Application of dihydropyridine compound or salt thereof in preparation of medicine for preventing and / or treating HPV (human papillomavirus) infection

Abstract

The invention relates to an application of a dihydropyridine compound or a salt thereof in preparation of a medicine for preventing and / or treating HPV infection, and belongs to the technical field of medicines and medicinal preparations. The invention provides application of a dihydropyridine compound or a salt thereof in preparation of a medicine for preventing and / or treating HPV (human papillomavirus) infection. The dihydropyridine compound is selected from one or more of nifedipine, amlodipine, lercanidipine, nimodipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine and azelnidipine. According to the scheme, the application of the dihydropyridine compound or the salt thereof in prevention and / or treatment of HPV is reported for the first time, and an in-vitro cell experiment is carried out through azelnidipine in the dihydropyridine compound; furthermore, the inhibition effect on the HPV virus is verified through mouse intradermal vaccination virus model drug delivery and mouse vaginal vaccination virus model drug delivery.

Description

Technical Field

[0001] The present application relates to the use of a dihydropyridine compound or a salt thereof in the preparation of a drug for preventing and / or treating HPV infection, and belongs to the technical field of medicine and pharmaceutical preparations. Background Art

[0002] Human papillomavirus (HPV) is a small, double-stranded, circular DNA virus that is divided into high-risk and low-risk types based on their pathogenicity. High-risk HPV types, such as HPV16, HPV18, HPV31, and HPV45, can cause head and neck cancers, including cervical cancer, bladder cancer, oral cancer, and laryngeal cancer. Low-risk HPV types, such as HPV1, HPV5, HPV6, HPV8, and HPV11, are primarily associated with genital warts and benign cervical lesions. The two most common high-risk HPV types are HPV16 and HPV18. Persistent high-risk HPV plays a crucial role in the development of cervical cancer and is a major risk factor for women's health.

[0003] In the medical field, the treatment of HPV infection has always been a research hotspot and a difficult topic. Currently, treatments for HPV infection mainly include surgery, physical therapy, and medication. Surgical treatments mainly include cervical cold knife conization and loop electrosurgical excision procedure, physical therapy mainly uses light / sound sensitizers combined with light / sound dynamic technology, and medications include interferon therapy, traditional Chinese medicine, and integrated Chinese and Western medicine. However, these treatments still have some shortcomings in terms of cost, efficacy, safety, onset time, ease of use, and treatment compliance.

[0004] HPV16 and HPV18 are closely related to the occurrence of cervical cancer. Since there is currently no specific medicine or effective treatment plan, finding new anti-HPV drugs is of great significance for preventing cervical cancer and improving the treatment effect of cervical cancer patients.

[0005] Dihydropyridine compounds (core structural chemical formula such as Figure 1 (as shown) are calcium channel blockers. Common dihydropyridine compounds include nifedipine, amlodipine, lercanidipine, nimodipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine and azelnidipine. Dihydropyridine compounds are currently mainly used as antihypertensive drugs.

[0006] Azelnidipine (English name: Azelnidipine, Chinese chemical name: 3-(1-diphenylmethylazetidin-3-yl) 5-isopropyl 2-amino-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, molecular formula: C 33 H 34 N4O6, molecular weight: 582.65, chemical structure formula Figure 2 (shown) is a new dihydropyridine calcium channel blocker that selectively acts on L-type calcium channels. Its chemical structure is similar to that of nifedipine. It is clinically used to treat systemic hypertension and angina pectoris. A few are used to treat congestive heart failure, protect the heart and kidneys, fight atherosclerosis, and improve insulin resistance.

[0007] There are currently no reports on the use of dihydropyridine compounds to prevent and / or treat HPV. Summary of the Invention

[0008] In order to solve the above problems, the present invention provides an application of a dihydropyridine compound or a salt thereof in the preparation of a drug for preventing and / or treating HPV infection. Specifically, an external preparation of a dihydropyridine compound or a salt thereof for preventing and / or treating high / low-risk HPV infection, a preparation method and an application thereof are provided. The preparation process of the provided azelnidipine preparation is simple and easy to industrialize. The cream and suppository of azelnidipine have stable performance, good in vitro release, significant effect in treating HPV, and are easy to use.

