A pharmaceutical composition for lichen sclerosus of the vulva and a method of preparing the same

Abstract

The present application provides a kind of pharmaceutical composition for vulvar sclerosing lichen and its preparation method, the pharmaceutical composition includes active ingredient and pharmaceutically acceptable vulva local administration adjuvant, active ingredient is made of 0.005-0.02 parts of mometasone furoate, 0.05-0.3 parts of calcineurin inhibitor, 0.5-2 parts of paeoniflorin, 0.8-3 parts of asiaticoside, 0.3-1.5 parts of recombinant collagen, 0.2-1 part of ginkgolide B.The present application is aimed at the core pathogenesis of disease, constructs multi-target whole course of disease synergistic therapy system, can realize anti-inflammatory regulation, anti-fibrosis, basement membrane repair, microcirculation improvement simultaneously, low-dose strong effect, greatly reduce side effect and recurrence rate;Liposome targeted delivery technology is used, the stability of active ingredient is guaranteed, process controllable adaptation industrialization production, and clinical application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a pharmaceutical composition for vulvar lichen sclerosus and its preparation method. Background Technology

[0002] Vulvar lichen sclerosus (VLS) is a chronic, progressive, inflammatory autoimmune skin and mucous membrane disease that commonly affects the female vulva. Women of reproductive age and postmenopausal women are at high risk, with a global incidence rate of approximately 1.7%. The core pathological features of this disease include progressive atrophy of the vulvar epidermis, rupture of the epidermal-dermal basement membrane band, loss of hemidesmosomes, hyalinization and fibrosis of superficial dermal collagen fibers, and extensive T-lymphocyte infiltration in the dermis. Clinically, it mainly manifests as persistent vulvar itching, pain, and dyspareunia. Prolonged illness can lead to vulvar anatomical deformities, vaginal stenosis, and even a 3%-5% risk of secondary vulvar squamous cell carcinoma, severely impacting the patient's physical health and quality of life.

[0003] Currently, the first-line treatment for vulvar lesions (VLS) is topical administration of high-potency corticosteroids (such as 0.05% clobetasol propionate cream). This approach only relieves itching symptoms through non-specific anti-inflammatory effects and cannot reverse the core pathological processes characteristic of VLS, such as epithelial atrophy, dermal fibrosis, and basement membrane destruction. Furthermore, long-term use can easily lead to serious adverse reactions such as vulvar skin atrophy, telangiectasia, secondary fungal infections, and hypothalamic-pituitary-adrenal axis suppression. The relapse rate after discontinuation of the drug is as high as 40%-60%. Calcineurin inhibitors are second-line treatments, mostly used for patients with hormone intolerance or hormone resistance. However, their anti-inflammatory effect as monotherapy is weaker than that of high-potency corticosteroids. In the initial stages of use, they can easily cause strong local irritation reactions such as burning and stinging of the vulvar mucosa. Similarly, they cannot reverse the fibrosis and epithelial atrophy process of VLS and cannot reduce the relapse rate at its root.

[0004] Existing adjunctive treatments for VLS, including physical therapy, external application of traditional Chinese medicine, and type I / III collagen moisturizing and repairing agents, can only alleviate symptoms and do not target the core pathogenesis of VLS. The application of collagen is limited to simply supplementing the dermal matrix with type I and III collagen, failing to target and repair the core pathological defects characteristic of VLS, such as hemidesmosome loss, basement membrane rupture, and disruption of epidermal stem cell homeostasis. This fails to break the vicious cycle of "barrier damage - increased inflammation - fibrosis progression - atrophy."

[0005] In summary, existing VLS treatment technologies have three major drawbacks: First, they can only treat the symptoms of inflammation and itching, but cannot reverse the core pathological process of VLS, and cannot fundamentally block disease progression or reduce recurrence rates; second, monotherapy requires high-dose administration, and long-term use results in significant side effects and poor patient compliance; third, there are no synergistic compound preparations targeting the entire pathological chain of VLS. Existing drug combinations are mostly used sequentially in clinical practice, without scientific formulation ratios and formulation process design, and cannot achieve synergistic therapeutic effects across multiple targets. Summary of the Invention

[0006] In view of this, the present invention proposes a pharmaceutical composition for vulvar lichen sclerosus and a method for preparing the same, thereby solving the above-mentioned problems.

[0007] The technical solution of this invention is implemented as follows:

[0008] A pharmaceutical composition for vulvar lichen sclerosus includes an active ingredient and excipients, wherein the active ingredient comprises, by weight, the following components: mometasone furoate 0.005-0.02 parts, calcineurin inhibitor 0.05-0.3 parts, paeoniflorin 0.5-2 parts, asiaticoside 0.8-3 parts, recombinant collagen 0.3-1.5 parts, and ginkgolide B 0.2-1 parts.

