Structure of high molecular weight hyaluronic acid, method for producing the same, and eye drop composition containing the same

Abstract

The present specification relates to a polymeric hyaluronic acid structure obtained by applying shear stress to a solution containing polymeric hyaluronic acid, a method for producing the same, and an eye drop composition containing the same.

Description

[Technical Field] 【0001】 This application claims priority under Korean Patent Application No. 10-2022-0023811 dated February 23, 2022, and incorporates all the contents disclosed in the said Korean Patent Application as part of this Specification. 【0002】 The present invention relates to a structure of high molecular weight hyaluronic acid, a method for producing the same, and an eye drop composition containing the same, and more particularly to a structure of high molecular weight hyaluronic acid obtained by applying shear stress to high molecular weight hyaluronic acid to reduce its viscosity, a method for producing the same, and an eye drop composition containing the same. [Background technology] 【0003】 Dry eye is a condition in which the tear gland malfunctions, resulting in insufficient tear production and excessive tear evaporation, causing the eyeball to dry out. This is a phenomenon of tear film drying, accompanied by increased osmotic pressure and inflammation of the eyeball surface, making it a disease that can cause inconvenience in daily life and, in severe cases, lead to blindness. 【0004】 Eye drops are the most commonly used treatment for dry eye, and commercially available eye drops are primarily developed to replenish moisture by replenishing the aqueous layer, which is the intermediate layer of tears, and play a role in preventing water evaporation through polymeric substances. Examples of such polymeric substances include hyaluronic acid, carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), and polyvinyl alcohol (PVA). 【0005】 Among these polymeric substances, hyaluronic acid is a biopolymer that exists in the human body, and is abundantly distributed in the skin, vitreous humor of the eye, synovial fluid, muscles, umbilical cord, and comb, performing various functions in different parts of the body. Furthermore, hyaluronic acid is highly soluble in water, making it easy to develop as an eye drop, and as a result, hyaluronic acid eye drops account for approximately 70% of the artificial tear market. 【0006】 Eye drops that are commercially available with hyaluronic acid as the main ingredient contain hyaluronic acid at concentrations of 0.1%, 0.15%, 0.18%, and 0.3%, and generally, products with low concentrations are commercially available. 【0007】 Hyaluronic acid exhibits different viscosity depending on its molecular weight, even at the same concentration; the higher the molecular weight of hyaluronic acid, the higher the viscosity. However, with conventional eye drops containing hyaluronic acid, while high viscosity provides greater symptom relief, it can also irritate the eyes of dry eye patients, cause stickiness or dryness when blinking, and blur vision. 【0008】 There is a growing demand for eye drops that contain high concentrations of hyaluronic acid, or that contain high molecular weight hyaluronic acid at the same concentration while maintaining adjustable viscosity. [Prior art documents] [Patent Documents] 【0009】 [Patent Document 1] Korean Published Patent No. 2021-0015377 (February 10, 2021), Method for producing low-viscosity hyaluronic acid [Overview of the project] [Problems that the invention aims to solve] 【0010】 The technical problem that the present invention aims to solve is to provide a structure of high molecular weight hyaluronic acid, a method for producing the same, and an eye drop composition containing the same. More specifically, the invention aims to provide a structure of high molecular weight hyaluronic acid with reduced viscosity, a method for producing the same, and an eye drop composition containing the same, without reducing the molecular weight by applying shear stress to high molecular weight hyaluronic acid. 【0011】 The technical problems that this invention aims to solve are not limited to those mentioned, and other technical problems not mentioned will be clearly understood by those skilled in the art to which this invention pertains from the following description. 【Means for Solving the Problems】 【0012】 In order to achieve the above technical problems, the viscosity of the solution containing the polymer hyaluronic acid structure according to one aspect of the present invention may be 1 to 60 cP. 【0013】 In one embodiment of the present invention, the molecular weight of the hyaluronic acid is 5.0×10 5 ~2.0×10 6 g / mol may also be in the range. 【0014】 In one embodiment of the present invention, the solution containing the structure may contain a hyaluronic acid content of 0.01% (w / v) to 0.5% (w / v). 【0015】 In one embodiment of the present invention, when the structure and the molecular weight and concentration of the hyaluronic acid, which is the precursor of the structure, are the same, a / b, which is the ratio of the viscosity (a) of the solution containing the structure and the viscosity (b) of the solution containing the hyaluronic acid, which is the precursor of the structure, may be 0.001 to 0.30. 【0016】 In one embodiment of the present invention, the intrinsic viscosity of the solution containing the structure may be 0.1 to 2.5 m 3 / kg. 【0017】 The present invention can provide an ophthalmic composition containing the polymer hyaluronic acid structure. 【0018】 In one embodiment of the present invention, the viscosity of the ophthalmic composition may be 1.0 to 30.0 cP. 【0019】 In one embodiment of the present invention, the ophthalmic composition may further contain an excipient. 【0020】 In one embodiment of the present invention, the excipient may be one or more selected from the group consisting of a buffer, a preservative, a tonicity regulator, and a pH regulator. 