Compound sodium hyaluronate solution as well as preparation method and application thereof

Abstract

The invention relates to the field of third-class medical instruments, in particular to a compound sodium hyaluronate solution as well as a preparation method and application thereof, the compound sodium hyaluronate solution comprises sodium hyaluronate, a pH regulator and water for injection; the sodium hyaluronate comprises medium molecular weight sodium hyaluronate with the molecular weight of 500 KDa to 1000 KDa and low molecular weight sodium hyaluronate with the molecular weight of 20 KDa to 200 KDa. The sodium hyaluronate in the bladder perfusion sodium hyaluronate liquid is grouped, so that instant repair of a damaged GAG layer of a human body is realized, and the duration of a repaired sodium hyaluronate physical protection layer can be maintained. Meanwhile, a part of low-molecular-weight sodium hyaluronate can enter an extracellular matrix while a GAG layer is repaired, so that the purposes of resisting inflammation and promoting angiogenesis are achieved.

Description

Technical Field

[0001] The present invention relates to the field of Class III medical devices, and in particular to a compound sodium hyaluronate solution and a preparation method and application thereof. Background Art

[0002] Nonbacterial cystitis is a chronic inflammatory condition of the bladder with complex etiology, difficult diagnosis, and incurable clinically. The main clinical manifestations are frequent urination, nocturia, urgency, and suprapubic bladder pain. Some patients may also experience urinary pain and hematuria, and no bacteria grow in urine culture. Studies have shown that the occurrence of nonbacterial cystitis is related to the damage of the protective layer of glycosaminoglycan (GAG) in the bladder mucosal barrier of the bladder epithelium.

[0003] After many clinical studies by researchers, it was found that intravesical injection of sterile sodium hyaluronate solution can significantly improve the clinical symptoms of patients and is of great help in the treatment of non-bacterial cystitis. Therefore, the development and application of sterile sodium hyaluronate solution is worth promoting and has great significance.

[0004] At first, Bioniche used the world's first bladder instillation of sodium hyaluronate solution (Cisita) to treat interstitial cystitis. Its efficacy has been confirmed by clinical trials, achieving the purpose of alleviating pain and relieving urinary urgency. To date, many domestic and foreign studies and clinical reports have shown that sodium hyaluronate solution (Cisita) has achieved good therapeutic effects. Even in China, most of the research is focused on Cisita, and there are still few studies on domestic sterile sodium hyaluronate solution.

[0005] The principle of the currently used sodium hyaluronate solution for bladder instillation is that it can form a physical protective layer on the bladder epithelium. Due to its large molecular weight, it can play a better barrier function. However, in actual use, we found that when the molecular weight of sodium hyaluronate increases to a certain level, its compatibility with the blood-urine barrier will decrease. Therefore, in actual use, high-molecular-weight sodium hyaluronate is easily washed away by urine, resulting in the defect of a shorter duration of the protective layer. Summary of the invention

[0006] The present invention aims to overcome the defects of the prior art sodium hyaluronate solution for bladder instillation, which has a short duration and an insignificant repair effect on bladder epithelial cells, and provides a compound sodium hyaluronate solution and a preparation method, a filling system and an application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the present invention is implemented by the following technical solutions: In a first aspect, the present invention first provides a compound sodium hyaluronate solution, which comprises Sodium hyaluronate, pH adjuster and water for injection; The sodium hyaluronate includes: medium molecular weight sodium hyaluronate with a molecular weight of 500KDa to 1000KDa, and low molecular weight sodium hyaluronate with a molecular weight of 20KDa to 200KDa.

[0008] Compared with the prior art which uses a bladder perfusion solution containing a single component of sodium hyaluronate, the present invention divides sodium hyaluronate into two different groups according to molecular weight in order to achieve better protection and repair of the glucosamine protective layer of the bladder epithelium.

