Hyaluronic acid derivatives or salts thereof, their production method and use

Abstract

The present invention provides a hyaluronic acid derivative or a salt thereof, a method for producing the same and a method for using the same, and a method for producing a matrix metalloproteinase comprising the steps of: The hyaluronic acid is modified with a methoxypolyethylene glycol epoxy derivative as the backbone. The polyethylene glycol derivative of hyaluronic acid is produced. By wrapping the hyaluronic acid skeleton using a linear polymer, it is resistant to enzymatic degradation. Masks glycosidic bonds in the pancreas, prolonging the circulation time of sodium hyaluronate in the body, It exerts a water-retaining effect. In addition, polyethylene glycol and sodium hyaluronate, As a synthetic and natural polymer material, it has excellent biocompatibility and biodegradability. The advantage of combining these materials is that it is possible to make better use of the properties of polymer materials.

Description

[Technical Field]

[0001] The present invention relates to the technical field of polymers, in particular to hyaluronic acid derivatives or salts thereof, and methods for producing the same. Regarding law and use. [Background technology]

[0002] Hyaluronic acid (HA) is found in the synovial fluid of animals and humans, in the eye, A naturally occurring organism found widely in connective tissues such as the vitreous body of the globule, skin, intercellular spaces, and cockscomb. It is a polymeric material and an important component of synovial fluid, vitreous body, skin and cartilage tissue. It has exceptional physical and chemical properties and a wide range of biological functions. It has biocompatibility, unique viscoelasticity, and excellent moisturizing and water-retaining properties, making it widely used in the beauty industry. Loved by many beauty enthusiasts, it is now used to reduce wrinkles, improve the appearance of skin and prevent skin aging. It can be injected into the skin of the face to relieve pain.

[0003] During production and use, hyaluronic acid exists mostly in the form of sodium hyaluronate. Natural sodium hyaluronate is easily biodegradable, has a short retention time in the body, and has low hardness. Its applications are limited due to its insufficient mechanical strength and viscosity. It is common to use a cross-linking agent to gel the sodium hyaluronate. A cross-linked modified gel that maintains biocompatibility and significantly improves resistance to enzymatic degradation has been developed. However, it is impossible to completely remove the cross-linking agent residues using conventional techniques. Most cross-linking agents are chemical reagents, and excessive cross-linking agents can lead to a decrease in biocompatibility. When injected into the human body, it is likely to trigger an immune response, irritate the skin, and even cause cell death. May cause toxicity.

[0004] In the study of non-crosslinked sodium hyaluronate, see Adrian Ranga, et al. Hyaluronic acid id hydrogels formed in situ by transglutaminase-catalyzed reaction[J].Biomacromo lecules, and Kazuteru Moriyama, etc.Hyaluronic acid grafted with poly(ethylene gl ycol) as a novel peptide formulation. Journal of Controlled Release To bind polyethylene glycol by reacting the carboxyl of the sodium salt molecule has been reported to be used.

[0005] LiteratureIrina M.Le-Deygen,etc.Poly(Ethylene Glycol)Interacts with Hyaluronan in Aque ous Media. Biomacromolecules contain hydroxyl and phosphate groups in the sodium hyaluronate molecule. It is possible to bond polyethylene glycol by utilizing hydrogen bonds between polyethylene glycols. It was reported.

[0006] In the prior art, the amino, carboxy and hydroxy groups of the sodium hyaluronate molecule are Reacting with polyethylene glycol to create polyethylene glycol hyaluronic acid with different properties However, currently, the hyaluronic acid sodium molecule is The epoxy was reacted with methoxypolyethylene glycol epoxy derivative to give hyaluronic acid nanoparticles. By modifying the thorium molecule with an ether bond, polyethylene glycol-coated hyaluronan can be obtained. There is no method to produce sodium phosphate. Summary of the Invention

[0007] The present invention overcomes the drawbacks of the prior art, and in a first aspect of the invention, provides a compound having a structure represented by formula (I): The present invention provides a hyaluronic acid derivative or a salt thereof having the following structure:

