A semilunar sustained-release drug film containing methylcatester and a preparation method thereof

Abstract

The present application provides the use of methylcatester for treating periodontitis. The present application also provides a method for preparing a sustained-release drug film containing methylcatester. The sustained-release drug film provided by the present application can inhibit the release of inflammatory factors, inhibit bacterial adhesion, and has a sustained-release time of up to the seventh day, has obvious effect on anti-periodontitis, and can effectively reduce the pathological changes of periodontal tissue.

Description

Technical Field

[0001] This invention relates to the use of methylcarboxyster, and more particularly to a sustained-release drug film containing methylcarboxyster, for the treatment of periodontitis. The invention also relates to a method for preparing the sustained-release drug film. Background Technology

[0002] Periodontitis is a chronic inflammatory disease caused by the combined effects of bacteria, host, and environment. The lesions affect the supporting tissues of the teeth, including the gingiva, periodontal ligament, alveolar bone, and cementum, ultimately leading to tooth loosening and loss, and is the leading cause of tooth loss in adults. Mechanical removal of plaque biofilm is the most basic and important method for treating periodontitis. However, due to the difficulty of scaling instruments reaching the bottom of deep periodontal pockets and their limited inhibitory effect on bacterial toxins and metabolites, mechanical methods cannot achieve ideal therapeutic effects for all cases of periodontitis. Clinically, antibiotics are often used as an adjunct to mechanical treatment of periodontitis. However, systemic administration faces many problems, including the risk of dysbiosis, induction of drug-resistant strains, low local drug concentrations, and poor patient compliance. Compared with systemic administration, local administration has advantages such as fewer side effects, higher safety, and stronger drug targeting. However, inappropriate selection of antibiotics and unsatisfactory carrier materials greatly limit its clinical application.

[0003] Currently, common oral topical drug delivery formulations on the market include sticks, ointments, sprays, and gels. We have found that stick formulations have a fixed volume, are prone to falling off, and cause significant discomfort; gel formulations are convenient to use, have stable drug activity, and are comfortable for patients, but are easily washed away by gingival crevicular fluid, resulting in low retention in periodontal pockets. Film formulations offer site-specific local delivery, eliminating or reducing systemic absorption. In recent years, the application of mucosal adhesion polymers in oral drug delivery has attracted considerable interest. Because the gingiva is a moist mucosal surface, mucosal adhesion polymer materials can interact with mucosal glycoproteins through hydrogen bonds, van der Waals forces, and electrostatic attraction. Once mucosal adhesion is achieved, the loaded drug will have greater retention at the application site, facilitating sustained release. Such film formulations also have the potential for self-administration. Therefore, sustained-release film formulations are an ideal route of drug delivery.

[0004] Integrins are transmembrane glycoprotein receptors widely distributed on the cell surface. Their main functions are adhesion and signal transduction, mediating cell-cell and cell-extracellular matrix interactions and participating in the regulation of cell proliferation, differentiation, adhesion, and migration. They play an important role in various pathological processes, such as chronic inflammation, thrombosis, and malignant tumors, all of which are related to integrin dysfunction. Integrin ligands include ECM (fibronectin, collagen, laminin) and other cell surface adhesion molecules, such as immunoglobulin superfamily members ICAM-1 and VCAM-1. Due to differences in their constituent subunits, the functions of various integrins differ. Integrin inhibitors have different mechanisms of action than anti-inflammatory drugs in suppressing inflammation. Currently, there are no reports of integrin inhibitors being formulated into suitable drug forms for the treatment of periodontitis. Summary of the Invention

[0005] In one aspect, the present invention provides the use of methylcalcester in the preparation of a medicament for treating periodontitis.

[0006] In some implementations, the drug is in the form of a sustained-release film.

[0007] On the other hand, the present invention provides a pharmaceutical composition for treating periodontitis, comprising the following components: methylcarboxyster, chitosan, sulfobetaine methacrylate (SBMA), β-cyclodextrin, glycerol, and potassium persulfate.