[0009] This includes medium molecular weight sodium hyaluronate with a molecular weight of 500KDa to 1000KDa. First of all, this part of sodium hyaluronate is close to the molecular weight of sodium hyaluronate in the normal GAG layer of the human body, so it can merge with the damaged GAG layer of the human body, thereby ensuring compatibility with the blood-urine barrier, making the combination between the two tighter, thereby preventing this part of sodium hyaluronate from being washed away by urine after use, and losing its protective effect on the damaged part of the bladder epithelial glucosamine protective layer.

[0010] At the same time, for this part of sodium hyaluronate, the inventors found that the molecular weight of sodium hyaluronate has a significant effect on the protective effect of the damaged part of the bladder epithelial glucosamine protective layer. When the molecular weight of sodium hyaluronate is too high (greater than 1000KDa), the degree of self-crosslinking will be higher, and the sodium hyaluronate will tend to be stable at this time, so it will affect the absorption and fusion effect with the GAG ​​layer when injected into the bladder, resulting in a shorter duration of the protective layer. When its molecular weight is too small (less than 500KDa), it is easily washed off, resulting in a weak barrier function for the GAG ​​layer, which is not conducive to alleviating pain and relieving urinary urgency.

[0011] In addition, the present invention also includes another part of low molecular weight sodium hyaluronate with a molecular weight of 20KDa to 200KDa. Compared with the above-mentioned medium molecular weight sodium hyaluronate, this part of sodium hyaluronate can be used as the main component of the extracellular matrix, thereby entering the extracellular matrix and combining with some receptors (ICAM-1, RHAMM, CD44) expressed by bladder epithelial cells, thereby regulating cell proliferation, differentiation and adhesion, and ultimately achieving anti-inflammatory, angiogenesis and other functions.

[0012] For this part of sodium hyaluronate, the inventors found that when the molecular weight of this part of sodium hyaluronate is too high (greater than 200KDa), its efficiency of entering cells is greatly reduced, so the effect of anti-inflammation and promoting angiogenesis will be greatly reduced. When its molecular weight is too small (less than 20KDa), it is more easily washed away by urine inside the bladder, so it is not conducive to achieving the purpose of anti-inflammation and promoting angiogenesis.

[0013] Therefore, in summary, the present invention divides the sodium hyaluronate in the sodium hyaluronate solution for bladder instillation into groups, which not only achieves immediate repair of the damaged GAG layer of the human body, but also maintains the duration of the physical protective layer of the repaired sodium hyaluronate. At the same time, while repairing the GAG ​​layer, a part of the low molecular weight sodium hyaluronate can also enter the extracellular matrix, thereby achieving the purpose of anti-inflammation and promoting angiogenesis.

[0014] Preferably, the composition comprises medium molecular weight sodium hyaluronate at a concentration of 0.6 to 0.9 mg / mL, low molecular weight sodium hyaluronate at a concentration of 0.2 to 0.5 mg / mL, and a pH adjuster at a concentration of 8.43 to 10.65 g / L.

[0015] Preferably, the pH adjuster includes sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride.

[0016] Preferably, the concentration of the sodium dihydrogen phosphate is 0.03-0.05 g / L, the concentration of the disodium hydrogen phosphate is 0.4-0.6 g / L, and the concentration of the sodium chloride is 8-10 g / L.

[0017] Preferably, the active ingredient is obtained by combining one or more of chondroitin, heparin, aminopolysaccharide and pentosan.

[0018] In addition to two kinds of sodium hyaluronate with different molecular weights, the present invention also adds a certain amount of active ingredients to the composition. These active ingredients can form extracellular space together with the small molecular weight sodium hyaluronate and the water contained therein, which is not only beneficial to the migration of epidermal cells and the entry and exit of immune cells in the epidermis, but also beneficial to the supply of nutrients and the discharge of cell metabolic products.

