CN111732675A - Hyaluronic acid-glucosamine graft copolymer, preparation method and application thereof - Google Patents
Hyaluronic acid-glucosamine graft copolymer, preparation method and application thereof Download PDFInfo
- Publication number
- CN111732675A CN111732675A CN202010833433.0A CN202010833433A CN111732675A CN 111732675 A CN111732675 A CN 111732675A CN 202010833433 A CN202010833433 A CN 202010833433A CN 111732675 A CN111732675 A CN 111732675A
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- CN
- China
- Prior art keywords
- hyaluronic acid
- glucosamine
- salt
- graft copolymer
- carbodiimide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- 229920000578 graft copolymer Polymers 0.000 title claims abstract description 109
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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Abstract
The invention discloses a hyaluronic acid-glucosamine graft copolymer, a preparation method and application thereof. The preparation method of the hyaluronic acid-glucosamine graft copolymer comprises the following steps: mixing a condensing agent, glucosamine or a salt thereof and water to obtain a mixed solution; the pH value of the mixed solution is adjusted, and then hyaluronic acid or salt thereof is added for amidation reaction to obtain the hyaluronic acid-glucosamine graft copolymer, so that the preparation method is simple and efficient; the method is carried out in aqueous solution, so that environmental protection pressure caused by using an organic solvent is avoided; the reaction condition is mild, the long chain and the molecular structure of the hyaluronic acid or the salt thereof in a reaction system are not damaged, and higher molecular weight can be maintained; the hyaluronic acid-glucosamine graft copolymer can directly restore the viscoelasticity of joint synovial fluid, has the effects of lubricating and nourishing joints and has slow absorbability when being injected into joint cavities.
Description
Technical Field
The invention relates to the technical field of hyaluronic acid modification, in particular to a hyaluronic acid-glucosamine graft copolymer, a preparation method and application thereof.
Background
Osteoarthritis is a relatively common joint disease and presents a significant risk to the patient. At present, more than half of the elderly aged over 60 years old in China are full of osteoarthritis. Meanwhile, osteoarthritis due to improper exercise and trauma in young people is also becoming more common. The disability rate of osteoarthritis is as high as 53%. Pain is the most common symptom of osteoarthritis, and the treatment of osteoarthritis aims to relieve pain, delay disease progression, correct deformity, improve or restore joint function, and improve the quality of life of patients.
The treatment of osteoarthritis is mainly divided into physical therapy, drug therapy and surgical therapy. The intra-articular drug injection therapy is a method of directly injecting drugs into a diseased region, and is widely applied due to the treatment characteristics of quick response and small wound, and the used drugs are mainly classified into western drug injections and Chinese drug injections. The western medicine injection mainly comprises novel preparations such as hyaluronic acid, glucocorticoid, platelet-rich plasma, medical ozone, hypertonic glucose, medical chitosan, mesenchymal liver cells, cytokines, gene therapy and the like. The Chinese medicinal injection mainly comprises chuanqizine injection, sinomenine hydrochloride, Shenmai injection, Salvia miltiorrhiza injection, Shuxuening injection, ginsenoside Rg1 and the like.
Hyaluronic acid is a commonly used drug for the treatment of osteoarthritis, especially gonarthritis. Hyaluronic acid is a major component of joint synovial fluid and is also one of the major components of cartilage matrix. The hyaluronic acid injection can lubricate synovial tissues, tissue planes, ligaments, collagen structures and the like in joints, reduce friction among tissues, achieve the effect of increasing elasticity, buffer the pressure of articular cartilage, and improve the inflammatory response of surrounding tissues, so that the healing and regeneration of the articular cartilage are promoted, the pain is relieved, and the motion function of the knee joint is recovered.
D-glucosamine, a basic substance for the synthesis of articular cartilage collagen and proteoglycan, is present in all connective tissues of the human body, and is present mostly in cartilage, synovium and synovial fluid. The most representative of the medicines for improving the disease condition is glucosamine medicines, wherein the effectiveness of glucosamine sulfate and sodium chloride double salt crystals is guaranteed to the greatest extent, so that the symptoms can be relieved, the function can be improved, and the structural progress of the disease can be delayed by long-term administration. Researches show that the glucosamine drug can accelerate the regeneration and repair of chondrocytes, stimulate the repair and reconstruction of cartilage elastic tissues, prevent the loss of elasticity of cartilage, control the balance of synovial secretion, relieve joint inflammation and realize the thorough treatment of joint diseases from symptom improvement type to structure improvement type.
Early osteoarthritis is generally treated conservatively, with the most common clinical treatments including oral administration of glucosamine and intra-articular injection of hyaluronic acid. Many researches show that the treatment effect of treating osteoarthritis by oral glucosamine and hyaluronic acid injection is better than that of single treatment of two methods, and the treatment effect is quicker and more obvious. However, compared with the method of directly injecting glucosamine into a lesion site, the oral administration of glucosamine for treating osteoarthritis has the process that glucosamine needs to be absorbed after being orally taken, then enters the systemic circulation to be distributed and metabolized, and finally reaches the lesion site, and the glucosamine playing the drug effect only accounts for a part of the content of the oral glucosamine and has slow effect. In order to exert the structural improvement effect of glucosamine on cartilage to a greater extent, CN105982911A discloses a preparation method of a high viscoelastic injection of a combination of glucosamine and hyaluronic acid. The injection mainly comprises glucosamine sulfate sodium chloride double salt and hyaluronic acid. The advantages of the glucosamine gel are that the characteristics of high hygroscopicity, easy oxidation and difficult storage of the glucosamine are changed, the stability of the product is improved, the curative effect is good, the drug resistance rate is low, and the adverse reaction is less. CN110950976A discloses glucosamine hyaluronate with a good treatment effect on osteoarthritis model rats by joint cavity injection and application thereof, wherein the preparation method of the glucosamine hyaluronate comprises the following steps: mixing glucosamine salt and hyaluronate, or dissolving them in water respectively, and performing ion exchange reaction in dialysis bag. The glucosamine is combined with negative ions (anions) of hyaluronic acid through positive ions (cations), and the solid powder finished product is obtained through concentration, precipitation by an organic solvent and spray drying.
As is well known, hyaluronic acid has good biocompatibility and biodegradability, is an excellent filler, but has a slightly poor stability, is sensitive to radicals and hyaluronidase in the human body, and is easily degraded. Therefore, it is necessary to improve the stability and other properties of hyaluronic acid by chemical modification while retaining the excellent properties of hyaluronic acid.
Disclosure of Invention
In order to solve the problems, the invention provides the hyaluronic acid-glucosamine graft copolymer which has good stability, good curative effect and less adverse reaction, and the hyaluronic acid-glucosamine graft copolymer is used for the bone joint injection, can prolong the acting time and reduce the injection frequency.