[0008] In some embodiments, the relative amounts of the components are: 0.5-1.5 g of methylcarboxyster, 2-3 g of chitosan, 0.3-0.6 g of sulfobetaine methacrylate (SBMA), 0.3-0.6 g of β-cyclodextrin, 1-2 mL of glycerol, and 0.1-0.2 mg of potassium persulfate.

[0009] In some embodiments, the pharmaceutical composition is prepared in the form of a sustained-release film.

[0010] On the other hand, the present invention provides a method for preparing a sustained-release drug film, comprising the following steps:

[0011] 1) Dissolve chitosan in a 2% (v / v) aqueous solution of acetic acid;

[0012] 2) Dissolve methylcalcet and β-cyclodextrin in water, and add glycerol;

[0013] 3) Mix the solutions obtained in steps 1) and 2), add sulfobetaine methacrylate (SBMA) and potassium persulfate, and stir until homogeneous to obtain a viscous liquid; and

[0014] 4) Allow the viscous liquid to dry into a film on the mold.

[0015] In some embodiments, step 1) of the method includes: dissolving 2-3 g of chitosan in 150 mL of 2% v / v acetic acid aqueous solution, and setting aside after the chitosan is completely dissolved; step 2) includes: adding 0.5-1.5 g of methylcalcesterol and 0.3-0.6 g of β-cyclodextrin to 100 mL of deionized water, and simultaneously adding 1-2 mL of glycerol, and stirring in a stirrer at 85 °C until completely dissolved; step 3) includes: mixing the solutions obtained in steps 1) and 2) respectively, and adding 0.3-0.6 g of SBMA and 0.1-0.2 mg of potassium persulfate, and stirring at 60 °C under nitrogen for no less than 3 h, for example 3-4 h.

[0016] In some implementations, step 4) further includes adjusting the pH of the viscous liquid to 6.5.

[0017] In some embodiments, the relative amounts of each component in each step are: 2.5 g chitosan, 150 mL of 2% (v / v) aqueous acetic acid solution, 1 g methylcalcesterol, 0.5 g β-cyclodextrin, 100 mL water, 1 mL glycerol and 0.5 g SBMA.

[0018] On the other hand, the present invention provides a sustained-release drug film prepared by the above method. Detailed Implementation

[0019] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0020] The term "or" refers to a single element among the listed optional elements, unless the context explicitly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed optional elements.

[0021] The terms “comprising,” “containing,” “having,” and similar expressions used herein do not exclude elements not listed. These terms also include cases where the text consists only of the listed elements.

[0022] The term "about" typically refers to a variation within 10% above or below a specified value, such as a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value. Unless the context otherwise requires, the use of the term "about" when referring to a specific value is intended to cover situations where the specific value is preceded by that value.

[0023] Carotegrast Methyl is a broad-spectrum integrin inhibitor that selectively inhibits integrin α4. On March 28, 2022, Carotegrast Methyl was approved by the Japanese PMDA as the first oral anti-integrin drug for the treatment of ulcerative colitis, overcoming the adverse effects of multidrug resistance. Integrin α4 often exists as an isoform of β1. α4β1 not only mediates the migration of immune cells but also facilitates the adhesion and colonization of periodontal bacteria due to its domain that binds to surface proteins of periodontal pathogens. Furthermore, α4β1 can transmit information from the extracellular matrix into the cell, activating intracellular signaling pathways related to inflammation and differentiation. Of particular note is the recent research on exosomes, which suggests that the exosomal pathway may play a unique and crucial role in the pathogenesis of periodontitis. It has been reported that proteins binding to α4β1 exist on the surface of the exosome membrane; therefore, through α4β1, the bioactive substances carried by exosomes affect receptor cells and alter their function. The inventors anticipated that methylcartester could potentially be used to treat periodontitis by binding to integrins, inhibiting their mediated adhesion and migration of pathogenic bacteria and immune cells, and blocking transmembrane conduction of the extracellular matrix and exosomes in the inflammatory microenvironment. The therapeutic effect was demonstrated by the examples described below.