[0019] Preferably, the molecular weight of the chondroitin is 10KDa to 40KDa, and the concentration is 0 to 0.4mg / mL; The molecular weight of the heparin is 5KDa to 20KDa, and its concentration is 0 to 0.5mg / mL; The glucosamine accounts for 0-1 wt% of the system mass.

[0020] In a second aspect, the present invention also provides a method for preparing the compound sodium hyaluronate solution, comprising the following steps: (1) Add all raw materials into a sterile batching tank and stir to mix evenly; (2) Pressurize under nitrogen protection until completely dissolved and stirred; (3) Move the batching tank to the filling room for filling.

[0021] Preferably, the pressure in step (1) is controlled at 0.1 MPa to 0.2 MPa; The stirring time is not less than 17 hours.

[0022] In a third aspect, the present invention further provides a semi-automatic filling system for filling the compound sodium hyaluronate solution, comprising: A batching tank, which is used to dissolve the components in the compound sodium hyaluronate solution, comprising a tank body, the tank body is provided with a stirring device for stirring the internal materials and a regulating frequency converter for controlling the stirring rate of the stirring device, the tank body is provided with a feeding port and a discharging port for feeding and discharging materials, and the tank body is also provided with a nitrogen inlet for introducing nitrogen; A filter, which is used to filter the prepared materials in the batching tank, and includes a first filter and a second filter connected in series, wherein the first filter is connected to the discharge port; The filling glass tube is connected with the second filter so as to be used for filling the material flowing out of the filter.

[0023] In a fifth aspect, the present invention also provides the use of the compound sodium hyaluronate solution in a drug or medical device for repairing the glucosamine protective layer of the bladder epithelium.

[0024] Therefore, the present invention has the following beneficial effects: (1) The compound sodium hyaluronate solution of the present invention can form a physical protective layer on the glucosamine protective layer of the bladder epithelium, thereby providing a long-lasting and more effective barrier function; (2) While protecting the bladder epithelium with glucosamine, it can also allow a portion of low molecular weight sodium hyaluronate to enter the extracellular matrix, thereby achieving the purpose of anti-inflammation and promoting angiogenesis; (3) The present invention also adds active ingredients to the compound sodium hyaluronate solution, so that it can form an extracellular space together with the small molecular weight sodium hyaluronate and the water contained therein, which is not only beneficial to the migration of epidermal cells and the entry and exit of immune cells in the epidermis, but also beneficial to the supply of nutrients and the discharge of cell metabolic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the structure of the semi-automatic filling system in the present invention.

[0026] Figure 2 Figure 3 shows the changes in body weight of experimental rats.

[0027] Figure 3 is the pain threshold of the rat bladder surface area.

[0028] Figure 4is the pain threshold of the bladder surface area of ​​rats in the control experiment.

[0029] Among them: a batching tank 100, a tank body 110, a stirring device 120, an adjusting frequency converter 130, a feeding port 140, a discharge port 150, a nitrogen inlet 160, a filter 200, a first filter 210, a second filter 220, and a filling glass tube 300. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0031] Example 1 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0032] Example 2 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0033] Example 3 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0034] Example 4 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0035] Example 5 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0036] Example 6 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0037] Example 7 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0038] Example 8 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0039] Example 9 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0040] Example 10 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0041] Embodiment 11 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0042] Example 12 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0043] Embodiment 13 A compound sodium hyaluronate solution, which is composed of materials with the following molecular weights and concentrations:

[0044] The preparation process of the compounded sodium hyaluronate solution in Examples 1 to 13 is as follows: Weigh the raw materials in Examples 1 to 13 and the compounded sodium hyaluronate solution in Examples 1 to 8 according to Table 1, and then slowly add them to the physiological balance solution in the sterile batching tank. Adjust the frequency converter to stir at 50±2 rpm. After the feeding is completed, close the feed port. Turn on the nitrogen to control the pressure at 0.1MPa to 0.2MPa. Stir for no less than 17 hours to completely dissolve into a colorless, transparent, clear liquid. After overnight, move the batching tank to the semi-automatic filling system in the filling room for filling, and the filling volume is 50 to 54 ml / bottle.

