Medicine formula for repairing eardrum calcification and preparation method

Abstract

The invention relates to a medicine formula for repairing eardrum calcification and a preparation method. The collagen peptide III and the citric acid-chitosan conjugate are combined to realize combination of active repair and active decalcification, so that a repair material is provided, the problem of calcium salt deposition is solved from the pathogenesis, and both symptoms and root causes are treated. In-situ gelation and long-acting slow release of the medicine are realized by utilizing the temperature-sensitive hydrogel, the defect of short residence time of a traditional liquid preparation is overcome, and the availability and the treatment effect of the medicine are improved. Through the nanoemulsion, the penetrability of the medicine components to eardrum cuticle is remarkably improved, and it is ensured that the active components can effectively reach the focus. The citric acid-chitosan conjugate can be degraded, and citric acid is an intermediate product of tricarboxylic acid circulation in vivo and is high in safety. The anti-inflammatory component, the repairing component and the decalcification component have a synergistic effect to jointly create a microenvironment beneficial to tissue regeneration. The product can be administrated through ear dripping or tympanic chamber injection, so that the treatment pain of a patient is greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a drug formula for repairing tympanic membrane calcification and a preparation method thereof. BACKGROUND

[0002] Tympanic membrane (tympanic membrane) calcification, also known as tympanic cavity sclerosis, is a white calcified plaque left on the tympanic membrane after the healing of chronic inflammation such as otitis media. These plaques are mainly composed of hydroxyapatite crystals, which can cause a significant decrease in tympanic membrane elasticity and an increase in mass, thereby causing conductive hearing loss, tinnitus and other symptoms. At present, for severe tympanic membrane calcification which seriously affects hearing, the main treatment in clinic is surgical treatment (such as tympanoplasty). However, surgery has problems such as large trauma, high cost, long postoperative recovery period and anesthesia risk.

[0003] In terms of non-surgical treatment, there is currently a lack of drugs that can effectively dissolve calcified plaques and simultaneously promote tympanic membrane tissue regeneration. Traditional ear drops are difficult to penetrate the stratum corneum of the tympanic membrane, and the drug stays in the ear canal for a short time, which cannot form an effective concentration at the lesion site. Some studies have attempted to use calcium ion chelators (such as EDTA), but they have poor biocompatibility, lack tissue targeting, can cause irritation to normal tissues, and cannot provide the necessary nutritional support for repair.

[0004] Therefore, there is an urgent need in the art to develop a new non-invasive drug that can effectively target and soften / dissolve calcified plaques, while providing support for the regeneration and repair of tympanic membrane tissue, and can achieve long-term retention of the drug at the lesion site. SUMMARY

[0005] In view of the problems existing in the prior art, the present application discloses a drug for repairing tympanic membrane calcification, the main components and weight ratio of which are as follows: 0.5-5wt% of type III collagen peptide, 1-10wt% of citric acid-chitosan conjugate, 10-25wt% of temperature-sensitive poloxamer 407, 1-5wt% of nanoemulsion, 0.01-0.1wt% of dexamethasone palmitate, and the rest is phosphate buffer solution with a pH of 6.5-7.4.

[0006] As a preferred scheme of the present application, the molecular weight of the type III collagen peptide ranges from 10-50kDa.

[0007] As a preferred scheme of the present application, the citric acid-chitosan conjugate is prepared by amidation reaction of citric acid and chitosan catalyzed by carbodiimide hydrochloride.

[0008] As a preferred scheme of the present application, the nanoemulsion is composed of oleic acid, Tween 80 and ethanol at a mass ratio of 1:2:1.