[0024] To reduce the adverse effects of adjuvant antibiotics in existing periodontitis treatments, while improving clinical efficiency and facilitating patient self-care, this application provides a crescent-shaped sustained-release drug film containing methylcarboxyster and its preparation method.

[0025] In some embodiments, this application provides a sustained-release drug film, prepared using the following technical solution.

[0026] The technical solution includes the use of the following raw materials in the following mass percentages: methyl carboxyster 0.5-1.5g, chitosan 2-3g, sulfobetaine methacrylate (SBMA) 0.3-0.6g, β-cyclodextrin 0.3-0.6g, glycerol 1-2mL, and potassium persulfate 0.1-0.2mg.

[0027] Chitosan possesses excellent biocompatibility and low toxicity, and can be degraded by enzymes in the human body. The linear sugar chains of chitosan contain numerous repeating active amino and hydroxyl groups, making it ideal for use as a polymeric carrier for drugs. Grafting or copolymerizing small-molecule drugs onto its backbone can yield polymers with strong sustained-release capabilities. Chitosan and its derivatives play unique roles in drug delivery, including stabilizing or protecting drug components, promoting drug absorption, and delaying or controlling drug release, making it a perfect tool for intelligent drug delivery.

[0028] SBMA (sulfobetaine methacrylate) is a biocompatible monomer with both cations and anions. Its zwitterionic structure not only endows SBMA with excellent antibacterial properties but also allows it to form ionic bonds with other substances, enhancing its mechanical and adhesive properties. Furthermore, it can act as a conductive ion, increasing electrical conductivity and facilitating the conversion of pressure and physiological signals into electrical signals. The double bonds in SBMA can undergo free radical polymerization, making it a promising candidate for applications in the biomedical field.

[0029] β-Cyclodextrin (β-CD) is a product of starch cyclization via acid hydrolysis. It can encapsulate various compound molecules, altering the physicochemical properties of the encapsulated substances. β-CD is widely used in the pharmaceutical excipient industry primarily to increase drug stability, prevent drug oxidation and decomposition, improve drug solubility and bioavailability, reduce drug toxicity, and mask off-odors and malodors.

[0030] Brief description of preparation method:

[0031] (1) Dissolve 2-3g of chitosan in 150mL of 2% v / v acetic acid aqueous solution and leave overnight until the chitosan is completely dissolved.

[0032] Preferably, 2.5 g of chitosan is completely dissolved in 150 mL of 2% v / v acetic acid aqueous solution without any flocculent precipitate.

[0033] (2) Dissolve 0.5-1.5g of methylcalcet and 0.3-0.6g of β-cyclodextrin in 100mL of deionized water, and add 1-2mL of glycerol at the same time. Stir the mixture in a magnetic stirrer at 85℃ and 400r / min for 1.5h until completely dissolved.

[0034] Preferably, when 1g of methylcastatin (Ea Pharma Co., Ltd. (30400AMX00186000)) and 0.5g of β-cyclodextrin are dissolved in 100mL of deionized water, methylcastatin exhibits the best solubility, while β-cyclodextrin is the most effective in maintaining drug stability and improving drug bioavailability.

[0035] Preferably, when 1 mL of glycerol is added, the mixture has moderate viscosity, and the film-forming solution achieves optimal thickness and film flexibility during film formation.

[0036] (3) Mix the two solutions and add 0.3-0.6 g of sulfobetaine methacrylate (SBMA) and 0.1-0.2 mg of potassium persulfate. Stir continuously with magnetic force for 3 h at 60 °C under nitrogen.

