a pharmaceutically effective amount of the complex or a composition comprising the complex. The appropriate total daily dosage may be determined by the treating physician within the scope of sound medical judgment, and may be administered once or in several divided doses. However, for the purposes of the present invention, it is preferable that a specific therapeutically effective amount for a specific patient be applied differently depending on various factors, including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0117] Meanwhile, but not limited thereto, the treatment method may be a combination therapy further comprising administering a compound or substance having therapeutic activity against one or more diseases.

[0118] In the present invention, the term "combination" should be understood to refer to simultaneous, separate, or sequential administration. If the administration is sequential or separate, the interval between administrations of the secondary components should be such that the beneficial effects of the combination are not lost.

[0119] In the present invention, the administration dose of the complex may be about 0.0001 μg to 500 mg per 1 kg of patient body weight, but is not limited thereto.

[0120]

[0121] 4. A polymer production method by synthesizing hyaluronic acid and diamine in a chlorinated organic solvent and N-hydroxysuccinimide (NHS).

[0122] In one embodiment of the present invention, a method for producing a polymer is provided, wherein hyaluronic acid and diamine are synthesized in a chlorinated organic solvent and N-hydroxysuccinimide (NHS).

[0123] In the present invention, as a non-limiting example of the “chlorine-based organic solvent”, one or a mixture of two or more selected from the group consisting of chloroform, methylene chloride, carbon tetrachloride, carbon dichloride, trichloroethane, vinyl chloride, ethylene dichloride, trichloroethylene, and tetrachloroethylene may be used, and preferably, ethylene dichloride (EDC) solvent may be used.

[0124]

[0125] In another embodiment of the present invention, a manufacturing method is provided, comprising: (a) dissolving the hyaluronic acid in triple-distilled water, and then adding diamine, a chlorinated organic solvent, and N-hydroxysuccinimide to the hyaluronic acid solution and reacting the same; (b) purifying the hyaluronic acid-diamine synthesized in step (a) using a dialysis membrane.

[0126] In the present invention, “N-hydroxysuccinimide” or “N-Hydroxysuccinimide, NHS” is an organic compound with the chemical formula (CH2CO)2NOH. It is a white solid that is generally used as a reagent for preparing an active ester in peptide synthesis, and can be synthesized by heating succinic anhydride with hydroxylamine or hydroxylamine hydrochloride.

[0127]

[0128] In another embodiment of the present invention, the diamine is a saturated C2-C 12 , a linear, branched, or cyclic diamine; wherein the amine group of the diamine is primary or secondary.

[0129] In another embodiment of the present invention, a manufacturing method is provided, characterized in that the hyaluronic acid is selected from the group consisting of sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, and tetrabutylammonium hyaluronate.

[0130] In another embodiment of the present invention, a manufacturing method is provided, wherein the chlorinated organic solvent is one or a mixture of two or more selected from the group consisting of chloroform, methylene chloride, carbon tetrachloride, carbon dichloride, trichloroethane, vinyl chloride, ethylene dichloride, trichloroethylene, and tetrachlorethylene.

[0131] In another embodiment of the present invention, a manufacturing method is provided, wherein the hyaluronic acid is represented by the following chemical formula 7.

[0132] [Chemical Formula 7]

[0133]

[0134] In the above chemical formula 7, n = an integer from 13 to 13,200, preferably n = an integer from 100 to 5,300 in the above chemical formula 7, or more preferably n = an integer from 260 to 4,000 in the above chemical formula 7, but is not limited thereto.

[0135]

[0136] In one specific embodiment of the present invention, a method for producing a polymer further comprises a gallic acid derivative.

[0137] In another specific embodiment of the present invention, the gallic acid is provided by a manufacturing method represented by the following chemical formula 2:

[0138] [Chemical Formula 2]

[0139]

[0140] In the above chemical formula 2, R2 is selected from the group consisting of a hydroxyl group, a carbonyl group, a carboxyl group, a hydroperoxy group, a carboxamide group, a primary amine group, and a secondary amine group.

[0141]

[0142] In one embodiment of the present invention, a hydrogel comprising a polymer to which a hyaluronic acid derivative is bound is provided.

[0143] In another embodiment of the present invention, a hydrogel is provided, characterized in that the hyaluronic acid is selected from the group consisting of sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, and tetrabutylammonium hyaluronate.