[0009] The present application provides the use of a dihydropyridine compound or a salt thereof in the preparation of a drug for preventing and / or treating HPV infection, wherein the dihydropyridine compound is selected from one or more of nifedipine, amlodipine, lercanidipine, nimodipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine and azelnidipine. The core structure of the dihydropyridine compound is as follows: Figure 1 shown.

[0010] Optionally, the dihydropyridine compound is azelnidipine.

[0011] Optionally, the HPV includes HPV16, HPV45, HPV6, HPV5, HPV8, HPV11, and HPV31.

[0012] Optionally, the dihydropyridine compound or its salt has the effect of inhibiting HPV activity.

[0013] Optionally, the drug is an ointment, cream, gel, capsule, effervescent tablet, microneedle, suppository, tablet, pill, foam, patch or lotion.

[0014] Optionally, the drug further comprises pharmaceutically acceptable excipients.

[0015] Optionally, the administration of the drug includes topical administration, transdermal administration, and intravaginal administration.

[0016] Optionally, the medicament is used to prevent and / or treat skin warts, vaginitis, cervical erosion, cervical cancer, cervical and / or vaginal intraepithelial neoplasia, polyps and warts caused by HPV infection;

[0017] The skin warts include common warts, flat warts, plantar warts, condyloma acuminata, epidermodysplasia verruciformis, anal and / or genital warts.

[0018] The present application provides a pharmaceutical composition for preventing and / or treating HPV infection, the pharmaceutical composition comprising a dihydropyridine compound or a salt thereof;

[0019] The dihydropyridine compound is selected from one or more of nifedipine, amlodipine, lercanidipine, nimodipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine and azelnidipine.

[0020] Optionally, the pharmaceutical composition is a cream, which includes, by weight: 0.1-10 parts of azelnidipine, 0.5-2 parts of skin penetration enhancer, 10-30 parts of oil phase matrix, 0.2-5 parts of emulsifier, 5-30 parts of moisturizer, 0.1-2 parts of antioxidant, 1-15 parts of emulsifier, 0.1-2 parts of pH adjuster, and 0.05-0.2 parts of preservative.

[0021] Optionally, the pharmaceutical composition is a suppository, which comprises, by weight: 0.1-10 parts of azelnidipine, 10-40 parts of an oily phase matrix, 0.1-5 parts of an absorption enhancer, 0.1-5 parts of a surfactant, 0.01-0.05 parts of an antioxidant, 0.01-0.05 parts of a preservative, and 0.01-0.05 parts of a hardener.

[0022] Optionally, the pharmaceutical composition is a microneedle, which comprises, by weight: 1 to 24 parts of azelnidipine, 20 to 40 parts of a biodegradable polymer, 1 to 15 parts of a plasticizer, 10 to 50 parts of an excipient, 0.1 to 5 parts of a surfactant, 0.1 to 2 parts of an antioxidant, and 0.01 to 0.05 parts of a preservative.

[0023] The beneficial effects of this application include but are not limited to:

[0024] 1. According to the use of dihydropyridine compounds or salts thereof in the preparation of drugs for preventing and / or treating HPV infection, this application reports for the first time the use of dihydropyridine compounds or salts thereof in preventing and / or treating HPV infection. There are currently no reports on the use of dihydropyridine compounds in preventing and / or treating HPV. This application provides a new approach to preventing and / or treating HPV.

[0025] 2. According to the use of the dihydropyridine compounds or their salts in the preparation of drugs for preventing and / or treating HPV infection, the present application has determined the inhibitory effect of dihydropyridine compounds or their salts on HPV through in vitro cell experiments combined with intradermal virus inoculation model administration in mice and vaginal virus inoculation model administration in mice, and the effect of treating HPV is significant.

