Hyaluronic acid complex polysaccharide composition, and preparation method and application thereof

Abstract

The present application relates to the technical field of biomedical materials, in particular to a hyaluronic acid composite polysaccharide composition, a preparation method and application thereof. Specifically, by introducing other high-molecular mucopolysaccharides on the basis of hyaluronic acid cross-linked chondroitin sulfate (HA-CS), the present application can form an interpenetrating network structure with HA-CS through electrostatic interaction, or linear polysaccharide chains can form hydrogen bonds with the hydroxyl and carboxyl groups of HA-CS, further forming a dense network structure, enhancing the adhesion of the material to the bladder mucosa, reducing the risk of loss with urine, and prolonging the retention time of the temporary protective layer; at the same time, the synergistic effect between the various composite polysaccharide components increases the stability of the material itself, improves the residence time of the composition on the surface of the bladder mucosa, enables it to continuously exert a protective and repairing effect, and effectively promotes the repair of bladder mucosal injury, thus having good practical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a hyaluronic acid-polysaccharide composition, its preparation method, and its application. Background Technology

[0002] The bladder epithelial glycosaminoglycan (GAG) protective layer is composed of various polysaccharides such as hyaluronic acid and chondroitin sulfate. As a physical barrier, it prevents harmful substances in urine from directly contacting the mucosa and maintains the integrity of the mucosa. The occurrence and development of chronic cystitis (such as interstitial cystitis and radiation cystitis) are usually closely related to the loss of GAG in the mucosal layer.

[0003] Currently, clinical treatments for chronic cystitis include oral medications, bladder irrigation, physical therapy, and surgery, but all have limitations. For example, traditional antibiotic treatment is only effective for infectious lesions and is prone to drug resistance; systemic medication may cause various adverse reactions.

[0004] Recent studies have shown that early repair of the bladder's protective GAG ​​layer can prevent the chronic progression of cystitis. Exogenous supplementation of GAG components can improve the permeability of the bladder mucosal epithelium and reduce inflammatory stimulation. Sterile sodium hyaluronate solution (Scistar) produced by Mylan can be used as a temporary replacement for a lack of the bladder's protective GAG ​​layer. Sodium hyaluronate is an important component of the GAG ​​layer, and intravesical instillation of this solution can effectively relieve symptoms caused by mucosal damage. However, this solution has a short residence time in the bladder and is easily flushed away by urine, making it difficult to achieve continuous and effective protection and repair. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a hyaluronic acid complex polysaccharide composition, its preparation method, and its application. Specifically, the present invention introduces other high-molecular-weight mucopolysaccharides into a hyaluronic acid-crosslinked chondroitin sulfate (HA-CS) base. These polysaccharides can form an interpenetrating network structure with HA-CS through electrostatic interactions, or the linear polysaccharide chains can form hydrogen bonds with the hydroxyl and carboxyl groups of HA-CS, further densifying the network structure, enhancing the material's adhesion to the bladder mucosa, reducing the risk of loss with urine, and prolonging the retention time of the temporary protective layer. Simultaneously, the synergistic effect between multiple complex polysaccharide components increases the stability of the material itself, improves the residence time of the composition on the bladder mucosa surface, enabling it to continuously exert its protective and repairing effects, and effectively promotes the repair of bladder mucosal damage. Based on the above research results, the present invention is thus completed.

[0006] Specifically, the present invention adopts the following technical solution: In a first aspect, the present invention provides a hyaluronic acid complex polysaccharide composition, wherein the effective components of the composition include hyaluronic acid cross-linked chondroitin sulfate and high molecular weight mucopolysaccharide; the mass ratio of the hyaluronic acid cross-linked chondroitin sulfate to the high molecular weight mucopolysaccharide is 1:4-4:1.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned hyaluronic acid complex polysaccharide composition, the method comprising: mixing and dissolving hyaluronic acid cross-linked chondroitin sulfate and high molecular weight mucopolysaccharide in phosphate buffer.

[0008] A third aspect of the present invention provides the application of the above-described hyaluronic acid complex polysaccharide composition in the preparation of products; said products can be used as a temporary substitute for the lack of a glycosaminoglycan protective layer in human mucosal tissues, or for the protection of damaged wounds. Specifically, this includes the protection of damaged or missing glycosaminoglycan protective layers caused by various diseases, mechanical injuries, or surgical wounds.

[0009] The beneficial effects of one or more of the above technical solutions: The above technical solution introduces other high-molecular-weight mucopolysaccharides into the basis of hyaluronic acid cross-linked chondroitin sulfate (HA-CS). These can form an interpenetrating network structure with HA-CS through electrostatic interactions, or the linear polysaccharide chains can form hydrogen bonds with the hydroxyl and carboxyl groups of HA-CS to further densify the network structure, enhance the adhesion of the material to the bladder mucosa, reduce the risk of loss with urine, and prolong the retention time of the temporary protective layer. At the same time, the synergistic effect between multiple complex polysaccharide components increases the stability of the material itself and improves the residence time of the composition on the bladder mucosa surface, enabling it to continuously exert its protective and repairing effects and effectively promote the repair of bladder mucosal damage.