[0009] Preferably, the active ingredients, by weight, include the following components: 0.01 parts mometasone furoate, 0.1 parts calcineurin inhibitor, 1.0 part paeoniflorin, 1.5 parts asiaticoside, 0.8 parts recombinant collagen, and 0.5 parts ginkgolide B.

[0010] Preferably, the calcineurin inhibitor is pimecrolimus or tacrolimus, with pimecrolimus being preferred; pimecrolimus has strong lipophilicity, excellent local retention on the vulvar mucosa, extremely low systemic absorption, and significantly lower local irritation than tacrolimus, making it more suitable for long-term use on the delicate vulvar mucosa.

[0011] Preferably, the recombinant collagen is recombinant human type XVII collagen, which can precisely repair the broken basement membrane band at the VLS lesion site, restore epidermal stem cell homeostasis, and reverse vulvar epithelial atrophy.

[0012] Preferably, the excipients include 10-20 parts of oil phase matrix, 2-5 parts of emulsifier, 5-15 parts of humectant, 0.5-3 parts of penetration enhancer, and 0.05-0.5 parts of antibacterial agent.

[0013] Further preferably, the oil phase matrix is ​​composed of stearic acid, white petrolatum, liquid paraffin, and glyceryl monostearate in a mass ratio of (2-4):(3-5):(3-6):(2-3);

[0014] The emulsifier is polysorbate 80, or a composite emulsifier composed of polysorbate 80 and Span 80 in a mass ratio of (3-5):1.

[0015] The humectant is composed of glycerol and 1,3-butanediol in a mass ratio of (2-4):1.

[0016] The penetration enhancer is composed of menthol and laurocapram at a mass ratio of 1:(1-3);

[0017] The antibacterial agent is phenoxyethanol.

[0018] Preferably, the dosage form of the pharmaceutical composition is a cream, ointment, gel, lotion, or vaginal suppository, with a preference for a cream; the cream is an O / W type cream with a fine texture, good spreadability, long retention time at the vulvar lesion, no stickiness, easy to wash off, and is fully suitable for local vulvar drug administration scenarios.

[0019] This invention also provides a method for preparing a pharmaceutical composition for vulvar lichen sclerosus, wherein the dosage form is a cream, comprising the following steps:

[0020] Preparation of S1 drug-loaded liposome suspension: Weigh the lipid components, mometasone furoate, calcineurin inhibitor, paeoniflorin, asiaticoside, and ginkgolide B, dissolve them in an organic solvent, remove the organic solvent by rotary evaporation under reduced pressure, and form a uniform lipid film on the inner wall of the container; add hydration solution for hydration, and after high-pressure homogenization, filter and sterilize with a microporous membrane to obtain a drug-loaded liposome suspension with an average particle size of 80-200 nm, for later use;

[0021] Preparation of S2 aqueous base solution: Weigh the humectant and antibacterial agent according to the ratio, add them to purified water, heat and stir until completely dissolved, sterilize and cool to below 40℃, add recombinant collagen, stir until completely dispersed, adjust the pH of the system to 4.0-6.0 with pH adjuster to obtain aqueous phase, keep warm at 35-40℃ for later use;

[0022] S3 oil phase preparation: Weigh the oil phase matrix and emulsifier according to the ratio, heat until completely melted, stir evenly, keep warm at 75-85℃ for later use, and obtain the oil phase;

[0023] S4 Emulsification and Mixing: The oil phase in a warm state is slowly added to the aqueous phase, and emulsified by high-speed stirring at a constant temperature of 75-85℃. When the temperature drops to below 38℃, the drug-loaded liposome suspension prepared in step S1 is added, and the mixture is stirred at low speed until homogeneous. The mixture is then cooled to room temperature to obtain the drug composition in cream form.

[0024] Preferably, the specific process parameters of the above preparation method are as follows:

[0025] In step S1, the lipid components are hydrogenated soybean lecithin and cholesterol in a mass ratio of (3-5):1, and the total mass of the two is in the ratio of (2-8):1 to the total mass of mometasone furoate, calcineurin inhibitor, paeoniflorin, asiaticoside, and ginkgolide B.

[0026] The organic solvent is a mixed solvent composed of anhydrous ethanol and dichloromethane in a volume ratio of 1:(2-4), and the ratio of the organic solvent to the lipid component is (15-25) mL:1g.

[0027] The hydration solution is a phosphate buffer solution with a pH of 5.0, and the ratio of the hydration solution to the lipid component is (8-12) mL:1 g. The hydration temperature is 35-45℃ and the hydration time is 30-60 min.