【0021】 The present invention can provide an eye drop composition for the prevention or treatment of dry eye, comprising the aforementioned eye drop composition. 【0022】 The present invention provides a kit comprising a container containing the eye drop composition, wherein the container includes a dispensing means suitable for topical administration of the eye drop composition. 【0023】 In one embodiment of the present invention, the dispensing means may provide the eye drop composition in a dropwise manner, dispensing droplets with a volume of 0.01 to 0.10 ml. [Effects of the Invention] 【0024】 This invention offers the advantage of significantly reducing viscosity and improving usability by producing a novel hyaluronic acid structure that utilizes intramolecular interactions such as ionic bonds, including hydrogen bonds, and / or interactions such as ionic clusters, while maintaining the same molecular weight. 【0025】 Furthermore, the eye drop composition containing the polymer hyaluronic acid structure of the present invention has the advantage of having a large amount of hyaluronic acid in the eye drop composition, which improves moisture retention capacity. [Brief explanation of the drawing] 【0026】 [Figure 1] This figure shows a polymer chain of hyaluronic acid, which is a precursor of a structure according to one embodiment of the present invention. [Figure 2] This figure shows a single polymer chain of hyaluronic acid, which is a precursor of a structure according to one embodiment of the present invention. [Figure 3] This is a scanning electron microscope (SEM) image of an aqueous solution containing a structure according to one embodiment of the present invention. [Figure 4] This is a scanning electron microscope (SEM) image of an aqueous solution containing a structure according to one embodiment of the present invention. [Figure 5] This is a transmission electron microscope (TEM) image of hyaluronic acid, a precursor of a structure according to one embodiment of the present invention. [Figure 6] This figure shows a comparison of HPLC measurement results according to one embodiment of the present invention. [Modes for carrying out the invention] 【0027】 Generally, polymers, unlike molecules with low molecular weight, have long-chain structures with regular repeating units formed by chemical reactions of monomeric molecules. The structure of long-chain polymers exists in a solid state, or, if in a liquid state, the chains are twisted. 【0028】 In very dilute polymer solutions, viscosity is related to volume. This is well known from Einstein's equation and can be shown in Equation 1 below: 【number】 (Here, η is the viscosity of the solution, η0 is the viscosity of the solvent, η rel (where φ represents relative viscosity, and φ represents volume ratio) 【0029】 The volume of a polymer solution increases with increasing molecular weight, greater affinity between the solvent and the polymer, and greater repulsive force between unit chain structures. 【0030】 Furthermore, in polymer solutions, as the concentration increases, the chains permeate each other, forming a very large continuum structure, while as the concentration of the polymer decreases, the interactions between the individual chains decrease. 【0031】 Unlike ionic polymers, where each chain exists independently, hyaluronic acid polymers in aqueous solution are known to form a twin helix structure, where two chains are bundled together. This is sometimes called a twin helix or macromolecular crowding. Figure 1 shows a schematic diagram of two chains of hyaluronic acid polymer intertwined to form a twin helix structure. 【0032】 Generally, eye drop compositions containing high molecular weight hyaluronic acid promote collagen synthesis, have a longer retention time in the eyeball, and exhibit superior moisture retention compared to eye drops containing low molecular weight hyaluronic acid. However, a problem arises when applying high molecular weight hyaluronic acid to the eyeball: it can feel sticky when blinking, resulting in an unpleasant user experience. To address these issues, numerous studies have been conducted to adjust the viscosity of high molecular weight solutions. 【0033】 However, these methods for adjusting viscosity, such as those that induce hydrolysis of the hyaluronic acid polymer chains through pH adjustment or catalyst addition, thereby reducing the molecular weight and lowering the viscosity of the polymer solution, had limitations in that they could not maintain the polymeric characteristics of hyaluronic acid. 【0034】 To solve the aforementioned problems, the inventors applied high shear stress to a pair of polymer chains of high-viscosity hyaluronic acid, converting them into a single helix structure, and confirmed that ion aggregates are formed through intramolecular interactions and / or interactions such as ion clusters between the single polymer chains. 【0035】 This resulted in a decrease in the volume of the high molecular weight hyaluronic acid chain, and consequently, a decrease in the volume of high molecular weight hyaluronic acid in the solution, leading to a significant decrease in viscosity. 【0036】 The following describes, first, a method for producing a polymer hyaluronic acid structure according to one embodiment of the present invention, and then, in detail, the polymer hyaluronic acid structure. 【0037】 Method for manufacturing structures of high molecular weight hyaluronic acid As used herein, the term "structure of high molecular weight hyaluronic acid" means a structure having a single polymer chain structure formed by applying shear stress to high molecular weight hyaluronic acid having a pair of polymer chain structures. 【0038】 As used herein, the term “precursor” means a precursor or precursor used to produce the polymer hyaluronic acid structure according to the present invention. That is, the precursor of the polymer hyaluronic acid structure according to the present invention means hyaluronic acid having a pair of polymer chain structures to which shear stress has not been applied. 