[0144] In another embodiment of the present invention, a hydrogel is provided, wherein the hyaluronic acid derivative is represented by the following chemical formula 1:

[0145] [Chemical Formula 1]

[0146]

[0147] A hydrogel in which in the above chemical formula 1, n is 1 to 20, and R is hydrogen or gallic acid or a gallic acid derivative.

[0148] In another embodiment of the present invention, the gallic acid derivative provides a hydrogel represented by the following chemical formula 2:

[0149] [Chemical Formula 2]

[0150]

[0151] In the above chemical formula 2, R2 is selected from the group consisting of a hydroxy group, a carbonyl group, a carboxyl group, a hydroperoxy group, and a carboxamide group.

[0152] In another embodiment of the present invention, a hydrogel is provided wherein the polymer is hyaluronic acid-ethylenediamine represented by the following chemical formula 3:

[0153] [Chemical Formula 3]

[0154]

[0155] In the above chemical formula 3, x:y is 99:1 to 50:50.

[0156] In another embodiment of the present invention, a hydrogel is provided, wherein in the chemical formula 3, x:y is 95:5 to 70:30.

[0157] In another embodiment of the present invention, a hydrogel is provided wherein the polymer is hyaluronic acid-hexamethylenediamine represented by the following chemical formula 4:

[0158] [Chemical Formula 4]

[0159]

[0160] In the above chemical formula 4, x:y is 99:1 to 50:50.

[0161] In another embodiment of the present invention, a hydrogel is provided, wherein in the chemical formula 4, x:y is 95:5 to 70:30.

[0162] In another embodiment of the present invention, a hydrogel is provided wherein the polymer is hyaluronic acid-decanedyneinamine represented by the following chemical formula 5:

[0163] [Chemical Formula 5]

[0164]

[0165] In the above chemical formula 5, x:y is 99:1 to 50:50.

[0166] In another embodiment of the present invention, a hydrogel is provided, wherein in the chemical formula 5, x:y is 95:5 to 70:30.

[0167] In another embodiment of the present invention, the polymer provides a hydrogel of hyaluronic acid-decanediamine-gallic acid represented by the following chemical formula 6:

[0168] [Chemical Formula 6]

[0169]

[0170] In the above chemical formula 6, x:y is 99:1 to 80:20.

[0171] In another embodiment of the present invention, a hydrogel is provided, wherein in the chemical formula 6, x:y is 99:1 to 90:10.

[0172] In one embodiment of the present invention, a composition for promoting angiogenesis comprising the hydrogel is provided.

[0173] In one embodiment of the present invention, a method for producing a polymer is provided, wherein hyaluronic acid and diamine are synthesized in a chlorinated organic solvent and N-hydroxysuccinimide (NHS).

[0174] In another embodiment of the present invention, a manufacturing method is provided, comprising: (a) dissolving the hyaluronic acid in triple-distilled water, and then adding diamine, a chlorinated organic solvent, and N-hydroxysuccinimide to the hyaluronic acid solution and reacting the same; (b) purifying the hyaluronic acid-diamine synthesized in step (a) using a dialysis membrane.

[0175] In another embodiment of the present invention, the diamine is a saturated C2-C 12 , a linear, branched, or cyclic diamine; wherein the amine group of the diamine is primary or secondary.

[0176] In another embodiment of the present invention, a manufacturing method is provided, characterized in that the hyaluronic acid is selected from the group consisting of sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, and tetrabutylammonium hyaluronate.

[0177] In another embodiment of the present invention, a manufacturing method is provided, wherein the chlorinated organic solvent is one or a mixture of two or more selected from the group consisting of chloroform, methylene chloride, carbon tetrachloride, carbon dichloride, trichloroethane, vinyl chloride, ethylene dichloride, trichloroethylene, and tetrachlorethylene.

[0178] In another embodiment of the present invention, a manufacturing method is provided, wherein the hyaluronic acid is represented by the following chemical formula 7:

[0179] [Chemical Formula 7]

[0180]

[0181] In the above chemical formula 7, n = an integer from 13 to 13,200.

[0182] In another embodiment of the present invention, a manufacturing method is provided, wherein n in the chemical formula 7 is an integer from 100 to 5,300.

