Application of 5-azacytosine nucleoside in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia and its microneedle patch

Abstract

This invention belongs to the field of biomedical technology and discloses the application of 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia (CIN) and its microneedle patch. In vitro and in vivo experiments demonstrate that 5-azacytosine nucleoside or pharmaceutically acceptable salts can significantly inhibit the growth and colony formation of CIN cells and promote apoptosis of CIN cells, thus it can be used in the preparation of drugs for the prevention and / or treatment of CIN. This invention also provides a microneedle patch loaded with 5-azacytosine nucleoside, which allows for precise local delivery and sustained release of 5-azacytosine nucleoside in the CIN lesion area. This overcomes the problems of easy hydrolysis and ring opening of 5-azacytosine nucleoside, high toxicity from systemic administration, and difficulty in penetrating the barrier to reach the lesion with traditional external administration. It has the advantages of being minimally invasive, highly safe, and having good patient compliance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia and their microneedle patches. Background Technology

[0002] Cervical intraepithelial neoplasia (CIN) is an essential stage in the development of cervical cancer, characterized by atypical proliferation of cells within the epithelial layer of the vaginal portion and endocervical canal of the cervix. Based on the depth of epithelial invasion, CIN is classified into three grades: CIN 1, CIN 2, and CIN 3. At the molecular level, persistent infection with high-risk HPV is the root cause of CIN.

[0003] The current gold standard treatment—loop electrosurgical excision procedure (LEEP) or cold knife conization—while effectively removing lesions, is significantly invasive. The surgery can cause permanent damage to the cervical anatomy, increasing the risk of premature birth, premature rupture of membranes, and cervical insufficiency in future pregnancies. Furthermore, the surgery cannot eliminate latent HPV in surrounding tissues, leading to a high recurrence rate. For CIN 1 and some CIN 2 patients, a "observation and follow-up" strategy is often adopted clinically, but this can place a significant psychological burden on patients.

[0004] Epigenetic therapy offers a new non-surgical approach to treating CIN. Epigenetic therapy is an innovative method that treats diseases by modifying the regulatory mechanisms of gene expression (such as DNA methylation and histone modification). Its key feature is that it does not directly alter the DNA sequence, but rather achieves its therapeutic goal by "switching" gene activity.

[0005] 5-aza-2'-deoxycytidine (also known as 5-azacytidine or azacytidine), with the chemical formula C8H 12 N4O5, a potent methyltransferase inhibitor (DNMTi), can be incorporated into the DNA of dividing cells. It forms a covalent complex with methyltransferases to block the maintenance of methylation, thereby reversing gene silencing and inducing apoptosis or differentiation in tumor cells. Clinically, it is used to treat breast cancer, colorectal cancer, melanoma, and acute myeloid leukemia. However, azacitidine has several drawbacks in clinical application: extremely low chemical stability (its half-life in aqueous solution is very short, making it prone to hydrolysis and ring-opening); and significant systemic toxicity (systemic administration can lead to severe bone marrow suppression and immunosuppression).

[0006] Traditional external drug delivery for CIN treatment, such as vaginal gels or suppositories, is often limited by the 10-100 μm thick mucus layer on the cervical surface, which contains a large amount of mucin. This mucin effectively blocks the penetration of large molecules and hydrophobic drugs, making it difficult for the drugs to reach the basal layer of the lesion site, resulting in low local delivery efficiency. Currently, there are no reports on the preparation of azacitidine as a drug for the prevention and / or treatment of cervical intraepithelial neoplasia. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide the use of 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia.

[0008] Another object of the present invention is to provide a demethylated microneedle patch loaded with 5-azacytosine nucleoside for the prevention and / or treatment of cervical intraepithelial neoplasia.

[0009] The objective of this invention is achieved through the following solution:

[0010] Use of 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia.

[0011] Furthermore, the 5-azacytosine nucleoside has the chemical formula C8H. 12 N4O5, the structural formula is shown below:

[0012] .

[0013] Furthermore, 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts can significantly inhibit the growth of cervical intraepithelial neoplasia cells.

[0014] Furthermore, 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts significantly inhibited the clonal formation of cervical intraepithelial neoplasia cells.

[0015] Furthermore, 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts promote apoptosis in cervical intraepithelial neoplasia cells.

[0016] Furthermore, the drugs, whether identical or different, include therapeutically effective amounts of 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts.

[0017] Furthermore, the same or different drugs can be prepared into various pharmaceutical dosage forms using conventional methods. These dosage forms include: tablets (sugar-coated tablets, film-coated tablets, enteric-coated tablets), capsules (hard capsules, soft capsules), pills, granules, oral liquids, tinctures, lozenges, powders, pills, suspensions, tinctures, drops, and other oral dosage forms, as well as injections, ointments, creams, patches, powders, and other dosage forms for administration other than oral administration.

[0018] Furthermore, the drugs, whether identical or different, may also contain one or more pharmaceutically acceptable carriers or excipients.

[0019] Furthermore, the carrier or excipient may include diluents, adhesives, surfactants, humectants, adsorbents, lubricants, fillers, disintegrants, preservatives, etc.