[0028] The high-pressure homogenization is carried out at a pressure of 800-1200 bar, and the number of cycles is 3-5; the microporous filter membrane is a 0.22 μm polyethersulfone antibacterial filter membrane.

[0029] In step S2, the heating and stirring temperature is 70-90℃, the heating and stirring time is 20-30 minutes, and the sterilization method is pasteurization.

[0030] In step S4, the constant temperature high-speed stirring speed is 3000-5000 rpm, and the emulsification time is 15-30 min; the low-speed stirring speed when adding the drug-loaded liposome suspension is 500-1000 rpm.

[0031] The above-mentioned pharmaceutical composition is used in the preparation of a medicament for the prevention and / or treatment of vulvar lichen sclerosus; the medicament is also used to reverse vulvar epithelial atrophy, repair the epidermal basement membrane barrier, reverse dermal collagen fibrosis, reduce the recurrence rate of the disease, and for the treatment of patients with hormone-intolerant or hormone-resistant vulvar lichen sclerosus and for maintenance therapy during the remission period.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] This invention scientifically combines low-dose mometasone furoate, calcineurin inhibitors, paeoniflorin, asiaticoside, ginkgolide B, and recombinant human type XVII collagen. Low-dose mometasone furoate combined with pimecrolimus achieves synergistic anti-inflammatory effects through a dual-pathway approach, rapidly relieving itching and controlling inflammation; paeoniflorin regulates immune balance and synergistically blocks basement membrane protein degradation; asiaticoside inhibits fibroblast proliferation and reverses collagen fibrosis; recombinant human type XVII collagen repairs hemidesmosomes and the basement membrane band, reversing epithelial atrophy and repairing the mucosal barrier; ginkgolide B improves local microcirculation, accelerates lesion healing, and reduces recurrence rates. This synergistic combination not only achieves potent anti-inflammatory and immunomodulatory effects through a dual-pathway approach, rapidly controlling symptoms, but more importantly, it simultaneously inhibits dermal fibrosis, improves microcirculation, and specifically repairs damaged epidermal basement membrane bands.

[0034] This invention employs a thin-film dispersion-high-pressure homogenization method to prepare drug-loaded liposomes, encapsulating five small-molecule active ingredients within a liposome bilayer structure. The average particle size of the liposomes is controlled between 80-200 nm, allowing for precise penetration of the stratum corneum barrier at vulvar lesions. The liposomes target and remain at the lesion sites in the epidermis and dermis, significantly increasing local drug concentration, reducing systemic absorption, and eliminating the risk of systemic immunosuppression and hormone-related adverse reactions. Furthermore, the liposome encapsulation technology solves the problems of large solubility differences among multiple active ingredients and easy degradation during storage, ensuring the long-term stability of the formulation.

[0035] The pharmaceutical composition of this invention can cover the entire course of VLS treatment scenarios. It can be used for symptom control in the acute phase, long-term maintenance treatment in the remission phase, and treatment of patients with hormone intolerance and hormone resistance. It fills the gap in the existing VLS treatment field where there is no compound preparation suitable for the entire course of the disease, and has broad clinical application prospects. Detailed Implementation

[0036] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0037] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0039] The recombinant collagen in this embodiment of the invention is recombinant human type XVII collagen, a white or off-white spongy solid with a molecular weight of 53.1 kDa, purchased from Zhuhai Jibaikang Biotechnology Co., Ltd., CAS: 9007-34-5, product number: P403.

[0040] In the following examples and comparative examples, all raw materials used were pharmaceutical grade. Specifically, recombinant human type XVII collagen was a recombinant humanized collagen containing an extracellular active domain, with a bioactivity ≥95%; the purity of mometasone furoate, pimecrolimus, paeoniflorin, asiaticoside, and ginkgolide B was ≥98%; and the remaining excipients met the pharmaceutical excipient standards of the 2025 edition of the Chinese Pharmacopoeia. All examples were based on the preparation of 100g of cream, and the unit of measurement for raw materials is g.

[0041]

[0042] Creams were prepared according to the component formulations of Examples 1-3 above using the following method:

[0043] Preparation of S1 drug-loaded liposome suspension: Weigh hydrogenated soybean lecithin, cholesterol, mometasone furoate, pimecrolimus, paeoniflorin, asiaticoside, and ginkgolide B, and dissolve them in 100 mL of anhydrous ethanol-dichloromethane mixed solvent (volume ratio 1:3). Remove all organic solvents by rotary evaporation under reduced pressure at 35 °C (process solvents are not included in the total weight of the finished product), forming a uniform, crystal-free lipid film on the inner wall of a flask. Add 50 mL of pH 5.0 phosphate buffer, hydrate at 40 °C for 45 min, homogenize three times under 1000 bar high pressure, and filter sterilize using a 0.22 μm polyethersulfone sterile filter membrane to obtain a drug-loaded liposome suspension with an average particle size of 120 nm and an encapsulation efficiency of 91.2%, for later use.