[0037] Preferably, when 0.5g of sulfobetaine methacrylate (SBMA) is added to the mixture of the two solutions, the graft polymerization reaction between SBMA and β-cyclodextrin is most complete, and the mass ratio with chitosan is 5:1, resulting in the best antibacterial properties and water absorption and swelling properties.

[0038] Preferably, 0.1 mg of potassium persulfate has no effect on the chitosan backbone structure and can increase the stability and adsorption capacity of the drug film.

[0039] (4) Obtain a viscous liquid and increase the pH to 6.5 by adding 1M NaOH aqueous solution.

[0040] Preferably, the film-forming solution is most stable when the pH is adjusted to 6.5.

[0041] (5) Lay a film on a 1.5cm×1.5cm glass mold and dry it to form a film (preferably prepared into a crescent shape).

[0042] (6) Sterilize the film by irradiating it under a UV lamp for 15 minutes.

[0043] (7) Packaging.

[0044] Topical application of sustained-release drug films can assist patients with advanced, chronic, and aggressive periodontitis who cannot rely on mechanical treatment alone, and sustained-release drug films are also suitable for refractory cases that do not respond well to traditional treatments. Since periodontitis is caused by multiple factors, including plaque biofilm and an overactive immune inflammatory response in the host, sustained-release drug films carrying integrin inhibitors can address both of these issues simultaneously.

[0045] A sustained-release drug film containing methylcastatin was prepared according to the preferred scheme of the above preparation method, and the following experiments were conducted.

[0046] Example 1 Bacterial Adhesion Experiment

[0047] The sustained-release membrane was pre-prepared into 10.0 mm × 13.0 mm sheet samples, and a glass slide of the same size was used as a control. 1.6 mL of a 1 × 10⁻⁶ solution was added to a 24-well bacterial culture plate. 3A suspension of *Porphyromonas gingivalis* (cfu / mL) was prepared, and sterilized slow-release film samples and control slides were placed in it. After gentle shaking, the samples were incubated at 37°C. After 3 hours of incubation, the samples were removed and rinsed three times with sterile phosphate buffer to remove unadhered bacteria. They were then placed in sterile liquid culture medium and sonicated at 40kHz for 2 minutes to detach bacteria from the material surface without affecting bacterial activity. The plate count method was used. The ultrasonically treated bacterial suspension was serially diluted with sterile physiological saline, and 0.1 mL of the diluted suspension was evenly spread on a pre-prepared solid culture medium. The plates were incubated at 37°C for 24 hours, and the colony count was calculated. The total number of bacteria adhering to the material surface was calculated based on the colony count results.

[0048] The results showed that the control slide surface exhibited high bacterial adsorption after three hours, while only a very small amount of Porphyromonas gingivalis was detected on the surface of the slow-release film, and its adhesion was significantly reduced.

[0049] Example 2: In vitro sustained-release experiment of drug film

[0050] Take a 1.5cm × 1.5cm sustained-release film sample. Using a dissolution assay apparatus with 900mL (pH 7.4) phosphate buffer as the release medium, a rotation speed of 100r / min, and a temperature of (37±0.5)℃, place the sample in a rotating basket. At predetermined time points, take 10mL of solution, and immediately replenish with the same volume and temperature of release medium. Filter the solution. Use the filtrate as the test solution and measure the absorbance at 276nm using UV-Vis spectrophotometry. Separately, accurately weigh an appropriate amount of methylcarbazide reference standard, dissolve it in pH 7.4 phosphate buffer, and quantitatively dilute it to prepare a solution containing approximately 20μg per mL. Measure the cumulative release rate using the same method.

[0051] The results showed that the drug-loaded membrane released drugs slowly in the first 30 minutes, and began to release drugs rapidly from the 60th minute. The cumulative release rate reached 50% on day 1, 67% on day 2, 79% on day 3, 88% on day 4, 93% on day 5, 97% on day 6, and more than 99% on day 7.