[0020] Based on the above applications, this invention also provides a demethylated microneedle patch loaded with 5-azacytosine nucleoside for the prevention and / or treatment of cervical intraepithelial neoplasia. This invention's microneedle patch, by loading 5-azacytosine nucleoside into a biodegradable microneedle structure, achieves local delivery and sustained release of the drug in the cervical lesion area, thereby improving therapeutic efficacy and reducing systemic adverse reactions, overcoming the challenges of the clinical application of 5-azacytosine nucleoside.

[0021] Furthermore, the demethylated microneedle patch structure includes a base layer and a microneedle array layer distributed on the surface of the base layer.

[0022] The substrate layer is used to support the microneedle array structure and ensure that the microneedle patch has sufficient mechanical stability and adhesion during use.

[0023] The microneedle array layer is composed of multiple regularly arranged microneedle units, each of which is set perpendicular or approximately perpendicular to the substrate layer.

[0024] The base layer and the microneedle array layer, which may be the same or different, are made of biodegradable polymer materials and can swell, degrade, or partially dissolve after being inserted into cervical epithelial tissue.

[0025] The biodegradable polymer material may be selected from at least one of the following: hyaluronic acid, polyvinylpyrrolidone, polyethylene glycol (PEG), gelatin, chitosan, and modified derivatives of the above materials; such as methacrylated hyaluronic acid (HAMA).

[0026] Furthermore, the biodegradable polymer material may be selected from at least two of the materials mentioned above, such as methacrylated hyaluronic acid and polyethylene glycol. Furthermore, the molecular weight of the polyethylene glycol may be within a conventional range, such as 10K.

[0027] Furthermore, the content of the biodegradable polymer material can be 1-20 wt%.

[0028] The height of the microneedle unit can be designed according to actual needs to ensure that it can penetrate the cervical epithelial barrier without damaging deeper tissues.

[0029] The microneedle unit is loaded with 5-azacytosine nucleoside, and the loading methods include, but are not limited to:

[0030] 5-azacytosine nucleoside was uniformly dispersed in the polymer matrix of the microneedle unit;

[0031] 5-azacytosine nucleoside was embedded in the internal structure of the microneedle unit;

[0032] A drug-containing coating is formed on the surface of the microneedle unit.

[0033] Furthermore, the loading of 5-azacytosine nucleoside in the microneedle patch can be 1-5 wt%.

[0034] The substrate layer and the microneedle array layer can be connected together by any conventional method in the art, and can be prepared by any conventional technique in the art.

[0035] For example, the above-mentioned biodegradable polymer material can be formulated into a precursor solution, and 5-azacytosine nucleoside can be dispersed in the precursor solution. With or without the addition of a crosslinking agent and / or photoinitiator, the material can be added to a mold, dried, and with or without photocuring, and then demolded to obtain a microneedle patch.

[0036] Furthermore, the crosslinking agent can be any reagent conventionally used in the art, such as calcium chloride. Furthermore, after drying, photocuring treatment such as ultraviolet light irradiation can further crosslink and form a stable microneedle patch.

[0037] Furthermore, in order to cooperate with the photocuring process, a photoinitiator is added to the precursor solution. The photoinitiator can be a reagent commonly used in the art, such as LAP, etc.; the amount used can be the conventional amount used in the art, such as 0.1-1 wt%.

[0038] The demethylated microneedle patch loaded with 5-azacytosine provided by this invention is applied to the cervical lesion area. The microneedle units penetrate the cervical epithelial tissue under external force. Subsequently, the microneedle units degrade or swell in the tissue fluid environment, causing the 5-azacytosine loaded in the microneedle units to be slowly released at the lesion site. This release acts on the abnormally methylated epithelial cells, reversing the abnormal epigenetic state and inhibiting lesion progression.

[0039] This invention utilizes 5-azacytosine nucleoside loaded onto microneedle patches, significantly reducing the toxic side effects such as bone marrow suppression and nausea associated with systemic administration of 5-azacytosine nucleoside. By employing local targeted delivery, the active drug is concentrated in the cervical lesion area, greatly reducing the proportion of drug entering the bloodstream, thus minimizing systemic toxicity while ensuring efficacy.

[0040] The microneedle patch of this invention is a non-surgical intervention that perfectly preserves fertility: Compared to the clinical gold standard LEEP surgery or cold knife conization, this invention is a non-invasive / minimally invasive therapy that does not cause permanent damage to the cervical anatomy. This has significant clinical value for women of childbearing age who wish to have children and are concerned about premature birth or infertility caused by cervical insufficiency due to surgery. Furthermore, the microneedle design avoids touching pain nerves in the dermis during puncture, making the drug delivery process virtually painless. This painless and convenient drug delivery method greatly alleviates patients' fear of traditional surgery and injections, and is conducive to increasing the willingness of CINI / II patients to actively seek treatment.

[0041] The microneedle patch of this invention effectively improves the chemical stability of 5-azacytosine nucleoside: 5-azacytosine nucleoside is readily hydrolyzed in aqueous solution. This invention utilizes a solid polymer matrix constructed from methacrylic acid, hyaluronic acid, and polyethylene glycol, which effectively locks the drug conformation and isolates it from moisture, significantly extending the drug's shelf life at room temperature and solving the problems of storage and distribution.