[0008] TIFF0007799154000001.tif8271 (where X is -(CR1R2) m -, -(CH2) m NH-, -NHCO(CH2) m -, -(CH2) m CONH- and -CO(CH2) m -One or more selected from A combination of several types, m is an integer from 1 to 10, R1 and R2 are independently H, alkyl, cycloalkyl, cycloalkylalkyl, Alkenyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, -CO R3, -C(O)OR3, -C(O)NR3R4, -CH=NR3, -CN, -OR3, -OC(O)R3, -S(O) n -R3, -NR3R4, -NR3C(O)R4, and Selected from the n is selected from 0, 1, and 2; R3 and R4 are each independently hydrogen, alkyl, cycloalkyl, alkenyl, or aryl. selected from aryl, heterocyclyl, and halogen; k is the degree of polymerization of the polyethylene glycol residue, and k is an integer of 40 to 4600 (for example, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 55, 60, 65 ,70,75,80,85,90,95,100,105,110,111,112,1 13, 114, 115, 116, 117, 118, 119, 120, 150, 200, 4 00, 500, 800, 1000, 2000, 3000, 4000, 4500), preferred Preferably, an integer of 45 to 4545, more preferably an integer of 75 to 4545, particularly preferably , an integer between 100 and 4545, t is the degree of polymerization of the hyaluronic acid residue, and t is an integer between 100 and 8000 (for example, 10 0, 105, 110, 115, 120, 125, 130, 135, 140, 145, 15 0, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 50 00, 5500, 6000, 5500, 7000, 7100, 7200, 7300, 74 00), preferably an integer of 120 to 7500, more preferably an integer of 700 to 6000. a number, particularly preferably an integer of 1000 to 4000; Preferably, X is -(CR1R2) m - and Preferably, m is an integer of 1 to 5, for example, 1, 2, 3, 4, or 5.

[0009] In one embodiment of the present invention, said m=1.

[0010] Preferably, R1 and R2 are independently H, C1-C6 alkyl, C3-C6 alkyl, is selected from cycloalkyl, -OR3, -NR3R4, and halogen.

[0011] Preferably, R3 and R4 are independently selected from hydrogen and C1-C3 alkyl. will be done.

[0012] In one embodiment of the present invention, R1 and R2 are both H.

[0013] In one preferred embodiment of the present invention, X is —CH 2 —.

[0014] The salts include sodium salts, potassium salts, calcium salts, magnesium salts, zinc salts, cobalt salts, and tetrabutylammonium salts, and preferably one or more selected from the group consisting of tetrabutylammonium salts. is the sodium salt.

[0015] In one embodiment of the present invention, the hyaluronic acid derivative has the following structure: TIFF0007799154000002.tif8073

[0016] In a second aspect of the present invention, A step of weighing hyaluronic acid, mixing hyaluronic acid with an alkaline solvent, and stirring the mixture ( 1) and Add the methoxypolyethylene glycol epoxy derivative to the solution obtained in step (1). and (2) reacting the The methoxypolyethylene glycol epoxy derivative has the structure of formula (III): The present invention provides a method for producing the hyaluronic acid derivative of formula (II) or a salt thereof. TIFF0007799154000003.tif1555

[0017] Preferably, the alkaline solvent in step (1) is Sodium hydroxide , Potassium hydroxide M , sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate It is a species or species.

[0018] In one preferred embodiment of the present invention, the alkaline solvent has a mass concentration of 0.25% to 0.8% Sodium hydroxide solution (e.g., 0.25%, 0.35%, 0.5%, 0.8 %), preferably a sodium hydride solution with a mass concentration of 0.35% to 0.5%.

[0019] The molecular weight of the sodium hyaluronate is 40,000 to 3,200,000 daltons, preferably 50,000 to 6,000 daltons. 3 million daltons, more preferably 280,000 to 2.4 million daltons, particularly preferably 40 It ranges from 10,000 to 1.6 million Daltons.

[0020] The mass ratio of the hyaluronic acid to the alkaline solvent is 1:5 to 30 (specifically, for example, 1 :5, 1:10, 1:15, 1:20, 1:25 or 1:30), preferably 1:10 is.

[0021] The molar ratio of the polymer units in the hyaluronic acid and methoxypolyethylene glycol epoxy derivatives is The ratio of the molten metal to the molten metal is 0.1 to 10:1 (specifically, for example, 0.1:1, 0.2:1, 0.3:1, , 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1 , 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 , 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1 , 9.5:1, 10:1).