[0045] The structure of the semi-automatic filling system is as follows Figure 1 As shown, it includes: A batching tank 100, which is used to dissolve the components in the compound sodium hyaluronate solution in Examples 1 to 13, comprises a tank body 110, the tank body 100 is provided with a stirring device 120 for stirring the internal material and a regulating frequency converter 130 for controlling the stirring rate of the stirring device 120, the tank body 110 is provided with a feeding port 140 and a discharging port 150 for feeding and discharging materials, and the tank body 110 is also provided with a nitrogen inlet 160 for introducing nitrogen; The filter 200 is used to filter the prepared materials in the batching tank 100, and includes a first filter 210 and a second filter 220 connected in series, wherein the first filter 210 is connected to the discharge port 150; The filling glass tube 300 is connected to the second filter 220 , so as to be used for filling the material flowing out of the filter 200 .

[0046]

Performance test

[0047]

Weight average molecular weight and its distribution test

[0048] Molecular weight of sodium hyaluronate was determined by using an 18-angle laser scattering instrument coupled with size exclusion chromatography (MALLS-GPC).

[0049] The test results are shown in Table 2 below: Table 2 [Product test results]

[0050] The product performance requirements of the compound sodium hyaluronate solution described in the present invention are shown in Table 4 below: Table 4

[0051] [Verification of the effectiveness of compound sodium hyaluronate solution for interstitial cystitis] 1. Basic Information Interstitial cystitis is a chronic inflammatory disease of the bladder, and chronic pain seriously affects the quality of life of patients. The main manifestations are frequent urination, urgency, bladder or pelvic pain, reduced urination, etc., and more than 80% of patients are women. Cyclophosphamide injection is a common irritant that causes interstitial cystitis in animal models. Cyclophosphamide or chemical irritants such as its liver metabolite acrolein can directly damage urothelial cells, causing varying degrees of bladder mucosal erosion, edema, bleeding and leukocyte infiltration. The compound sodium hyaluronate solution in this application can be used as a protective agent for interstitial cystitis and defects in the bladder epithelial glucosamine protective layer caused by various reasons.

[0052] 2. Test products, reference products and solvents 2.1 Test products Name: Compound sodium hyaluronate solution Batch number: 23101721 Content: 40mg / 50ml / bottle Valid until: 2026.10.16 Storage conditions: Store at room temperature, do not freeze Dosage form: liquid preparation.

[0053] 2.2 Reference substances 2.2.1 Negative Control Name: Sodium Chloride Injection Batch number: A2305231 Content: 0.9% Valid until: 2025.05 Storage conditions: 10~30℃, sealed Dosage form: liquid preparation.

[0054] 2.2.2 Marketed reference substances Name: Sterile sodium hyaluronate solution (Sista) Batch number: 221215 Content: 40mg / 50ml Valid until: 2025.12.08 Storage conditions: Store at room temperature, do not freeze Dosage form: liquid preparation.

[0055] 2.3 Solvents and Modeling Reagents 2.3.1 Solvents Name: Sodium Chloride Injection Batch number: A2305231 Content: 0.9% Ingredients: The main ingredient is sodium chloride Physical and chemical properties: Colorless clear liquid Valid until: April 2025 Storage conditions: 10~30℃, sealed Production unit: Hangzhou Minsheng Pharmaceutical Co., Ltd.

[0056] 2.3.2 Modeling reagents Name: Cyclophosphamide Batch number: JS238602 Content: 97%, BR Ingredients: The main ingredient is cyclophosphamide Physical and chemical properties: white powder Storage conditions: 2~8℃, sealed Production unit: Yuanye Biotechnology Co., Ltd.