[0010] The hyaluronic acid-polysaccharide composite prepared by the above technical solution can be used as a temporary substitute for the glycosaminoglycan protective layer in human mucosal tissues when it is lacking, or for the protection of damaged wounds. Due to the composition's good adhesion, it can reduce the loss of temporary glycosaminoglycan components caused by urine flushing, increase the product's retention time on the damaged bladder mucosa surface, and provide an opportunity for the self-repair of the damaged mucosa, thus achieving a more comprehensive mucosal repair effect. It solves the problems of large drug side effects, unsatisfactory effects, and easy recurrence of chronic inflammation in traditional treatment methods, and therefore has good practical application value. Detailed Implementation

[0011] The following detailed description is exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0012] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0013] Because it is composed of a single component of sodium hyaluronate or two polysaccharides covalently cross-linked, the number of intermolecular interaction sites is limited. Under the dynamic environment of urine flushing and organ peristalsis in the bladder, it is prone to insufficient adhesion and it is difficult to maintain the integrity of the protective layer for a long time.

[0014] In view of this, in a typical embodiment of the present invention, a hyaluronic acid complex polysaccharide composition is provided, wherein the effective components of the composition include hyaluronic acid cross-linked chondroitin sulfate and high molecular weight mucopolysaccharide; the mass ratio of hyaluronic acid cross-linked chondroitin sulfate to high molecular weight mucopolysaccharide is 1:4-4:1, preferably 1:3-3:1, more preferably 1:2-2:1, and most preferably 1:1.

[0015] The hyaluronic acid-crosslinked chondroitin sulfate is obtained by crosslinking hyaluronic acid and chondroitin sulfate. The crosslinking agent selected can be a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), or genipin. When EDC and NHS are used, their molar ratio is 1:1 to 1:4, and the ratio of the total mass of the crosslinking agent to the total mass of hyaluronic acid and chondroitin sulfate is 1:5 to 1:100.

[0016] The mass ratio of hyaluronic acid to chondroitin sulfate is 1:9-9:1, preferably 4:6-8:2, more preferably 5:5-6:4, and most preferably 5.5:4.5.

[0017] The hyaluronic acid comprises hyaluronic acid and its salts. Preferably, the hyaluronic acid is sodium hyaluronate or zinc hyaluronate, and most preferably sodium hyaluronate. The molecular weight of the hyaluronic acid is 300,000 to 2,500,000 Daltons, preferably 500,000 to 2,000,000 Daltons, and most preferably 1,000,000 to 1,500,000 Daltons.

[0018] The chondroitin sulfate is preferably sodium chondroitin sulfate, with a molecular weight of 20,000-60,000 Daltons, more preferably 30,000-60,000 Daltons, and most preferably 30,000-40,000 Daltons.

[0019] The high molecular weight polysaccharides include, but are not limited to, one or more of chitosan, sodium alginate, xanthan gum, guar gum, sodium carboxymethyl cellulose, and gellan gum.

[0020] The molecular weight of the high-molecular-weight mucopolysaccharide is 5-3 million Daltons, preferably 8-2 million Daltons, more preferably 10-1.5 million Daltons, and even more preferably 30-1 million Daltons.

[0021] The chitosan is preferably carboxymethyl chitosan with a degree of deacetylation of not less than 60% and a molecular weight of not less than 300,000 Daltons, preferably with a degree of deacetylation of 60%-90% and a molecular weight of not less than 500,000 Daltons, and most preferably with a degree of deacetylation of 80% and a molecular weight of 800,000-1,000,000 Daltons.

[0022] The hyaluronic acid-crosslinked chondroitin sulfate and high molecular weight mucopolysaccharide are mixed and dissolved in phosphate buffer; further, the osmotic pressure of the phosphate buffer is 270-350 mOsm / L, and even further, the osmotic pressure of the phosphate buffer is 290-320 mOsm / L. This effectively simulates the physiological environment, reduces irritation or damage to mucosal tissues, and helps maintain the normal morphology and function of mucosal epithelial cells, avoiding cell edema due to hypotonicity or cell dehydration due to hypertonicity, thereby protecting damaged bladder mucosa; improving the stability and adhesion of the composition, and ensuring the clinical safety and tolerability of the product.

[0023] In actual application, the hyaluronic acid complex polysaccharide composition is in liquid form. Preferably, the concentration of the hyaluronic acid complex polysaccharide composition is 0.002-0.05 g / mL, more preferably 0.003-0.03 g / mL, more preferably 0.004-0.02 g / mL, and most preferably 0.01 g / mL.

[0024] In another specific embodiment of the present invention, a method for preparing the above-mentioned hyaluronic acid complex polysaccharide composition is provided, the method comprising: mixing and dissolving hyaluronic acid cross-linked chondroitin sulfate and high molecular weight mucopolysaccharide in phosphate buffer.

[0025] The preparation method of the hyaluronic acid crosslinked chondroitin sulfate includes: dissolving sodium hyaluronate or zinc hyaluronate and sodium chondroitin sulfate in sodium chloride solution, adding a crosslinking agent and reacting to obtain the product.

[0026] The concentration of the sodium chloride solution is 0.5-1.5%, preferably 0.9%.

[0027] The crosslinking agent can be a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), or genipin; when EDC and NHS are used, their molar ratio is 1:1-1:4, and the ratio of the total mass of the crosslinking agent to the total mass of hyaluronic acid and chondroitin sulfate is 1:5-1:100, preferably 1:50-1:10, more preferably 1:30-1:10, and most preferably 1:20.