【0039】 A polymer hyaluronic acid structure according to one embodiment of the present invention can be produced by applying shear stress to a solution containing hyaluronic acid, which is a precursor of the structure. 【0040】 The shear stress applied to the solution containing hyaluronic acid, which is a precursor of the aforementioned structure, may be either mechanical shear stress or ultrasonic application. 【0041】 The mechanical shear stress can be applied by attaching a solution containing hyaluronic acid, which is a precursor of the structure, to the inlet of a high-pressure disperser and dispersing it under high pressure. Alternatively, the solution containing hyaluronic acid, which is a precursor of the structure, can be injected into a mill such as a ball mill, pin mill, or roll mill, and mechanical shear stress can be applied. The mechanical shear stress will be explained in detail below. When the solution containing the hyaluronic acid precursor passes through the nozzle of the high-pressure disperser at high speed, the hyaluronic acid precursor of the structure is subjected to very high shear stress due to passing through a physically narrow area. 【0042】 The aforementioned shear stress may be applied using ultrasound. The application of ultrasound will be explained in detail below. 【0043】 According to one embodiment of the present invention, the shear stress may be applied by applying ultrasound to the solution containing the hyaluronic acid. 【0044】 When the ultrasound is added to the solution containing the hyaluronic acid, a pressure wave is generated, and this pressure wave can apply shear stress to the hyaluronic acid, which is a precursor of the structure. 【0045】 The intensity of the applied ultrasonic wave may be 200 J / sec to 800 J / sec or 400 J / sec to 600 J / sec. 【0046】 The energy added per unit volume of the applied ultrasound can be calculated by multiplying the ultrasound intensity (J / sec) by the duration of application (sec) and the measured volume (ml). 【0047】 According to one embodiment of the present invention, the energy per unit volume of ultrasound applied to the solution containing hyaluronic acid may be 100 J / ml to 90 kJ / ml. If the ultrasound energy is less than 100 J / ml, sufficient shear stress may not be applied to the solution containing hyaluronic acid, making it difficult to form a structure. If the ultrasound energy exceeds 90 kJ / ml, excessive heat may be applied to the solution containing hyaluronic acid, making it difficult to form a structure. 【0048】 The ultrasound can be applied at a temperature of 10°C to 80°C for 10 seconds to 500 minutes. If the ultrasound is applied at a temperature below 10°C, there is no change in the solution containing the hyaluronic acid. If the ultrasound is applied at a temperature exceeding 80°C, a phase change occurs in the solution containing the hyaluronic acid, making it difficult to form the polymer hyaluronic acid structure according to the present invention. Also, if the ultrasound is applied for less than 10 seconds, there is no change in the solution containing the hyaluronic acid. If the ultrasound is applied for a time exceeding 500 minutes, the structure in the solution containing the hyaluronic acid is deformed, making it impossible to form the polymer hyaluronic acid structure according to the present invention. 【0049】 Structure of high molecular weight hyaluronic acid The polymer hyaluronic acid structure according to the present invention can be produced by the manufacturing method described above. Specifically, the polymer hyaluronic acid structure can be produced by applying shear stress to hyaluronic acid, which is a precursor of the structure, to form a single polymer chain. 【0050】 The aforementioned single polymer chain may have a structure that includes the chemical structure of hyaluronic acid as shown in Figure 2. The chemical structure of hyaluronic acid has one carboxyl anion (-COO) for every two cyclic hexoses. - It can be a single polymer chain having a ) group. 【0051】 The aforementioned single polymer chains can form ionic aggregates through intramolecular interactions and / or interactions such as ion clusters, as the distance between the single chains decreases. The aforementioned intramolecular interactions can be carried out via intramolecular hydrogen bonds, ionic bonds, etc. The aforementioned interactions such as ion clusters occur in sodium hyaluronate salts, where carboxyl anions (-COO δ- ) and sodium cation (Na δ+ This can be done through the interaction between the partial charges of ). 【0052】 As shown in FIGS. 3 and 4, the ion complex can include a spherical structure. The ion complex may have a spherical structure with a diameter of 1 to 20 nm, 2 to 18 nm, or 3 to 16 nm due to the interaction such as the intramolecular interaction or ion cluster. 【0053】 On the other hand, hyaluronic acid, which is a precursor of the structure according to the present invention, has a general polymer chain structure and does not contain the ion complex having the spherical structure. FIG. 5 is a TEM photograph of hyaluronic acid, which is a precursor of the structure according to the present invention. Only lines are observed as traces generated during the freezing process for TEM photography of the hyaluronic acid, and the shape of the ion complex having a spherical structure is not observed. 【0054】 Molecular weight of high molecular weight hyaluronic acid structure The molecular weight of the polymer hyaluronic acid structure according to the present invention is 5.0×10 5 ~2.0×10 6 g / mol, 5.5×10 5 ~1.95×10 6 g / mol, or 6.0×10 5 ~1.90×10 6 g / mol may be acceptable. 【0055】 The structure of the polymer hyaluronic acid according to the present invention is the same as the molecular weight of hyaluronic acid, which is the precursor of the structure. That is, even if a shear stress is applied to a solution containing hyaluronic acid, which is the precursor of the structure, to produce a structure of polymer hyaluronic acid, the same molecular weight as that of hyaluronic acid, which is the precursor of the structure before the application of the shear stress, can be maintained, and a structure with a low viscosity can be produced. 