[0022] In step (1), the stirring time is 5 to 50 minutes, preferably 10 to 30 minutes. n (e.g., 15min, 16min, 17min, 18min, 19min, 20min n), and more preferably 10 to 20 minutes.

[0023] In step (2), the polyethylene glycol epoxy derivative is a monomethoxypoly ethylene glycol ethylene oxide, and preferably the monomethoxy polyethylene The molecular weight of the glycol ethylene oxide is 2000 to 200,000, for example, 2000 , 3000, 4000, 5000, 10000, 20000, 30000, 50000, It is 100,000.

[0024] In step (2), the reaction includes stirring and then allowing to stand.

[0025] Preferably, in step (2), the reaction time is 10 to 24 hours, preferably 13 to 24 hours. 21 hours, more preferably 17 to 19 hours.

[0026] Preferably, the method for producing a hyaluronic acid derivative or a salt thereof includes a step of purifying the product. and purifying the product by adding an acidic reagent to the reaction mixture in step (2). Adjusting the resulting solution to neutrality and / or adding a precipitant to precipitate the product. and / or further comprising adding a washing agent to wash the precipitate.

[0027] The acidic reagent is selected from a dilute hydrochloric acid solution, a dilute sulfuric acid solution, a dilute acetic acid solution, and a dilute hypochlorous acid solution. It is one or more types.

[0028] In one embodiment of the invention, the acidic reagent is a dilute hydrochloric acid solution.

[0029] The precipitating agent is acetonitrile or ethanol.

[0030] In one embodiment of the invention, the precipitating agent is acetonitrile.

[0031] The cleaning agent is ethanol or water.

[0032] In one embodiment of the invention, the cleaning agent is ethanol.

[0033] In one embodiment of the present invention, the method for producing the hyaluronic acid derivative or a salt thereof comprises: Weigh out the acid, dissolve it in the alkaline solvent, and stir for 10 to 30 minutes until it becomes homogeneous. Add the methoxypolyethylene glycol epoxy derivative and mix the ingredients until they are uniformly mixed. Stir for 10 to 24 hours until the mixture is dissolved, then leave it at room temperature for 15 to 18 hours, and then add an acidic reagent to neutralize the pH of the system. Then, a precipitant is added to cause precipitation, and the resulting particles are stirred vigorously to make them uniform. Centrifuge and let stand, remove the supernatant, and continue precipitation with a precipitant for 2 to 5 times. The precipitate was washed with detergent 1 to 3 times, and finally the obtained precipitate product was placed in a vacuum drying oven. Put it in and dry.

[0034] In a third aspect of the present invention, the use of a compound of formula (I) or (II) in the manufacture of pharmaceutical, medical cosmetology and cosmetic products is provided. (II) Use of the hyaluronic acid derivative or a salt thereof.

[0035] Preferably, the use is the use of the compound of formula (I) or (II) in the manufacture of a medical cosmetic product. The use of a benzophenone derivative or a salt thereof.

[0036] Preferably, the medical cosmetic product is a soft tissue filler.

[0037] The present invention relates to a method for treating soft tissue comprising administering to a subject the hyaluronic acid derivative of formula (I) or (II) of the present invention or a salt thereof. A woven filler is further provided.

[0038] In the present invention, a polyethylene glycol derivative and hyaluronic acid or a salt thereof are bonded by an ether bond. The reaction product is not a cross-linked gel but a composite polymer material that is soluble in aqueous solution. The produced hyaluronic acid derivative or its salt maintains the water-retaining and moisturizing properties of hyaluronic acid. It not only improves the skin's resistance to hyaluronidase degradation in vivo, but also reduces fine lines on the face and neck. When used in restorations, it reduces the frequency of injections, improves patient comfort, and reduces costs. do. [Brief explanation of the drawings]

[0039] [Figure 1] 1 shows the relationship between the molecular weight of the control sample and time after enzymatic decomposition. [Figure 2] 1 shows the relationship between the molecular weight of enzymatic decomposition and time for Sample 8. DETAILED DESCRIPTION OF THE INVENTION

[0040] Unless otherwise defined, all scientific and technical terms used in this invention are has the same meaning as commonly understood by a person skilled in the relevant technical field. "Alkyl" refers to a straight or branched hydrocarbon chain radical that does not contain unsaturated bonds, and In the specification, C1-C6 alkyl includes methyl, ethyl, n-propyl, isopropyl, 1-6 such as n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, etc. alkyl containing 1 to 3 carbon atoms, preferably C1 to C3 alkyl (methyl, ethyl, n "Cycloalkyl" refers to alicyclic hydrocarbons. Representative cycloalkyls contain 1 to 4 single and / or fused rings, and 3 to about 18 rings. In the present invention, C3-C6 cycloalkyl includes cyclopropyl, Refers to cycloalkyl containing 3 to 6 carbon atoms, such as cyclopentyl and cyclohexyl. .