[0028] The specific conditions for the reaction are as follows: the reaction temperature is 18-35℃, further 20℃-35℃, preferably 25℃-35℃, more preferably 25℃-30℃, and most preferably 30℃; The pH of the reaction system is 5.0-7.0, preferably 5.5-7, more preferably 5.5-6.5, and most preferably 6.0; the stirring reaction time is 2-12 hours, further 3-12 hours, preferably 3-10 hours, more preferably 4-8 hours, and most preferably 4-5 hours.

[0029] The phosphate buffer solution can be prepared using conventional methods, specifically by adding sodium dihydrogen phosphate dihydrate or sodium dihydrogen phosphate or potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate or disodium hydrogen phosphate, sodium chloride and / or potassium chloride to water, stirring until fully dissolved, and then filtering the solution through a filter membrane (e.g., 0.22 μm).

[0030] In another specific embodiment of the present invention, the phosphate buffer solution is prepared by dissolving 8g sodium chloride, 0.198g potassium chloride, 1.36g disodium hydrogen phosphate, and 0.27g potassium dihydrogen phosphate in 1000ml of water for injection; or the phosphate buffer solution in the composition of the present invention is prepared by dissolving 8g sodium chloride, 1.36g disodium hydrogen phosphate, and 0.24g sodium dihydrogen phosphate in 1000ml of water for injection; or the phosphate buffer solution in the composition of the present invention is prepared by dissolving 8g sodium chloride, 0.198g potassium chloride, 2.87g disodium hydrogen phosphate dodecahydrate, and 0.27g potassium dihydrogen phosphate in 1000ml of water for injection; or the phosphate buffer solution in the composition of the present invention is prepared by dissolving 8g sodium chloride, 2.87g disodium hydrogen phosphate dodecahydrate, and 0.312g sodium dihydrogen phosphate dihydrate in 1000ml of water for injection.

[0031] The mass ratio of hyaluronic acid cross-linked chondroitin sulfate to high molecular weight mucopolysaccharide is 1:4-4:1, preferably 1:3-3:1, more preferably 1:2-2:1, and most preferably 1:1.

[0032] In this invention, the pH value of the hyaluronic acid complex polysaccharide composition is 6.5-8.0, preferably 6.5-7.5.

[0033] In this invention, the preparation method further includes a step of sterilizing the hyaluronic acid complex polysaccharide composition, wherein the sterilization is preferably moist heat sterilization, and the sterilization parameters are a temperature of 115-126°C and a sterilization time of 6-30 min.

[0034] In another specific embodiment of the present invention, the application of the above-mentioned hyaluronic acid complex polysaccharide composition in the preparation of a product is provided; the product can be used as a temporary substitute when the glycosaminoglycan protective layer is lacking in human mucosal tissue, or for the protection of damaged wounds. Specifically, this includes: damage or loss of the glycosaminoglycan protective layer caused by various diseases, mechanical injuries, or surgical wounds.

[0035] Individuals with conditions such as interstitial cystitis, radiation cystitis, chronic recurrent urinary tract infection, and overactive bladder.

[0036] Mechanical injury, such as trauma during cystoscopy and catheter insertion or placement, balloon compression, and friction and irritation of the mucosa from long-term indwelling catheters.

[0037] Patients who have suffered mucosal damage and partial loss or detachment of glycosaminoglycans due to surgery, such as those with mucosal damage caused by tumor surgery.

[0038] The hyaluronic acid complex polysaccharide composition provided by this invention forms a protective film on the surface of mucosal tissue through physical action, serving as an isolation barrier to protect damaged wounds and temporarily replace or supplement the defects of the glycosaminoglycan protective layer.

[0039] The product may be a drug.

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0041] The raw materials used in the following examples are all commercially available products that can be purchased.

[0042] Example 1: Preparation method of hyaluronic acid complex polysaccharide composition (1) Cross-linking reaction: Weigh 45g of sodium hyaluronate (molecular weight 300,000 Daltons) and 5g of sodium chondroitin sulfate (molecular weight 60,000 Daltons) and dissolve them in 5L of 0.9% sodium chloride solution. Add 0.5g of genipin and stir the reaction for 10 hours at a constant temperature of 35℃. Terminate the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid cross-linked chondroitin sulfate.

[0043] (2) Preparation of phosphate buffer: Weigh 8.0264g sodium chloride, 0.1983g potassium chloride, 1.3607g disodium hydrogen phosphate and 0.2708g potassium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir at room temperature for 10min to fully dissolve the solution and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0044] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.05 g / mL by mixing 8 g of hyaluronic acid crosslinked chondroitin sulfate and 4 g of carboxymethyl chitosan (molecular weight 1.2 million Daltons, degree of deacetylation 68%) with 240 mL of phosphate buffer from step (2) and stir at room temperature for 120 min to fully dissolve it.

[0045] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 121℃ for 20 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0046] Example 2: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: Weigh 45g of sodium hyaluronate (molecular weight 500,000 Daltons) and 5g of sodium chondroitin sulfate (molecular weight 60,000 Daltons) and dissolve them in 3L of 0.9% sodium chloride solution. Adjust the pH to 6 with dilute hydrochloric acid solution, add EDC (0.5g) and NHS (0.5g), and stir the reaction for 4 hours under constant temperature of 25℃. Then stop the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0047] (2) Preparation of phosphate buffer: Weigh 8.0014g sodium chloride, 1.3581g disodium hydrogen phosphate and 0.2406g sodium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir at room temperature for 10min to fully dissolve them and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0048] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.05 g / mL by mixing 4 g of hyaluronic acid crosslinked chondroitin sulfate and 8 g of sodium carboxymethyl cellulose (molecular weight 600,000 Daltons) prepared in step (1) with 240 mL of phosphate buffer in step (2) and stir at room temperature for at least 80 min to ensure complete dissolution.