[0057] 2.4 Preparation, labeling and storage of drug preparations 2.4.1 Preparation of drug delivery preparations Negative control substances, marketed control substances, and test substances are tested in stock form without the need for preparation.

[0058] Preparation of modeling reagents: Weigh cyclophosphamide and prepare it into a 175 mg / 10 mL concentration solution with normal saline.

[0059] 2.4.2 Labeling of drug preparations Negative control substances, marketed control substances and test substances are packaged in-house, stored in airtight containers and opened for immediate use.

[0060] The modeling preparation uses a white label, marked as 175 mg / kg / 10 mL, and is prepared for use immediately.

[0061] 2.4.3 Storage conditions of drug preparations Negative control substances, marketed control substances, and test substances should be sealed before administration and stored at room temperature.

[0062] The modeling preparations were stored at room temperature before administration.

[0063] 2.5 Disposal of test articles and drug preparations The remaining preparations after the test will be discarded by the experimental operator on the day when the administration is completed.

[0064] Unused positive control samples will be disposed of by the sample room after the experiment and handed over to the providing unit for disposal after the project is completed.

[0065] 3. Main instruments and reagents 3.1 Main instruments

[0066] 3.2 Reagents

[0067] 4. Experimental system 4.1 Experimental animals 4.1.1 Species, strains and grades Species: Rat Strain: Sprague-Dawley Grade: SPF level.

[0068] 4.1.2 Gender and quantity of animals Quantity to be purchased: 115 female animals.

[0069] Planned number of animals to be used: 80 female animals.

[0070] 4.1.3 Age and Weight Application age range: 6-8 weeks old.

[0071] Weight range for application: 200g~220g.

[0072] Age range at the start of the experiment: 6-8 weeks old.

[0073] Body weight range at the beginning of the experiment: 237.3g~272.5g.

[0074] 4.1.4 Animal sources Laboratory Animal Production Unit: Animal Center of Hangzhou Medical College Production License No.: SCXK(Zhejiang)2019-0002 Laboratory Animal Quality Certificate No.: 20240705Aazz000018663 4.1.5 Animal Species SD rats and dogs are frequently used as model animals. According to the requirements of the client, this test system selected SD rats as the test subjects.

[0075] 4.1.6 Animal identification methods Identification during the adaptation period: During the quarantine adaptation period, white cage cards and tail tags are used as animal identification marks, and the animal's gender, cage number, project leader, project number, etc. are indicated.

[0076] Identification during the experiment: Use white cage cards and ear tags as animal identification marks, and indicate the experimental animal number, cage number, group, experimental animal gender, project leader, project number, etc. on the cage card.

[0077] 4.2 Animal feeding and management 4.2.1 Rearing environment Temperature: Control the temperature at 20-26℃.

[0078] Relative humidity: Control relative humidity at 40% to 70%.

[0079] Lighting: Automatic lighting, 12 / 12h light / dark alternation.

[0080] 4.2.2 Feeding SD rats were housed in a barrier facility environment, with ≤5 rats per cage, and the experimental animals had free access to water and food.

[0081] 4.2.3 Veterinary care Veterinarians monitored the animals' health throughout the experiment; no animals received veterinary treatment during the experiment.

[0082] 4.2.4 Feed Feed name: SPF maintenance feed for mice and rats.

[0083] Manufacturer: Keao Xieli (Tianjin) Feed Co., Ltd.

[0084] 4.2.5 Drinking water Drinking water for experimental animals.

[0085] 4.3 Animal health screening before the experiment 4.3.1 Animal quarantine and health screening After arrival, the animals will be acclimated and raised in the barrier environment. Before the test, the test personnel will conduct pre-test observations. A pre-test animal health summary will be conducted based on the animal health screening recommendations made by the veterinarian during the quarantine period and the animal observation results during the acclimatization period.

[0086] 4.3.2 Adaptive feeding Adaptation period: 3 days, during which the experimenter shall complete cage observation. The observation results during the adaptation period indicate that the animals are in good health and can be used for the experiment.