【0056】 In FIG. 6, (a) is a diagram showing a molecular weight measurement curve of sodium hyaluronate, which is a precursor of the structure according to the present invention, and (b) is a diagram showing a molecular weight measurement curve of the polymer hyaluronic acid structure according to the present invention. 【0057】 As shown in Figure 6, it was confirmed that the molecular weight measurement curve of hyaluronic acid, which is a precursor of the structure according to the present invention, and the molecular weight measurement curve of the polymer hyaluronic acid structure according to the present invention are almost identical with virtually no change. Therefore, the polymer hyaluronic acid structure according to the present invention can provide a polymer hyaluronic acid structure with reduced viscosity without decreasing molecular weight, even when shear stress is applied to the hyaluronic acid that is a precursor of the structure. 【0058】 The polymer hyaluronic acid structure according to the present invention maintains the characteristics of high molecular weight hyaluronic acid, such as its excellent water retention capacity and epithelial regeneration ability, while reducing viscosity to eliminate stickiness when applied to the eyeball, improving usability, and enhancing water retention. 【0059】 Viscosity of high molecular weight hyaluronic acid structures A 5.0 × 10 5 ~2.0×10 6 The viscosity of a solution containing a structure of high molecular weight hyaluronic acid having a molecular weight of g / mol and a concentration of 0.1-0.5% may be 1-60 cP. 【0060】 According to one embodiment of the present invention, when shear stress is applied to a solution containing polymer hyaluronic acid, which is a precursor of polymer hyaluronic acid structures, the physical structure of hyaluronic acid can be deformed from a twin helix structure to a single polymer chain, thereby reducing viscosity while maintaining the high molecular weight of hyaluronic acid. 【0061】 The reason why the viscosity of the solution containing the polymer hyaluronic acid structure according to the present invention is significantly reduced is that by applying shear stress to the solution containing hyaluronic acid, which is a precursor of the structure, a single polymer chain is formed, and an ion aggregate is formed within the single polymer chain, thereby reducing the volume of the polymer chain, and consequently reducing the volume of polymer hyaluronic acid in the solution. 【0062】 According to one embodiment of the present invention, when the molecular weight and concentration of the polymer hyaluronic acid structure and the hyaluronic acid precursor of the structure are the same, the ratio a / b, which is the viscosity of the solution containing the structure (a) and the viscosity of the solution containing the hyaluronic acid precursor of the structure (b), may be 0.001 to 0.30. 【0063】 Contact angle of high molecular weight hyaluronic acid structures The contact angle of a solution containing a polymer hyaluronic acid structure produced according to one embodiment of the present invention may be 20° to 30° on a hydrophilic glass surface, or 80° to 100° on a hydrophobic glass surface. 【0064】 The contact angle measurement method may include the sessile drop method for measuring static contact angle and the tilting drop method, captive drop method, wilhelmy plate method, etc. for measuring dynamic contact angle, and preferably the sessile drop method may be used. 【0065】 The hydrophilic glass surface may be a glass surface containing untreated silica material. 【0066】 The hydrophobic glass surface may be a glass surface containing a material coated with a Teflon emulsion. 【0067】 Intrinsic viscosity of high molecular weight hyaluronic acid structures Intrinsic viscosity refers to the volume per unit mass of a polymer when there is no viscous effect in the polymer solution. 【0068】 The aforementioned method for measuring intrinsic viscosity involves measuring the reduced viscosity of the aqueous solution to be measured according to its concentration, extrapolating this to obtain the reduced viscosity at a concentration of 0, thereby determining the intrinsic viscosity. The intrinsic viscosity can be calculated using the following formula 4. 【0069】 Formula 2 below is for calculating relative viscosity, and formula 3 below is for calculating reduced viscosity (where η is the viscosity of the solution, η0 is the viscosity of the solvent, and c is the concentration of the solution). Generally, the concentration of the solution (c) is expressed in units of (w / v), which is because η red This is because the physical meaning of this is the volume per unit weight of polymer in the solution. The intrinsic viscosity is obtained by plotting the reduced viscosity as a graph according to the concentration and extrapolating linearly to the point where the concentration becomes 0, as described above. 【0070】 【number】 (Here, η represents the viscosity of the solution, and η0 represents the viscosity of the solvent.) 【0071】 【number】 (Here, η represents the viscosity of the solution, η0 represents the viscosity of the solvent, and c represents the concentration of the solution.) 【0072】 【number】 (Here, η re (where c represents relative viscosity, and c represents the concentration of the solution.) 【0073】 The intrinsic viscosity of a solution containing the polymer hyaluronic acid structure according to the present invention can be calculated based on the above formulas 2 to 4. Specific intrinsic viscosity measurements of the solution containing the polymer hyaluronic acid structure according to the present invention are described in Experimental Example 8 below. 