[0042] The microneedle patch of the present invention also has excellent tissue retention and sustained-release properties: the mucosal adhesive polymer introduced into the patch can form a hydrogen bond network with cervical mucin, ensuring that the patch has good adhesion in the moist vaginal environment, preventing the drug from being washed away by secretions too quickly, and realizing the continuous and stable release of the drug.

[0043] Compared with the prior art, the present invention has the following significant advantages:

[0044] (1) This invention provides a novel application of 5-azacytosine nucleoside in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia. In vitro and in vivo experiments of this invention demonstrate that 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts can significantly inhibit the growth and colony formation of cervical intraepithelial neoplasia cells and promote apoptosis of cervical intraepithelial neoplasia cells, thus showing promising application prospects in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia.

[0045] (2) This invention uses microneedle patches to apply 5-azacytosine nucleoside to the CIN lesion area, achieving local precise delivery and continuous release. It overcomes the problems of easy hydrolysis and ring opening of 5-azacytosine nucleoside, high toxicity and side effects caused by systemic administration, and difficulty in penetrating the barrier to reach the lesion for permeation administration by traditional external administration. It has the advantages of being minimally invasive, highly safe, and having good compliance. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 This image shows the effect of 5-azacytosine nucleoside on inhibiting the growth of cervical intraepithelial neoplasia cells in vitro.

[0048] Figure 2 This image illustrates the effect of 5-azacytosine nucleoside on inhibiting clonal formation of cervical intraepithelial neoplasia cells in vitro.

[0049] Figure 3 This figure shows the effect of 5-azacytosine on human cervical epithelial cells.

[0050] Figures 4-5 The image shows the effect of 5-azacytosine nucleoside on promoting apoptosis in cervical intraepithelial neoplasia cells in vitro.

[0051] Figures 6-8 This is a graph showing the inhibitory effect of 5-azacytosine nucleoside on precancerous cervical lesions in vivo.

[0052] Figure 9 This is a graph showing the changes in body weight of the experimental mice.

[0053] Figure 10 This is a diagram showing the mechanical properties of the microneedle patch.

[0054] Figure 11 This is a diagram showing the stability of the microneedle patch.

[0055] Figure 12 This is a picture of a microneedle patch.

[0056] Figure 13 This image shows the effect of microneedle patches in a mouse model of precancerous cervical lesions.

[0057] Figure 14 This is a graph showing the changes in body weight of mice in the microneedle patch experiment.

[0058] In the figure, statistical differences are marked as * p < 0.05, ** p < 0.01. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0060] In specific embodiments of the present invention, the dosage of the drug of the present invention can be varied according to factors such as formulation method, manner, patient's age, weight, gender, pathological condition, diet, time, route, and responsiveness.

[0061] In specific embodiments of the present invention, the therapeutically effective amount refers to an amount that can produce a therapeutic effect on humans and / or animals and is acceptable to humans and / or animals. For example, a therapeutically or pharmaceutically effective amount refers to the amount of drug required to produce the desired therapeutic effect, which can be reflected by the results of clinical trials, animal model studies, and / or in vitro studies. A pharmaceutically effective amount depends on several factors, including but not limited to: the characteristics of the treatment subject (such as the height, weight, sex, age, and medication history of the treatment subject), and the severity of the disease.

[0062] In specific embodiments of the present invention, the administration methods of the drug include, but are not limited to: oral administration, non-gastrointestinal administration, administration via inhalation spray, local administration, rectal administration, nasal administration, buccal administration, vaginal administration, or administration via an implanted drug storage device.

[0063] In specific embodiments of the present invention, any orally acceptable dosage form may be used, including but not limited to capsules (hard capsules, soft capsules), tablets (sugar-coated tablets, film-coated tablets, enteric-coated tablets), aqueous suspensions, or solutions.

[0064] In specific embodiments of the present invention, liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.

[0065] In specific embodiments of the present invention, solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0066] In specific embodiments of the invention, the medicament further includes one or more pharmaceutically acceptable carriers or excipients. The pharmaceutically acceptable carriers or excipients may contain inert components that do not unduly inhibit the biological activity of the compound. The pharmaceutically acceptable carriers or excipients should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or free from other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical formulation techniques can be used.

[0067] In specific embodiments of the invention, pharmaceutically acceptable carriers or excipients include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, disintegrants, preservatives, etc. These substances are used as needed to aid in the stability of the formulation or to contribute to its activity or bioavailability, or to produce an acceptable taste or odor when taken orally. Formulations that can be used in such pharmaceutical compositions may be in the form of the original compound itself or optionally in the form of its pharmaceutically acceptable salts. Such formulated pharmaceutical compositions may be administered in any suitable manner known to those skilled in the art as needed.

[0068] In specific embodiments of the present invention, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, and water.

[0069] In specific embodiments of the present invention, the adhesive includes, but is not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose and hydroxypropylmethylcellulose.

[0070] In specific embodiments of the present invention, the surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol.

[0071] In specific embodiments of the present invention, the humectant includes, but is not limited to, glycerin and starch.