[0026] 3. According to the use of the dihydropyridine compounds or their salts in the preparation of drugs for preventing and / or treating HPV infection in the present application, the preparation process of the azelnidipine preparation provided in the present application is simple and easy to industrialize. The creams and suppositories of azelnidipine have stable performance, good in vitro release, significant effect in treating HPV, and are easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0028] Figure 1 The core structural chemical formula diagram of the dihydropyridine compound involved in this application;

[0029] Figure 2 The chemical structural formula of azelnidipine involved in this application;

[0030] Figure 3 This is a graph showing the inhibition of HPV16 by different concentrations of azelnidipine in the human cervical cancer Hela cell line involved in Example 5 of the present application;

[0031] Figure 4 This is a graph showing the inhibition of HPV16 by nimodipine at different concentrations in the human cervical cancer Hela cell line as described in Example 6 of the present application;

[0032] Figure 5 This is a graph showing the inhibition of HPV16 by amlodipine at different concentrations in the human cervical cancer Hela cell line as described in Example 6 of the present application;

[0033] Figure 6 This is a photographic result of the skin irritation reaction of azelnidipine on guinea pigs involved in Example 7 of the present application;

[0034] Figure 7 This is a graph showing the effect of azelnidipine cream applied to mice on intradermal HPV16PsV in Example 8 of the present application;

[0035] Figure 8 This is a graph showing the results of fluorescein expression in mice after vaginal administration of the azelnidipine suppository involved in Example 9 of the present application. DETAILED DESCRIPTION

[0036] The present application is described in detail below with reference to examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and reagents in the examples of the present application are purchased through commercial channels.

[0037] Example 1 Cream and its preparation method

[0038] The cream comprises, by weight, 0.1-10% of azelnidipine, 0.5-2% of a skin penetration enhancer, 10-30% of an oil phase matrix, 0.2-5% of an emulsifier, 5-30% of a moisturizer, and may further comprise 0.1-2% of an antioxidant, 1-15% of an emulsifier, 0.1-2% of a pH regulator, 0.05-0.2% of a preservative, with the balance being water.

[0039] The skin penetration enhancer is selected from the group consisting of surfactants, dimethyl sulfoxide and its analogs, azone compounds, alcohol compounds, fatty acid compounds, or combinations thereof; preferably, the skin penetration enhancer includes azone compounds, such as laurocapram.

[0040] The emulsifier is a nonionic surfactant, an anionic surfactant, or a combination thereof; preferably, the emulsifier is selected from the following group: polyoxyethylene sorbitan fatty acids, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, or a combination thereof.

[0041] The antioxidant is selected from the group consisting of natural flavonoid antioxidants, phenolic antioxidants, active polysaccharide antioxidants, vitamin antioxidants, saponins and tannin antioxidants, or a combination thereof.

[0042] The emulsifier is selected from the following groups: alkanolamide thickeners, fatty alcohol and fatty acid thickeners, ether thickeners, ester thickeners, amine oxide thickeners, cellulose and its modified thickeners, polyacrylamide thickeners, natural rubber and its modified thickeners, hydrophobic associating thickeners, or a combination thereof.

[0043] The moisturizing agent is selected from the following group: polyol moisturizing agents, natural moisturizing factors, amino acid moisturizing agents, polymer biochemical moisturizing agents, or a combination thereof.

[0044] The preservative is selected from the group consisting of parabens, imidazolidinyl urea, kathones and hydroxymethyldimethylhydantoin, or a combination thereof.

[0045] In a specific embodiment, the formula of azelnidipine cream is shown in Table 1 below.

[0046] Table 1 Azelnidipine cream formula composition

[0047]

[0048] In a specific embodiment, the preparation method of azelnidipine cream comprises the following steps:

[0049] 1) Preparation of oil phase: 10 g of white vaseline, 4 g of stearic acid, 3 g of glyceryl monostearate, 18 g of silicone oil, 1 g of vitamin E, and 2 g of laurocapram were placed in a beaker and mixed uniformly under stirring at 80° C. to prepare an oil phase.