【0074】 Eye drop composition containing a high molecular weight hyaluronic acid structure According to one embodiment of the present invention, an eye drop composition containing the polymer hyaluronic acid structure can be provided. 【0075】 The aforementioned eye drop composition may contain the structural characteristics of the high molecular weight hyaluronic acid according to the present invention. 【0076】 The description of the structure of the aforementioned high molecular weight hyaluronic acid is the same as described above. Furthermore, the eye drop composition according to the present invention may contain one or more other common ingredients such as pharmaceutically acceptable buffers, preservatives, tonicity modifiers, pH modifiers, etc. The pH of the eye drop composition according to one embodiment of the present invention is preferably 5.5 as the lower limit, more preferably 6, and even more preferably greater than 6, and the upper limit is preferably 8, more preferably 7.5, and even more preferably 7. Within this pH range, the hyaluronic acid or its salt in the eye drop composition is stabilized, and the eye drop composition can be used in a low-irritation manner. 【0077】 An eye drop composition according to one embodiment of the present invention may further contain a buffer suitable for maintaining the preferred pH range described above. Examples of the buffer include, but are not limited to, bicarbonate buffer, acetate buffer, citrate buffer, phosphate buffer, borate buffer, or tromethamine (TRIS, 2-amino-2-hydroxymethyl-1,3-propanediol) buffer, and combinations thereof. The amount (concentration) of the buffer added is not particularly limited, as long as it can maintain the pH of the eye drop composition according to one embodiment of the present invention within the preferred range described above. 【0078】 An eye drop composition according to one embodiment of the present invention may further contain a preservative suitable for preventing microbial contamination. The preservative may include any compound or substance. Examples of the preservative can be selected from the group including: persalts such as perborates and percarbonates; alcohols such as benzyl alcohol and chlorobutanol; preservatives containing quaternary ammonium salts such as benzalkonium chloride, benzalkonium bromide and polyquaternium; guanidine preservatives such as polyhexamethylene biguanidine (PHMB) and chlorhexidine; mercury preservatives such as thimerosal, phenylmercuric acetate and phenylmercuric nitrate; metal chlorides such as alkali metal and alkaline earth metal chlorites; ophthalmologically acceptable salts such as sorbic acid and potassium sorbate and mixtures; and oxidative preservatives such as stabilized oxychloro complexes (e.g., Purite (a registered trademark of Allergan, Inc.)). The amount of preservative varies relatively widely depending on the specific preservative used. If no preservatives are added to the eye drop composition, the eye drop composition can be used as a single-use eye drop, and the eye drop composition is consumed in one dose. Otherwise, the eye drop composition can be used as a multi-use eye drop, for example, contained in a container equipped with a filter attached to the nozzle of the container for dispensing the eye drops, or contained in an airless dispensing system device. 【0079】 An eye drop composition according to one embodiment of the present invention may further contain a tonicity modifier that adjusts the osmotic pressure of the eye drop to be similar to the osmotic pressure inside the eyeball, thereby eliminating irritation and pain caused by the osmotic pressure difference during instillation. Examples of the tonicity modifier may be in ionic and / or nonionic forms. Ionic tonicity modifiers are, for example, one or more alkali metal or earth metal halides: calcium chloride, potassium chloride, sodium chloride, lithium chloride, potassium bromide, sodium bromide, sodium iodide, sodium phosphate, potassium phosphate, sodium sulfate and potassium, sodium bicarbonate and potassium, and boric acid. Nonionic tonicity modifiers are, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, dextrose, or combinations thereof. Glycerin, sodium chloride, and mannitol are the most preferred tonicity modifiers. The amount of tonicity modifier can be varied depending on whether an isotonic, hypertonic, or hypotonic liquid is required. The compositions of the present invention generally have an osmotic pressure in the range of 150 to 1500 mOsm / kg, preferably 150 to 500 mOsm / kg, and most preferably 180 to 250 mOsm / kg. 【0080】 The eye drop composition according to one embodiment of the present invention may further contain a pH adjusting agent to adjust the pH to the preferred range described above. The pH adjusting agent is not particularly limited as long as it can adjust the pH of the eye drop composition according to one embodiment of the present invention, but specific examples include dilute hydrochloric acid and sodium hydroxide. The amount (concentration) of the pH adjusting agent added is not particularly limited as long as it can adjust the pH of the eye drop composition according to one embodiment of the present invention to the preferred range described above. 【0081】 An eye drop composition according to one embodiment of the present invention can be prepared by dissolving the components in an aqueous medium. Deionized water is a preferred aqueous medium that can contain small amounts of other hydrophilic solvents, such as glycols and / or polyols. The composition can be prepared by preparing a solution of one or more components and then adding the remaining one or more components, or by preparing two or more separate solutions, each containing one or more components, and then mixing all of these solutions together. 【0082】 An eye drop composition according to one embodiment of the present invention may be an eye drop composition for the prevention or treatment of dry eye. 