[0009] A preparation method of a drug for repairing tympanic membrane calcification, comprising the following steps: Step one, preparation of citric acid-chitosan conjugate Step 1.1, 1 part by weight of chitosan is dissolved in 1% acetic acid solution to prepare a 2wt% chitosan solution; Step 1.2, under stirring, 0.5 parts by weight of citric acid and 0.1 parts by weight of carbodiimide hydrochloride are sequentially added to the chitosan solution to obtain a mixed solution; Step 1.3, the pH of the mixed solution is adjusted to 5.5, and the reaction is carried out at room temperature for 12-24 hours in the dark, and the reaction liquid is obtained after the reaction is completed; Step 1.4, the reaction liquid is transferred to a dialysis bag with a molecular weight cut-off of 3.5kDa, and dialyzed with deionized water for 48 hours to remove unreacted impurities; Step 1.5, the solution after dialysis is freeze-dried to obtain white citric acid-chitosan conjugate solid, which is ready for use; Step two, preparation of nanoemulsion Step 2.1, oleic acid, Tween 80 and anhydrous ethanol are accurately weighed according to the mass ratio of 1:2:1 and uniformly mixed as an oil phase; Step 2.2, the oil phase is slowly added dropwise into 5 times the volume of phosphate buffer under magnetic stirring; Step 2.3, a probe-type ultrasonic instrument is used for ultrasonic treatment at a power of 200W for 10 minutes to obtain a clear or translucent nanoemulsion primary product; Step 2.4, the nanoemulsion primary product is sterile filtered through a microporous filter membrane to obtain the nanoemulsion, which is stored at 4°C in a sealed state; Step three, preparation of composite hydrogel Step 3.1, 10-25 parts by weight of poloxamer 407 is slowly added to a phosphate buffer solution pre-cooled to 4°C, and magnetic stirring is carried out at 2-8°C until complete dissolution to obtain a clear poloxamer base solution; Step 3.2, under the conditions of continuous stirring and low temperature, the poloxamer base solution is sequentially added with the citric acid-chitosan conjugate prepared in step one, collagen type III peptide, nanoemulsion prepared in step two and dexamethasone palmitate according to the weight ratio to carry out concentration adjustment, and a mixed solution is obtained; Step 3.3, the mixed solution is continuously stirred at 2-8°C in the dark for at least 2 hours until a uniform and transparent pre-gel solution is formed, and the pre-gel solution is sterilely packaged to obtain the drug for repairing tympanic membrane calcification.

[0010] As a preferred scheme of the present application, the microporous filter membrane in step 2.4 is a 0.22μm microporous filter membrane.

[0011] As a preferred embodiment of the present invention, the stirring speed for dissolving poloxamer 407 in step 3.1 is 300-500 rpm.

[0012] As a preferred embodiment of the present invention, the drug for repairing eardrum calcification is stored at 2-8°C.

[0013] The beneficial effects of this invention are as follows: This invention combines type III collagen peptides and citric acid-chitosan conjugates to achieve a combination of active repair and active decalcification, providing not only repair materials but also addressing the root cause of calcium salt deposition, thus achieving a comprehensive treatment. The use of thermosensitive hydrogels enables in-situ gelation and long-lasting release of the drug, overcoming the short retention time of traditional liquid formulations and improving drug utilization and therapeutic efficacy. Nanoemulsions significantly enhance the penetration of drug components into the keratinocyte of the eardrum, ensuring that the active ingredients can effectively reach the lesion. The citric acid-chitosan conjugate itself is biodegradable, and citric acid is an intermediate product of the tricarboxylic acid cycle in the body, ensuring high safety. The anti-inflammatory components work synergistically with the repair and decalcification components to create a microenvironment conducive to tissue regeneration. This product can be administered via ear drops or intratympanic injection, avoiding surgical trauma and greatly reducing patient suffering, risks, and costs, providing a new conservative treatment option for eardrum calcification. Detailed Implementation

[0014] Example 1

[0015] The present invention provides a medicine for repairing calcification of the eardrum, the main components and their weight ratios being as follows: Active repair ingredient: 0.5wt% type III collagen peptides, with a molecular weight range of 10-50kDa. Type III collagen is an important component of the middle layer (fibrous layer) of the eardrum. Its small molecule peptides are easily utilized by cells, promoting the migration, proliferation and secretion of fibroblasts, and are the core material for active tissue repair. Calcium ion chelating component: 1 wt% citric acid-chitosan conjugate. This conjugate is prepared by amidation reaction of citric acid and chitosan under the catalysis of carbodiimide hydrochloride. Chitosan has good biocompatibility, film-forming properties, and hemostatic effects, while the covalently linked citrate anion is a natural, safe, and potent calcium ion chelator in vivo, specifically binding to calcium in hydroxyapatite, dissolving it, thereby softening and eliminating calcification spots at their source. Matrix composition: 10 wt% thermosensitive poloxamer 407. This polymer solution is liquid at low temperatures and rapidly transforms into a three-dimensional network hydrogel at body temperature (37°C), achieving in-situ gelation and controlled sustained release of the drug, greatly prolonging the drug's action time on the eardrum surface. Penetration-enhancing component: 1 wt% nanoemulsion system, wherein the nanoemulsion is composed of oleic acid, Tween 80 and ethanol in a mass ratio of 1:2:1. This nanoemulsion can effectively disrupt the lipid bilayer structure of the stratum corneum, significantly enhance the membrane permeability of both hydrophilic and hydrophobic drug components, and ensure that the active ingredients can reach the lesion site. Bioactive ingredient: 0.01 wt% dexamethasone palmitate. As a prodrug, it is highly lipid-soluble, easily taken up by cells, and hydrolyzed by intracellular enzymes into dexamethasone, which has a strong anti-inflammatory effect. It can effectively inhibit the local inflammatory response during calcification and create a favorable microenvironment for tissue repair. Solvent: 87.49 wt% phosphate buffer at pH 6.5, providing a stable physiological pH environment.