[0050] 2) Preparation of the aqueous phase: 2g of the emulsifier sodium fatty alcohol polyoxyethylene ether sulfate, 1g of Tween 80, 5g of the active ingredient, and 10g of glycerol were added to water and mixed thoroughly with stirring at 80°C to prepare an aqueous phase. 0.1% methylparaben dissolved in an appropriate amount of anhydrous ethanol was then added to the aqueous phase, and triethanolamine was added dropwise to a pH of 7.

[0051] 3) Cream Preparation: The prepared oil phase and water phase were mixed, and the water phase was maintained at 80°C. The water phase was slowly added to the oil phase while continuously stirring to fully emulsify the oil phase and the water phase. After stirring evenly, the cream was allowed to cool naturally to room temperature. Stirring was continued during the cooling process to ensure the uniformity of the cream.

[0052] 4) Packaging and Storage: The cooled cream is quality checked to ensure that its appearance, texture, pH value, etc. meet the requirements, and then packaged into suitable packaging containers and sealed for storage.

[0053] Example 2 Suppository and its preparation method

[0054] The suppository comprises, by weight, 0.1 to 10 parts of azelnidipine, 10 to 40 parts of an oily phase base, 0.1 to 5 parts of an absorption promoter, 0.1 to 5 parts of a surfactant, 0.01 to 0.05 parts of an antioxidant, 0.01 to 0.05 parts of a preservative, and 0.01 to 0.05 parts of a hardener.

[0055] The oil phase matrix is selected from the group consisting of cocoa butter, semi-synthetic fatty acid esters, semi-synthetic fatty acid glycerides, or combinations thereof.

[0056] The absorption enhancer is selected from the group consisting of laurocapram, isopropyl myristate, taurine, glycine, menthol, or a combination thereof.

[0057] The surfactant is selected from the group consisting of glyceryl monostearate, polysorbate 80, Span 80, sodium lauryl sulfate, or a combination thereof.

[0058] The antioxidant is vitamin E, sodium sulfite, sodium thiosulfate, or a combination thereof.

[0059] The preservative is selected from the group consisting of methylparaben, sodium benzoate, potassium sorbate, benzalkonium chloride, or a combination thereof.

[0060] The hardener is selected from the group consisting of white wax, cetyl alcohol, carnauba wax, or a combination thereof.

[0061] In a specific embodiment, the formula of azelnidipine suppository is shown in Table 2 below.

[0062] Table 2 Azelnidipine suppository formula composition

[0063]

[0064] In a specific embodiment, the preparation method of azelnidipine suppository comprises the following steps:

[0065] 1) Preparation of the oil phase matrix: The weighed amount of cocoa butter was placed in a beaker and melted in a 50°C water bath with continuous stirring until completely melted and evenly mixed. To the melted oil phase matrix, the weighed amounts of azelnidipine, laurocapram, glyceryl monostearate, Span 80, vitamin E, methylparaben, benzalkonium chloride, and carnauba wax were added in sequence with continuous stirring at 50°C to allow the ingredients to fully dissolve and be evenly dispersed in the oil phase matrix.

[0066] 2) Injection molding: The prepared mixture is injected into a suppository mold while it is still hot. After the injection molding is completed, the mold is allowed to cool naturally at room temperature to solidify the suppository.

[0067] 3) Demolding and packaging: After the suppository is completely solidified, carefully demold it and inspect its appearance to ensure that the surface is smooth and free of cracks, bubbles, and other defects. Qualified suppositories should be packaged and sealed for storage.

[0068] Example 3 Microneedle preparation and preparation method thereof

[0069] The microneedle preparation comprises, by weight, 1 to 24 parts of azelnidipine, 20 to 40 parts of a biodegradable polymer, 1 to 15 parts of a plasticizer, 10 to 50 parts of an excipient, 0.1 to 5 parts of a surfactant, 0.1 to 2 parts of an antioxidant, and 0.01 to 0.05 parts of a preservative.