[0052] Example 3 Antibacterial Zone Experiment

[0053] Dilute the Porphyromonas gingivalis bacterial suspension to 3×10⁻⁶. 5CFU / mL. Take 1 mL of bacterial suspension and add it evenly dropwise onto sterilized solid culture medium. Prepare a 1×1 cm² glass slide with a coating on one side, and invert it onto the solid culture medium with the coated side facing the bacterial suspension. Incubate in this state at 37°C for 1, 3, 5, and 7 days. After the incubation period, observe whether any areas without any colonies appear around the coating. Compare the sustained-release membrane containing sulfobetaine methacrylate (SBMA) with that without sulfobetaine methacrylate (SBMA).

[0054] The results showed that on day 1, the antibacterial rate without SBMA was less than 10%, while the antibacterial rate with SBMA reached 40%; on day 3, the antibacterial rate without SBMA was about 20%, while the antibacterial rate with SBMA reached 75%; on day 5, the antibacterial rate without SBMA was about 18%, while the antibacterial rate with SBMA reached 95%; and on day 7, the antibacterial rate without SBMA was about 15%, while the antibacterial rate with SBMA reached 98%.

[0055] Example 4: Performance Testing Experiment of Sustained-Release Membrane

[0056] This embodiment investigates the effect of sulfobetaine methacrylate (SBMA) content on the swelling degree of the film and the effect of stirring time of the sustained-release film-forming solution on the drug loading of the film.

[0057] The sustained-release membranes were prepared according to the preparation method described above, with the only modification being the amount of SBMA used. The experimental procedure is briefly described below. Four types of membranes were obtained by graft polymerization of 0.3g, 0.4g, 0.5g, and 0.6g of SBMA with 0.5g of β-cyclodextrin, and cut into 1.5cm × 1.5cm pieces for later use. After thoroughly drying the membranes, they were weighed using an analytical balance and then placed in a petri dish containing 5mL of buffer solution for 24 hours. After removal, the residual buffer solution on the surface was quickly absorbed with filter paper, and the membranes were weighed again to calculate the swelling degree. The results showed that the swelling degrees of the four membranes were 12.12%, 13.01%, 13.43%, and 22.6%, respectively. There was no significant difference in the swelling degree of the film containing 0.3g, 0.4g, and 0.5g SBMA. Since the graft polymerization reaction between 0.5g SBMA and 0.5g β-cyclodextrin was sufficient, the film-forming solution preferably contains 0.5g SBMA.

[0058] Films were formed under continuous magnetic stirring at 60℃ and nitrogen atmosphere for 1h, 2h, 3h and 4h respectively. The results showed that the drug loading of methylcarbenicillin reached its maximum value after stirring for 3h. There was no significant difference in drug loading between stirring for 3h and 4h. When the stirring time was 1h and 2h, the drug loading of methylcarbenicillin was about 65%-70% of that after stirring for 3h.

[0059] Example 4: Rat Periodontitis Model Experiment

[0060] A rat model of periodontitis in the left maxillary first molar was established using the silk suture ligation method. A sustained-release drug film, appropriately cut and applied, was placed in the buccal-palatal periodontal pocket of the left maxillary first molar (no drug film was placed in the control group). The drug film was monitored for shedding every one day, and replaced weekly. Six rats were sacrificed for Micro-CT scans and histopathological staining to assess inflammatory factor expression and bone repair. The drug film was replaced until week 4, at which point the experiment concluded.

[0061] The results show:

[0062] Compared with the control group in the first week, the drug-coated group showed a 88% reduction in IL-1β expression, a 90% reduction in TNF-α expression, and an 87% reduction in NLRP3 inflammatory factor expression; the volume and number of bone trabeculae increased by 30%.

[0063] In the second week, compared with the control group, the drug-coated group showed a 91% reduction in IL-1β expression, a 93% reduction in TNF-α expression, and a 92% reduction in NLRP3 inflammatory factor expression; the volume and number of bone trabeculae increased by 70%.