[0016] A method for preparing a drug for repairing calcification of the eardrum includes the following steps: Step 1: Preparation of citric acid-chitosan conjugate Step 1.1: Dissolve 1 part by weight of chitosan in a 1% acetic acid solution to prepare a 2 wt% chitosan solution; Step 1.2: While stirring, add 0.5 parts by weight of citric acid and 0.1 parts by weight of carbodiimide hydrochloride to the chitosan solution to obtain a mixed solution; Step 1.3: Adjust the pH of the mixed solution to 5.5, and react at room temperature in the dark for 12-24 hours. After the reaction is complete, the reaction solution is obtained. Step 1.4: Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyze with deionized water for 48 hours to remove unreacted impurities; Step 1.5: Freeze-dry the dialyzed solution to obtain a white citric acid-chitosan conjugate solid for later use; Step 2: Preparation of Nanoemulsions Step 2.1: Weigh oleic acid, Tween 80 and anhydrous ethanol precisely in a mass ratio of 1:2:1, and mix them evenly to form the oil phase; Step 2.2: Slowly add the oil phase dropwise to 5 times its volume of phosphate buffer solution under magnetic stirring; Step 2.3: Use a probe-type ultrasonic instrument to sonicate at 200W power for 10 minutes to obtain a clear or semi-transparent nanoemulsion primary product; Step 2.4: The nanoemulsion primary product is aseptically filtered through a 0.22μm microporous membrane to obtain the nanoemulsion, which is then sealed and stored at 4℃. Step 3: Preparation of composite hydrogel Step 3.1: Slowly add 10 parts by weight of poloxamer 407 to phosphate buffer pre-cooled to 4°C, and magnetically stir at 2°C until completely dissolved at a stirring speed of 300 rpm to obtain a clear poloxamer base solution. Step 3.2: Under continuous stirring and low temperature conditions, add the following ingredients sequentially to the poloxamer base solution according to the weight ratio: citric acid-chitosan conjugate prepared in step one, type III collagen peptide, nanoemulsion prepared in step two, and dexamethasone palmitate, and adjust the concentration to obtain a mixed solution. Step 3.3: Stir the prepared mixture continuously at 2°C and in the dark for at least 2 hours until a homogeneous and transparent pregel solution is formed. Dispense the solution under aseptic conditions to obtain the drug for repairing calcification of the eardrum, and store it at 2°C.

[0017] Example 2

[0018] The present invention discloses a drug for repairing calcification of the tympanic membrane, the main components and their weight ratios being: 5 wt% type III collagen peptide, 10 wt% citric acid-chitosan conjugate, 25 wt% thermosensitive poloxamer 407, 5 wt% nanoemulsion, 0.1 wt% dexamethasone palmitate, and 54.9 wt% phosphate buffer solution at pH 7.4 as solvent.