[0044] Preparation of S2 aqueous phase base solution: Weigh glycerol, 1,3-butanediol, phenoxyethanol, and polysorbate 80, add them to purified water, heat and stir at 80℃ for 25 min until completely dissolved, pasteurize and cool to 38℃, add recombinant human type XVII collagen, stir until completely dispersed, add citrate-sodium citrate buffer to adjust the pH of the system to 5.2, obtain the aqueous phase, and keep warm at 38℃ for later use;

[0045] S3 oil phase preparation: Weigh stearic acid, white petrolatum, liquid paraffin, glyceryl monostearate, and Span 80, heat to 80°C until completely melted, stir evenly, and keep warm at 80°C for later use to obtain the oil phase;

[0046] S4 Emulsification and Mixing: Slowly add the oil phase kept at 80℃ to the aqueous phase kept at 38℃, emulsify at 80℃ and 4000rpm for 20 minutes, and when the temperature drops below 38℃, add the drug-loaded liposome suspension prepared in step S1, stir at 800rpm until uniform, and cool to room temperature to obtain a 100g cream dosage form of the drug composition.

[0047] Controlled Trial

[0048] Comparative Example 1: Commercially Available Comparative Drug

[0049] Commercially available 0.05% clobetasol propionate cream (VLS), 10g / tube.

[0050] Comparative Example 2: Commercially Available Comparative Drug

[0051] Commercially available 1% pimecrolimus cream, 10g / tube.

[0052] Comparative Example 3: Formula lacking recombinant human type XVII collagen

[0053] Except for removing recombinant human type XVII collagen and replenishing the weight to 100g with purified water, the rest of the formulation and preparation method are completely consistent with Example 1.

[0054] Comparison Example 4: Formulation without liposome encapsulation

[0055] Without preparing drug-loaded liposomes, mometasone furoate, pimecrolimus, paeoniflorin, asiaticoside, and ginkgolide B were directly added to the oil phase. The rest of the formulation and preparation method were completely consistent with Example 1. The total weight of the finished product was 100g.

[0056] Experimental Example 1: Formulation Quality and Stability Testing

[0057] 1. The creams prepared in Examples 1-3 were subjected to quality testing. The testing items included appearance, pH value, particle size, liposome particle size and encapsulation efficiency, related substances, and microbial limits. Accelerated stability testing was also conducted (6 months at 40℃±2℃ and 75%±5% relative humidity). The test results are as follows:

[0058]

[0059] 2. Five small molecule active ingredients (mometasone furoate, pimecrolimus, paeoniflorin, asiaticoside, and ginkgolide B): High performance liquid chromatography (HPLC) was used for detection, in accordance with General Chapter 0512 of the 2025 edition of the Chinese Pharmacopoeia. Standard curves were established for each ingredient, the measured content of each active ingredient in the sample was calculated, and the content retention rate was calculated by comparing it with the initial content at day 0.

[0060] Recombinant human type XVII collagen: The total protein content was determined by BCA protein quantification method, and the binding activity of its extracellular active domain was determined by enzyme-linked immunosorbent assay (ELISA). The protein content retention rate and bioactivity retention rate were calculated respectively, with bioactivity retention rate as the core judgment indicator.

[0061]

[0062] The results showed that the creams prepared in Examples 1-3 of this invention met all pharmaceutical standards, showed no significant changes after 6 months of accelerated stability testing, had high retention rates of active ingredients, and exhibited excellent formulation stability.

[0063] Experimental Example 2: Pharmacodynamic Test

[0064] 1. Experimental Materials

[0065] 1.1 Laboratory Animals

[0066] SPF-grade female BALB / c mice, 6-8 weeks old and weighing 18-22g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Housing environment: temperature 22℃±2℃, relative humidity 50%±5%, 12h light-dark cycle, free access to food and water. Experiments began after one week of acclimatization. All procedures in this experiment complied with the requirements of the "Guidelines for Ethical Review of Laboratory Animals".

[0067] 1.2 Reagents and Instruments

[0068] Main reagents: estradiol benzoate injection, benzo[a]pyrene, anhydrous ethanol, 4% paraformaldehyde, HE staining kit, Masson's trichrome staining kit, mouse IL-6, IL-17, IFN-γ, TGF-β1 ELISA kit; type XVII collagen primary antibody, α-SMA primary antibody, GAPDH primary antibody; BCA protein quantification kit, SDS-PAGE gel preparation kit.

[0069] Main instruments: electronic analytical balance, paraffin microtome, optical microscope, image analysis system, microplate reader, Western blotting electrophoresis transfer system, high-speed refrigerated centrifuge.