The specific technical scheme of the invention is as follows:
1. a preparation method of a hyaluronic acid-glucosamine graft copolymer comprises the following steps:
mixing a condensing agent, glucosamine or a salt thereof and water to obtain a mixed solution;
adjusting the pH value of the mixed solution, and then adding hyaluronic acid or salt thereof to perform amidation reaction to obtain the hyaluronic acid-glucosamine graft copolymer.
2. The production method according to item 1, wherein the condensing agent is a complex condensing agent, preferably, the complex condensing agent is a complex condensing agent composed of carbodiimides and succinimides; or
The composite condensing agent is a composite condensing agent consisting of carbodiimides and benzotriazoles; or
The composite condensing agent is composed of carbodiimides, succinimides and benzotriazoles;
preferably a complex condensing agent composed of carbodiimides and succinimides.
3. The production method according to item 2, wherein,
the carbodiimide is carbodiimide or a carbodiimide salt, preferably the carbodiimide is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide;
preferably, the succinimide is N-hydroxysuccinimide or N-hydroxythiosuccinimide;
preferably, the benzotriazole is N-hydroxybenzotriazole, 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazole or 1-hydroxy-7-azabenzotriazole.
4. The production method according to item 3, wherein the carbodiimide salt is carbodiimide hydrochloride; preferably, the composite condensing agent is a composite condensing agent consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.
5. The production method according to any one of claims 1 to 4, wherein the pH of the mixed solution is adjusted to 7 to 10, preferably 8 to 9.
6. The method of any one of claims 1-5, wherein the molecular weight of the hyaluronic acid or salt thereof is 900k-3000kDa, preferably 900k-1500 kDa.
7. The production method according to any one of claims 2 to 6, wherein the molar ratio of the carboxyl group of the hyaluronic acid or the salt thereof, the carbodiimide group and the succinimide group is 1:0.5 to 2, preferably 1:1 to 2.
8. The production process according to any one of items 1 to 7, wherein the molar ratio of the carboxyl group of the hyaluronic acid or a salt thereof to the amino group of the glucosamine or a salt thereof is 1 or less, preferably 0.2 to 1, and more preferably 0.2 to 0.5.
9. The production process according to any one of claims 1 to 8, wherein the amidation reaction time is 2 to 8 hours, preferably 4 to 8 hours.
10. The production method according to any one of claims 1 to 9, wherein the concentration of the hyaluronic acid or the salt thereof is 10 to 200g/L, preferably 50 to 100 g/L.
11. The production method according to any one of claims 1 to 10, wherein a step of adding an organic solvent for precipitation after diluting by adding water is further included after the amidation reaction.
12. The production method according to claim 11, wherein the organic solvent is one or more selected from methanol, ethanol, propanol, isopropanol, propylene glycol, and acetone.
13. A hyaluronic acid-glucosamine graft polymer prepared by the preparation method of any one of items 1 to 12.
14. The hyaluronic acid-glucosamine graft polymer of item 13, wherein the hyaluronic acid-glucosamine graft polymer has an electrical conductivity of 800 μ S/cm or less, preferably 2 to 400 μ S/cm, and more preferably 2 to 200 μ S/cm.
15. A hyaluronic acid-glucosamine graft polymer according to claim 13 or 14, wherein 20-40% of the total carboxyl groups of hyaluronic acid form amide bonds, preferably 25-40% of the carboxyl groups form amide bonds.
16. A hyaluronic acid-glucosamine graft polymer according to any of claims 13-15, wherein the molecular weight of the hyaluronic acid-glucosamine graft polymer is between 900k and 3500kDa, preferably between 900k and 1800 kDa.
17. A hyaluronic acid-glucosamine graft polymer, wherein the hyaluronic acid-glucosamine graft polymer has an electrical conductivity of 800. mu.S/cm or less, preferably 2 to 400. mu.S/cm, and more preferably 2 to 200. mu.S/cm.
18. The hyaluronic acid-glucosamine graft polymer of claim 17, wherein the hyaluronic acid-glucosamine graft polymer is formed by linking carboxyl groups of hyaluronic acid and amino groups of glucosamine through covalent bonds to form amide bonds.
19. A hyaluronic acid-glucosamine graft polymer according to claim 17 or 18, wherein 20-40% of the total carboxyl groups of hyaluronic acid form amide bonds, preferably 25-40% of the carboxyl groups form amide bonds.
20. A hyaluronic acid-glucosamine graft polymer according to any of claims 17-19, wherein the molecular weight of the hyaluronic acid-glucosamine graft polymer is between 900k and 3500kDa, preferably between 900k and 1800 kDa.
21. An osteoarticular injection comprising the hyaluronic acid-glucosamine graft polymer prepared by the preparation method of any one of claims 1 to 12 or the hyaluronic acid-glucosamine graft polymer of any one of claims 13 to 20, wherein the concentration of the hyaluronic acid-glucosamine graft polymer is 0.5 to 2.0% (g/ml).
ADVANTAGEOUS EFFECTS OF INVENTION
The invention provides a hyaluronic acid-glucosamine graft copolymer and a preparation method thereof, and a hyaluronic acid derivative grafted with glucosamine is used for preparing an osteoarticular injection. The hyaluronic acid-glucosamine graft copolymer can directly recover the viscoelasticity of joint synovial fluid, has the effects of lubricating and nourishing joints and slow absorbability when being injected into a joint cavity, and after being injected into a knee joint, the hyaluronic acid can stay in the joint cavity for a long time, so that the drug effect is kept, the mutual friction is reduced, the adhesion of the joint and surrounding tissues is prevented, the sustained release effect on the glucosamine is realized, the long-acting action mechanism of the glucosamine can be maintained, and the preparation method is simple and efficient; the method is carried out in aqueous solution, so that the environmental protection pressure caused by using an organic solvent is avoided; the reaction condition is mild, the long chain and the molecular structure of the hyaluronic acid or the salt thereof in a reaction system are not damaged, and higher molecular weight can be maintained; the hyaluronic acid and the glucosamine are connected by a stable covalent bond through the mediation of a condensing agent.
Drawings
FIG. 1 is a graph showing a comparison of enzymatic hydrolysis curves of a hyaluronic acid-glucosamine graft copolymer, hyaluronic acid, and glucosamine hyaluronate in Experimental example 1.