[0072] In specific embodiments of the present invention, the adsorption carrier includes, but is not limited to, starch, lactose, bentonite, silica gel, kaolin, and bentonite.

[0073] In specific embodiments of the present invention, the lubricant includes, but is not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

[0074] In specific embodiments of the present invention, the fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0075] In specific embodiments of the present invention, the disintegrants include, but are not limited to, crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, and soybean polysaccharides.

[0076] Example 1: 5-azacytosine nucleoside inhibits the growth of cervical intraepithelial neoplasia cells in vitro.

[0077] I. Experimental Methods

[0078] This embodiment used the cervical intraepithelial neoplasia cell line (S12) (regulated by Dicer1, influencing the tumor biological characteristics of cervical cancer cells, *Modern Journal of Urogenital Oncology*; provided by Tongji Medical College, Huazhong University of Science and Technology), and human cervical epithelial cells (HUCEC), human immortalized keratinocytes (HaCaT), and human renal epithelial cells (293T) as controls. Cells were treated with culture media containing different concentrations of 5-azacytidine (5-Aza-C). To analyze the effect of 5-Aza-C on the proliferation of the above cells, CCK8 assays and colony formation assays were used to detect changes in cell proliferation and colony formation ability after 5-Aza-C treatment.

[0079] 1. Cell Culture: S12 cells were cultured in DMEM-F12 medium containing 5% fetal bovine serum and 1% penicillin / streptomycin. The medium was also supplemented with the following components: cholera toxin 8.4 ng / mL, insulin 5 μg / mL, adenine 24.3 μg / mL, hydrocortisone 0.5 μg / mL, and epidermal growth factor 10 ng / mL. HUCEC cells, HaCaT cells, and 293T cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were cultured in a cell culture incubator at 37°C and 5% CO2. When the cell confluence reached 70-80%, the cells were passaged at a ratio of 1:2-4. During passage, the old medium was aspirated, and the cells were carefully washed twice with sterile phosphate-buffered saline (PBS), discarded, and then digested with 0.25% trypsin. The cells were then seeded into culture flasks for further culture or plated.

[0080] 2. Effects of CCK8 assay on the proliferation of 5-azacytosine nucleoside in cervical intraepithelial neoplasia cells and control cells.

[0081] S12 cells, HUCEC cells, HaCaT cells, and 293T cells in good growth condition were selected, digested with trypsin, and prepared into cell suspensions. 2000 cells / 100 μL / well were seeded into 96-well plates and incubated at 37℃, 5% CO2 for 8 hours. After observing uniform cell plating and good adhesion, the original culture medium was aspirated, and corresponding culture media containing different concentrations of 5-azacytosine were added. The plates were then incubated at 37℃, 5% CO2 for 96 hours.

[0082] After 96 hours, add 10 μL of CCK-8 solution directly to each well, taking care to avoid generating air bubbles during the addition process. Incubate the culture plate with CCK-8 at 37°C and 5% CO2 for 1-4 hours. Remove the 96-well plate and measure the OD value of each well at 450 nm using a microplate reader. Analyze the data and calculate the half-maximal inhibitory concentration (IC50).

[0083] 3. Detection of the effect of 5-azacytosine on the clonogenic ability of cervical intraepithelial neoplasia cells and cervical epithelial cells.

[0084] S12 and HUCEC cells in good growth condition were selected, digested with trypsin, and prepared into cell suspensions. 1000 cells / 2000 μL / well were seeded into 6-well plates and incubated at 37℃, 5% CO2 for 8 hours. After observing uniform cell distribution and good adhesion, the original culture medium was aspirated, and corresponding culture media containing different concentrations of 5-azacytosine nucleoside were added. The plates were then incubated at 37℃, 5% CO2, with the culture medium and drugs replaced every 72 hours. After 7-10 days of culture, when visible clones appeared in the culture plates, the culture was terminated, the supernatant was discarded, and the cells were carefully washed twice with PBS.

[0085] Cells were fixed with 4% paraformaldehyde for 15 min, then the fixative was discarded. Cells were stained with 1 mL of crystal violet-methanol solution, then slowly washed away with running water and air-dried. The number of clones was counted by photographing with a digital camera, and the colony formation rate was calculated.

[0086] II. Experimental Results

[0087] To analyze the effects of 5-azacytosine nucleoside on the proliferation of cervical intraepithelial neoplasia cells and control epithelial cells, this invention performed a CCK8 assay. Gradual concentrations of 5-azacytosine nucleoside were added to the culture medium of cervical intraepithelial neoplasia cells and control epithelial cells, and the inhibitory effect was detected. Figure 1 As shown, the IC50 values ​​of 5-azacytosine nucleoside against S12 cells, HUCEC cells, HaCaT cells, and 293 cells were 359.6 nM, 10159 nM, 3395 nM, and 6168 nM, respectively. It can be seen that the inhibitory effect of 5-azacytosine nucleoside on the growth of cervical intraepithelial neoplasia cells is far superior to that on control epithelial cells, demonstrating its highly effective and low-toxicity characteristics. Furthermore, as... Figures 2-3 As shown, 5-Aza-C can effectively inhibit the clonal formation of cervical intraepithelial neoplasia cells, while having a very weak effect on cervical epithelial cells. These results indicate that 5-azacytosine nucleoside has a selective inhibitory effect on the proliferation of cervical intraepithelial neoplasia cells under in vitro conditions, providing experimental evidence for its use as a local treatment for precancerous cervical lesions.