[0070] The biodegradable polymer is selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), chitosan, hyaluronic acid, or a combination thereof.

[0071] The plasticizer is selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, triethyl citrate (TEC), or a combination thereof.

[0072] The excipient is selected from the group consisting of lactose, mannitol, sodium chloride, sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP), or a combination thereof.

[0073] The surfactant is selected from the group consisting of Tween-80, Span-80, poloxamer, or a combination thereof.

[0074] The antioxidant is selected from the following group: vitamin E, vitamin C, propyl gallate, or a combination thereof.

[0075] The preservative is selected from the group consisting of methylparaben, sodium benzoate, benzalkonium chloride, or a combination thereof.

[0076] In a specific embodiment, the formula of the azelnidipine microneedle preparation is shown in Table 3 below.

[0077] Table 3 Azelnidipine microneedle formulation composition

[0078]

[0079] In a specific embodiment, the preparation method of the azelnidipine microneedle preparation comprises the following steps:

[0080] 1) Polymer Solution Preparation: Weigh the prescribed amount of PLGA and add it to an appropriate amount of dichloromethane. Stir to fully dissolve. Add the prescribed amounts of azelnidipine, glycerol, lactose, mannitol, Tween-80, vitamin E, propyl gallate, and methylparaben to the polymer solution in sequence. Stir continuously during addition to ensure that all ingredients are evenly dispersed in the solution.

[0081] 2) 3D Printing Model Design: Design a 3D model of the azelnidipine microneedles using computer-aided design (CAD) software. Determine parameters such as microneedle shape, length, diameter, and array layout.

[0082] 3) 3D printing microneedles: Pour the prepared mixed solution into the barrel of the 3D printer, set the printing parameters such as printing speed, temperature, layer height, fill rate, etc., and print according to the designed model.

[0083] 4) Microneedle Post-Processing: After printing is complete, the microneedles are carefully removed from the printing platform to remove any excess solution or impurities from the surface. Vacuum drying is used to remove organic solvents from the microneedles, allowing the microneedles to solidify and form. Qualified azelnidipine microneedles are packaged and stored.

[0084] Example 4 Cytotoxicity Evaluation of Azelnidipine

[0085] Human cervical cancer cells HeLa and human skin fibroblast-like cells HSF were cultured in DMEM high-glucose medium containing 10% FBS (fetal bovine serum), 100 U / ml penicillin, and 100 μg / ml streptomycin. Human immortalized epidermal cells HaCat were cultured in MEM high-glucose medium containing 10% FBS (fetal bovine serum), 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells in the logarithmic growth phase were seeded at 7000 / well (180 μl) in 96-well plates. After 24 hours of culture, various concentrations of azelnidipine (2.5, 5, 10, 20, 50, and 100 μM) were added. A blank control group received an equal volume of culture medium. Four replicate wells were set up for each concentration and cultured for an additional 72 hours.

[0086] After 72 hours of azelnidipine treatment, 50% (m / v) cold trichloroacetic acid (TCA) was added to each well to fix the cells. After staining with SRB, 150 μl / well of Tris solution was added and the OD value at 540 nm was measured on a microplate reader.

[0087] The cell growth inhibition rate was calculated according to the following formula:

[0088] Inhibition rate (%) = [(OD 540对照孔 -OD 540给药孔 ) / OD 540对照孔 ]×100%.

[0089] The results are shown in Tables 3 to 5 below.

[0090] Table 3 Effects of different concentrations of azelnidipine on Hela cell proliferation after direct contact with the cells for 72 h in vitro

[0091]

[0092] Table 4 Effects of different concentrations of azelnidipine on HaCat cell proliferation after direct contact with the cells for 72 h in vitro

[0093]

[0094] Table 5 Effects of different concentrations of azelnidipine on HSF cell proliferation after direct contact with the cells for 72 h in vitro

[0095]

[0096] According to the results in the above table, azelnidipine showed no obvious cytotoxicity to human cervical cancer cells Hela, human skin fibroblast-like cells HSF and human immortalized epidermal cells HaCat in the concentration range of 2.5-100 μM.