Detailed Description
The present invention is described in detail in the following description of embodiments with reference to the figures, in which like numbers represent like features throughout the figures. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
The invention provides a preparation method of a hyaluronic acid-glucosamine graft copolymer, which comprises the following steps:
mixing a condensing agent, glucosamine or a salt thereof and water to obtain a mixed solution;
adjusting the pH value of the mixed solution, and then adding hyaluronic acid or salt thereof to perform amidation reaction to obtain the hyaluronic acid-glucosamine graft copolymer.
The condensing agent is not particularly limited as long as the carboxyl group of hyaluronic acid or a salt thereof can be amidated with the amino group of glucosamine or a salt thereof, and for example, a complex condensing agent composed of a carbodiimide-based substance, which may be a carbodiimide or a carbodiimide salt, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, or a salt thereof, or a complex condensing agent composed of a carbodiimide-based substance and a succinimide-based substance, or a complex condensing agent composed of a carbodiimide-based substance and a benzotriazole-based substance, or a complex condensing agent composed of a carbodiimide-based substance, a benzotriazole-based substance, and a succinimide-based substance, preferably a complex condensing agent composed of a carbodiimide-based substance and a succinimide-based substance, which is a condensing agent used for the condensation acylation reaction of carboxylic acid or amine, which is commonly used in the prior art, is used, 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide (ETC) or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (CMC), which may be, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
The succinimide species may be, for example, N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide (Sulfo-NHS).
The benzotriazole-based substance may be, for example, N-hydroxybenzotriazole (HOBt), 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazole (HOOBt), or 1-hydroxy-7-azabenzotriazole (HAt).
Preferably, the composite condensing agent of the present invention is a composite condensing agent consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
The invention greatly improves the condensation efficiency and degree by combining the two composite condensing agents.
The principle of the invention for preparing the hyaluronic acid-glucosamine graft copolymer by adopting the carbodiimide and succinimide composite condensing agent is as follows:
and carrying out amidation reaction on carboxyl of the hyaluronic acid and amino of glucosamine under the mediation of EDC and NHS under the alkaline condition to form the hyaluronic acid-glucosamine graft copolymer.
The reaction sequence is represented as follows:
the glucosamine is an important hexosamine, is formed by replacing one hydroxyl of glucose with amino, is easy to dissolve in water and hydrophilic solvent, is commonly called aminosugar, is called glucosamine for short, and is also called glucosamine. Such as glucosamine hydrochloride, glucosamine sulfate or glucosamine phosphate.
As shown in the above reaction, the product obtained by grafting (graft product) is represented by the following formula (I), wherein the hyaluronic acid-glucosamine graft polymer is formed by forming an amide bond by covalently linking carboxyl groups of hyaluronic acid and amino groups of glucosamine, 20 to 40% of all carboxyl groups of hyaluronic acid in the resulting graft polymer are amidated with amino groups of glucosamine to form an amide bond by covalently linking, thereby forming a graft product, and some of the graft polymer of the present invention are in the form of a graft product, and some of the graft polymer is still hyaluronic acid itself, that is, the graft ratio is the number of disaccharide repeating units to which hyaluronic acid has been grafted/total number of disaccharide repeating units of hyaluronic acid.
In a preferred embodiment of the present invention, wherein the pH of the mixed solution is adjusted before adding hyaluronic acid or a salt thereof to perform the amidation reaction so that the reaction is performed under a neutral or alkaline environment, which is favorable for avoiding protonation of the primary amino group of glucosamine or a salt thereof, the pH of the mixed solution is preferably adjusted to 7 to 10, preferably 8 to 9, for example, the pH of the mixed solution is adjusted to 7, 7.5, 8, 8.5, 9, 9.5, 10 or any range therebetween.
The pH of the mixed solution can be adjusted by using an acid or an alkali, and the acid can be an acid known to those skilled in the art, and can be, for example, a common inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, etc.; the base may be a base known to those skilled in the art, and may be, for example, a conventional inorganic base such as an alkali metal hydroxide, for example, sodium hydroxide.
The molecular weight of hyaluronic acid or a salt thereof is not particularly required in the present invention, and may be selected according to the application field, action, etc. of the product, for example, the molecular weight of hyaluronic acid or a salt thereof may be 900k-3000kDa, preferably 900k-1500kDa, for example, the molecular weight of hyaluronic acid or a salt thereof may be 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa, 2600kDa, 2700kDa, 2800kDa, 2900kDa, 3000kDa, or any range therebetween.
The hyaluronic acid salt may be, for example, a sodium salt, a potassium salt, a calcium salt, a zinc salt, or the like of hyaluronic acid, and is preferably a sodium salt of hyaluronic acid.
In a preferred embodiment of the present invention, the molar ratio of the carboxyl group, the carbodiimide group and the succinimide group of the hyaluronic acid or the salt thereof is 1:0.5-2:0.5-2, preferably 1:1-2:1-2, and for example, the molar ratio of the carboxyl group, the carbodiimide group and the succinimide group of the hyaluronic acid or the salt thereof may be 1:0.5:0.5, 1:0.5:1, 1:0.5:1.5, 1:0.5:2, 1:1:0.5, 1:1: 1:1, 1:1:1.5, 1:1:2, 1:1.5:0.5, 1:1.5:1.5, 1:1: 1.5: 2:2, 1:2:1, 1:2:1.5, 1:2:2, 1:1: 2:1.5, 1:2:2 or any range therebetween.
The molar ratio of carboxyl, carbodiimide and succinimide of the hyaluronic acid or the salt thereof is the molar ratio of carboxyl/carbodiimide/succinimide of the hyaluronic acid or the salt thereof.
In a preferred embodiment of the present invention, wherein the molar ratio of the carboxyl group of the hyaluronic acid or a salt thereof to the amino group of the glucosamine or a salt thereof is 1 or less, preferably 0.2 to 1, and more preferably 0.2 to 0.5, for example, the molar ratio of the carboxyl group of the hyaluronic acid or a salt thereof to the amino group of the glucosamine or a salt thereof may be 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or the like.
The molar ratio of the carboxyl groups of the hyaluronic acid or the salt thereof to the amino groups of the glucosamine or the salt thereof is the molar ratio of the carboxyl groups of the hyaluronic acid or the salt thereof to the amino groups of the glucosamine or the salt thereof.
In a preferred embodiment of the present invention, wherein the amidation reaction time is 2-8 hours, preferably 4-8 hours, for example, the amidation reaction time may be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours or any range therebetween.
In order to allow the glucosamine or a salt thereof to react with the hyaluronic acid or a salt thereof more sufficiently, the hyaluronic acid or a salt thereof may be maintained at about 4 ℃ for 10 to 30min before the addition of the hyaluronic acid or a salt thereof for amidation reaction, so that the hyaluronic acid or a salt thereof is sufficiently swollen, and then the reaction may be performed at room temperature.