[0070] 1.3 Test Drug

[0071] Test substances 1-3: Creams prepared in Examples 1, 2, and 3 of this invention;

[0072] Control drug 1: Commercially available 0.05% clobetasol propionate cream;

[0073] Control drug 2: Commercially available 1% pimecrolimus cream;

[0074] Control drug 3: Control Example 3 cream lacking recombinant human type XVII collagen;

[0075] Control drug 4: Cream of control example 4 without liposome encapsulation;

[0076] Blank matrix cream: Cream excipients without any active ingredients, with a formulation consistent with Example 1.

[0077] 2. Experimental Methods

[0078] 2.1 Construction of VLS mouse model

[0079] A mouse VLS model was constructed using a combination of estradiol and benzo[a]pyrene skin application induction method. Specific procedures are as follows:

[0080] (1) Before modeling, remove the vulvar hair of the mice to expose the vulvar skin and mucous membrane, with an area of ​​about 1 cm².

[0081] (2) Apply 20 μL of 0.01% estradiol benzoate anhydrous ethanol solution to the vulva of each mouse daily for 7 consecutive days, and apply 20 μL of 0.5% benzo[a]pyrene olive oil solution every other day for 8 consecutive weeks.

[0082] (3) After the modeling was completed, six mice were randomly selected to take vulvar skin tissue. The results were verified by HE staining and Masson staining: the vulva of the mice showed epithelial atrophy, superficial collagen hyalinization and a large number of lymphocytes infiltrating the dermis, which were consistent with the typical pathological features of VLS. The modeling was deemed successful.

[0083] 2.2 Animal grouping and administration

[0084] The 108 mice that successfully developed the model were randomly divided into 9 groups of 12 mice each. At the same time, a normal control group of 12 mice that did not develop the model was set up. The grouping is as follows:

[0085] Normal control group (NC group): No model was established, and 20mg of blank matrix cream was applied to the vulva daily.

[0086] Model control group (M group): Modeling was successful, and 20mg of blank matrix cream was applied to the vulva daily.

[0087] Example 1 Group (E1 Group): Modeling was successful. Apply 20mg of Example 1 cream to the vulva daily.

[0088] Example 2 group (E2 group): Modeling was successful. Apply 20mg of Example 2 cream to the vulva daily.

[0089] Example 3 Group (E3 Group): Modeling was successful. Apply 20mg of Example 3 cream to the vulva daily.

[0090] Control group 1 (D1 group): Modeling was successful, and 20mg of 0.05% clobetasol propionate cream was applied to the vulva daily.

[0091] Control group 2 (D2 group): Modeling was successful, and 20mg of 1% pimecrolimus cream was applied to the vulva daily.

[0092] Control group 3 (D3 group): Modeling was successful, and 20mg of Control Group 3 cream was applied to the vulva daily.

[0093] Control group 4 (D4 group): Modeling was successful. Apply 20mg of Control Group 4 cream to the vulva daily.

[0094] All groups were administered the drug once daily for 4 consecutive weeks. During the administration period, mice had free access to food and water, and their general condition, itching behavior, and skin lesion changes were recorded.

[0095] 2.3 Detection Indicators and Methods

[0096] 2.3.1 Skin lesion appearance score

[0097] Skin lesion appearance scoring: Vulvar skin lesions in mice were scored using a single-blind method before drug administration, at 2 weeks after drug administration, and at 4 weeks after drug administration. Scoring criteria:

[0098] 0 points: The skin and mucous membranes of the vulva are normal in color, without hypopigmentation, atrophy, or erosion, and the anatomical structure is normal;

[0099] 1 point: Mild hypopigmentation, no itching or atrophy, normal anatomical structure;

[0100] 2 points: Significant hypopigmentation, mild itching, no obvious atrophy, and no anatomical deformation;

[0101] 3 points: Depigmentation accompanied by mild atrophy of the skin and mucous membranes, moderate itching, and no obvious deformation of the anatomical structure;

[0102] 4 points: Severe depigmentation, mucosal atrophy with vaginal stenosis, severe itching, erosion, and obvious anatomical deformation.

[0103] 2.3.2 Histopathological examination

[0104] Four weeks after drug administration, six mice in each group were sacrificed, and full-thickness vulvar skin tissue was harvested. The tissue was fixed in 4% paraformaldehyde for 24 hours, then dehydrated in a gradient manner, cleared, and embedded in paraffin. 4 μm thick sections were prepared and stained with hematoxylin and eosin (HE) and Masson's trichrome. Tissue morphology was observed under an optical microscope, and quantitative analysis was performed using an image analysis system.