[0097] Example 5 In vitro antiviral effect of azelnidipine

[0098] Cervical cancer cells Hela and human skin fibroblast-like cells HSF were cultured in DMEM high-glucose medium containing 10% FBS (fetal bovine serum), 100 U / ml penicillin, and 100 μg / ml streptomycin. Human immortalized epidermal cells HaCat were cultured in MEM high-glucose medium containing 10% FBS (fetal bovine serum), 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells in the logarithmic growth phase were cultured at 1×10 4 85 μl of virus were inoculated into each well of a 96-well plate. When the cell confluency reached approximately 50%, 5 μl of HPV of different genotypes (HPV16, HPV5, HPV6, HPV8, HPV11, HPV31, HPV45) and 10 μl of azelnidipine solution at different concentrations (0.625, 1.25, 2.5, 5, and 10 μM) were added. For the blank control group, equal volumes of virus and culture medium were added, with three replicates set for each concentration. Culture was continued for 48 hours.

[0099] After 48 hours, the culture medium was aspirated and 100 μl of lysis buffer was added to each well. The cells were shaken on ice at 180 rpm for 15 minutes. 80 μl of the liquid from the 96-well plate was transferred to a white chromogenic plate and 80 μl of chromogenic buffer was added to each well. Relative light units (RLU) were measured using a microplate reader.

[0100] Inhibition rate (%) = (RLU of experimental group - RLU of blank group) / (RLU of virus group - RLU of blank group) × 100%, and IC50 was calculated based on the inhibition rate of different concentrations.

[0101] The inhibitory effects of different concentrations of azelnidipine on HPV16 in human cervical cancer Hela cell line are shown in Table 6 and Figure 3 shown.

[0102] Table 6 Anti-HPV16 activity of Hela cells

[0103]

[0104] The inhibitory effects of azelnidipine on different genotypes of HPV in different cell lines are summarized and the results are shown in Table 7 below.

[0105] Table 7 IC50 values of azelnidipine against different genotypes of HPV in different cell lines

[0106]

[0107] According to the results in the above table, azelnidipine has good inhibitory activity against different HPV genotypes in human cervical cancer cells Hela, human skin fibroblast-like cells HSF and human immortalized epidermal cells HaCat.

[0108] Example 6 In vitro antiviral effects of other dihydropyridine compounds

[0109] Cervical cancer cells Hela were cultured in DMEM high-glucose medium containing 10% FBS (fetal bovine serum), 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells in the logarithmic growth phase were plated at 1×10 4 Inoculate 85 μl of virus per well (96-well plate) and, when the cell confluency reaches approximately 50%, add 5 μl of HPV16 and 10 μl of nimodipine / amlodipine solutions (5 and 10 μM, respectively). For the blank control group, add equal volumes of virus and culture medium, with four replicates per well for each concentration, and continue incubation for 48 hours.

[0110] After 48 hours, the culture medium was aspirated and 100 μl of lysis buffer was added to each well. The cells were shaken on ice at 180 rpm for 15 minutes. 80 μl of the liquid from the 96-well plate was transferred to a white chromogenic plate and 80 μl of chromogenic buffer was added to each well. Relative light units (RLU) were measured using a microplate reader.

[0111] Inhibition rate (%) = (RLU of experimental group - RLU of blank group) / (RLU of virus group - RLU of blank group) × 100%, and IC50 was calculated based on the inhibition rate of different concentrations.

[0112] The inhibitory effects of different concentrations of nimodipine and amlodipine on HPV16 in human cervical cancer Hela cell line, such as Figure 4 and Figure 5 shown.

[0113] according to Figure 4 and Figure 5 The results showed that both nimodipine and amlodipine had good inhibitory activity against HPV16 in human cervical cancer cells Hela.