In a preferred embodiment of the present invention, the method further comprises the step of adding water for dilution and then adding an organic solvent for precipitation after the amidation reaction, i.e. adding water for dilution and then adding an organic solvent for precipitation.
The organic solvent may be, for example, methanol, ethanol, propanol, isopropanol, propylene glycol or acetone.
After adding organic solvent for precipitation, adding inorganic acid to adjust the pH value to be neutral, and obtaining white precipitate.
The inorganic acid may be, for example, hydrochloric acid, sulfuric acid, nitric acid, or the like.
In a more preferred embodiment of the present invention, the white precipitate is obtained by washing with an organic solvent, dehydrating, removing residual small molecular impurities, and drying.
The organic solvent may be, for example, ethanol, methanol, propanol, isopropanol, propylene glycol, acetone, or the like.
In a preferred embodiment of the present invention, the concentration of the hyaluronic acid or the salt thereof is 10-200g/L, preferably 50-100g/L, for example, the concentration of the hyaluronic acid or the salt thereof may be 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L, 160g/L, 170g/L, 180g/L, 190g/L, 200g/L or any range therebetween.
Wherein the concentration of hyaluronic acid refers to the final concentration of hyaluronic acid in the whole reaction system.
The concentration of the hyaluronic acid or the salt thereof is within the range, the raw materials can be well dissolved, the viscosity of a reaction system is not too high, and the reaction can be carried out quickly. If the molecular weight of hyaluronic acid or a salt thereof is large, a small concentration may be selected for facilitating dissolution and reaction, and if the molecular weight of hyaluronic acid or a salt thereof is small, a large concentration may be selected.
The invention provides a hyaluronic acid-glucosamine graft polymer, which is prepared by the preparation method.
Compared with hyaluronic acid, the hyaluronic acid-glucosamine graft polymer has the property of resisting hyaluronidase degradation, so that the hyaluronic acid-glucosamine graft polymer can prolong the in-vivo retention time, play a role in lubrication for a long time, slowly release glucosamine and play dual effects.
The hyaluronic acid-glucosamine graft polymer utilizes an amide bond in which an amino group of glucosamine and a carboxyl group of hyaluronic acid form a covalent bond, and the conductivity of an aqueous solution of the hyaluronic acid-glucosamine graft polymer is lower than that of hyaluronic acid. Therefore, when the bone joint injection is prepared, the influence of different concentrations on the osmotic pressure, the conductivity and the ionic strength of the injection can be ignored.
In a preferred embodiment of the present invention, the hyaluronic acid-glucosamine graft polymer has an electrical conductivity of 800. mu.S/cm or less, preferably 2 to 400. mu.S/cm, and more preferably 2 to 200. mu.S/cm.
For example, the hyaluronic acid-glucosamine graft polymer may have an electrical conductivity of 2. mu.S/cm, 5. mu.S/cm, 15. mu.S/cm, 20. mu.S/cm, 25. mu.S/cm, 30. mu.S/cm, 35. mu.S/cm, 40. mu.S/cm, 45. mu.S/cm, 50. mu.S/cm, 55. mu.S/cm, 60. mu.S/cm, 65. mu.S/cm, 70. mu.S/cm, 75. mu.S/cm, 80. mu.S/cm, 85. mu.S/cm, 90. mu.S/cm, 95. mu.S/cm, 100. mu.S/cm, 110. mu.S/cm, 120. mu.S/cm, 130. mu.S/cm, 140. mu.S/cm, 150. mu.S/cm, 160. mu.S/cm, 170. mu.S/cm, 180. mu.S/cm, 200. mu.S/cm, 210. mu.S/cm, 220. mu.S/cm, 230. mu.S/cm, 240. mu.S/cm, 250. mu.S/cm, 260. mu.S/cm, 270. mu.S/cm, 280. mu.S/cm, 290. mu.S/cm, 300. mu.S/cm, 310. mu.S/cm, 320. mu.S/cm, 330. mu.S/cm, 340. mu.S/cm, 350. mu.S/cm, 360. mu.S/cm, 370. mu.S/cm, 380. mu.S/cm, 390. mu.S/cm, 400. mu.S/cm, 410. mu.S/cm, 420. mu.S/cm, 430. mu.S/cm, 440. mu.S/cm, 450. mu.S/cm, 460. mu.S/cm, 470. mu.S/cm, 480. mu.S/cm, 490. mu.S/cm, 500. mu.S/cm, 510, 530 μ S/cm, 540 μ S/cm, 550 μ S/cm, 560 μ S/cm, 570 μ S/cm, 580 μ S/cm, 590 μ S/cm, 600 μ S/cm, 610 μ S/cm, 620 μ S/cm, 630 μ S/cm, 640 μ S/cm, 650 μ S/cm, 660 μ S/cm, 670 μ S/cm, 680 μ S/cm, 690 μ S/cm, 700 μ S/cm, 710 μ S/cm, 720 μ S/cm, 730 μ S/cm, 740 μ S/cm, 750 μ S/cm, 760 μ S/cm, 770 μ S/cm, 780 μ S/cm, 790 μ S/cm, 800 μ S/cm or any range therebetween.
The conductivity of the hyaluronic acid-glucosamine graft polymer was measured by preparing the hyaluronic acid-glucosamine graft polymer into a 0.5% (after dried) aqueous solution using ultrapure water, i.e., the conductivity measured when preparing a 0.5% aqueous solution. There is no limitation on the instrument for measuring the conductivity, which characterizes the ability of the solution to conduct current, and thus, any instrument that can measure the current of the solution is sufficient.
In a specific embodiment, the conductivity is measured using a conductivity meter model DDSJ-308A. The conductivity meter is used to determine the ability to conduct current in a solution of a certain concentration, for example, in the present invention, the conductivity meter is used to determine the ability to conduct current in a hyaluronic acid-glucosamine graft copolymer solution at a concentration of 0.5%.
In a preferred embodiment of the present invention, 20 to 40% of all carboxyl groups of hyaluronic acid form amide bonds, and preferably 25 to 40% of all carboxyl groups form amide bonds.
For example, the amount of carboxyl groups of hyaluronic acid involved in formation of amide bonds may be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or any range therebetween.
The formation of amide bonds by 20-40% of all carboxyl groups of hyaluronic acid refers to the formation of amide bonds by amidation reaction of 20-40% of all carboxyl groups of hyaluronic acid with amino groups of glucosamine in a reaction system, and can also be referred to as a grafting ratio.
For the grafting rate, the hyaluronic acid-glucosamine graft copolymer is subjected to enzymolysis to obtain an enzymolysis product, and then the obtained enzymolysis product is measured to obtain the grafting rate.