[0049] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 126℃ for 6 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0050] Example 3: Preparation method of hyaluronic acid complex polysaccharide composition (1) Cross-linking reaction: Weigh 18g of zinc hyaluronic acid (molecular weight 500,000 Daltons) and 2g of sodium chondroitin sulfate (molecular weight 30,000 Daltons) and dissolve them in 2L of 0.9% sodium chloride solution. Add 1.0g of genipin and stir the reaction for 8 hours at a constant temperature of 30℃. Terminate the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid cross-linked chondroitin sulfate.

[0051] (2) Preparation of phosphate buffer: Weigh 8.0264g sodium chloride, 0.1983g potassium chloride, 1.3607g disodium hydrogen phosphate and 0.2708g potassium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir at room temperature for 10min to fully dissolve the solution and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0052] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.002 g / mL by mixing 0.8 g of hyaluronic acid cross-linked chondroitin sulfate and 0.2 g of xanthan gum (molecular weight 3 million Daltons) prepared in step (1) with 500 mL of phosphate buffer in step (2), and stir at room temperature for at least 30 min to fully dissolve it.

[0053] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 119°C for 30 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0054] Example 4: Preparation method of hyaluronic acid complex polysaccharide composition (1) Cross-linking reaction: Weigh 2g of sodium hyaluronate (molecular weight 1.5 million Daltons) and 18g of sodium chondroitin sulfate (molecular weight 20,000 Daltons) and dissolve them in 2L of 0.9% sodium chloride solution. Add 1.6g of genipin and stir the reaction at a constant temperature of 30℃ for 12 hours. Terminate the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid cross-linked chondroitin sulfate.

[0055] (2) Preparation of phosphate buffer: Weigh 8.0013g sodium chloride, 2.8704g disodium hydrogen phosphate dodecahydrate and 0.3132g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0056] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.002 g / mL by mixing 0.3 g of hyaluronic acid crosslinked chondroitin sulfate and 0.2 g of sodium alginate (molecular weight 200,000 Daltons) prepared in step (1) with 250 mL of phosphate buffer in step (2) and stir at room temperature for at least 60 min to ensure complete dissolution.

[0057] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 122℃ for 16 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0058] Example 5: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: Weigh 18g of zinc hyaluronic acid (molecular weight 450,000 Daltons) and 12g of sodium chondroitin sulfate (molecular weight 60,000 Daltons) and dissolve them in 1.5L of 0.9% sodium chloride solution. Adjust the pH to 6 with dilute hydrochloric acid solution, add EDC (1.5g) and NHS (1.5g), and stir the reaction for 8 hours under constant temperature of 20℃. Then stop the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0059] (2) Preparation of phosphate buffer: Weigh 8.0013g sodium chloride, 2.8704g disodium hydrogen phosphate dodecahydrate and 0.3132g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0060] (3) Dissolving and mixing: 2.4g of hyaluronic acid crosslinked chondroitin sulfate and 9.6g of gellan gum (molecular weight 1 million Daltons) prepared in step (1) are mixed with 400mL of phosphate buffer in step (2) to prepare a solution with a mass-volume concentration of 0.03g / mL. Stir at room temperature for 180min to fully dissolve the solution.

[0061] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 121℃ for 20 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0062] Example 6: Preparation method of hyaluronic acid complex polysaccharide composition (1) Cross-linking reaction: Weigh 24g of sodium hyaluronate (molecular weight 2 million Daltons) and 6g of sodium chondroitin sulfate (molecular weight 30,000 Daltons) and dissolve them in 3L of 0.9% sodium chloride solution. Adjust the pH to 5 with dilute hydrochloric acid solution, add EDC (0.5g) and NHS (1.0g), and stir the reaction for 6 hours under constant temperature of 25℃. Then stop the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid cross-linked chondroitin sulfate.

[0063] (2) Preparation of phosphate buffer: Weigh 8.0136g sodium chloride, 1.3529g disodium hydrogen phosphate and 0.2417g sodium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir for 10min at room temperature to fully dissolve them and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0064] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.03 g / mL by mixing 2 g of hyaluronic acid crosslinked chondroitin sulfate and 4 g of carboxymethyl chitosan (molecular weight 600,000 Daltons, degree of deacetylation 85%) with 200 mL of phosphate buffer from step (2) and stir at room temperature for 90 min to fully dissolve it.

[0065] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 123℃ for 12 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0066] Example 7: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: Weigh 24g of sodium hyaluronate (molecular weight 2 million Daltons) and 6g of sodium chondroitin sulfate (molecular weight 20,000 Daltons) and dissolve them in 3L of 0.9% sodium chloride solution. Add 0.6g of genipin and stir the reaction for 8 hours at a constant temperature of 30℃. Terminate the reaction and add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0067] (2) Preparation of phosphate buffer: Weigh 8.0027g sodium chloride, 2.8712g disodium hydrogen phosphate dodecahydrate and 0.3126g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0068] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.003 g / mL by mixing 1.5 g of hyaluronic acid cross-linked chondroitin sulfate and 0.5 g of sodium alginate (molecular weight 50,000 Daltons) prepared in step (1) with 1000 mL of phosphate buffer in step (2) and stir at room temperature for at least 40 min to ensure complete dissolution.