[0183] In another embodiment of the present invention, a manufacturing method is provided, wherein n in the chemical formula 7 is an integer from 260 to 4,000.

[0184] In another embodiment of the present invention, a method for preparing a polymer further comprises a gallic acid derivative.

[0185] In another embodiment of the present invention, the gallic acid is provided by a manufacturing method represented by the following chemical formula 2:

[0186] [Chemical Formula 2]

[0187]

[0188] In the above chemical formula 2, R2 is selected from the group consisting of a hydroxyl group, a carbonyl group, a carboxyl group, a hydroperoxy group, a carboxamide group, a primary amine group, and a secondary amine group.

[0189]

[0190] The various hyaluronic acid derivatives and hydrogels containing hyaluronic acid derivatives developed in the present invention can be utilized for various diseases requiring angiogenic effects, and can be directly utilized as wound healing agents or wound dressings through inflammation relief and angiogenic effects. Furthermore, because the present hyaluronic acid derivatives have the ability to generate blood vessels, they can be utilized in various drug delivery systems, tissue engineering, and regenerative medicine. For example, when cells are cultured in vitro or tissues are regenerated and transplanted, nutrients can be supplied through diffusion from blood vessels surrounding the transplanted tissue. However, if blood vessels are not formed in the surrounding tissue, successful tissue regeneration cannot occur due to a lack of oxygen and nutrients. Developing drug delivery systems or tissue engineering carriers based on the present hyaluronic acid derivatives, or adding the present hyaluronic acid derivatives to existing biomaterials, can promote blood vessel formation in the surrounding tissues, supplying oxygen and nutrients, thereby inducing successful tissue regeneration. Therefore, it is believed that the technology developed through the present invention can be applied and utilized in various fields of biomedicine, such as drug delivery and tissue engineering.

[0191] Figure 1 schematically illustrates the behavior of Pluronic F127 hydrogel containing a hyaluronic acid derivative at or above the lower critical solution temperature (LCST).

[0192] Figure 2 shows the results of an in ovo chick Chorioallantoic membrane (CAM) assay for analyzing the angiogenic effect of hyaluronic acid derivatives.

[0193] Figure 3 illustrates the sample processing process for analyzing the angiogenic effect.

[0194] Figure 4 shows the results of angiogenic effect analysis of hyaluronic acid derivatives (200 kDa) (left: sample photo, middle: appearance of blood vessels through image processing, right: image processing for quantification).

[0195] Figure 5 shows the results of quantitative vascular analysis of hyaluronic acid derivatives (200 kDa) through image processing.

[0196] Figure 6 shows the results of angiogenic effect analysis of hyaluronic acid derivatives (1.5 MDa).

[0197] Figure 7 shows the results of quantitative vascular analysis of a hyaluronic acid derivative (1.5 MDa) through image processing.

[0198]

[0199] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0200]

[0201] Manufacturing example

[0202] Manufacturing Example 1. Synthesis of hyaluronic acid-ethylenediamine (HA-EDA)

[0203] Hyaluronic acid (500 mg) was dissolved in 50 mL of distilled and deionized water, and ethylenediamine (EDA) (439 mg), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (126 mg), and n-hydroxysuccinimide (NHS) (76 mg) were slowly added to the hyaluronic acid solution, and the mixture was reacted for 12 hours. The pH was maintained between 4.5 and 6.5 during the reaction. The synthesized hyaluronic acid-ethylenediamine was purified using a dialysis membrane (Dialysis membrane, MWCO = 12-14 kDa) and dialyzed against distilled water for 3 days. The finally purified hyaluronic acid-ethylenediamine was freeze-dried and stored.

[0204]

[0205] Through the above manufacturing method, hyaluronic acid-ethylenediamine (HA-EDA) represented by the following chemical formula 10 was synthesized.

[0206] [Chemical Formula 10]

[0207]

[0208] In the above chemical formula 1, n = an integer of 13 to 13,200, preferably n = an integer of 100 to 5,300, and more preferably n = an integer of 260 to 4,000. In addition, in the above chemical formula 1, x:y is 99:1 to 50:500, preferably x:y is 99:1 to 85:15, and even more preferably x:y is 99:3 to 90:10 in the above chemical formula 1, but is not limited thereto.