In a preferred embodiment of the present invention, the hyaluronidase, preferably bacterial hyaluronidase, is used to carry out complete enzymatic hydrolysis of the hyaluronic acid-glucosamine graft copolymer, and then the enzymatic hydrolysis product is measured to obtain the grafting ratio, for example, the enzymatic hydrolysis product is measured by size exclusion chromatography and calculated from the peak area ratio of the grafted fragment in the size exclusion chromatogram according to the area normalization method; preferably, the size exclusion chromatography is an Agilent-1260 high performance liquid chromatography system, and the chromatographic conditions can be, for example: superdex Peptide 10/300GL, mobile phase 10mmol ammonium acetate, flow rate 0.5ml/min, sample amount 25 μ l, column temperature 25 deg.C, detector as Variable Wavelength Detector (VWD), and detection wavelength 232 nm.
In a preferred embodiment of the present invention, wherein the molecular weight of the hyaluronic acid-glucosamine graft polymer is 900k-3500kDa, preferably 900k-1800 kDa.
For example, the hyaluronic acid-glucosamine graft polymer may have a molecular weight of 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa, 2600kDa, 2700kDa, 2800kDa, 2900kDa, 3000kDa, 3100kDa, 3200kDa, 3300kDa, 3400kDa, 3500kDa, or any range therebetween.
The molecular weight of the hyaluronic acid-glucosamine graft polymer can be controlled by an apparatus including a multi-angle laser light scattering instrumentDetermining, preferably by combining a multi-angle laser light scattering instrument with a liquid chromatography system to determine the molecular weight of the hyaluronic acid-glucosamine graft copolymer; preferably, the chromatographic conditions for determining the molecular weight of the hyaluronic acid-glucosamine graft copolymer can be: the mobile phase was 0.2mol/L NaCl (containing 0.02% NaN)3) (ii) a The flow rate is 0.6 ml/min; the sample concentration is 0.05 mg/ml; the column temperature was 35 ℃; the injection volume was 500. mu.l.
The invention provides a hyaluronic acid-glucosamine graft polymer, wherein the electrical conductivity of the hyaluronic acid-glucosamine graft polymer is below 800 mu S/cm, preferably 2-400 mu S/cm, and more preferably 2-200 mu S/cm.
Preferably, the hyaluronic acid-glucosamine graft polymer is formed by linking carboxyl groups of hyaluronic acid and amino groups of glucosamine through covalent bonds to form amide bonds.
Preferably, 20 to 40% of all carboxyl groups of hyaluronic acid form amide bonds, preferably 25 to 40% of carboxyl groups form amide bonds; preferably, the molecular weight of the hyaluronic acid-glucosamine graft polymer is 900k-3500kDa, preferably 900k-1800 kDa.
The invention provides a bone joint injection, which comprises the hyaluronic acid-glucosamine graft polymer prepared by the preparation method or the hyaluronic acid-glucosamine graft polymer, wherein the concentration of the hyaluronic acid-glucosamine graft polymer is 0.5-2.0% (g/ml), for example, the concentration of the injection is 0.5% (g/ml), 1% (g/ml), 1.5% (g/ml), 2% (g/ml) or any range therebetween.
The hyaluronic acid-glucosamine graft copolymer is injected into the joint cavity, so that the viscoelasticity of joint synovial fluid can be directly recovered, the joint synovial fluid has the effects of lubricating and nourishing joints, and has slow absorbability, after the hyaluronic acid-glucosamine graft copolymer is injected into the knee joint, the hyaluronic acid can stay in the joint cavity for a long time, the drug effect is kept, the mutual friction is reduced, the adhesion of the joints and surrounding tissues is prevented, the sustained release effect on glucosamine is realized, and the long-acting action mechanism of the glucosamine can be maintained.
The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
TABLE 1 table of sources of raw materials used in examples and comparative examples
| Raw materials | Purity of | Manufacturer of the product |
| Hyaluronic acid | 99.9％ | BLOOMAGE BIOTECH Co.,Ltd. |
| Glucosamine sulfate salt | 99％ | Hebei Chuang Source Biotechnology Ltd |
| Glucosamine | 99％ | Hebei Chuang Source Biotechnology Ltd |
| EDC | 98％ | SINOPHARM CHEMICAL REAGENT Co.,Ltd. |
| NHS | 98％ | SINOPHARM CHEMICAL REAGENT Co.,Ltd. |
Example 1
(1) At room temperature, 4.79g of EDC, 2.88g of NHS and 13.86g of glucosamine sulfate are sequentially added into 100ml of purified water, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 9.0 by using NaOH solution, adding 10g of hyaluronic acid (1500kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 4h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol for precipitation, adjusting the pH value of the precipitate suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing the precipitate by using ethanol, dehydrating for 3 times, finally removing the liquid by suction filtration to obtain wet powder, and putting the wet powder into a vacuum drier at 40 ℃ to dry, thereby finally obtaining the hyaluronic acid-glucosamine graft copolymer.
The molecular weight of the obtained hyaluronic acid-glucosamine graft copolymer was measured by the following method:
the hyaluronic acid-glucosamine graft copolymer was measured using a multi-angle laser light scattering instrument DAWN EOS (Wyota, USA) in combination with a liquid chromatography system (Agilent, USA). The chromatographic conditions are as follows: the mobile phase was 0.2mol/L NaCl (containing 0.02% NaN 3); the flow rate is 0.6 ml/min; the sample concentration is 0.05 mg/ml; the column temperature was 35 ℃; the injection volume was 500. mu.l.
The graft ratio was determined as follows:
the method comprises the steps of carrying out complete enzymolysis on a hyaluronic acid-glucosamine graft copolymer by using bacterial hyaluronidase at 37 ℃ and 5mmol/L in the presence of a phosphate buffer solution with pH of 6.0 as a medium, measuring an enzymolysis product by using a size exclusion chromatography, and calculating the grafting rate according to an area normalization method by using the peak area ratio of a grafting fragment in the size exclusion chromatogram. An Agilent-1260 type high performance liquid chromatography system, the chromatographic conditions are as follows: superdex Peptide 10/300GL, mobile phase 10mmol ammonium acetate, flow rate 0.5ml/min, sample amount 25 μ l, column temperature 25 deg.C, detector as Variable Wavelength Detector (VWD), and detection wavelength 232 nm.
The conductivity was measured as follows:
the prepared hyaluronic acid-glucosamine graft copolymer is dried, prepared into 0.5 percent (g/ml) solution by ultrapure water, and the conductivity of the solution is measured by a DDSJ-308A conductivity meter.