[0041] The following clearly and completely describes the technical means in the embodiments of the present invention. It is clear that the examples are only some of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments, all other embodiments that a person skilled in the art can obtain without making creative efforts. are within the protection scope of the present invention. Example 1

[0042] Weigh out 380 mg of HA (600,000) and use a 0.25% Sodium hydroxide solution Dissolve in 3.8 ml Stir vigorously for 10 to 30 minutes until homogenous, and then mix with M-PEG2000-EPOX( TIFF0007799154000004.tif1334, from Beijing Keykai Technology Co., Ltd. (same below) Add 1g and mix the materials evenly The mixture was stirred vigorously until the mixture was dissolved, and then left at room temperature for 18 hours. The next day, 1 ml of 0.25 N HCl was added. Adjust the pH of the system to about 7, then add 20 ml of acetonitrile to precipitate, and stir vigorously. The product was stirred to make the particles uniform, centrifuged, allowed to settle, and the supernatant was removed. The precipitate was washed four times with 20 ml of ethanol, and then washed twice with 20 ml of absolute ethanol. The product was dried in a vacuum oven to obtain Sample 1 (0.25% NaOH). Multi-angle optical differential detection and nuclear magnetic detection were performed using sampling. Example 2

[0043] Weigh out 570 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 5.7 ml Stir vigorously for 10-30 minutes until homogenous, then add M-PEG2000-EPOX 1 0.5g was added, and the mixture was stirred vigorously until the ingredients were uniformly mixed, and then left at room temperature for 18 hours. The next day, add 1.5 ml of 0.35N HCl to adjust the pH of the system to about 7. Add 30 ml of acetonitrile to precipitate, stir vigorously to homogenize the product particles, and then centrifuge. The mixture was separated and allowed to stand, and the supernatant was removed. The mixture was precipitated four times with 30 ml of acetonitrile. The product was further washed twice with 30 ml of absolute ethanol and then dried in a vacuum oven. Sample 2 (0.35% NaOH) was obtained. Nuclear magnetic detection was performed. Example 3

[0044] Weigh out 380 mg of HA (600,000) and add 0.5% Sodium hydroxide solution Dissolve in 3.8 ml Stir vigorously for 10-30 minutes until homogenous, then add 1g of M-PEG2000-EPOX. The mixture was then stirred vigorously until the ingredients were uniformly mixed, and the mixture was left to stand at room temperature for 18 hours. 1 ml of 0.5N HCl was added to adjust the pH of the system to about 7, and then acetonitrile was added. Add 20 ml of the solution to precipitate, stir vigorously to homogenize the product particles, then centrifuge and let it settle. The supernatant was removed. The precipitation was continued four times with 20 ml of acetonitrile, and then with anhydrous ethanol. The product was washed twice with 20 ml of ethanol, and then dried in a vacuum oven. (0.5% NaOH) was obtained. Sampling was performed using multi-angle optical differential detection and nuclear magnetic detection. It was. Example 4

[0045] Weigh out 380 mg of HA (600,000) and use a 0.8% Sodium hydroxide solution Dissolve in 3.8 ml Stir vigorously for 10-30 minutes until homogenous, then add 1g of M-PEG2000-EPOX. The mixture was then stirred vigorously until the ingredients were uniformly mixed, and the mixture was left to stand at room temperature for 18 hours. 1 ml of 0.8N HCl was added to adjust the pH of the system to about 7, and then acetonitrile was added. Add 20 ml of the solution to precipitate, stir vigorously to homogenize the product particles, then centrifuge and let it settle. The supernatant was removed. The precipitation was continued four times with 20 ml of acetonitrile, and then with anhydrous ethanol. The product was washed twice with 20 ml of ethanol and dried in a vacuum oven. Sample 4 (0.8% NaOH) was obtained. Sampling was performed using multi-angle optical differential detection and nuclear magnetic detection. It was. Example 5