[0087] 5. Pre-experimental plan 5.1 Modeling scheme The model was established by intraperitoneal injection of 175 mg / kg cyclophosphamide solution. Cyclophosphamide was prepared with normal saline and used immediately after preparation.

[0088] 5.2 Dosage regimen According to the clinical administration route of the test article, normal saline, test article or marketed control article is instilled into the bladder.

[0089] 5.3 Animal grouping The 80 screened female rats were divided into 8 groups according to their body weight using a stratified random method, namely A (normal group), C (model group), E (test sample treatment group in Example 1 once), F (test sample treatment group in Example 1 twice), G (test sample treatment group in Example 1 three times), H (marketed reference sample treatment group once), I (marketed reference sample treatment group twice), and J (marketed reference sample treatment group three times), with 10 rats in each group.

[0090] 5.4 Route of administration and reasons for selection According to the intended clinical route of use of the test article, the drug is administered by intravesical instillation.

[0091] 5.5 Administration Bladder instillation.

[0092] 5.6 Dosing frequency, duration and reasons for selection According to the planned clinical administration frequency of the test article, the drug is administered once every two days for a total of three times, and the test article stays in the bladder for at least 30 minutes.

[0093] 5.7 Dosing volume 0.5mL / piece.

[0094] 6. Test Methods 6.1 Health Observation and Weight Recording Animals were observed daily, and the individual status of mice, pathological manifestations, animal body shape and coat color, etc. were recorded. If severe adverse reactions or dead animals were found, their status was recorded immediately or gross dissection was performed, and the number and status of severe adverse reactions or animal deaths in each group of animals were counted.

[0095] After modeling, body weight was tested every day, and the body weight change curve was used to observe whether there were any differences in body weight changes among the model animals, test product group animals, and competitor group animals.

[0096] 6.2 Pain measurement behavior Interstitial cystitis has symptoms such as pain, and the product can be evaluated from the perspective of pain relief. VonFrey was used to stimulate the bladder surface area just below the lower abdomen and pelvis of the rat, so that the fiber slowly bent and presented an "s" shape. After stimulation, the rat showed abdominal contraction, jumping and bending of the trunk, which was counted as a positive reaction. Otherwise, the weight of the fiber was increased and the above method was repeated. The fiber stimulation can only be performed when the rat is not walking, not eating, and is quiet, and all four limbs are on the ground. The measurement time interval is 10 minutes, and at least 3 measurements are taken to take the average value. Pain measurement was performed 72 hours after modeling (Day 3), 24 hours after the first bladder instillation (Day 4), 24 hours after the second bladder instillation (Day 6), and 24 hours after the third bladder instillation (Day 8).

[0097] 6.3 Urine leukocyte and nitrite detection Interstitial cystitis is a non-bacterial cystitis. Leukocyte and nitrite tests are used to exclude bacterial infection. Urine was collected 72 hours after modeling (Day 3) and 24 hours after the third bladder instillation (Day 8) for urine leukocyte and nitrite tests to exclude the influence of bacterial infection on the experimental results.

[0098] 6.4 Sample Storage After the biological samples are collected, they are stored in a -80℃ ultra-low temperature refrigerator. The storage of biological samples is monitored and recorded in accordance with current regulations. After the sample test is completed, the test samples need to be stored for a certain period of time in accordance with the regulations or the SOP of the sample testing unit. After the storage period, they can be destroyed only with the written consent of the client.

[0099] 6.5 Animal treatment Euthanasia: After the experiment, the animals were euthanized by bleeding under anesthesia.