[0105] (1) Epithelial thickness measurement: In HE stained sections, 5 fields of view were randomly selected from each section to measure the vertical distance from the epidermal basement membrane to the stratum corneum, and the average value was taken as the epithelial thickness;

[0106] (2) Inflammatory cell infiltration score: In HE-stained sections, the degree of inflammatory cell infiltration in the dermis was assessed using a single-blind method. The scoring criteria were as follows:

[0107] 0 points: No inflammatory cell infiltration;

[0108] 1 point: A small number of scattered inflammatory cells infiltrated the superficial dermis;

[0109] 2 points: Diffuse inflammatory cell infiltration in the dermis, without lymphoid follicle formation;

[0110] 3 points: Extensive inflammatory cell infiltration throughout the entire dermis, accompanied by the formation of lymphoid follicles.

[0111] (3) Collagen fibrosis scoring: In Masson trichrome stained sections, the degree of collagen fiber proliferation and hyalinization in the dermis was assessed by a single-blind method, and the scoring criteria were as follows;

[0112] 0 points: Collagen fibers are neatly arranged, without hyperplasia or hyaline degeneration;

[0113] 1 point: Mild hyperplasia of superficial dermal collagen fibers, slightly disordered arrangement, no hyalinization;

[0114] 2 points: Moderate proliferation of collagen fibers in the dermis, disordered arrangement, and focal hyalinization;

[0115] 3 points: Severe proliferation and fusion of collagen fibers in the entire dermis, extensive hyalinization, accompanied by superficial dermal edema.

[0116] 2.3.3 Detection of tissue inflammatory factors and fibrotic factors;

[0117] Four weeks after administration, vulvar skin tissue was collected from six mice in each group. After weighing, the tissue homogenate was prepared by adding pre-cooled PBS buffer. The homogenate was centrifuged at 12,000 r / min for 15 min at 4°C. The supernatant was collected and the contents of interleukin-6 (IL-6), interleukin-17 (IL-17), interferon-γ (IFN-γ), and transforming growth factor-β1 (TGF-β1) in the tissue were detected according to the ELISA kit instructions. The results were expressed as pg / mg protein.

[0118] 2.3.4 Detection of tissue protein expression

[0119] The protein expression levels of type XVII collagen and α-smooth muscle actin (α-SMA, a core marker of fibrosis) in mouse vulvar skin tissue were detected by Western blotting. Mouse vulvar skin tissue was collected, total protein was extracted, quantified using the BCA method, and subjected to SDS-PAGE electrophoresis, membrane transfer, and blocking. Primary antibodies (type XVII collagen 1:1000, α-SMA 1:1000, GAPDH 1:5000) were added and incubated overnight at 4°C. After washing, secondary antibody was added and incubated at room temperature for 1 h. After development, the gray values ​​of the bands were measured using an image analysis system. The relative protein expression level was expressed as the ratio of the gray values ​​of the target protein to the internal control GAPDH.

[0120] 2.3.5 Observation of relapse rate after drug withdrawal

[0121] After 4 weeks of drug administration, the drug was discontinued and the mice were observed for another 4 weeks. There were 6 mice remaining in each group. The appearance of skin lesions was scored weekly. The criteria for relapse were: the skin lesion score increased by ≥2 points after drug discontinuation compared to the time of drug discontinuation, or severe itching, depigmentation, or atrophy reappeared. The relapse rate of each group was calculated.

[0122] Relapse rate = number of mice that relapsed / total number of mice in the group × 100%.

[0123] 3. Experimental Results

[0124] 3.1 Effects on the appearance of skin lesions in mice

[0125] Before administration, there was no significant difference in the skin lesion appearance score among the model groups (P>0.05), and all scores were significantly higher than those of the normal control group (P<0.01). After 2 and 4 weeks of administration, the skin lesion scores of the mice in each administration group were significantly lower than those of the model control group (P<0.05 or P<0.01). The specific results are shown in Table 3.

[0126]

[0127] The results showed that after 4 weeks of administration, the skin lesion improvement effect of groups 1 and 3 of the present invention was significantly better than that of control group 1 of the commercially available positive drug (P<0.01), and the effect of group 2 of the present invention was better than that of control group 1 (P<0.05); the skin lesion improvement effect of control group 3 was significantly weaker than that of group 1 of the present invention, proving that type XVII collagen is the core component for reversing skin lesion atrophy; the effect of control group 4 was significantly weaker than that of group 1 of the present invention, proving that liposome targeted delivery technology can significantly improve drug efficacy.