[0046] Weigh out 190 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 5.7 ml Stir vigorously for 10-30 minutes until homogenous, then add M-PEG2000-EPOX 5 00 mg was added, and the mixture was stirred vigorously until the materials were uniformly mixed, and then left to stand at room temperature for 18 hours. The next day, 1 ml of 0.35N HCl was added to adjust the pH of the system to about 7, and then acetone was added. Add 10 ml of nitrile to precipitate, stir vigorously to homogenize the product particles, and then centrifuge. The mixture was left to stand, and the supernatant was removed. The mixture was precipitated four times with 10 ml of acetonitrile. The product was then washed twice with 10 ml of absolute ethanol and dried in a vacuum oven. Sample 5 (0.35% NaOH) was obtained. After sampling, multi-angle optical differential detection and nuclear analysis were performed. Magnetic detection was performed. Example 6

[0047] Weigh out 760 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 7.6 ml Stir vigorously for 10 to 30 minutes until homogenous, then add M-PEG2000-EPOX 2 g was added, and the mixture was stirred vigorously until the materials were uniformly mixed, and then the mixture was left to stand at room temperature for 15 hours. On the day of the test, 2 ml of 0.35 N HCl was added to adjust the pH of the system to about 7, and then acetonitrile was added. Add 40 ml of ethanol to precipitate the product, stir vigorously to homogenize the product particles, and then centrifuge to separate the product. The supernatant was removed. The precipitation was continued four times with 40 ml of acetonitrile. Wash twice with 40 ml of aqueous ethanol, then place the product in a vacuum oven to dry, and then sample Sample 6 (0.35% NaOH) was obtained. It was sampled and subjected to multi-angle optical differential detection and nuclear magnetic detection. I went out. Example 7

[0048] Weigh out 760 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 7.6 ml Stir vigorously for 10 to 30 minutes until homogenous, then add M-PEG2000-EPOX 2 g was added, and the mixture was stirred vigorously until the materials were uniformly mixed, and then the mixture was left to stand at room temperature for 21 hours. On the day of the test, 2 ml of 0.35 N HCl was added to adjust the pH of the system to about 7, and then acetonitrile was added. Add 40 ml of ethanol to precipitate the product, stir vigorously to homogenize the product particles, and then centrifuge to separate the product. The supernatant was removed. The precipitation was continued four times with 40 ml of acetonitrile. Wash twice with 40 ml of aqueous ethanol, then place the product in a vacuum oven to dry, and then sample Sample 6 (0.35% NaOH) was obtained. It was sampled and subjected to multi-angle optical differential detection and nuclear magnetic detection. I went out. Example 8

[0049] Weigh out 570 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 5.7 ml Stir vigorously for 10 to 30 minutes until homogenous, then add M-PEG5000-EPOX 2 Add 750 mg, stir vigorously until the ingredients are uniformly mixed, and leave at room temperature for 18 hours. The next day, 2 ml of 0.35 N HCl was added to adjust the pH of the system to about 7. Add 30 ml of acetonitrile to precipitate, stir vigorously to homogenize the product particles, and then centrifuge. The mixture was separated and allowed to stand, and the supernatant was removed. The mixture was precipitated four times with 30 ml of acetonitrile. The product was further washed twice with 30 ml of absolute ethanol and then dried in a vacuum oven. Sample 8 (0.35% NaOH) was obtained. Nuclear magnetic detection was performed. Example 9

[0050] Weigh out 570 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 5.7 ml Stir vigorously for 10-30 minutes until homogenous, then add M-PEG5000-EPOX 1 Add 875 mg, stir vigorously until the ingredients are uniformly mixed, and leave at room temperature for 18 hours. The next day, 2 ml of 0.35 N HCl was added to adjust the pH of the system to about 7. Add 30 ml of acetonitrile to precipitate, stir vigorously to homogenize the product particles, and then centrifuge. The mixture was separated and allowed to stand, and the supernatant was removed. The mixture was precipitated four times with 30 ml of acetonitrile. The product was further washed twice with 30 ml of absolute ethanol and then dried in a vacuum oven. Sample 9 (0.35% NaOH) was obtained. Nuclear magnetic detection was performed. Example 10