[0100] 7. Data Collection and Analysis After the test was completed, the values ​​of each index were counted, and the average values ​​of multiple experimental data were calculated. GraphPad Prism 8 software was used to summarize and analyze the data, make statistical graphs, analyze significant differences, and issue a test report. The qualitative analysis results such as urine test, blood routine test, and HE pathological staining test were presented in the form of charts. The data were expressed as Mean ± SEM, and GraphPad Prism 8 software was used to perform statistical Two-way ANOVA Tukey's multiple comparisons test to evaluate the data. * is P < 0.05, ** is P < 0.01, and *** is P < 0.001, all of which are significantly different.

[0101] 8. Experimental results 8.1 Validation of cyclophosphamide modeling results After intraperitoneal administration of 175 mg / kg of cyclophosphamide solution, urine was collected on Day 3 for urine nitrite detection, and blood was collected for leukocyte detection from animals in Group A (normal), Group C (model), Group E (one treatment with the test product in Example 1), Group F (two treatments with the test product in Example 1), Group G (three treatments with the test product in Example 1), Group H (one treatment with the marketed control product), Group I (two treatments with the marketed control product), and Group J (three treatments with the marketed control product).

[0102] The test results showed that 93.75% of the experimental animals did not show positive reactions in urine nitrite and leukocyte tests, indicating that the model was successfully established and has research significance.

[0103] 8.2 Effect of sterile sodium hyaluronate solution on weight changes in interstitial cystitis model animals Cyclophosphamide, as a broad-spectrum anti-tumor drug, will affect the animal body and cause the animal weight to decrease. The weight monitoring data during the experiment are as follows Figure 2 Compared with the C (model) group, there was no significant change in body weight in each treatment group.

[0104] 8.3 Effect of sterile sodium hyaluronate solution on pain threshold of bladder surface in interstitial cystitis model animals Use Von Frey to stimulate the bladder surface area just below the lower abdomen and pelvis of rats, record the weight of the fiber when pain occurs, repeat 3 times, each time with an interval of 10 minutes, and take the average value of 3 times as the pain threshold. Figure 3 .

[0105] Compared with the C (model) group, 24 days after the third bladder instillation on Day 8, the pain thresholds of the F (secondary treatment with the test product of Example 1) group (P < 0.01), the G (three-time treatment with the test product of Example 1) group (P < 0.001), and the J (three-time treatment with the listed control product) group (P < 0.01) increased significantly, and the sterile sodium hyaluronate solution and the positive listed product can relieve the bladder pain caused by interstitial cystitis in a dose-dependent manner (secondary instillation, three-time instillation). It is suggested that the sterile sodium hyaluronate solution of the test product and the positive listed product have a certain effect on relieving the bladder pain caused by interstitial cystitis after three treatments.

[0106] 8.4 Control experiment Experimental purpose: To explore the effect of high molecular weight sterile sodium hyaluronate solution on the pain threshold of the bladder surface of interstitial cystitis model animals. The groups in the experiment were the same as the formal experiment. Methods: 8-week-old female SD rats were selected and randomly divided into a normal group (n=8), a blank group (n=12), a model group (175 mg / kg cyclophosphamide) (n=16), a control test group (molecular weight of a single 1500KDa) (n=16), and a positive drug group (n=16).

[0107] The weight changes during the whole experiment were recorded, the skin pain in the bladder area of ​​the mice was analyzed using a von Frey fiber analgesia instrument, urodynamic analysis was performed using a multichannel physiological instrument, urine routine tests were used to qualitatively analyze the urine leukocyte and nitrite content, HE pathological staining was used for semi-quantitative analysis of bladder tissue pathological damage, and methylcobalamin blue staining was used to analyze mast cell counts.

[0108] Conclusion of the control experiment: The weight of the mice began to recover after the second week, and it is speculated that the damage caused by cyclophosphamide may be reduced after two weeks. Figure 4 As can be seen from the data chart, the control test product sterile sodium hyaluronate and the positive drug Cistanche both have a protective effect on the pain damage caused by cyclophosphamide, but the control test product has a weaker protective effect on the pain damage of the positive drug Cistanche. Urodynamics is not statistically significant. However, during the experiment, it was also found that the mortality rate of the control test product group was extremely high, and it was speculated whether there was an injury-aggravating effect or sample contamination or a high dose of cyclophosphamide.