[0088] Example 2: 5-azacytosine promotes apoptosis in cervical intraepithelial neoplasia cells

[0089] I. Experimental Methods

[0090] This embodiment further analyzes the effect of 5-azacytosine on apoptosis in cervical intraepithelial neoplasia cells. Cervical intraepithelial neoplasia cells S12 were selected as the experimental subject. Cells were treated with culture media containing different concentrations of 5-azacytosine, and the effect of 5-azacytosine on the apoptosis level of cervical intraepithelial neoplasia cells was evaluated through apoptosis detection experiments.

[0091] 1. Cell Culture: S12 cells were cultured in DMEM-F12 medium containing 5% fetal bovine serum and 1% penicillin-streptomycin. The medium was also supplemented with the following additional components: cholera toxin 8.4 ng / mL, insulin 5 μg / mL, adenine 24.3 μg / mL, hydrocortisone 0.5 μg / mL, and epidermal growth factor 10 ng / mL. Cell culture was conducted at 37°C in a 5% CO2 incubator. When cell confluence reached 70-80%, cells were passaged at a ratio of 1:2-4. During passage, the old medium was aspirated, and the cells were carefully washed twice with sterile phosphate-buffered saline (PBS), discarded, and then digested with 0.25% trypsin. Cells were then seeded into culture flasks for further culture or plated.

[0092] 2. 5-azacytosine nucleoside treatment procedure: Select S12 cells in good growth condition, digest them with trypsin to prepare a cell suspension, and seed 10,000 cells / 1 mL / well in 12-well plates. Incubate at 37℃ and 5% CO2 for 8 hours. Observe that the cells are evenly spread and adhere well. Then, remove the original culture medium and add culture medium containing 0.2 and 1 μM 5-azacytosine nucleoside as an experimental control group. Incubate at 37℃ and 5% CO2 for 72 hours, changing the medium every 24 hours.

[0093] 3. Flow cytometry detection of apoptosis: S12 cells treated with 5-azacytosine nucleoside for 72 hours were collected. Adherent cells were digested with trypsin, collected, and washed twice with pre-cooled PBS. Subsequently, following the instructions of the apoptosis detection kit, cells were resuspended in binding buffer, stained with Annexin V-FITC and propidium iodide (PI) in the dark, and incubated at room temperature for a certain period before flow cytometry was used to detect apoptosis. Early and late apoptotic cells were analyzed and statistically analyzed based on Annexin V and PI staining results.

[0094] II. Experimental Results

[0095] To analyze the effect of 5-azacytosine on apoptosis in cervical intraepithelial neoplasia cells, this example describes the treatment of S12 cells with the drug, followed by flow cytometry analysis to detect the level of apoptosis. The results are as follows: Figures 4-5 As shown, compared with the untreated control group, the proportion of apoptosis in S12 cells treated with 5-azacytosine nucleoside was significantly increased, and the degree of apoptosis increased with increasing drug concentration. These results indicate that 5-azacytosine nucleoside can effectively induce apoptosis in cervical intraepithelial neoplasia cells under in vitro conditions, providing experimental evidence for its use as a local treatment for cervical precancerous lesions.

[0096] Example 3: 5-azacytosine nucleoside inhibits the growth of cervical intraepithelial neoplasia cells in vivo.

[0097] This embodiment further verifies the inhibitory effect of 5-azacytosine on the growth of cervical intraepithelial neoplasia cells under in vivo conditions. The in vivo inhibitory effect of 5-azacytosine was evaluated using a subcutaneous tumor animal model of cervical intraepithelial neoplasia cells S12.

[0098] I. Experimental Methods

[0099] Sixteen female 4–5 week old NSG mice (purchased from Jicui Pharmaceutical, Guangzhou, China) were randomly divided into a control group (Ctrl group) and an experimental group (5-Aza-C group), with 8 mice in each group. After one week of acclimatization, the mice were subcutaneously inoculated with S12 cell line. Starting from day 8 post-inoculation, mice in the 5-Aza-C group received a subcutaneous injection of 5-azacytosine (2 mg / kg, PBS as solvent) around the tumor, while mice in the Ctrl group received an equal volume of the solvent, once every three days. Tumor volume and body weight were measured every three days during the experiment. At the end of the experiment on day 32, the mice were sacrificed, and the tumors were photographed, weighed, and analyzed.

[0100] II. Experimental Results

[0101] To analyze the inhibitory effect of 5-azacytosine on cervical intraepithelial neoplasia cells in vivo, an animal model was used for evaluation. Compared with the control group, the tumor volume in the 5-Aza-C group was significantly reduced ( Figure 6 The tumor growth curves of the two groups showed significant differences. Figure 7 The endpoint tumor weight was statistically significantly lower in the Ctrl group than in the Ctrl group. Figure 8 P < 0.01. Throughout the experiment, there was no significant difference in body weight between the two groups of mice (P < 0.01). Figure 9The results indicate that subcutaneous injection of 5-Aza-C as a local treatment method did not cause significant toxic side effects. In summary, these results demonstrate that 5-Aza-C can significantly inhibit the growth of cervical intraepithelial neoplasia cells in animal models and exhibits good safety. This further validates its feasibility for treating precancerous cervical lesions and provides experimental evidence for the subsequent application of 5-Aza-C in minimally invasive local drug delivery formulations.