[0051] Weigh out 570 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 5.7 ml Stir vigorously for 10-30 minutes until homogenous, then add M-PEG5000-EPOX 7 Add 500 mg, stir vigorously until the ingredients are uniformly mixed, and leave at room temperature for 18 hours. The next day, 2 ml of 0.35 N HCl was added to adjust the pH of the system to about 7. Add 30 ml of acetonitrile to precipitate, stir vigorously to homogenize the product particles, and then centrifuge. The mixture was separated and allowed to stand, and the supernatant was removed. The mixture was precipitated four times with 30 ml of acetonitrile. The product was further washed twice with 30 ml of absolute ethanol and then dried in a vacuum oven. Sample 10 (0.35% NaOH) was obtained. Nuclear magnetic detection was performed. Example 11

[0052] Weigh out 570 mg of HA (600,000) and use a 0.35% Sodium hydroxide solution Dissolve in 5.7 ml The mixture was stirred vigorously for 10 to 30 minutes until homogenized, and then left at room temperature for 18 hours. Add 2 ml of 35N HCl to adjust the pH of the system to about 7, then add 3 ml of acetonitrile. Add 0 ml of water to precipitate, stir vigorously to homogenize the product particles, centrifuge and let stand. The supernatant was removed. Precipitation was continued four times with 30 ml of acetonitrile, and then with absolute ethanol. The product was then dried in a vacuum oven. (0.35% NaOH) was obtained. Sampling was performed using multi-angle optical differential detection and nuclear magnetic detection. Multi-angle optical detection revealed that the molecular weight was 6.366 × 10 5 Da, and sample 11 is To calculate the number of polyethylene glycols attached to the sodium hydroxybenzoate backbone This was used as a comparative sample. Example 12

[0053] The molecular weight information obtained by multi-angle optical differential detection of the products in Examples 1 to 4 was Shown in Table 1.

[0054] [Table 1]

[0055] As can be seen from the molecular weight data in Table 1, the alkali concentration of the reaction solution can be either too low or too high. If the concentration of the alkaline solution is too low, the reactivity of the raw material will not be high, and the alkaline solution will If the liquid concentration is too high, the raw material sodium hyaluronate may be decomposed by alkali. There are more.

[0056] Molecular weights obtained by multi-angle optical differential detection for the products in Examples 2 and 5 The information is shown in Table 2.

[0057] [Table 2]

[0058] As can be seen from the molecular weight data in Table 2, too much solvent makes the system too dilute and the raw material The alkaline hydrolysis of sodium hyaluronate is accelerated.

[0059] The products in Examples 2, 6, and 7 were subjected to multi-angle optical differential detection. The molecular weight information obtained is shown in Table 3.

[0060] [Table 3]

[0061] As can be seen from the molecular weight data in Table 3, reaction time is also a limiting factor in product performance. If the reaction time is too long, there is a high risk of the raw material sodium hyaluronate deteriorating.

[0062] Multi-angle optical differential detection was performed on the products in Examples 8, 9, and 10. The molecular weight information obtained is shown in Table 4.

[0063] [Table 4]

[0064] As can be seen from the molecular weight data in Table 4, HA and methoxypolyethylene glycol epoxy The feed ratio of the derivatives also has a certain effect on the performance of the product. Example 13

[0065] Taking Sample 2 and Sample 11 as examples, polyethylene glycol-modified sodium hyaluronate The method for calculating the ratio of the number of sodium hyaluronate disaccharides to the number of PEG in the product is shown below. (The molecular weight of one disaccharide unit is 380).

[0066] The average number of disaccharide units in sample 11 is 6.366×105 / 380=1675.

[0067] The number of PEG2000 in sample 2 is (12.02-6.366)105 / 20 00=283.

[0068] The ratio of the number of disaccharides to the number of PEG2000 in sample 2 is 1675 / 283 = 6:1 .

[0069] In vitro enzymatic hydrolysis experiments

[0070] Experimental steps for enzymatic hydrolysis For example, take sample 8. Weigh out 3.4 mg of sample 8 and add 242 μl of sterile water for injection. After vortexing for 20 minutes, the solution was injected with 10 U / ml hyaluronidase. Add 242 μl of sterile aqueous solution, mix the sample evenly, and place in a constant temperature water bath at 37°C. , sampling every hour to test for changes in molecular weight by multi-angle optical differential detection method did.