[0128] 3.2 Effects on the pathological characteristics of mouse vulvar tissue

[0129] HE staining results showed that: in the normal control group, the vulvar epithelium of mice was clear and uniform in thickness, with neatly arranged collagen in the dermis and no inflammatory cell infiltration; in the model control group, the vulvar epithelium of mice was significantly atrophied and thinned, the epithelial feet disappeared, the superficial dermal collagen underwent hyalinization, and a large number of inflammatory cells infiltrated the dermis; all treatment groups showed varying degrees of improvement, with the epithelial thickness of mice in Examples 1-3 basically returning to normal, the collagen neatly arranged, and the inflammatory cell infiltration almost disappearing, showing significantly better improvement than the control groups. Specific quantitative results are shown in Table 4.

[0130]

[0131] The results showed that the 1-3 groups of the present invention could significantly reverse the epithelial atrophy, dermal inflammatory infiltration and collagen fibrosis characteristic of VLS. Among them, the epithelial thickness recovery and anti-fibrosis effect of the 1 and 3 groups were significantly better than those of the commercial control group 1 (P<0.01); while the control group 1 could only reduce inflammatory infiltration and could not reverse epithelial atrophy and collagen fibrosis, which is completely consistent with the clinical defects of the prior art.

[0132] 3.3 Effects on inflammation and fibrosis factors in mouse vulvar tissue

[0133] Four weeks after administration, compared with the model control group, the contents of IL-6, IL-17, IFN-γ and TGF-β1 in the tissues of each administration group were significantly reduced (P<0.01); among them, the levels of inflammatory factors and fibrotic factors in the groups of Examples 1-3 were significantly lower than those in the control group (P<0.05 or P<0.01), and the specific results are shown in Table 5.

[0134]

[0135] The results showed that the embodiment group of the present invention can inhibit the activation of the VLS-characteristic Th1 / Th17 inflammatory pathway from the root, and at the same time significantly downregulate the expression of the fibrosis core factor TGF-β1, achieving a synergistic effect of anti-inflammatory + anti-fibrotic, which is significantly better than existing monotherapy.

[0136] 3.4 Effects on the expression of type XVII collagen and α-SMA protein in mouse vulvar tissue

[0137] Western Blot results showed that, compared with the normal control group, the expression of type XVII collagen in the vulvar tissue of mice in the model control group was significantly downregulated and the expression of α-SMA was significantly upregulated (P<0.01); after 4 weeks of drug administration, the expression of type XVII collagen in groups 1-3 was significantly increased and the expression of α-SMA was significantly decreased, and the effect was significantly better than that of each control group (P<0.01). The specific results are shown in Table 6.

[0138]

[0139] The results showed that the expression of type XVII collagen missing in the VLS lesion group could be significantly restored, and the expression of the fibrosis marker α-SMA was significantly downregulated. This verified the core technical effect of the present invention in accurately repairing the basement membrane barrier and reversing dermal fibrosis at the molecular level. However, the control group 3 did not have this effect, and the commercially available control drugs in control groups 1 and 2 could not restore the expression of type XVII collagen, proving that the present invention fills the target gap in the prior art.

[0140] 3.5 Relapse rate results after drug discontinuation

[0141] The relapse rates for each group were observed for 4 weeks after medication was discontinued. The results are shown in Table 7.

[0142]

[0143] The results showed that the relapse rate after drug withdrawal in the embodiment group of the present invention was significantly lower than that of the commercially available control drug (P<0.01), proving that the present invention can block the progression of VLS from the root cause, rather than just relieving symptoms, thus solving the core problem of high relapse rate after drug withdrawal in the prior art.

[0144] Test Example 3: Skin Irritation Test

[0145] The rabbit skin irritation test method was used. The test substances of Examples 1-3, Control Example 1, and Control Example 2 were applied to intact and broken skin of rabbits once a day for 7 consecutive days. Local skin reactions were observed and irritation scores were calculated.

[0146] The results showed that in the cases of rabbits in groups 1-3, there were no erythema or edema reactions on intact or damaged skin, and the irritation score was 0, indicating that the skin was non-irritating. In the control group 1, 2 rabbits showed mild erythema, and in the control group 2, 3 rabbits showed obvious erythema and mild edema, both of which were mild to moderate irritants.

[0147] Experimental conclusion: The pharmaceutical composition of the present invention is non-irritating to the delicate skin and mucous membranes of the vulva.

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for vulvar lichen sclerosus, characterized in that, The product includes active ingredients and excipients. The active ingredients, by weight, include the following components: mometasone furoate 0.005-0.02 parts, calcineurin inhibitor 0.05-0.3 parts, paeoniflorin 0.5-2 parts, asiaticoside 0.8-3 parts, recombinant collagen 0.3-1.5 parts, and ginkgolide B 0.2-1 parts.