The molecular weight of the hyaluronic acid-glucosamine graft copolymer obtained by the above method was 1607kDa, the graft ratio was measured to be 27%, and the conductivity of the 0.5% solution was measured to be 148. mu.S/cm.
Example 2
(1) At room temperature, 9.58g of EDC, 5.75g of NHS and 13.86g of glucosamine sulfate are sequentially added into 100ml of purified water, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 8.0 by using NaOH solution, adding 10g of hyaluronic acid (900kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 8h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally removing the liquid by suction filtration to obtain wet powder, putting the wet powder into vacuum drying at 40 ℃ to finally obtain the hyaluronic acid-glucosamine graft copolymer, measuring the molecular weight, the grafting rate and the conductivity of a 0.5% ultrapure water solution by using the same method as that of the example 1, the molecular weight is 994kDa, the grafting rate is 33 percent, and the conductivity is 101 mu S/cm.
Example 3
(1) At room temperature, 4.79g of EDC, 2.88g of NHS and 13.86g of glucosamine sulfate are sequentially added into 100ml of purified water, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 10.0 by using NaOH solution, adding 20g of hyaluronic acid (900kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 2h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally removing the liquid by suction filtration to obtain wet powder, putting the wet powder into vacuum drying at 40 ℃ to finally obtain a hyaluronic acid-glucosamine graft copolymer, measuring the molecular weight, the grafting rate and the conductivity of a 0.5% ultrapure water solution by using the same method as that in example 1, the molecular weight is 972kDa, the grafting rate is 24 percent and the conductivity is 290 mu S/cm.
Example 4
(1) At room temperature, 0.96g of EDC, 0.58g of NHS and 3.47g of glucosamine sulfate are added into 100ml of purified water in sequence, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 7.0 by using NaOH solution, adding 1g of hyaluronic acid (3000kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 4h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally removing the liquid by suction filtration to obtain wet powder, putting the wet powder into vacuum drying at 40 ℃ to finally obtain a hyaluronic acid-glucosamine graft copolymer, measuring the molecular weight, the grafting rate and the conductivity of a 0.5% ultrapure water solution by using the same method as that in example 1, the obtained product has the molecular weight of 3230kDa, the grafting rate of 26 percent and the conductivity of 133 mu S/cm.
Example 5
(1) At room temperature, 7.19g of EDC, 4.32g of NHS and 11.19 g of glucosamine are sequentially added into 100ml of purified water, and are completely dissolved to obtain a mixed solution;
(2) adjusting the pH value of the mixed solution to 8.5 by using NaOH solution, adding 10g of sodium hyaluronate (1200kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 6h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally removing the liquid by suction filtration to obtain wet powder, putting the wet powder into a vacuum chamber at 40 ℃ for drying to finally obtain the hyaluronic acid-glucosamine graft copolymer, measuring the molecular weight, the grafting rate and the conductivity of 0.5% ultrapure water solution by using the same method as that in example 1, the obtained product has a molecular weight of 1372kDa, a grafting rate of 35 percent and a conductivity of 90 mu S/cm.
Example 6
(1) At room temperature, 3.59g of EDC, 4.32g of NHS and 26.85g of glucosamine are sequentially added into 100ml of purified water, and are completely dissolved to obtain a mixed solution;
(2) adjusting the pH value of the mixed solution to 7.0 by using NaOH solution, adding 15g of sodium hyaluronate (2000kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 3h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally removing the liquid by suction filtration to obtain wet powder, putting the wet powder into a vacuum chamber at 40 ℃ for drying to finally obtain the hyaluronic acid-glucosamine graft copolymer, measuring the molecular weight, the grafting rate and the conductivity of a 0.5% ultrapure water solution by using the same method as that of the example 1, the obtained product has the molecular weight of 2019kDa, the grafting rate of 22 percent and the conductivity of 322 mu S/cm.
Example 7
(1) At room temperature, 3.83g of EDC, 1.15g of NHS and 27.73g of glucosamine sulfate are sequentially added into 100ml of purified water, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 10.0 by using NaOH solution, adding 8g of hyaluronic acid (2400kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 4h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally removing the liquid by suction filtration to obtain wet powder, putting the wet powder into vacuum drying at 40 ℃ to finally obtain a hyaluronic acid-glucosamine graft copolymer, measuring the molecular weight, the grafting rate and the conductivity of a 0.5% ultrapure water solution by using the same method as in example 1, the molecular weight of the obtained product is 2481kDa, the grafting rate is 21 percent, and the conductivity is 303 mu S/cm.
Example 8
(1) At room temperature, 9.58g of EDC, 5.75g of NHS and 13.86g of glucosamine sulfate are sequentially added into 100ml of purified water, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 6 by using NaOH solution, adding 10g of hyaluronic acid (900kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 8h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing the precipitate with ethanol, dehydrating for 3 times, finally performing suction filtration to remove the liquid to obtain wet powder, putting the wet powder into a vacuum chamber at 40 ℃ for drying to obtain solid powder, measuring the molecular weight, the grafting rate and the conductivity of 0.5% ultrapure water solution of the hyaluronic acid-glucosamine graft copolymer obtained by adopting the same method as the embodiment 1 to obtain the molecular weight of 830, the grafting rate of 4kDa, the conductivity was 790. mu.S/cm.
Example 9
(1) At room temperature, 9.58g of EDC, 5.75g of NHS and 13.86g of glucosamine sulfate are sequentially added into 100ml of purified water, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 11 by using NaOH solution, adding 10g of hyaluronic acid (900kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 8h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the diluent to precipitate, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally performing suction filtration to remove liquid to obtain wet powder, putting the wet powder into a vacuum chamber at 40 ℃ for drying to obtain solid powder, measuring the molecular weight, the grafting rate and the conductivity of 0.5% ultrapure water solution of the hyaluronic acid-glucosamine graft copolymer obtained by adopting the same method as the example 1 to obtain the molecular weight of 412 with the grafting rate of 14 kDa, the conductivity was 733. mu.S/cm.
TABLE 2 raw material usage tables used in examples and comparative examples
Comparative example 1
(1) At room temperature, 9.58g of EDC, 5.75g of NHS and 13.86g of glucosamine sulfate are sequentially added into 100ml of purified water, and a mixed solution is obtained after complete dissolution;
(2) adjusting the pH value of the mixed solution to 4.5 by using NaOH solution, adding 10g of hyaluronic acid (900kDa) into the mixed solution while stirring, uniformly dispersing, activating at 4 ℃ for 10-30min, taking out, reacting at room temperature for 8h, adding 200ml of purified water to dilute the reaction solution after the reaction is finished, adding 2.5 times of ethanol in volume of the dilution solution for precipitation, adjusting the pH value of the precipitated suspension to 7.0 by using hydrochloric acid, standing for a period of time, removing the supernatant, repeatedly washing and dehydrating the precipitate by using ethanol for 3 times, finally performing suction filtration to remove liquid to obtain wet powder, putting the wet powder into a vacuum chamber at 40 ℃ for drying to obtain solid powder, measuring the molecular weight, the grafting rate and the conductivity of 0.5% ultrapure water solution of the hyaluronic acid-glucosamine graft copolymer obtained by adopting the same method as the embodiment 1 to obtain the molecular weight of 801kDa and the grafting rate of 0kDa, the conductivity was 820. mu.S/cm.