[0209]

[0210] Manufacturing Example 2. Synthesis of hyaluronic acid-hexamethylene diamine (HA-HDA)

[0211] 500 mg of hyaluronic acid was dissolved in 50 mL of distilled water, and 503 mg of hexamethylene diamine (HDA), 126 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and 76 mg of n-hydroxysuccinimide (NHS) were slowly added to the hyaluronic acid solution, and the mixture was reacted for 12 hours. The pH was maintained between 4.5 and 6.5 during the reaction. The synthesized hyaluronic acid-hexamethylene diamine was purified using a dialysis membrane (Dialysis membrane, MWCO = 12-14 kDa) and dialyzed against distilled water for 3 days. The finally purified hyaluronic acid-hexamethylene diamine was freeze-dried and stored.

[0212]

[0213] Through the above manufacturing method, hyaluronic acid-hexamethylene diamine (HA-HDA) represented by the following chemical formula 11 was synthesized.

[0214] [Chemical Formula 11]

[0215]

[0216] In the above chemical formula 2, n = an integer of 13 to 13,200, preferably n = an integer of 100 to 5,300, and more preferably n = an integer of 260 to 4,000. In addition, in the above chemical formula 2, x : y = 99 : 1 to 50 : 50, and more preferably x : y = 95 : 5 to 70 : 30.

[0217]

[0218] Manufacturing Example 3. Synthesis of hyaluronic acid-decane diamine (HA-DDA)

[0219] 500 mg of hyaluronic acid was dissolved in 50 mL of distilled water, and 809 mg of decane diamine (DAD), 126 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and 76 mg of n-hydroxysuccinimide (NHS) were slowly added to the hyaluronic acid solution, and the mixture was reacted for 12 hours. The pH was maintained between 4.5 and 6.5 during the reaction. The synthesized hyaluronic acid-decane diamine was purified using a dialysis membrane (Dialysis membrane, MWCO = 12-14 kDa) and dialyzed against distilled water for 3 days. The finally purified hyaluronic acid-decane diamine was freeze-dried and stored.

[0220]

[0221] Through the above manufacturing method, hyaluronic acid-decane diamine (HA-DDA) represented by the following chemical formula 12 was synthesized.

[0222] [Chemical Formula 12]

[0223]

[0224] In the above chemical formula 3, n = an integer of 13 to 13,200, preferably n = an integer of 100 to 5,300, and more preferably n = an integer of 260 to 4,000. In addition, in the above chemical formula 3, x : y = 99 : 1 to 50 : 50, and more preferably x : y = 95 : 5 to 70 : 30.

[0225]

[0226] Manufacturing Example 4. Synthesis of hyaluronic acid-decane diamine-gallic acid (HA-DDA-GA)

[0227] In Manufacturing Example 3, 200 mg of hyaluronic acid-decanediamine prepared was dissolved in 20 mL of triple-distilled water, and 809 mg of gallic acid (GA), 126 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and 76 mg of NHS (n-hydroxysuccinimide) were slowly added to the hyaluronic acid solution, and the mixture was reacted for 12 hours. The pH was maintained between 4.5 and 6.5 during the reaction. The synthesized hyaluronic acid-decanediamine-gallic acid was purified using a dialysis membrane (Dialysis membrane, MWCO = 12-14 kDa), dialyzed against pH 2 NaCl solution for 2 days, and then dialyzed against triple-distilled water for 4 hours. The finally purified hyaluronic acid-decanediamine-gallic acid was freeze-dried and stored.

[0228]

[0229] Through the above manufacturing method, hyaluronic acid-decane diamine-gallic acid (HA-DDA-GA) represented by the following chemical formula 13 was synthesized.

[0230] [Chemical Formula 13]

[0231]

[0232] In the above chemical formula 4, n = an integer of 13 to 13,200, preferably n = an integer of 100 to 5,300, and more preferably n = an integer of 260 to 4,000. In addition, in the above chemical formula 4, x:y = 99:1 to 80:20, and more preferably x:y = 99:1 to 90:10.

[0233]

[0234] Manufacturing Example 5. Manufacturing of Pluronic F127 hydrogel carrier containing hyaluronic acid derivative.