2. The pharmaceutical composition for vulvar lichen sclerosus as described in claim 1, characterized in that, The active ingredients, by weight, include the following components: 0.01 parts mometasone furoate, 0.1 parts calcineurin inhibitor, 1.0 part paeoniflorin, 1.5 parts asiaticoside, 0.8 parts recombinant collagen, and 0.5 parts ginkgolide B.

3. The pharmaceutical composition for vulvar lichen sclerosus as described in claim 1, characterized in that, The calcineurin inhibitor is pimecrolimus or tacrolimus.

4. The pharmaceutical composition for vulvar lichen sclerosus as described in claim 1, characterized in that, The recombinant collagen is recombinant human type XVII collagen.

5. The pharmaceutical composition for vulvar lichen sclerosus as described in claim 1, characterized in that, The excipients include 10-20 parts of oil phase matrix, 2-5 parts of emulsifier, 5-15 parts of humectant, 0.5-3 parts of penetration enhancer, and 0.05-0.5 parts of antibacterial agent.

6. The pharmaceutical composition for vulvar lichen sclerosus as described in claim 1, characterized in that, The oil phase matrix is ​​composed of stearic acid, white petrolatum, liquid paraffin, and glyceryl monostearate in a mass ratio of (2-4):(3-5):(3-6):(2-3); The emulsifier is polysorbate 80, or a composite emulsifier composed of polysorbate 80 and Span 80 in a mass ratio of (3-5):

1. The humectant is composed of glycerol and 1,3-butanediol in a mass ratio of (2-4):

1. The penetration enhancer is composed of menthol and laurocapram at a mass ratio of 1:(1-3); The antibacterial agent is phenoxyethanol.

7. The pharmaceutical composition for vulvar lichen sclerosus as described in claim 1, characterized in that, The dosage form of the drug is cream, ointment, gel, lotion, or vaginal suppository.

8. A method for preparing a pharmaceutical composition for vulvar lichen sclerosus as described in any one of claims 1-6, characterized in that, The dosage form is a cream, and the steps include: Preparation of S1 drug-loaded liposome suspension: Weigh the lipid components, mometasone furoate, calcineurin inhibitor, paeoniflorin, asiaticoside, and ginkgolide B, dissolve them in an organic solvent, remove the organic solvent by rotary evaporation under reduced pressure, and form a uniform lipid film on the inner wall of the container; add hydration solution for hydration, and after high-pressure homogenization, filter and sterilize with a microporous membrane to obtain a drug-loaded liposome suspension with an average particle size of 80-200 nm, for later use; Preparation of S2 aqueous base solution: Weigh the humectant and antibacterial agent according to the ratio, add them to purified water, heat and stir until completely dissolved, sterilize and cool to below 40℃, add recombinant collagen, stir until completely dispersed, adjust the pH of the system to 4.0-6.0 with pH adjuster to obtain aqueous phase, keep warm at 35-40℃ for later use; S3 oil phase preparation: Weigh the oil phase matrix and emulsifier according to the ratio, heat until completely melted, stir evenly, keep warm at 75-85℃ for later use, and obtain the oil phase; S4 Emulsification and Mixing: The oil phase in a warm state is slowly added to the aqueous phase, and emulsified by high-speed stirring at a constant temperature of 75-85℃. When the temperature drops to below 38℃, the drug-loaded liposome suspension prepared in step S1 is added, and the mixture is stirred at low speed until homogeneous. The mixture is then cooled to room temperature to obtain the drug composition in cream form.

9. The preparation method according to claim 8, characterized in that, In step S1, the lipid component is hydrogenated soybean lecithin and cholesterol in a mass ratio of (3-5):1, and the total mass ratio of the two to the total mass of mometasone furoate, calcineurin inhibitor, paeoniflorin, asiaticoside, and ginkgolide B is (2-8):1; the organic solvent is a mixed solvent of anhydrous ethanol and dichloromethane in a volume ratio of 1:(2-4), and the ratio of the organic solvent to the lipid component is (15-25) mL:1g; the hydration solution is a phosphate buffer solution with a pH of 5.0, and the ratio of the hydration solution to the lipid component is (8-12) mL:1g, the hydration temperature is 35-45℃, and the hydration time is 30-60min; the high-pressure homogenization pressure is 800-1200 bar, and the number of cycles is 3-5; the microporous filter membrane is a 0.22μm polyethersulfone sterilization filter membrane. In step S2, the heating and stirring temperature is 70-90℃; In step S4, the constant temperature high-speed stirring speed is 3000-5000 rpm, and the emulsification time is 15-30 min; when adding the drug-loaded liposome suspension, the low-speed stirring speed is 500-1000 rpm.

10. Use of the pharmaceutical composition according to any one of claims 1-7 in the preparation of a medicament for the prevention or treatment of vulvar lichen sclerosus.

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