[0114] Example 7: Skin irritation test of Azelnidipine cream

[0115] Four guinea pigs, 4 to 8 weeks old, half male and half female, weighing 200 to 300 g, were used as experimental mice. Hair was removed from both sides of the dorsal spine, with the removed area larger than 3 x 3 cm. The day after hair removal, 0.5 mL (g) of the test sample was directly applied to the skin of symmetrical 2.5 x 2.5 cm areas selected from the same experimental animals. Azelnidipine cream was then applied to one side of the skin, while a blank cream was applied to the other side. A blank area was left as a control. The drug was removed 4 hours after administration. Photographs were taken 1, 24, 48, and 72 hours after drug removal to evaluate the skin irritation of a single dose. Skin erythema and edema reactions to a single dose were scored according to the "Technical Guidelines for Drug Irritation, Allergy, and Hemolytic Studies 2014."

[0116] Table 8 Skin irritation reaction scoring standard

[0117] stimulus response Score erythema No erythema 0 Mild erythema (barely visible) 1 Moderate erythema (clearly visible) 2 Severe erythema 3 Purple-red erythema to mild eschar formation 4 edema No edema 0 Mild edema (barely visible) 1 Moderate edema (obvious swelling) 2 Severe edema (skin bulge of 1 mm with clear contours) 3 Severe edema (skin swelling greater than 1 mm and expanding) 4 Highest total score 8

[0118] The results are as follows Figure 6 and as shown in Table 9 below.

[0119] Table 9 Azelnidipine skin irritation score of guinea pigs

[0120]

[0121] As shown in the images and table, a single dose of azelnidipine cream in the guinea pig model showed no signs of edema, scabs, or erythema. This indicates that a single dose of azelnidipine was non-irritating to guinea pig skin. This suggests that a single dose of azelnidipine did not irritate guinea pig skin, demonstrating its safety.

[0122] Example 8 Inhibitory activity of azelnidipine cream against HPV16 PsV virus intradermally inoculated mice

[0123] Balb / c nude mice, 6-8 weeks old, female, weighing 18-20 g, were used as experimental mice. On day 0, the nude mice were infected intradermally with 100 μL of HPV1 6PsV. Each mouse was then injected with 50 μL of pseudovirus solution (2 cm x 2 cm) at a fixed area (viral titer of 500,000). On day 2, observations were made using a small animal imaging device and grouped for drug administration based on the results of semi-quantitative analysis of in vivo fluorescence expression. Twelve nude mice were randomly divided into two groups: a model group and a drug administration group, with 6 mice in each group. The model group was infected only with pseudovirus. The drug administration groups were topically administered 0.5 h after virus infection and were divided into low (2 mg / kg), medium (5 mg / kg), and high (10 mg / kg) dose groups. On day 5, luciferase expression in the mice in the different groups was measured, and the antiviral effect of azelnidipine cream was recorded using in vivo small animal imaging.

[0124] Luciferase chromogenic substrate (XenoLight D-Luciferin) was dissolved in saline to 15 mg / ml, and 25 μL of the substrate was injected intraperitoneally into each mouse using a positive displacement pipette. After injection, the mouse was placed in an isoflurane anesthesia chamber for induction of anesthesia, which took approximately 3 minutes to reach stable state. The animal was then transferred to the examination chamber of the Xenogen In Vivo Imaging System (IVIS) and maintained under anesthesia. Image acquisition was performed using LivingImage 2.0 software with the following parameters: Bin = 8, f / stop = 1, and Exposure time = 60 s. A region of strong signal was selected in the image, and the signal value within the selected region was acquired and recorded.

[0125] The results are shown in Table 10 and Figure 7 shown.

[0126] Table 10 Semi-quantitative analysis of fluorescein expression in mice after administration of azelnidipine cream

[0127] Group Fluorescence intensity Virus group <![CDATA[2.9*10 5 ]]> High-dose group (10 mg / kg) <![CDATA[6.2*10 4 ]]> Medium dose group (5 mg / kg) <![CDATA[8.4*10 4 ]]> Low-dose group (2 mg / kg) <![CDATA[1.2*10 5 ]]>

[0128] The results in the figures and tables show that after five days of administration, the high-dose group showed a significant decrease in luciferin expression (P<0.0001) compared to the model group, indicating a significant decrease in viral expression, indicating that azelnidipine cream has a significant inhibitory effect on HPV16PsV. This suggests that azelnidipine has an inhibitory effect on HPV16PsV inoculated intradermally in nude mice.