[0071] The sample information for testing the enzymatic hydrolysis experiment is as follows:

[0072] Control sample: HA 1.2 million Da. The relationship between enzymatic decomposition molecular weight and time is shown in Figure 1.

[0073] Sample 2: The ratio of the number of HA disaccharides to the number of PEG2000 is 6:1.

[0074] Sample 12: The ratio of the number of HA disaccharides to the number of PEG2000 is 17:1.

[0075] Sample 8: The ratio of the number of HA disaccharides to the number of PEG5000 was 13:1, and the enzyme-degraded molecules The dose-time relationship diagram is shown in FIG.

[0076] Sample 9: The ratio of the number of HA disaccharides to the number of PEG5000 is 22:1.

[0077] Sample 13: The ratio of the number of HA disaccharides to the number of PEG5000 is 42:1.

[0078] Enzymatic degradation data of PEG5000-HA sample

[0079] [Table 5]

[0080] [Table 6] Enzymatic degradation data of PEG2000-HA sample TIFF0007799154000010.tif50152

[0081] As can be seen from the data in Tables 5 and 6, when the molecular weight of PEG is 5000, the HA and PEG When the ratio of PEG to HA was between 13:1 and 42:1, PEG did not affect the resistance of HA to enzymatic degradation. The larger the ratio, the more pronounced the effect. When the molecular weight of PEG is 2000, It has a significant effect on the resistance of HA to enzymatic degradation.

Claims

1. A hyaluronic acid derivative having a structure represented by formula (I) or a salt thereof. (wherein X is —(CR 1 R 2 ) m —, m is an integer from 1 to 10; R １ and R ２ are independently selected from H, a C 1 -C 6 alkyl group, a C 3 -C 6 cycloalkyl group; Selected, k is the degree of polymerization of the polyethylene glycol residue, and k is an integer of 40 to 4600. 、 t is the degree of polymerization of the hyaluronic acid residue, and t is an integer from 100 to 8000.

2. The k is an integer of 45 to 4545, The hyaluronan according to claim 1, wherein t is an integer of 120 to 7500. Phosphoric acid derivatives or their salts.

3. The X is -(CR １ R ２ ) ｍ -, wherein m is an integer of 1 to 5. The hyaluronic acid derivative or its salt according to claim 1.

4. The R １ and R ２ and (iii) are both H. Acid derivatives or their salts.

5. The hyaluronic acid derivative or the hyaluronic acid derivative according to claim 1, wherein X is —CH 2 —. is the salt.

6. The salts include sodium salts, potassium salts, calcium salts, magnesium salts, zinc salts, cobalt salts, and tetrabutylammonium salts. The hyaluronic acid derivative or its salt according to claim 1.

7. 7. The salt according to claim 1, wherein the salt is a sodium salt having the structure of formula (II): The hyaluronic acid derivative or its salt according to any one of the preceding claims. (II)

8. A step of weighing hyaluronic acid, mixing hyaluronic acid with an alkaline solvent, and stirring the mixture ( 1) and Add the methoxypolyethylene glycol epoxy derivative to the solution obtained in step (1). and (2) reacting the The alkaline solvent in step (1) is a solution of sodium hydroxide in a mass concentration of 0.25% to 0.8%. sodium solution, The methoxypolyethylene glycol epoxy derivative has the structure of formula (III): The method for producing the hyaluronic acid derivative or its salt according to claim 1 .

9. further comprising purifying the product; The step of purifying the product comprises: adding an acidic reagent to adjust the solution obtained in step (2) to neutral; and / or adding a precipitating agent to precipitate the product, and / or adding a washing agent to wash the precipitate. The method of claim 8, further comprising the step of: Manufacturing method.

10. The mass ratio of the hyaluronic acid to the alkaline solvent is 1:5 to 1:

30.

9. A method for producing a hyaluronic acid derivative or a salt thereof according to claim 8.

11. 8. In step (2), the reaction time is 10 to 24 hours. A method for producing the hyaluronic acid derivative or salt thereof described in

12. The hyaluronic acid derivative of claim 1 in the manufacture of pharmaceutical, medical-cosmetic and cosmetic products. Use of the body or its salts.

13. Use of a hyaluronic acid derivative or a salt thereof in the manufacture of a product for medical cosmetic use.

13. The use according to claim 12.

14. 13. The use according to claim 12, wherein the product is a soft tissue filler.

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