[0235] As illustrated in Fig. 1, Pluronic F127, a thermosensitive polymer, is a polymer with phase transition characteristics that exist in a sol form at low temperatures starting from the lower critical solution temperature, and change into a gel form at temperatures higher than the lower critical solution temperature. Accordingly, by mixing Pluronic and a hyaluronic acid derivative at room temperature (20°C) and raising the temperature to 37°C, which is the body temperature, Pluronic F127 containing a hyaluronic acid derivative can be produced.

[0236]

[0237] Specifically, the method for preparing the Pluronic F127 hydrogel containing the hyaluronic acid derivative is as follows. For rapid dissolution of Pluronic F127, Pluronic F127 (20 wt%) was dissolved in pH 7.4 PBS in a 4°C refrigerator, and the hyaluronic acid derivative was added at room temperature to a final concentration of 2 mg / mL to prepare a hyaluronic acid-derivative / Pluronic F127 solution.

[0238]

[0239] Example

[0240] Example 1. In ovo chick Chorioallantoic membrane (CAM) assay for analyzing the angiogenic effect of hyaluronic acid derivatives

[0241] As shown in Figures 2 and 3, after incubating the eggs for 48 hours, the window was opened and 20 uL of Pluronic F127 hydrogel loaded with a hyaluronic acid derivative was dropped onto the egg embryo, followed by incubation for 12 hours again and angiogenic effects were analyzed from the location where the hydrogel was dropped.

[0242]

[0243] Example 2. Analysis of the angiogenic effect of hyaluronic acid derivatives

[0244] As shown in Fig. 4, compared to the untreated group and the group treated with Pluronic, more blood vessels were generated in the group treated with hyaluronic acid (200 kDa), and more blood vessels were generated in the group treated with synthetic hyaluronic acid derivatives (HA-EDA, HA-HDA, HA-DDA, HA-DDA-GA) than in the group treated with hyaluronic acid. Image processing was performed to quantify this.

[0245]

[0246] Example 3. Quantitative vascular analysis of hyaluronic acid derivatives using image processing.

[0247] As shown in Figure 5, it was confirmed that the most blood vessels were created in the groups treated with hyaluronic acid-hexamethylenediamine and hyaluronic acid-decanediamine-gallic acid, and significant blood vessel creation was confirmed compared to the group that was not treated with anything, the group treated with only pluronic hydrogel, and the group treated with pluronic hydrogel containing hyaluronic acid (P < 0.01).

[0248]

[0249] In addition, as shown in FIGS. 6 and 7, the hyaluronic acid derivative (1.5 MDa) synthesized through the present invention showed significant angiogenesis compared to the group that was not treated with anything or the group treated only with Pluronic hydrogel (P < 0.01).

[0250]

[0251] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A hydrogel containing a hyaluronic acid derivative polymer, The above hyaluronic acid derivative is a hydrogel represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, n is 1 to 20, R is selected from the group consisting of gallic acid or a gallic acid derivative, The above gallic acid derivative is represented by the following chemical formula 2: R of the following chemical formula 2 2 A hydrogel in which R of the above chemical formula 1 is covalently bonded to nitrogen: [Chemical formula 2] In the above chemical formula 2, R2 is selected from the group consisting of a hydroxy group, a carbonyl group, a carboxyl group, a hydroperoxy group, and a carboxamide group.

2. In paragraph 1, The above polymer is a hydrogel, which is hyaluronic acid-ethylenediamine represented by the following chemical formula 3: [Chemical Formula 3] In the above chemical formula 3, x:y is 99:1 to 50:

50.

3. In paragraph 2, A hydrogel wherein in the chemical formula 3, x:y is 95:5 to 70:

30.

4. In paragraph 1, The above polymer is a hydrogel, which is hyaluronic acid-hexamethylenediamine represented by the following chemical formula 4: [Chemical Formula 4] In the above chemical formula 4, x:y is 99:1 to 50:

50.

5. In paragraph 4, A hydrogel wherein in the chemical formula 4, x:y is 95:5 to 70:

30.

6. In paragraph 1, The above polymer is a hydrogel, which is hyaluronic acid-decanedyneinamine represented by the following chemical formula 5: [Chemical Formula 5] In the above chemical formula 5, x:y is 99:1 to 50:

50.

7. In paragraph 6, A hydrogel wherein in the chemical formula 5, x:y is 95:5 to 70:

30.