Comparative example 1 is different from example 2 only in pH and the same conditions are applied, but the molecular weight and the graft ratio of the hyaluronic acid-glucosamine graft copolymer are greatly different, and at pH 4.5, the two graft reactions do not occur, which means that the hyaluronic acid or the salt thereof and the glucosamine or the salt thereof can only undergo the graft reaction within a specific pH range.
Comparative example 2 glucosamine hyaluronate was prepared according to the technical scheme of glucosamine hyaluronate described in chinese patent application CN110950976A
Weighing 5g of hyaluronic acid with molecular weight of 900KDa and 5g of glucosamine sulfate, dissolving in water, dialyzing in a dialysis bag with molecular weight cutoff of 7000Da for 3 days, removing small molecular salts, collecting the product of the cutoff macromolecule, performing rotary evaporation and concentration, precipitating with ethanol, performing suction filtration, and performing vacuum drying to obtain glucosamine hyaluronate. The molecular weight, the graft ratio and the conductivity of 0.5% ultrapure water solution of the obtained glucosamine hyaluronate were measured by the same method as in example 1 to obtain molecular weight of 893kDa, graft ratio of 0% and conductivity of 847. mu.S/cm.
It can be seen that the hyaluronic acid and the glucosamine sulfate do not undergo a grafting reaction, and it can be clearly understood that the hyaluronic acid and the glucosamine sulfate exist in an ionic bond form.
Application example 1
The hyaluronic acid-glucosamine graft copolymer obtained in example 1 was mixed with phosphate-glycerol buffer to prepare 1.0% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe and used as an injection for joint cavity after autoclaving.
Application example 2
The hyaluronic acid-glucosamine graft copolymer obtained in example 2 was mixed with phosphate-glycerol buffer to prepare 1.5% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe and used as an injection for joint cavity after autoclaving.
Application example 3
The hyaluronic acid-glucosamine graft copolymer obtained in example 3 was mixed with phosphate-NaCl buffer solution to prepare 2% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe, and used as an injection for joint cavity after autoclaving.
Application example 4
The hyaluronic acid-glucosamine graft copolymer obtained in example 4 was mixed with phosphate-NaCl buffer solution to prepare 0.5% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe, and used as an injection for joint cavity after autoclaving.
Application example 5
The hyaluronic acid-glucosamine graft copolymer obtained in example 5 was mixed with phosphate-NaCl buffer to prepare 1.5% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe, and used as an injection for joint cavities after autoclaving.
Application example 6
The hyaluronic acid-glucosamine graft copolymer obtained in example 6 was mixed with phosphate-NaCl buffer solution to prepare 2% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe, and used as an injection for joint cavity after autoclaving.
Application example 7
The hyaluronic acid-glucosamine graft copolymer obtained in example 7 was mixed with phosphate-NaCl buffer to prepare 1.8% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe and used as an injection for joint cavities after autoclaving.
Application example 8
The hyaluronic acid-glucosamine graft copolymer obtained in example 8 was mixed with phosphate-NaCl buffer solution to prepare 0.5% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe, and used as an injection for joint cavity after autoclaving.
Application example 9
The hyaluronic acid-glucosamine graft copolymer obtained in example 9 was mixed with phosphate-NaCl buffer solution to prepare 2% (g/ml) hyaluronic acid-glucosamine graft copolymer gel, which was filled in a prefilled syringe and used as an injection for joint cavity after autoclaving.
Comparative example 3
Hyaluronic acid with molecular weight of 950kDa is prepared into 1.5% (g/ml) hyaluronic acid gel by phosphate-glycerol buffer solution, and the hyaluronic acid gel is filled into a pre-filled syringe and can be used for joint cavity injection after autoclaving.
Comparative example 4
Taking the glucosamine hyaluronate obtained in the comparative example 2, preparing 1.5% (g/ml) glucosamine hyaluronate gel by using phosphate-glycerol buffer solution, filling the glucosamine hyaluronate gel into a pre-filled syringe, and performing autoclaving to obtain the glucosamine hyaluronate gel which can be used for joint cavity injection.
Experimental example 1 enzymolysis study of hyaluronic acid-glucosamine graft copolymer against hyaluronidase
The bacterial hyaluronidase can specifically degrade hyaluronic acid, double bonds can appear in enzymolysis products, and ultraviolet absorption is realized at 232 nm. Therefore, A232 can reflect enzymolysis, and the larger the value of A232, the more degradation products containing double bonds are, the stronger the enzymolysis is.
The hyaluronic acid-glucosamine graft copolymer prepared in example 1, the glucosamine hyaluronate prepared in comparative example 2 and hyaluronic acid were respectively prepared into 0.2% solutions as substrates, bacterial hyaluronidase was added, enzymatic hydrolysis was performed in a phosphate buffer solution at 37 ℃, 5mmol/L, ph6.0, and heated to boil for 2 minutes to terminate the enzymatic hydrolysis. The results of the two enzymolysis curves are shown in fig. 1 by measuring the ultraviolet absorption value of the enzymolysis product at 232nm under different reaction times.
As can be seen from FIG. 1, the A of hyaluronic acid and glucosamine hyaluronate occurred with the lapse of enzymolysis time232The value growth rate is obviously higher than that of the hyaluronic acid-glucosamine graft copolymer, and the hyaluronic acid and the glucosamine hyaluronate are permeatedThe hyaluronic acid-glucosamine graft copolymer has obvious effect of resisting hyaluronidase degradation, which shows that the hyaluronic acid and the glucosamine of the hyaluronic acid-glucosamine are bonded together by covalent bonds and are relatively stable, while the hyaluronic acid and the glucosamine of the glucosamine hyaluronate in the comparative example 2 are bonded together by ionic bonds and are easy to be dissociated in aqueous solution and have relatively weak stability, so that the hyaluronic acid-glucosamine graft copolymer is easy to be degraded by hyaluronidase.
The hyaluronic acid-glucosamine graft copolymers obtained in examples 2 to 10 have similar technical effects.