[0069] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 124℃ for 10 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0070] Example 8: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: Weigh 12g of sodium hyaluronate (molecular weight 1 million Daltons) and 18g of sodium chondroitin sulfate (molecular weight 20,000 Daltons) and dissolve them in 3L of 0.9% sodium chloride solution. Add 1.0g of genipin and stir the reaction for 10 hours at a constant temperature of 25℃. Add dilute hydrochloric acid to adjust the pH of the system to 6.5 to terminate the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0071] (2) Preparation of phosphate buffer: Weigh 8.0027g sodium chloride, 2.8712g disodium hydrogen phosphate dodecahydrate and 0.3126g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0072] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.003 g / mL by mixing 0.4 g of hyaluronic acid crosslinked chondroitin sulfate and 0.2 g of guar gum (molecular weight 1.5 million Daltons) prepared in step (1) with 200 mL of phosphate buffer in step (2). Stir at room temperature for at least 30 min to ensure complete dissolution.

[0073] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 125℃ for 8 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0074] Example 9: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: Weigh 10g of sodium hyaluronate (molecular weight 800,000 Daltons) and 10g of sodium chondroitin sulfate (molecular weight 50,000 Daltons) and dissolve them in 2L of 0.9% sodium chloride solution. Adjust the pH to 5-6 with dilute hydrochloric acid solution, add EDC (0.5g) and NHS (0.5g), and stir the reaction for 8 hours under constant temperature of 20℃. Then stop the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze dry it under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0075] (2) Preparation of phosphate buffer: Weigh 8.0104g sodium chloride, 2.8713g disodium hydrogen phosphate dodecahydrate and 0.3127g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0076] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.02 g / mL by mixing 2 g of hyaluronic acid crosslinked chondroitin sulfate and 3 g of sodium carboxymethyl cellulose (molecular weight 500,000 Daltons) prepared in step (1) with 250 mL of phosphate buffer in step (2) and stir at room temperature for 120 min to fully dissolve it.

[0077] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 125℃ for 8 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0078] Example 10: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: 12g of sodium hyaluronate (molecular weight 1.5 million Daltons) and 8g of sodium chondroitin sulfate (molecular weight 30,000 Daltons) were weighed and dissolved in 4L of 0.9% sodium chloride solution. The pH was adjusted to 5-6 with dilute hydrochloric acid solution. EDC (0.3g) and NHS (0.6g) were added. The reaction was stirred for 4 hours under constant temperature of 25℃ and the reaction was terminated. Three times the volume of anhydrous ethanol was added to obtain a precipitate. The precipitate was washed three times with purified water and anhydrous ethanol alternately. After being dissolved in 10 times the mass of water for injection, it was vacuum freeze-dried for 24h to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0079] (2) Preparation of phosphate buffer: Weigh 8.0029g sodium chloride, 2.8701g disodium hydrogen phosphate dodecahydrate and 0.3119g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0080] (3) Dissolving and mixing: 1g of hyaluronic acid crosslinked chondroitin sulfate and 3g of carboxymethyl chitosan (molecular weight 1.6 million Daltons, degree of deacetylation 90%) prepared in step (1) are mixed with 200mL of phosphate buffer in step (2) to prepare a solution with a mass-volume concentration of 0.02g / mL. Stir at room temperature for 180min to fully dissolve the solution.

[0081] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 121℃ for 20 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0082] Example 11: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: 24g of sodium hyaluronate (molecular weight 500,000 Daltons) and 16g of sodium chondroitin sulfate (molecular weight 30,000 Daltons) were weighed and dissolved in 2L of 0.9% sodium chloride solution. The pH was adjusted to 8.0 with sodium hydroxide solution. 0.8g of genipin was added and the reaction was stirred for 6 hours under constant temperature of 30℃. The pH of the system was adjusted to 6.5 with dilute hydrochloric acid to terminate the reaction. Three times the volume of anhydrous ethanol was added to obtain a precipitate. The precipitate was washed three times with purified water and anhydrous ethanol alternately. After being dissolved in 10 times the mass of water for injection, it was freeze-dried under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0083] (2) Preparation of phosphate buffer: Weigh 8.0006g sodium chloride, 1.3521g disodium hydrogen phosphate and 0.2419g sodium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir at room temperature for 10min to fully dissolve them and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0084] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.004 g / mL by mixing 0.8 g of hyaluronic acid crosslinked chondroitin sulfate and 0.2 g of gellan gum (molecular weight 800,000 Daltons) prepared in step (1) with 250 mL of phosphate buffer in step (2) and stir at room temperature for 50 min to fully dissolve it.

[0085] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 123℃ for 12 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0086] Example 12: Preparation method of hyaluronic acid complex polysaccharide composition (1) Crosslinking reaction: Weigh 20g of sodium hyaluronate (molecular weight 1.8 million Daltons) and 20g of sodium chondroitin sulfate (molecular weight 50,000 Daltons) and dissolve them in 2L of 0.9% sodium chloride solution. Add 1.0g of genipin and stir the reaction for 6 hours at a constant temperature of 30℃. Add dilute hydrochloric acid to adjust the pH of the system to 6.5 to terminate the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0087] (2) Preparation of phosphate buffer: Weigh 8.0009g sodium chloride, 1.3536g disodium hydrogen phosphate and 0.2412g sodium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir at room temperature for 10min to fully dissolve them and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0088] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.004 g / mL by mixing 0.8 g of hyaluronic acid cross-linked chondroitin sulfate and 0.6 g of sodium alginate (molecular weight 80,000 Daltons) prepared in step (1) with 350 mL of phosphate buffer in step (2) and stir at room temperature for at least 40 min to ensure complete dissolution.