[0109] Therefore, in summary, the present invention divides the sodium hyaluronate in the sodium hyaluronate solution for bladder instillation into groups, which not only achieves immediate repair of the damaged GAG layer of the human body, but also maintains the duration of the physical protective layer of the repaired sodium hyaluronate. At the same time, while repairing the GAG ​​layer, a portion of the low molecular weight sodium hyaluronate can also enter the extracellular matrix, thereby achieving the purpose of dose-dependently relieving the pain caused by cyclophosphamide-induced interstitial cystitis in SD rats.

Claims

1. A compound sodium hyaluronate solution, characterized in that: include Sodium hyaluronate, pH adjuster and water for injection; The sodium hyaluronate includes: medium molecular weight sodium hyaluronate with a molecular weight of 500KDa to 1000KDa, and low molecular weight sodium hyaluronate with a molecular weight of 20KDa to 200KDa.

2. A compound sodium hyaluronate solution according to claim 1, characterized in that: The invention comprises a medium molecular weight sodium hyaluronate with a concentration of 0.6 to 0.9 mg / mL, a low molecular weight sodium hyaluronate with a concentration of 0.2 to 0.5 mg / mL, and a pH adjuster with a concentration of 8.43 to 10.65 g / L.

3. A compound sodium hyaluronate solution according to claim 1, characterized in that: The pH adjuster includes sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride.

4. A compound sodium hyaluronate solution according to claim 3, characterized in that: The concentration of the sodium dihydrogen phosphate is 0.03-0.05 g / L, the concentration of the disodium hydrogen phosphate is 0.4-0.6 g / L, and the concentration of the sodium chloride is 8-10 g / L.

5. A compound sodium hyaluronate solution according to any one of claims 1 to 4, characterized in that: It also includes active ingredients obtained from one or more combinations of chondroitin, heparin, aminopolysaccharide, and pentosan.

6. A compound sodium hyaluronate solution according to claim 5, characterized in that: The molecular weight of the chondroitin is 10KDa to 40KDa, and the concentration is 0 to 0.4mg / mL; The molecular weight of the heparin is 5KDa to 20KDa, and its concentration is 0 to 0.5mg / mL; The glucosamine accounts for 0-1 wt% of the system mass.

7. A method for preparing the compound sodium hyaluronate solution according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Add all raw materials into a sterile batching tank and stir to mix evenly; (2) Pressurize under nitrogen protection until completely dissolved and stirred; (3) Move the batching tank to the filling room for filling.

8. The method according to claim 7, characterized in that In the step (1), the pressure is controlled at 0.1 MPa to 0.2 MPa; The stirring time is not less than 17 hours.

9. A semi-automatic filling system for filling the compound sodium hyaluronate solution according to any one of claims 1 to 6, characterized in that: include: A batching tank (100) for dissolving the components in the composite sodium hyaluronate solution according to any one of claims 1 to 6, comprising a tank body (110), the tank body (100) being provided with a stirring device (120) for stirring the internal material and a regulating frequency converter (130) for controlling the stirring rate of the stirring device (120), the tank body (110) being provided with a feeding port (140) and a discharging port (150) for feeding and discharging materials, and the tank body (110) being further provided with a nitrogen inlet (160) for introducing nitrogen; A filter (200) is used to filter the prepared materials in the batching tank (100), and comprises two first filters (210) and a second filter (220) connected in series, wherein the first filter (210) is connected to the discharge port (150); The filling glass tube (300) is connected to the second filter (220) so as to be used for filling the material flowing out of the filter (200).

10. Use of the compound sodium hyaluronate solution according to any one of claims 1 to 6 in a drug or medical device for repairing the glucosamine protective layer of the bladder epithelium.

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