Experimental example 2 Effect of hyaluronic acid-glucosamine graft copolymer on the expression levels of inflammatory factors IL-1 beta and TNF-alpha
40 healthy adult rabbits (purchased from academy of agricultural science of Shandong province) with the weight of 2.5-3.0 kg and unlimited male and female parts. Randomly 8 blank control groups were selected, and the remaining molds were made. Rabbits in each group were fed free water under identical conditions.
A0.4% papain solution is prepared in advance before an experiment, and the preparation method comprises the following steps: dissolving papain 4.0mg in 1ml physiological saline, adding cysteine hydrochloride 50mg, and filtering with 0.22 μm filter membrane after complete dissolution.
The molding process comprises the following steps: the rabbit was fixed on the test bed in a supine position after anesthesia by intravenous injection at the edge of the rabbit ear with 2.0% pentobarbital solution, the hair at the knee joint was scraped off, the rabbit was disinfected conventionally, 0.3ml of 0.4% papain solution was injected at the right knee joint, and the left side was used as a normal control. The placebo rabbits were not treated. The model rabbits were randomly divided into 4 groups of 8 rabbits, each of which was a model group (0.3ml of physiological saline), a hyaluronic acid group (0.3ml of the comparative example 3 injection, joint cavity injection), a glucosamine hyaluronate group (0.3ml of the comparative example 4 injection, joint cavity injection), and a test sample group (0.3ml of the application example 2 injection). For the 7 th day after molding, the joint cavity was injected with 0.3ml/joint once a week for 5 weeks.
After 7d of the last administration, the test is finished, the rabbits are fasted overnight before the test without water prohibition, anesthetized by 2.0% pentobarbital solution, blood is taken from abdominal aorta, and serum is prepared and stored at-80 ℃ for detecting serum inflammatory factors IL-1 beta and TNF-alpha. IL-1 beta and TNF-alpha are respectively determined by adopting an IL-1 beta enzyme-linked immunoassay kit and a TNF-alpha enzyme-linked immunoassay kit. The results are shown in Table 3.
TABLE 3 comparison of inflammatory factor content in rabbit sera of various groups
| Group of | IL-1 β concentration (pg/mL) | TNF- α concentration (pg/mL) |
| Blank control group | 47.1±5.8 | 38.4±9.1 |
| Model set | 83.3±4.6 | 60.7±7.8 |
| Group of hyaluronic acids | 58.4±3.4 | 44.5±6.0 |
| Glucosamine hyaluronate group | 50.0±3.5 | 48.1±7.2 |
| Test article group | 40.2±5.4 | 29.6±7.5 |
The experimental result shows that the levels of inflammatory factors IL-1 beta and TNF-alpha in rabbit serum of a model group are obviously higher than those of a blank control group, which indicates that the rabbit osteoarthritis molding is successful; compared with the model group, the levels of inflammatory factors IL-1 beta and TNF-alpha in rabbit serum of the hyaluronic acid group, the glucosamine hyaluronic acid salt group and the test sample group are all obviously reduced; compared with the hyaluronic acid group and the glucosamine hyaluronate group, the level of inflammatory factors IL-1 beta and TNF-alpha in serum is reduced more obviously, which shows that the expression level of the inflammatory factors IL-1 beta and TNF-alpha can be reduced obviously by injecting the hyaluronic acid-glucosamine graft copolymer into the joint cavity, and the effect is better than that of the hyaluronic acid and the glucosamine hyaluronate.
The injection solution of application example 1 and the injection solutions of application examples 3 to 9 have similar technical effects.
In conclusion, the hyaluronic acid-glucosamine graft copolymer disclosed by the invention can directly recover the viscoelasticity of joint synovial fluid in a joint cavity after being injected into the joint cavity, has the effects of lubricating and nourishing joints, and has slow absorbability, so that hyaluronic acid can stay in the joint cavity for a long time after being injected into a knee joint, the drug effect is kept, the mutual friction is reduced, the adhesion of the joints and surrounding tissues is prevented, the sustained release effect on glucosamine is realized, and the long-acting action mechanism of glucosamine can be maintained.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (10)
1. A preparation method of a hyaluronic acid-glucosamine graft copolymer comprises the following steps:
mixing a condensing agent, glucosamine or a salt thereof and water to obtain a mixed solution;
adjusting the pH value of the mixed solution, and then adding hyaluronic acid or salt thereof to perform amidation reaction to obtain the hyaluronic acid-glucosamine graft copolymer.
2. The production method according to claim 1, wherein the condensing agent is a complex condensing agent, preferably, the complex condensing agent is a complex condensing agent composed of carbodiimides and succinimides; or
The composite condensing agent is composed of carbodiimides and benzotriazoles; or
The composite condensing agent is composed of carbodiimides, succinimides and benzotriazoles;
preferably a complex condensing agent composed of carbodiimides and succinimides.
3. The production method according to claim 2, wherein the carbodiimide compound is a carbodiimide or a carbodiimide salt,
preferably, the carbodiimide is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide;
preferably, the succinimide is N-hydroxysuccinimide or N-hydroxythiosuccinimide;
preferably, the benzotriazole is N-hydroxybenzotriazole, 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazole or 1-hydroxy-7-azabenzotriazole.
4. The production method according to claim 3, wherein the carbodiimide salt is carbodiimide hydrochloride; preferably, the composite condensing agent is a composite condensing agent consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.
5. The production method according to any one of claims 1 to 4, wherein the pH of the mixed solution is adjusted to 7 to 10, preferably 8 to 9.
6. The method of any one of claims 1-5, wherein the molecular weight of the hyaluronic acid or salt thereof is 900k-3000kDa, preferably 900k-1500 kDa.
7. The production method according to any one of claims 2 to 6, wherein the molar ratio of the carboxyl group, the carbodiimide group and the succinimide group of the hyaluronic acid or the salt thereof is 1:0.5 to 2, preferably 1:1 to 2.
8. The production method according to any one of claims 1 to 7, wherein the molar ratio of the carboxyl group of the hyaluronic acid or a salt thereof to the amino group of the glucosamine or a salt thereof is 1 or less, preferably 0.2 to 1, and more preferably 0.2 to 0.5.
9. The production method according to any one of claims 1 to 8, wherein the amidation reaction time is 2 to 8 hours, preferably 4 to 8 hours.
10. The production method according to any one of claims 1 to 9, wherein the concentration of the hyaluronic acid or the salt thereof is 10 to 200g/L, preferably 50 to 100 g/L.
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| CN112138138A (en) * | 2020-11-19 | 2020-12-29 | 乐康珍泰（天津）生物技术有限公司 | A pharmaceutical composition for treating gout and preparation method thereof |
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