[0089] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 124℃ for 10 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0090] Example 13: Preparation method of hyaluronic acid complex polysaccharide composition (1) Cross-linking reaction: Weigh 5.5g of sodium hyaluronate (molecular weight 1.2 million Daltons) and 4.5g of sodium chondroitin sulfate (molecular weight 40,000 Daltons) and dissolve them in 2L of 0.9% sodium chloride solution. Add 0.5g of genipin and stir the reaction at 30℃ for 5 hours. Terminate the reaction and add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid cross-linked chondroitin sulfate.

[0091] (2) Preparation of phosphate buffer: Weigh 8.0009g sodium chloride, 1.3536g disodium hydrogen phosphate and 0.2412g sodium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir at room temperature for 10min to fully dissolve them and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0092] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.01 g / mL by mixing 5 g of hyaluronic acid crosslinked chondroitin sulfate and 5 g of carboxymethyl chitosan (molecular weight 1 million Daltons, degree of deacetylation 80%) with 1000 mL of phosphate buffer from step (2) and stir at room temperature for 200 min to fully dissolve it.

[0093] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 126℃ for 6 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0094] Example 14: Preparation method of hyaluronic acid complex polysaccharide composition (1) Cross-linking reaction: Weigh 4.5g of sodium hyaluronate (molecular weight 1.5 million Daltons) and 5.5g of sodium chondroitin sulfate (molecular weight 20,000 Daltons) and dissolve them in 2L of 0.9% sodium chloride solution. Adjust the pH to 6.0 with dilute hydrochloric acid solution, add EDC (0.4g) and NHS (0.8g), and stir the reaction for 6 hours under constant temperature of 25℃. Then terminate the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain hyaluronic acid cross-linked chondroitin sulfate.

[0095] (2) Preparation of phosphate buffer: Weigh 8.0143g sodium chloride, 0.1978g potassium chloride, 2.8714g disodium hydrogen phosphate dodecahydrate and 0.2709g potassium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir at room temperature for 10min to fully dissolve the solution and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0096] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.01 g / mL by mixing 2.5 g of hyaluronic acid crosslinked chondroitin sulfate and 7.5 g of xanthan gum (molecular weight 50,000 Daltons) prepared in step (1) with 1000 mL of phosphate buffer in step (2) and stir at room temperature for 120 min to fully dissolve it.

[0097] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 119°C for 30 minutes to obtain hyaluronic acid complex polysaccharide composition.

[0098] Comparative Example 1 (1) Crosslinking reaction: Weigh 5.5g of sodium hyaluronate (molecular weight 1 million Daltons) and 4.5g of sodium chondroitin sulfate (molecular weight 30,000 Daltons) and dissolve them in 2L of 0.9% sodium chloride solution. Adjust the pH to 5 with dilute hydrochloric acid solution, add EDC (0.6g) and NHS (0.4g), and stir the reaction for 6 hours under constant temperature of 25℃. Then stop the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze dry it under vacuum for 24 hours to obtain hyaluronic acid crosslinked chondroitin sulfate.

[0099] (2) Preparation of phosphate buffer: Weigh 8.0104g sodium chloride, 2.8723g disodium hydrogen phosphate dodecahydrate and 0.3119g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0100] (3) Dissolving and mixing: Prepare a solution by mixing the cross-linked polysaccharide prepared in step (1) with the phosphate buffer solution in step (2) at a ratio of 0.002 g / mL, and stir at room temperature for 30 min to dissolve it completely.

[0101] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 124℃ for 10 minutes.

[0102] Comparative Example 2 (1) Preparation of phosphate buffer: Weigh 8.0011g sodium chloride, 1.3516g disodium hydrogen phosphate and 0.2423g sodium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir for 10min at room temperature to fully dissolve them and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0103] (2) Dissolving and mixing: 5g of sodium hyaluronate (molecular weight 1.5 million Daltons) and 5g of carboxymethyl chitosan (molecular weight 800,000 Daltons, degree of deacetylation 75%) were mixed with 100mL of phosphate buffer from step (1) to prepare a solution with a mass-volume concentration of 0.1g / mL. The solution was stirred at room temperature for 200min to ensure complete dissolution.

[0104] (3) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 123℃ for 12 minutes.

[0105] Comparative Example 3 (1) Preparation of phosphate buffer: Weigh 8.0025g sodium chloride, 1.3521g disodium hydrogen phosphate and 0.2419g sodium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir for 10min at room temperature to fully dissolve them and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0106] (2) Dissolving and mixing: Weigh 3g of sodium hyaluronate (molecular weight 1 million Daltons), 7g of sodium chondroitin sulfate (molecular weight 20,000 Daltons), and 5g of xanthan gum (molecular weight 1.5 million Daltons) and mix with 750mL of phosphate buffer from step (1) to prepare a solution with a mass-volume concentration of 0.02g / mL. Continue stirring at room temperature for 30min to ensure complete dissolution. (3) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 121℃ for 20 minutes.