[0102] Example 4: Preparation and Mechanical Properties of Microneedle Patches

[0103] To facilitate the application of 5-azacytosine nucleoside in the treatment of precancerous cervical lesions and to address the problem of low local delivery efficiency at the application level, this invention provides a microneedle patch with good mechanical properties and structural stability, wherein the microneedles are loaded with 5-azacytosine nucleoside.

[0104] I. Experimental Methods

[0105] 1. Preparation of microneedle patches

[0106] Using hyaluronic acid and its modified derivatives as the main matrix materials, multiple microneedle patches with different formulations were prepared by adjusting the type, concentration, and crosslinking method of the materials. These formulations include, but are not limited to, unmodified hyaluronic acid (HA) systems, methacrylated hyaluronic acid (HAMA) systems, and systems in which polyethylene glycol (PEG) of different mass fractions is introduced as a modifying component. In some formulations, a photoinitiator system is used in conjunction with ultraviolet light irradiation or the introduction of calcium ions for crosslinking treatment to improve the structural stability and mechanical strength of the microneedle patches.

[0107] The specific operation is as follows: dissolve the matrix material in water to prepare a precursor solution (the concentration of the matrix material can be 1-20wt%), add or not add crosslinking agent / photoinitiator and mix evenly, inject into the microneedle mold, centrifuge to make the solution fully fill the mold needle cavity, after filling is completed, dry the mold, and perform or not perform photocuring treatment, demold to obtain microneedle patch.

[0108] 2. Mechanical property testing of microneedle patches

[0109] The mechanical properties of microneedle patches with different formulations were evaluated using a pressure-displacement test method. The microneedle patches were placed on a mechanical testing platform, and a gradually increasing vertical pressure was applied to the microneedle array under controlled conditions. The pressure-deformation behavior during compression was recorded, and pressure-displacement curves were obtained. By analyzing the load-bearing capacity, deformation trend, and structural integrity of microneedle patches with different formulations during compression, their mechanical properties were comprehensively evaluated.

[0110] II. Experimental Results

[0111] The results showed that microneedle patches with different formulations exhibited significant differences in pressure-displacement curves. Microneedle patches without cross-linking treatment had lower overall load-bearing capacity and were prone to deformation under pressure; HAMA-based microneedle patches with photocross-linking treatment showed significantly improved mechanical properties and were able to withstand greater external forces.

[0112] Further comparison revealed that introducing an appropriate amount of PEG component into the HAMA system helps enhance the structural stability and compressive strength of the microneedle patch. Specifically, some microneedle patches containing PEG and treated with UV crosslinking exhibited higher maximum load-bearing capacity and better deformation stability in pressure-displacement tests.

[0113] Based on the combined mechanical test results of various microneedle patch formulations, the microneedle patch prepared by UV crosslinking of 8-12% HAMA, 0.5-1.5% PEG, and 0.1-1% LAP exhibited the best mechanical properties. It maintained good structural integrity under high pressure conditions and withstood external forces exceeding 2N in pressure-displacement tests, demonstrating potential as a local cervical drug delivery carrier. Partial mechanical property test results of different polymer material combinations are shown below. Figure 10 As shown.

[0114] Example 5: Comparison of sustained-release performance between HAMA photocurable microneedles and conventional HA microneedles

[0115] To compare the differences in dissolution behavior and sustained-release performance between photocurable HAMA microneedle patches and conventional hyaluronic acid (HA) microneedle patches, this embodiment constructed microneedle patches with different material systems and observed and compared their dissolution processes under the same conditions. Specific methods and results are as follows:

[0116] I. Experimental Methods

[0117] 1. Preparation of different microneedle patches

[0118] The following three groups of microneedle patches were prepared using the same method as in Example 4:

[0119] To compare the differences in dissolution behavior and sustained-release performance of microneedle patches with different material systems, the following experimental groups were set up in this embodiment:

[0120] Group 1: 10% HA + 1% PEG group:

[0121] The microneedle patch, constructed using conventional hyaluronic acid (HA) and polyethylene glycol (PEG 10K), does not undergo photocuring.

[0122] Group 2: 10% HAMA + 1% PEG group:

[0123] The microneedle patch, constructed using methacrylated hyaluronic acid (HAMA) and polyethylene glycol (PEG 10K), was not subjected to photocuring.

[0124] Group 3: 10% HAMA + 1% PEG + UV group:

[0125] Based on the HAMA system in Group 2, 0.5% LAP photoinitiator was added and photocured by ultraviolet light to form a cross-linked microneedle structure.

[0126] Each group of microneedle patches was placed under the same conditions, and their dissolution and structural integrity were observed and recorded at given time points (0.5h and 8h) to compare the dissolution rate and structural retention ability of microneedle patches of different material systems.