[0019] A method for preparing a drug for repairing calcification of the eardrum includes the following steps: Step 1: Preparation of citric acid-chitosan conjugate Step 1.1: Dissolve 1 part by weight of chitosan in a 1% acetic acid solution to prepare a 2 wt% chitosan solution; Step 1.2: While stirring, add 0.5 parts by weight of citric acid and 0.1 parts by weight of carbodiimide hydrochloride to the chitosan solution to obtain a mixed solution; Step 1.3: Adjust the pH of the mixed solution to 5.5, and react at room temperature in the dark for 12-24 hours. After the reaction is complete, the reaction solution is obtained. Step 1.4: Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyze with deionized water for 48 hours to remove unreacted impurities; Step 1.5: Freeze-dry the dialyzed solution to obtain a white citric acid-chitosan conjugate solid for later use; Step 2: Preparation of Nanoemulsions Step 2.1: Weigh oleic acid, Tween 80 and anhydrous ethanol precisely in a mass ratio of 1:2:1, and mix them evenly to form the oil phase; Step 2.2: Slowly add the oil phase dropwise to 5 times its volume of phosphate buffer solution under magnetic stirring; Step 2.3: Use a probe-type ultrasonic instrument to sonicate at 200W power for 10 minutes to obtain a clear or semi-transparent nanoemulsion primary product; Step 2.4: The nanoemulsion primary product is aseptically filtered through a 0.22μm microporous membrane to obtain the nanoemulsion, which is then sealed and stored at 4℃. Step 3: Preparation of composite hydrogel Step 3.1: Slowly add 25 parts by weight of poloxamer 407 to phosphate buffer pre-cooled to 4°C, and magnetically stir at 8°C until completely dissolved at a stirring speed of 500 rpm to obtain a clear poloxamer base solution. Step 3.2: Under continuous stirring and low temperature conditions, add the following ingredients sequentially to the poloxamer base solution according to the weight ratio: citric acid-chitosan conjugate prepared in step one, type III collagen peptide, nanoemulsion prepared in step two, and dexamethasone palmitate, and adjust the concentration to obtain a mixed solution. Step 3.3: Stir the prepared mixture continuously at 8°C and in the dark for at least 2 hours until a homogeneous and transparent pregel solution is formed. Dispense the solution under aseptic conditions to obtain the drug for repairing calcification of the eardrum and store it at 8°C.

[0020] Example 3

[0021] The present invention discloses a drug for repairing calcification of the tympanic membrane, the main components and weight ratios of which are: 3 wt% type III collagen peptide, 5 wt% citric acid-chitosan conjugate, 18 wt% thermosensitive poloxamer 407, 3 wt% nanoemulsion, 0.05 wt% dexamethasone palmitate, and 70.95 wt% pH 7.0 phosphate buffer as solvent.

[0022] A method for preparing a drug for repairing calcification of the eardrum includes the following steps: Step 1: Preparation of citric acid-chitosan conjugate Step 1.1: Dissolve 1 part by weight of chitosan in a 1% acetic acid solution to prepare a 2 wt% chitosan solution; Step 1.2: While stirring, add 0.5 parts by weight of citric acid and 0.1 parts by weight of carbodiimide hydrochloride to the chitosan solution to obtain a mixed solution; Step 1.3: Adjust the pH of the mixed solution to 5.5, and react at room temperature in the dark for 12-24 hours. After the reaction is complete, the reaction solution is obtained. Step 1.4: Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyze with deionized water for 48 hours to remove unreacted impurities; Step 1.5: Freeze-dry the dialyzed solution to obtain a white citric acid-chitosan conjugate solid for later use; Step 2: Preparation of Nanoemulsions Step 2.1: Weigh oleic acid, Tween 80 and anhydrous ethanol precisely in a mass ratio of 1:2:1, and mix them evenly to form the oil phase; Step 2.2: Slowly add the oil phase dropwise to 5 times its volume of phosphate buffer solution under magnetic stirring; Step 2.3: Use a probe-type ultrasonic instrument to sonicate at 200W power for 10 minutes to obtain a clear or semi-transparent nanoemulsion primary product; Step 2.4: The nanoemulsion primary product is aseptically filtered through a 0.22μm microporous membrane to obtain the nanoemulsion, which is then sealed and stored at 4℃. Step 3: Preparation of composite hydrogel Step 3.1: Slowly add 18 parts by weight of poloxamer 407 to phosphate buffer pre-cooled to 4°C, and magnetically stir at 5°C until completely dissolved at a stirring speed of 400 rpm to obtain a clear poloxamer base solution. Step 3.2: Under continuous stirring and low temperature conditions, add the following ingredients sequentially to the poloxamer base solution according to the weight ratio: citric acid-chitosan conjugate prepared in step one, type III collagen peptide, nanoemulsion prepared in step two, and dexamethasone palmitate, and adjust the concentration to obtain a mixed solution. Step 3.3: Stir the prepared mixture continuously at 5°C and in the dark for at least 2 hours until a homogeneous and transparent pregel solution is formed. Dispense the solution under aseptic conditions to obtain the drug for repairing calcification of the eardrum, and store it at 5°C.