8. In paragraph 1, The above polymer is a hydrogel of hyaluronic acid-decanediamine-gallic acid represented by the following chemical formula 6: [Chemical formula 6] In the above chemical formula 6, x:y is 99:1 to 80:

20.

9. In paragraph 8, A hydrogel wherein in the chemical formula 6, x:y is 99:1 to 90:

10.

10. A pharmaceutical composition for promoting blood vessel formation, comprising a hydrogel containing a hyaluronic acid derivative polymer as an active ingredient, The above hyaluronic acid derivative is a pharmaceutical composition represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, n is 1 to 20 and R is hydrogen.

11. A pharmaceutical composition for promoting angiogenesis comprising the hydrogel of claims 1 to 9.

12. A pharmaceutical composition for the prevention or treatment of angiogenesis-dependent diseases, comprising the hydrogel of clauses 1 to 9 as an effective ingredient, A pharmaceutical composition wherein the above angiogenesis-dependent disease is an ischemic disease.

13. A pharmaceutical composition for the prevention or treatment of angiogenesis-dependent diseases, comprising a hydrogel containing a hyaluronic acid derivative polymer as an active ingredient, The above angiogenesis-dependent disease is an ischemic disease, The above hyaluronic acid derivative is a pharmaceutical composition represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, n is 1 to 20 and R is hydrogen.

14. In paragraph 13, A pharmaceutical composition for preventing or treating angiogenesis-dependent disease, wherein the ischemic disease is any one selected from the group consisting of cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, angina pectoris, myocardial infarction, heart failure, cardiomegaly, retinal ischemia, ischemic colitis, ischemic acute renal failure, ischemic stroke, cerebrovascular dementia, brain trauma, and neonatal hypoxia.

15. A polymer production method comprising synthesizing hyaluronic acid and diamine in a chlorinated organic solvent and N-hydroxysuccinimide (NHS). (a) a step of dissolving the hyaluronic acid in triple-distilled water, then adding diamine, a chlorinated organic solvent, and N-hydroxysuccinimide to the hyaluronic acid solution and causing a reaction; (b) a step of purifying the hyaluronic acid-diamine synthesized in step (a) using a dialysis membrane; A method for manufacturing a polymer, further comprising a step of reacting a gallic acid derivative in the step (a) above, The above gallic acid derivative is prepared by the following chemical formula 2: [Chemical formula 2] In the above chemical formula 2, R2 is selected from the group consisting of a hydroxy group, a carbonyl group, a carboxyl group, a hydroperoxy group, a carboxamide group, a primary amine group, and a secondary amine group.

16. In paragraph 15, The above diamine is saturated C2-C 12 , linear, branched, or cyclic diamines; A manufacturing method wherein the amine group of the above diamine is primary or secondary.

17. In paragraph 16, A manufacturing method, wherein the above chlorinated organic solvent is one or a mixture of two or more selected from the group consisting of chloroform, methylene chloride, carbon tetrachloride, carbon dichloride, trichloroethane, vinyl chloride, ethylene dichloride, trichloroethylene, and tetrachlorethylene.

18. In paragraph 17, The above hyaluronic acid is represented by the following chemical formula 7, manufacturing method: [Chemical formula 7] In the above chemical formula 7, n = an integer from 13 to 13,200.

19. In paragraph 18, A manufacturing method, wherein n in the chemical formula 7 is an integer from 100 to 5,300.

20. In paragraph 19, A manufacturing method, wherein n in the chemical formula 7 is an integer from 260 to 4,000.

21. A method for preventing or treating angiogenesis-dependent disease, which comprises administering the hydrogel of clauses 1 to 9 to a subject.

22. In paragraph 21, A method for preventing or treating an angiogenesis-dependent disease, wherein the angiogenesis-dependent disease is any one selected from the group consisting of ischemic disease, wound, burn, psoriasis, chronic ulcer, cardiovascular hemorrhage, cerebral hemorrhage, bedsores, diabetes, retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, diabetic foot ulcer, and pulmonary hypertension.

23. In paragraph 22, A method for preventing or treating angiogenesis-dependent disease, wherein the ischemic disease is any one selected from the group consisting of cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, angina pectoris, myocardial infarction, heart failure, cardiomegaly, retinal ischemia, ischemic colitis, ischemic acute renal failure, ischemic stroke, cerebrovascular dementia, brain trauma, and neonatal hypoxia.

Images