【0083】 The present invention provides a kit comprising a container containing the eye drop composition. The container containing the eye drop composition includes a dispensing means suitable for topically administering the eye drop composition to a patient's eyeball. The dispensing means can dispense the eye drop composition in a dropwise manner in droplets of volume from 0.01 to 0.10 ml, but is not limited to such volumes and can typically include the volume per droplet applied to the eyeball. [Examples] 【0084】 Hereinafter, embodiments of the present invention will be described in detail so that they can be easily implemented by a person with ordinary skill in the art to which the present invention pertains. However, the present invention can be embodied in a variety of different forms and is not limited to the embodiments described herein. 【0085】 Manufacturing of high molecular weight hyaluronic acid structures [Example 1] Molecular weight is 9.0 × 10 5 0.5 g of g / mol sodium hyaluronate (a product of Shiseido Nippon Co., Ltd.) was placed in a graduated cylinder, and distilled water was added to bring the total volume to 100 ml. The solution was stirred at 25°C until homogeneous to prepare a 0.5% (w / v) sodium hyaluronate aqueous solution. 【0086】 The prepared sodium hyaluronate aqueous solution was placed in the inlet of a high-pressure disperser (ILSHIN AUTOCLAVE's NLM 100) and dispersed under high pressure. After stabilizing at room temperature for 1 hour, an aqueous solution containing a high-molecular-weight hyaluronic acid structure was produced. 【0087】 [Example 2] An aqueous solution containing a polymer hyaluronic acid structure was produced in the same manner as in Example 1, except that the aqueous solution of sodium hyaluronate produced in Example 1 was subjected to high-pressure dispersion two and three times using a high-pressure disperser. 【0088】 [Example 3] Molecular weight is 1.2 × 10 6 An aqueous solution containing a polymer hyaluronic acid structure was prepared in the same manner as in Example 1, except that 0.5 g of sodium hyaluronate (a product of Shiseido Nippon Co., Ltd.) was used in a concentration of g / mol. 【0089】 [Example 4] An aqueous solution containing a polymer hyaluronic acid structure was produced in the same manner as in Example 2, except that the aqueous solution of sodium hyaluronate produced in Example 3 was subjected to high-pressure dispersion two, three, and four times using a high-pressure disperser. 【0090】 [Example 5] Molecular weight is 9.0 × 10 5 0.3g of g / mol sodium hyaluronate (a product of Shiseido Nippon Co., Ltd.) was placed in a graduated cylinder, and distilled water was added to bring the total volume to 100ml. The solution was stirred at 25°C until homogeneous to prepare a 0.3% (w / v) sodium hyaluronate aqueous solution. 【0091】 The aforementioned sodium hyaluronate aqueous solution was subjected to ultrasonic stimulation for approximately 20 minutes at 25°C using an ultrasonic homogenizer (BANDELIN HD4200, Germany) to produce an aqueous solution containing a polymer hyaluronic acid structure. The total energy of the applied ultrasonic waves varied with time, with approximately 1.5 kJ to 2.5 kJ of energy being added per minute. 【0092】 [Comparative Example 1] Molecular weight is 9.0 × 10 5 0.5 g of g / mol sodium hyaluronate (a product of Shiseido Nippon Co., Ltd.) was placed in a graduated cylinder, and distilled water was added to bring the total volume to 100 ml. The solution was stirred at 25°C until homogeneous to prepare a 0.5% (w / v) sodium hyaluronate aqueous solution. 【0093】 [Comparative Example 2] Molecular weight is 1.2 × 10 6 0.5 g of g / mol sodium hyaluronate (a product of Shiseido Nippon Co., Ltd.) was placed in a graduated cylinder, and distilled water was added to bring the total volume to 100 ml. The solution was stirred at 25°C until homogeneous to prepare a 0.5% (w / v) sodium hyaluronate aqueous solution. 【0094】 Experimental Example 1: Viscosity Comparison of Examples 1-4 and Comparative Examples 1 and 2 The viscosity of the aqueous solutions of Examples 1-4 and Comparative Examples 1 and 2 was measured at 25°C using a Brookfield DV2T viscometer. The measured viscosities are shown in Tables 1 and 2 below. Examples 2-1 and 2-2 show the aqueous solutions produced by repeating high-pressure dispersion using a high-pressure disperser two and three times in Example 2, respectively. Examples 4-1, 4-2, and 4-3 show the aqueous solutions produced by repeating high-pressure dispersion using a high-pressure disperser two, three, and four times in Example 4, respectively. 【0095】 [Table 1] 【0096】 [Table 2] 【0097】 The results in Table 1 above confirm that the viscosity of the aqueous solutions containing the polymer hyaluronic acid structures produced in Examples 1 and 2 is significantly lower than the viscosity of the sodium hyaluronate salt solution, which is a precursor of the structures produced in Comparative Example 1. Furthermore, it was confirmed that the viscosity decreased to approximately 10 cP with each repeated high-pressure dispersion. 【0098】 The results in Table 2 above confirm that the viscosity of the aqueous solutions containing the polymer hyaluronic acid structures produced in Examples 3 and 4 is significantly lower than the viscosity of the sodium hyaluronate salt solution, which is a precursor of the structures produced in Comparative Example 2. Furthermore, it was confirmed that the viscosity decreased to approximately 10 cP with each repeated high-pressure dispersion. 【0099】 This revealed that when a specific shear stress is applied to the precursor sodium hyaluronate salt, the twin helix structure of hyaluronic acid is converted to a single helix structure, and a polymer hyaluronic acid structure is produced in which an ionic aggregate is formed within the single polymer chain. The reason why the viscosity of the solution containing the polymer hyaluronic acid structure according to the present invention decreased significantly is that the volume of the polymer chain can be reduced by the formation of an ionic aggregate within the single polymer chain, thereby reducing the volume of polymer hyaluronic acid in the solution. 