[0107] Comparative Example 4 (1) Cross-linking reaction: 54g of sodium hyaluronate (molecular weight 1.5 million Daltons) and 6g of sodium alginate (molecular weight 100,000 Daltons) were weighed and dissolved in 2L of 0.9% sodium chloride solution. The pH was adjusted to 8 with sodium hydroxide solution. 20g of genipin was added and the reaction was stirred for 5 hours under constant temperature of 30℃. The reaction was terminated by adding dilute hydrochloric acid to adjust the pH of the system to 6.5. Three times the volume of anhydrous ethanol was added to obtain a precipitate. The precipitate was washed three times alternately with purified water and anhydrous ethanol. After being dissolved in 10 times the mass of water for injection, it was vacuum freeze-dried for 24 hours to obtain cross-linked complex polysaccharide.

[0108] (2) Preparation of phosphate buffer: Weigh 8.0105g sodium chloride, 0.1972g potassium chloride, 2.8709g disodium hydrogen phosphate dodecahydrate and 0.2713g potassium dihydrogen phosphate and dissolve them in 1000ml water for injection. Stir for 10min at room temperature to ensure complete dissolution, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0109] (3) Dissolving and mixing: Prepare a solution with a mass-volume concentration of 0.004 g / mL by mixing 0.8 g of the cross-linked polysaccharide prepared in step (1) and 0.6 g of sodium chondroitin sulfate (molecular weight 30,000 Daltons) with 350 mL of phosphate buffer in step (2), and stir at room temperature for at least 40 min to ensure complete dissolution.

[0110] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 119℃ for 30 minutes.

[0111] Comparative Example 5 (1) Cross-linking reaction: Weigh 10g of chondroitin sulfate sodium (molecular weight 30,000 Daltons) and 90g of guar gum (molecular weight 1,000,000 Daltons) and add them to 1L of 0.9% sodium chloride solution. Adjust the pH to 5.5 with dilute hydrochloric acid solution, add EDC (0.05g) and NHS (0.05g), and stir the reaction for 4 hours under constant temperature of 25℃. Then stop the reaction. Add three times the volume of anhydrous ethanol to obtain a precipitate. Wash the precipitate three times with purified water and anhydrous ethanol alternately. Then dissolve it with 10 times the mass of water for injection and freeze-dry it under vacuum for 24 hours to obtain cross-linked complex polysaccharide.

[0112] (2) Preparation of phosphate buffer: Weigh 8.0056g sodium chloride, 2.8709g disodium hydrogen phosphate dodecahydrate and 0.3124g sodium dihydrogen phosphate dihydrate, stir at room temperature for 10min to dissolve them completely, and filter through a 0.22μm filter membrane to obtain phosphate buffer.

[0113] (3) Dissolving and mixing: 4g of the cross-linked polysaccharide prepared in step (1) and 8g of carboxymethyl chitosan (molecular weight 1 million Daltons, degree of deacetylation 80%) are mixed with 240mL of phosphate buffer in step (2) to prepare a solution with a mass-volume concentration of 0.05g / mL. Stir at room temperature for at least 80min to ensure complete dissolution.

[0114] (4) Filling and sterilization: Fill into glass bottles, cover with rubber stoppers, and use aluminum-plastic composite caps to seal; perform moist heat sterilization at 125℃ for 8 minutes.

[0115] The adhesiveness of the compositions of Examples 1-14, the samples of Comparative Examples 1-5, and the sterile sodium hyaluronate solution (Scitech) produced by Mylan Institutional, Ireland, was tested in an in vitro model experiment. The results are shown in Table 1. The application effect of the above samples in the temporary replacement of mucosal defects in animal interstitial cystitis was also studied. The results are shown in Table 2.

[0116] Test Example 1: In Vitro Model Test of Adhesion Experimental protocol: Using a fresh canine isolated bladder model at 37°C, to simulate cystoscopic stent instillation, samples were instilled via a single 5F ureteral catheter. Sample adhesion was then assessed by rinsing with PBS buffer and compared with commercially available sterile sodium hyaluronate solution (Scistar). The content of active substances in the initial instilled sample, the emptied sample, and the washing solution in the canine bladder was measured. Based on the active substance content, the mass fraction of the residual amount of sample adhering to the bladder mucosa after the rinsing test was calculated relative to the original amount adhered (rinsing adhesion rate).

[0117] Experimental procedure: Add PBS buffer to a beaker and keep it at a constant temperature of 37°C. Place the animal's bladder with the opening facing upwards in the buffer solution. External force can be used to submerge the outer wall of the bladder below the liquid surface to maintain the infiltration state of the outer wall of the bladder tissue.

[0118] Empty bladder instillation: Using a disposable syringe connected to a 5F single-use ureteral catheter, 30 mL of sample was injected into an isolated canine bladder, maintaining contact between the sample and the bladder mucosa for 30 min. After the fixed time, the instilled sample was poured out and collected. The initial amount of sample adhering to the mucosa was detected and calculated. The isolated canine bladder was then rinsed with PBS buffer: 30 mL of PBS buffer was injected into the bladder to simulate the physiological state of a full bladder. After standing for 15 minutes, the buffer solution was collected in a test tube and stored at 2-8℃. This cycle was repeated twice (using a total of 3 rinses with PBS buffer). The mass fraction of the sample remaining on the bladder mucosa after the rinsing test relative to the original amount of adhering sample was detected and calculated (rinsing adhesion rate).

[0119] Experimental results: Table 1 shows the results of the wash-adhesion rate test of each sample on isolated canine bladder mucosa tissue after rinsing with PBS buffer 3 times.