[0127] II. Experimental Results

[0128] The results are as follows Figure 11 As shown, conventional HA microneedle patches dissolve rapidly upon contact with a liquid environment, with the microneedle structure softening and gradually disappearing within a short period. HAMA non-photocured microneedle patches exhibit a slower dissolution rate compared to conventional HA microneedles, but still show a relatively rapid dissolution trend, with the microneedle structure undergoing significant deformation within a short time. In contrast, HAMA photocured microneedle patches maintain a relatively intact microneedle structure for a longer period under the same conditions, with a significantly slower dissolution process, demonstrating better structural stability.

[0129] The above results indicate that, compared with conventional HA microneedle patches, photocured HAMA-based microneedle patches exhibit slower dissolution behavior and better structure retention, which is beneficial for achieving sustained drug release. Therefore, the HAMA photocuring system is more suitable as a matrix material for cervical local drug delivery microneedle patches, providing a technical basis for the sustained-release delivery of demethylated drugs.

[0130] Example 6: Construction and characterization of HAMA-PEG@5-Aza-C demethylated microneedles

[0131] Based on the aforementioned microneedle patch formulation screening results, this embodiment uses the optimal microneedle system obtained from the screening to construct a microneedle patch loaded with 5-Aza-C, and characterizes its structural morphology and basic properties.

[0132] I. Experimental Methods

[0133] 1. Preparation of HAMA-PEG@5-Aza-C microneedle precursor solution: Hyaluronic acid methacrylated (HAMA) was dissolved in sterile deionized water to prepare a HAMA solution with a predetermined mass fraction. A certain proportion of polyethylene glycol (PEG) was added as a modifying component, specifically 8-12 wt% HAMA + 0.5-1.5 wt% PEG. Under stirring, 0.1-1 wt% of photoinitiator LAP was added to the above solution to ensure complete dissolution and form a homogeneous polymer precursor solution. Subsequently, 5-Aza-C was added to the precursor solution at a content of 1-5 wt%, and the mixture was thoroughly mixed to obtain the drug-containing microneedle precursor solution.

[0134] 2. Molding and Construction of HAMA-PEG@5-Aza-C Microneedle Patches: The drug-containing microneedle precursor solution was injected into a microneedle mold, and the solution was centrifuged to fully fill the mold cavity. After filling, the mold was dried and then irradiated with ultraviolet light under predetermined conditions (e.g., photocuring at a 40W ultraviolet light source, with the patch 5cm away from the light source, for 5 minutes) to induce a cross-linking reaction of HAMA, forming a stable microneedle structure. After photocuring, the microneedle patch was demolded from the mold to obtain the HAMA-PEG@5-Aza-C demethylated microneedle patch. The microneedle patch includes a base layer and a regularly arranged microneedle array layer, with each microneedle unit having a conical or near-conical structure.

[0135] 3. Characterization of HAMA-PEG@5-Aza-C microneedle patches: The overall structure and microneedle array morphology of HAMA-PEG@5-Aza-C demethylated microneedle patches were characterized using a microscopic imaging system and a scanning electron microscope. Specifically, the overall appearance and array arrangement of the microneedle patches were observed using a microscopic imaging system, and the microscopic morphology and tip structure of the microneedles were further characterized using a scanning electron microscope.

[0136] II. Experimental Results

[0137] Figure 12 A schematic illustration shows the application scenario of microneedle patches in the cervix, indicating that the microneedle system can directly act on the cervical lesion area to achieve local drug delivery. Figure 12 B is a physical image of the completed microneedle patch. The patch has a complete overall structure, regular shape, good mechanical integrity, and is easy to operate and apply.

[0138] To further observe the macroscopic arrangement characteristics of the microneedle array, a stereomicroscope was used to observe the surface of the microneedle patch. Figure 12 C). The results showed that the microneedles were distributed in a regular array on the substrate, with uniform arrangement and consistent morphology of each needle. No obvious collapse or defects were observed, indicating that the microneedle preparation process had good repeatability and stability.

[0139] Subsequently, the microstructure of the microneedles was finely characterized using scanning electron microscopy (SEM). Figure 12 D shows the overall SEM morphology of the microneedle array. The microneedles are vertically arranged on the substrate surface with uniform spacing between them. The array structure is clear, and no obvious structural distortion was observed. Figure 12 E is a magnified side view of the microneedle, showing a regular conical or pyramidal structure with clear edges and a relatively smooth surface, indicating good molding quality. Further high-magnification observation of the needle tip structure... Figure 12 F), the results showed that the microneedle tip was sharp and the tip structure was intact, which helped to reduce the resistance of skin or mucous membrane puncture and improve tissue penetration ability.

[0140] The results above indicate that the microneedle patch prepared in this study exhibits good structural integrity and morphological consistency at both the macroscopic and microscopic levels, providing a structural basis for its use as a local drug delivery carrier and offering reliable material support for subsequent drug loading and biological function verification experiments.

[0141] Example 7: Validation of the efficacy of HAMA-PEG@5-Aza-C demethylated microneedle patches in a mouse model of precancerous cervical lesions

[0142] This embodiment uses an NSG mouse subcutaneous xenograft model to verify the local inhibitory effect of HAMA-PEG@5-Aza-C microneedle patch loaded with demethylating drugs on the growth of cervical precancerous lesions under in vivo conditions.