【0100】 Therefore, even if the solution contains high molecular weight hyaluronic acid, it is possible to produce a solution with reduced viscosity without a change in molecular weight. 【0101】 Experimental Example 2: Change in viscosity due to ultrasonic application time The viscosity of the aqueous solution containing the polymer hyaluronic acid structure produced in Example 5 was measured. The change in viscosity of the aqueous solution with respect to the ultrasonic application time is shown in Table 3 below. 【0102】 [Table 3] 【0103】 In the results shown in Table 3, it was confirmed that when ultrasound was applied to the aqueous solution containing the precursor high molecular weight hyaluronic acid in Example 5, the viscosity decreased as the duration of ultrasound application increased. Furthermore, it was confirmed that the viscosity decreased rapidly within the initial time of 1-2 seconds after ultrasound application began, and that the viscosity converged to approximately 3 cP after 10 minutes. 【0104】 Experimental Example 3: Measurement of molecular weight changes before and after ultrasonic application Molecular weight is 1.2 × 10 6 0.02 g of g / mol sodium hyaluronate salt was placed in a graduated cylinder, and 0.9% (w / v) NaCl was added to dissolve it until the total volume was 20 mL, thereby preparing an aqueous solution of sodium hyaluronate salt. 0.8 mL of this aqueous solution was taken, 0.1 M Na2SO4 was added, and a solution was prepared until the total volume was 10 mL. Subsequently, distilled water was added to the solution to dilute it 2-fold, and a sample for high-performance liquid chromatography (HPLC) analysis was prepared. 【0105】 A solution prepared in the same manner as in Example 5 was placed in an ultrasonic generator, and ultrasound was applied to produce an aqueous solution containing a polymer hyaluronic acid structure. A sample for gel permeation chromatography (GPC) measurement was prepared using the prepared aqueous solution. 【0106】 For concentration and molecular weight analysis of the HPLC and GPC samples, an HPLC (e2695, waters) and a GPC column (OHPak SB-802.5 HQ 8.0×300mm, 6um, Shodex) were used. A 0.1M sodium sulfate (Na2SO4) aqueous solution, prepared by dissolving 14.2g of anhydrous sodium sulfate (DAEJUNG) in 1L of purified water, was used as the mobile phase. The column temperature was maintained at 40°C and the oven temperature at 25°C. 100% 0.1M sodium sulfate (Na2SO4) was added, and the samples were stabilized in the oven at a flow rate of 1.0 mL / min for 1 hour. After filtering the samples through a syringe filter (PVDF, 0.2 μm, Φ 13 mm), 20 μL was injected into the GPC column and analyzed at a UV wavelength of 210 nm for 16 minutes. 【0107】 In Figure 6, (a) shows the molecular weight of sodium hyaluronate salt, which is a precursor of the structure according to the present invention, and (b) shows the molecular weight of the polymer hyaluronic acid structure according to the present invention. 【0108】 We confirmed that the molecular weight of hyaluronic acid, which is a precursor of the structure according to the present invention, and the molecular weight of the high molecular weight hyaluronic acid structure according to the present invention are almost identical with virtually no change. 【0109】 Experimental Example 4: Preparation of Viscosity-Adjusted Solutions In Example 5, an aqueous solution (a) containing a polymer hyaluronic acid structure was prepared by applying ultrasound to an aqueous solution of sodium hyaluronate salt, a precursor, at 25°C for 16 minutes using an ultrasonic homogenizer. An aqueous solution (b) of sodium hyaluronate salt without ultrasound application was also prepared and mixed with aqueous solution (a) to produce a solution. 【0110】 The viscosities of the solutions obtained by mixing aqueous solution (a) and aqueous solution (b) were compared and are shown in Table 4 below. 【0111】 [Table 4] 【0112】 The results in Table 4 above show that when an aqueous solution (a) containing the polymer hyaluronic acid structure according to the present invention is mixed with an aqueous solution (b) that has not been subjected to ultrasonic treatment at a concentration of approximately 10%, a solution with a viscosity of 3 to 6 cP can be produced. 【0113】 Experimental Example 5: Preparation of Viscosity-Adjusted Eye Drop Compositions Molecular weight is 1.0 × 10 6 0.3g of g / mol sodium hyaluronate (a product of Shiseido Nippon Co., Ltd.) was placed in a graduated cylinder, and distilled water was added to bring the total volume to 100ml. The solution was stirred at 25°C until homogeneous to prepare a 0.3% (w / v) sodium hyaluronate aqueous solution. 【0114】 The 0.3(w / v)% sodium hyaluronate aqueous solution produced was treated in the same manner as in Examples 1 to 5 to produce an aqueous solution containing a polymer hyaluronic acid structure. 【0115】 Additives were added to the aqueous solution containing the polymer hyaluronic acid structure produced as described above, and eye drop compositions containing the aqueous solution containing the polymer hyaluronic acid structure were prepared as shown in Table 5 below. The viscosity and osmotic pressure of the prepared eye drop compositions were measured and are shown in Table 5 below. 【0116】 [Table 5] 【0117】 In Table 5 above, one or more of the following were used as excipients: phosphate-buffered saline (PBS), sodium chloride (NaCl), and additives. The additives, as a mixture, contained 0.01% ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), 0.19% aminocaproic acid, 0.69% sodium chloride (NaCl), 0.15% potassium chloride (KCl), and distilled water. 【0118】 The results in Table 5 above confirm that by adding various excipients to an aqueous solution containing the polymer hyaluronic acid structure according to the present invention, an eye drop composition having a viscosity in the range of 1 to 30 cP can be provided. 