[0120] Table 1. Results of the flushing adhesion rate test of each sample on isolated canine bladder mucosa.

[0121] In Comparative Example 2, the sample viscosity was too high to pass through the 5F catheter, so the adhesion test could not be performed and no corresponding data were available.

[0122] The data in Table 1 shows that the final adhesion rate of the product compositions in the example group of the hyaluronic acid complex polysaccharide composition of the present invention on isolated canine bladder mucosa is higher than that of the comparative example product and the marketed control product. This proves that the hyaluronic acid complex polysaccharide composition of the present invention can improve the adhesion of the product on the mucosal surface, and the composition of Example 13 has the highest final adhesion rate after rinsing test, which still adheres to the bladder mucosa.

[0123] Test Example 2: Animal Experiment A rat model of interstitial cystitis was established by a single intraperitoneal injection of cyclophosphamide (CYP) at a dose of 150 mg / kg. Forty-eight hours after the intraperitoneal injection of cyclophosphamide, the rat models were divided into a blank control group, a marketed control group, and a combination group (Examples 1-14, Comparative Examples 1-5).

[0124] Rats were anesthetized and fixed. A 3F catheter was inserted into the rat bladder via the urethra, and a 1ml syringe was properly connected. In the blank control group, only urethral cannulation and extubation were performed, once every other day for three consecutive times, followed by sacrifice. In the market control group and the composition group, each animal was instilled with 0.5ml of the sample, retained for 30 minutes, once every other day for three consecutive times, followed by sacrifice. HE staining of bladder tissue showed mononuclear inflammatory cells, and toluidine blue staining showed mast cells. Table 2 shows the number of mononuclear inflammatory cells and mast cells in each group.

[0125] Table 2. Number of mononuclear inflammatory cells and mast cells in rats of each sample group

[0126] In Comparative Example 2, the sample viscosity was too high to complete bladder instillation through the 3F catheter, so no animal experiments were conducted and no corresponding data were available.

[0127] As can be seen from the data in Table 2, the compositions of each embodiment can reduce bacterial adhesion and inflammatory response, which is significantly better than the comparative example group and the marketed control group. This is because the cross-linking and synergistic effect of the composite polysaccharide composition affects the adhesion and flushing resistance of the composition product to the bladder mucosa tissue. It also proves that the hyaluronic acid composite polysaccharide composition prepared by the present invention can improve the temporary replacement effect of the product as a glycosaminoglycan protective layer.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hyaluronic acid complex polysaccharide composition for bladder instillation, characterized in that, The effective components of the hyaluronic acid complex polysaccharide composition include hyaluronic acid cross-linked chondroitin sulfate and high molecular weight mucopolysaccharide; the mass ratio of hyaluronic acid cross-linked chondroitin sulfate to high molecular weight mucopolysaccharide is 1:4-4:1; The hyaluronic acid-crosslinked chondroitin sulfate is obtained by crosslinking hyaluronic acid and chondroitin sulfate. The crosslinking agent used is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or genipin. When 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are used, the molar ratio of the two is 1:1 to 1:4, and the ratio of the total mass of the crosslinking agent to the total mass of hyaluronic acid and chondroitin sulfate is 1:5 to 1:

100. The mass ratio of hyaluronic acid to chondroitin sulfate is 1:9-9:1; The hyaluronic acid is sodium hyaluronate or zinc hyaluronate; the molecular weight of the hyaluronic acid is 300,000 to 2,000,000 Daltons. The chondroitin sulfate is sodium chondroitin sulfate, with a molecular weight of 20,000-60,000 Daltons; The high molecular weight polysaccharide includes one or more of chitosan, sodium alginate, xanthan gum, guar gum, sodium carboxymethyl cellulose, and gellan gum; The molecular weight of the high-molecular-weight mucopolysaccharide is 5-3 million Daltons; The hyaluronic acid crosslinked with chondroitin sulfate and the high molecular weight mucopolysaccharide were mixed and dissolved in phosphate buffer.

2. The method for preparing the hyaluronic acid complex polysaccharide composition for bladder instillation according to claim 1, characterized in that, The preparation method includes: mixing and dissolving hyaluronic acid-crosslinked chondroitin sulfate and high molecular weight mucopolysaccharide in phosphate buffer; The preparation method of the hyaluronic acid crosslinked chondroitin sulfate includes: dissolving sodium hyaluronate or zinc hyaluronate and sodium chondroitin sulfate in sodium chloride solution, adding a crosslinking agent and reacting to obtain the product; The specific conditions for the crosslinking reaction are: reaction temperature 18-35℃; The pH of the cross-linking reaction system is 5.0-7.0; the stirring time for the cross-linking reaction is 2-12 hours.

3. The method for preparing the hyaluronic acid complex polysaccharide composition for bladder instillation as described in claim 2, characterized in that, The concentration of the sodium chloride solution is 0.5-1.5%.

4. The method for preparing the hyaluronic acid complex polysaccharide composition for bladder instillation as described in claim 2, characterized in that, The preparation method further includes a step of sterilizing the hyaluronic acid complex polysaccharide composition.

5. The method for preparing the hyaluronic acid complex polysaccharide composition for bladder instillation as described in claim 4, characterized in that, The sterilization is performed by moist heat sterilization.

6. The use of the hyaluronic acid complex polysaccharide composition according to claim 1 in the preparation of a product, wherein the product is used for interstitial cystitis, radiation cystitis, chronic recurrent urinary tract infection, and overactive bladder.

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