[0143] I. Experimental Methods

[0144] 1. Establishment of a subcutaneous cervical precancerous lesion model in NSG mice: Ten healthy female NSG mice (purchased from Jicui Pharmaceutical, Guangzhou, China) were selected. Human cervical intraepithelial neoplasia cells S12 were digested and prepared into a single-cell suspension, which was then inoculated subcutaneously into NSG mice to construct a subcutaneous transplantation model of cervical precancerous lesions. After palpable subcutaneous lesion tissue formed at the inoculation site, the experimental animals were randomly divided into groups of 5 for subsequent local drug treatment.

[0145] 2. Local administration of HAMA-PEG@5-Aza-C microneedle patches (formulation: 10% HAMA + 1% PEG 10K + 0.5% LAP + 2% 5-Aza-C + UV): Administration began on the seventh day after tumor implantation. The prepared HAMA-PEG@5-Aza-C demethylated microneedle patches were applied to the subcutaneous lesion surface. Under appropriate external force, the microneedle array pierced into the lesion area, achieving local delivery of the demethylated drug to the lesion site. The microneedle patches were replaced every three days, i.e., on days 7, 10, 13, 16, and 19 of the experiment.

[0146] 3. Evaluation of local application effect: During the administration process, the growth of subcutaneous lesion tissue was monitored regularly. At the end of the experiment, the experimental animals were sacrificed, and the subcutaneous lesion tissue was removed. Its volume and weight were compared and analyzed to evaluate the local inhibitory effect of HAMA-PEG@5-Aza-C microneedle patch.

[0147] II. Experimental Results

[0148] In vivo experimental results showed that, compared with the control group, the tumor volume in the HAMA-PEG@5-Aza-C group was significantly reduced ( Figure 13 A) There were significant differences in the tumor growth curves between the two groups. Figure 13 B). The endpoint tumor weight was statistically significantly lower in the Ctrl group ( Figure 13 C, P < 0.01. Throughout the experiment, there was no significant difference in body weight between the two groups of mice (C, P < 0.01). Figure 14 The results indicate that HAMA-PEG@5-Aza-C microneedling treatment did not cause significant toxic side effects.

[0149] The above results demonstrate that the HAMA-PEG@5-Aza-C demethylated microneedle patch provided by this invention can achieve local delivery of demethylated drugs in a mouse subcutaneous cervical precancerous lesion model and effectively inhibit the in vivo growth of lesion tissue. These results further verify the feasibility and effectiveness of the demethylated microneedle patch of this invention for the local treatment of cervical precancerous lesions.

[0150] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

Use of 1,5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of cervical intraepithelial neoplasia.

2. The application according to claim 1, characterized in that... The 5-azacytosine nucleoside has the chemical formula C8H. 12 N4O5, the structural formula is shown below: 。 Use of 3,5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs that inhibit the growth of cervical intraepithelial neoplasia cells. The use of 4,5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs that inhibit the formation of clonal cells of cervical intraepithelial neoplasia. Use of 5,5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts in the preparation of drugs that promote apoptosis of cervical intraepithelial neoplasia cells.

6. The application according to any one of claims 1-5, characterized in that... The drugs mentioned, whether identical or different, include therapeutically effective amounts of 5-azacytosine nucleoside or its derivatives or pharmaceutically acceptable salts.

7. A demethylated microneedle patch loaded with 5-azacytosine nucleoside for the prevention and / or treatment of cervical intraepithelial neoplasia, used in any one of the applications described in claims 1-5, characterized in that: The demethylated microneedle patch structure includes a base layer and a microneedle array layer distributed on the surface of the base layer; the microneedle array layer is composed of multiple regularly arranged microneedle units, each microneedle unit being arranged perpendicularly or approximately perpendicularly to the base layer; in the microneedle patch, the loading amount of 5-azacytosine nucleoside is 1-5 wt%.

8. The demethylated microneedle patch according to claim 7, characterized in that: The substrate layer and the microneedle array layer, which may be the same or different, are respectively prepared from biodegradable polymer materials; the biodegradable polymer materials include at least one of hyaluronic acid, polyvinylpyrrolidone, polyethylene glycol, gelatin, chitosan, and modified derivatives of the above materials; the content of the biodegradable polymer materials is 1-20 wt%.

9. The demethylated microneedle patch according to claim 7, characterized in that... The microneedle unit is loaded with 5-azacytosine nucleoside, and the loading method includes at least one of the following: 5-azacytosine nucleoside was uniformly dispersed in the polymer matrix of the microneedle unit; 5-azacytosine nucleoside was embedded in the internal structure of the microneedle unit; A drug-containing coating is formed on the surface of the microneedle unit.

10. A method for preparing a demethylated microneedle patch according to any one of claims 7-9, characterized in that... By preparing a precursor solution of biodegradable polymer material, dispersing 5-azacytosine nucleoside in the precursor solution, adding or not adding a crosslinking agent and / or photoinitiator, adding the material to a mold, drying, and performing or not performing photocuring, and then demolding, a microneedle patch is obtained.

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