【0119】 Experimental Example 6: Evaluation of the water retention capacity of aqueous solutions of Examples 1, 3, 5 and Comparative Example 1 The water retention capacity of aqueous solutions from Examples 1, 3, and 5 and Comparative Example 1 was measured and evaluated. One g of each sample was taken, placed in a 10 cc vial, and left at 25°C for one hour. The change in weight was measured, and the results are shown in Table 6 below. 【0120】 [Table 6] 【0121】 The results in Table 6 above confirm that the aqueous solution of sodium hyaluronate treated with a high-pressure disperser or ultrasonic homogenizer showed lower viscosity and less weight change compared to an aqueous solution of pure sodium hyaluronate (Comparative Example 1). This indicates that the aqueous solution containing the hyaluronic acid structure according to the present invention has low viscosity and excellent water retention capacity. This result suggests that it is suitable for use in artificial tears and other applications where water retention capacity is required. 【0122】 Experimental Example 7: Measurement of Contact Angle of Aqueous Solutions of Examples 1, 3, 5 and Comparative Example 1 The contact angles were measured for the aqueous solutions prepared in Examples 1, 3, and 5 and Comparative Example 1. The contact angles were measured on both hydrophilic and hydrophobic glass surfaces. The measured contact angles are shown in Table 7 below. 【0123】 [Table 7] 【0124】 As shown in the results in Table 7 above, the contact angles of the aqueous solutions produced in Examples 1, 3, and 5 were larger on hydrophilic glass surfaces than in Comparative Example 1, and the contact angles of the aqueous solutions produced in Examples 1, 3, and 5 were smaller on hydrophobic glass surfaces than in Comparative Example 1. Therefore, it was found that the aqueous solutions produced in Examples 1, 3, and 5 are relatively lipophilic. 【0125】 Experiment Example 8: Intrinsic Viscosity Experiment The method for measuring intrinsic viscosity involves measuring the reduced viscosity of the aqueous solution to be measured according to its concentration, and then extrapolating this to obtain the reduced viscosity at a concentration of 0. This is called the intrinsic viscosity. As mentioned above, intrinsic viscosity means the volume per unit mass of polymer when there is no interaction between polymer chains in the solvent. 【0126】 The prepared 0.5(w / v)% sodium hyaluronate aqueous solution is diluted with distilled water to concentrations of 0.3(w / v)%, 0.1(w / v)%, 0.05(w / v)%, 0.01(w / v)%, and 0.005(w / v)%, respectively. The viscosity at 25°C is measured using a Brookfield DV2T viscometer, and this is calculated as the reduced viscosity. The intrinsic viscosity is then extrapolated. In the case of sodium hyaluronate with a molecular weight of 1.2 million, the value is 2.3m. 3 In the case of sodium hyaluronate salt having an intrinsic viscosity of / kg and a molecular weight of 900,000, the viscosity is 1.8m 3 It has an intrinsic viscosity of / kg. 【0127】 The aqueous solution containing the hyaluronic acid structure having a molecular weight of 900,000 produced in Example 5 contains approximately 0.8 ml 3 It was calculated to have an intrinsic viscosity of / kg.

Claims

[Claim 1] It is a structure derived from high molecular weight hyaluronic acid, The aforementioned structure has a single polymer chain, The single polymer chain forms a spherical aggregate with a diameter of 1 to 20 nm. The molecular weight of the aforementioned structure is 5.0 × 10 5 ~2.0×10 6 It is in the range of g / mol. When producing an aqueous solution containing the aforementioned structure, the viscosity of the aqueous solution is 1 to 60 cP. A structure of high molecular weight hyaluronic acid, characterized in that the viscosity of the solution is measured at 25 degrees Celsius using a Brookfield DV2T viscometer, and the solvent of the solution is distilled water. [Claim 2] The polymer hyaluronic acid structure according to claim 1, characterized in that when an aqueous solution containing the structure is produced, the aqueous solution contains a hyaluronic acid content of 0.01% (w / v) to 0.5% (w / v). [Claim 3] The polymer hyaluronic acid structure according to Claim 1, characterized in that when an aqueous solution containing the structure is produced, the ratio (a / b) of the viscosity of the aqueous solution containing the structure to the viscosity (b) of the aqueous solution containing hyaluronic acid (a precursor of the structure) having the same molecular weight and concentration as the structure is 0.001 to 0.

30. [Claim 4] The intrinsic viscosity of the solution containing the aforementioned structure is 0.1 to 2.5 m 3 The structure of high molecular weight hyaluronic acid according to claim 1, characterized in that it is / kg. [Claim 5] An eye drop composition comprising a polymer hyaluronic acid structure according to claim 1. [Claim 6] The eye drop composition according to claim 5, characterized in that the viscosity of the eye drop composition is 1.0 to 30.0 cP. [Claim 7] The eye drop composition according to claim 5, further comprising an excipient. [Claim 8] The eye drop composition according to claim 7, characterized in that the excipient is one or more selected from the group consisting of buffers, preservatives, tonicity modifiers, and pH modifiers. [Claim 9] The eye drop composition according to any one of claims 5 to 8, wherein the eye drop composition is for the prevention or treatment of dry eye. [Claim 10] A kit comprising a container containing an eye drop composition according to any one of claims 5 to 9, The container is a kit that includes a dispensing means suitable for topical administration of an eye drop composition. [Claim 11] The kit according to claim 10, wherein the dispensing means provides the eye drop composition in a dropwise manner, with a volume of 0.01 to 0.10 ml.

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