[0023] Components not described in detail in this article are existing technologies.

[0024] While the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and modifications or variations without creative effort are still within the protection scope of the present invention.

Claims

1. A drug for repairing calcification of the eardrum, characterized in that, Its main components and their weight ratios are as follows: 0.5-5 wt% type III collagen peptides, 1-10 wt% citric acid-chitosan conjugate, 10-25 The solution consists of wt% thermosensitive poloxamer 407, 1-5 wt% nanoemulsion, 0.01-0.1 wt% dexamethasone palmitate, and the balance being phosphate buffer at pH 6.5-7.

4.

2. The drug for repairing otitis media calcification according to claim 1, characterized in that: The molecular weight range of the type III collagen peptide is 10-50 kDa.

3. The drug for repairing otitis media calcification according to claim 1, characterized in that: The citric acid-chitosan conjugate is prepared by amidation reaction of citric acid and chitosan under the catalysis of carbodiimide hydrochloride.

4. The drug for repairing calcification of the eardrum according to claim 1, characterized in that: The nanoemulsion is composed of oleic acid, Tween 80 and ethanol in a mass ratio of 1:2:

1.

5. A method for preparing the medicament for repairing otitis media according to any one of claims 1-4, characterized in that, The steps include the following: Step 1: Preparation of citric acid-chitosan conjugate Step 1.1: Dissolve 1 part by weight of chitosan in a 1% acetic acid solution to prepare a 2 wt% chitosan solution; Step 1.2: While stirring, add 0.5 parts by weight of citric acid and 0.1 parts by weight of carbodiimide hydrochloride to the chitosan solution to obtain a mixed solution; Step 1.3: Adjust the pH of the mixed solution to 5.5, and react at room temperature in the dark for 12-24 hours. After the reaction is complete, the reaction solution is obtained. Step 1.4: Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyze with deionized water for 48 hours to remove unreacted impurities; Step 1.5: Freeze-dry the dialyzed solution to obtain a white citric acid-chitosan conjugate solid for later use; Step 2: Preparation of Nanoemulsions Step 2.1: Weigh oleic acid, Tween 80 and anhydrous ethanol precisely in a mass ratio of 1:2:1, and mix them evenly to form the oil phase; Step 2.2: Slowly add the oil phase dropwise to 5 times its volume of phosphate buffer solution under magnetic stirring; Step 2.3: Use a probe-type ultrasonic instrument to sonicate at 200W power for 10 minutes to obtain a clear or semi-transparent nanoemulsion primary product; Step 2.4: The nanoemulsion primary product is aseptically filtered through a microporous membrane to obtain the nanoemulsion, which is then sealed and stored at 4°C. Step 3: Preparation of composite hydrogel Step 3.1: Slowly add 10-25 parts by weight of poloxamer 407 to phosphate buffer pre-cooled to 4°C, and stir magnetically at 2-8°C until completely dissolved to obtain a clear poloxamer base solution. Step 3.2: Under continuous stirring and low temperature conditions, add the following ingredients sequentially to the poloxamer base solution according to the weight ratio: citric acid-chitosan conjugate prepared in step one, type III collagen peptide, nanoemulsion prepared in step two, and dexamethasone palmitate, and adjust the concentration to obtain a mixed solution. Step 3.3: Stir the prepared mixture continuously at 2-8℃ in the dark for at least 2 hours until a homogeneous and transparent pregel solution is formed. Then, dispense the solution under aseptic conditions to obtain the drug for repairing calcification of the eardrum.

6. The method for preparing a drug for repairing otitis media calcification according to claim 5, characterized in that: The microporous filter membrane mentioned in step 2.4 is a 0.22μm microporous filter membrane.

7. The method for preparing a drug for repairing otitis media calcification according to claim 5, characterized in that: In step 3.1, the stirring speed for dissolving poloxamer 407 is 300-500 rpm.

8. The method for preparing a drug for repairing otitis media calcification according to claim 5, characterized in that: The medication for repairing eardrum calcification should be stored at 2-8℃.