[0064] Compared with the control group in the third week, the drug-coated group showed a 96% reduction in IL-1β expression, a 94% reduction in TNF-α expression, and a 95% reduction in NLRP3 inflammatory factor expression; the volume and number of bone trabeculae increased by 88%.

[0065] In the fourth week, compared with the control group, the drug-coated group showed a 95% reduction in IL-1β expression, a 93% reduction in TNF-α expression, and a 95% reduction in NLRP3 inflammatory factor expression; the volume and number of bone trabeculae increased by 90%.

[0066] Because this invention incorporates methylcarboxyster into the drug film, it can achieve an inhibition rate of 89%-95% against inflammatory factors and 90%-98% against bacterial adhesion, with a sustained-release time lasting up to day 7. In rat experiments, it demonstrated significant anti-periodontal inflammation effects and effectively reduced pathological changes in periodontal tissues.

[0067] The improvements of the present invention compared to the prior art include at least the following:

[0068] 1. This is the first time that an integrin inhibitor has been added to a film-forming solution for the treatment of periodontal inflammation;

[0069] 2. In addition to chitosan, the excipients also include β-cyclodextrin, which can promote the dissolution of methylcarboxyster, improve the antibacterial properties of the drug film, and improve the taste, as well as sulfobetaine methacrylate (SBMA), which has a zwitterionic structure, giving the drug film excellent mechanical and adhesive properties.

[0070] References:

[0071] 1. Ma Yuan, Tang Shanhu, Li Sining, et al. Preparation and performance evaluation of oregano oil sustained-release membranes supported on β-cyclodextrin / chitosan / polyvinyl alcohol, Food Science, 2023.

[0072] 2. Lazaridou M, Nanaki S, Zamboulis A, et al. Super absorbent chitosan-based hydrogel sponges as carriers for caspofungin antifungaldrug. International Journal of Pharmaceutics, 2021.

[0073] 3. Song Fei, Xiang Yingying, Mao Zhijian, et al. Comparison of drug release characteristics of paclitaxel-chitosan sustained-release membranes prepared by two different methods. Journal of Kunming Medical University, 2017.

[0074] 4. Xuan Hongyun, Qian Jiaxi, Li Biyun, et al. Preparation and characterization of an antibacterial self-healing chitosan thin film material. Progress in Biomedical Engineering, 2022.

[0075] 5. Jiang Zhuoling, Jiang Changzhao, Ye Jincui, et al. Research progress and overview of commercially available oral films, China Journal of Traditional Chinese Medicine, 2020.

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[0081] 11. Huang, Zhengmei. Preparation and in vitro properties of antibacterial drug films for the treatment of periodontitis, Master's thesis, Shanghai Jiao Tong University School of Medicine, 2017.

Claims

1. A method for preparing a sustained-release drug film, characterized in that, Includes the following steps: Step 1: Dissolve 2-3g of chitosan in 150 mL of 2% v / v acetic acid aqueous solution; Step 2: Add 0.5-1.5g of methylcalcester and 0.3-0.6g of β-cyclodextrin to 100 mL of deionized water, and add 1-2 mL of glycerol at the same time. Stir at 85℃ until completely dissolved. Step 3: Mix the solutions obtained in Step 1 and Step 2, add 0.3-0.6g of sulfobetaine methacrylate (SBMA) and 0.1-0.2mg of potassium persulfate, and stir at 60℃ under nitrogen protection for no less than 3 hours to obtain a viscous liquid; Step 4: Adjust the pH of the viscous liquid to 6.5, and then dry it on the mold to form a film.

2. The method according to claim 1, wherein the amounts of each component are: 2.5 g chitosan, 1 g methylcalcet, 0.5 g β-cyclodextrin, 1 mL glycerol, and 0.5 g SBMA.

3. A sustained-release drug film, characterized in that, It is prepared by the method described in claim 1.

4. The use of the sustained-release drug film according to claim 3 in the preparation of a drug for treating periodontitis.