[0129] Example 9 Inhibitory activity of azelnidipine suppositories against HPV16 PsV virus inoculated vaginally in mice

[0130] Balb / c nude mice, 6-8 weeks old, female, weighing 18-20 g, were used as experimental mice for a mouse vaginal model of HPV 16PsV infection. Hormonal induction was achieved with a subcutaneous injection of 30 mg / ml Depo-Provera (medroxyprogesterone acetate). Three days later, 20 μl of N-9 (final concentration of 4%) was injected into the mouse vagina to induce chemical injury. Six hours later, a cytobrush was inserted into the vagina and rotated 10 times clockwise and then 10 times counterclockwise to induce physical injury, causing vaginal bleeding. Twelve nude mice were randomly divided into two groups: a model group and a drug-treated group, with six mice in each group. The model group was infected with only the pseudovirus. The drug-treated groups received an HPV 16PsV pseudovirus inoculum and azelnidipine suppository injected into the vagina at low (2 mg / kg), medium (5 mg / kg), and high (10 mg / kg) doses. Luciferase expression in the mice was measured on day 4. In vivo imaging of small animals was used to record the anti-HPV1 6 virus infection ability of azelnidipine suppositories in mice after vaginal administration.

[0131] Luciferase chromogenic substrate (XenoLight D-Luciferin) was dissolved in saline to 15 mg / ml, and 25 μL of the substrate was injected intraperitoneally into each mouse using a positive displacement pipette. After injection, the mouse was placed in an isoflurane anesthesia chamber for induction of anesthesia, which took approximately 3 minutes to reach stable state. The animal was then transferred to the examination chamber of the Xenogen In Vivo Imaging System (IVIS) and maintained under anesthesia. Image acquisition was performed using LivingImage 2.0 software with the following parameters: Bin = 8, f / stop = 1, and Exposure time = 60 s. A region of strong signal was selected in the image, and the signal value within the selected region was acquired and recorded.

[0132] The results are shown in Table 11 and Figure 8 shown.

[0133] Table 11 Semi-quantitative analysis of fluorescein expression in mice after administration of azelnidipine suppositories

[0134] Group Fluorescence intensity Virus group <![CDATA[2.4*10 6 ]]> High-dose group (10 mg / kg) <![CDATA[6.2*10 4 ]]> Medium dose group (5 mg / kg) <![CDATA[2.5*10 5 ]]> Low-dose group (2 mg / kg) <![CDATA[1.3*10 6 ]]>

[0135] As shown in the images and table, after four days of administration, the high-dose group showed a significant decrease in luciferin expression (P<0.05) compared to the model group, indicating a significant decrease in viral expression, indicating that azelnidipine suppositories have a significant inhibitory effect on HPV16 PsV. This suggests that azelnidipine has an inhibitory effect on HPV16 PsV inoculated vaginally in nude mice.

[0136] The foregoing is merely an embodiment of the present application, and the scope of protection of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the technical ideas and principles of the present application should be included in the scope of protection of the present application.

Claims

1. Use of a dihydropyridine compound or a salt thereof in the preparation of a drug for preventing and / or treating HPV infection, characterized in that: The dihydropyridine compound is selected from one or more of nifedipine, amlodipine, lercanidipine, nimodipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine and azelnidipine.

2. The use according to claim 1, characterized in that The dihydropyridine compound is azelnidipine.

3. The use according to claim 1, characterized in that The HPVs include HPV16, HPV45, HPV6, HPV5, HPV8, HPV11, and HPV31.

4. The use according to claim 1, characterized in that The dihydropyridine compound or the salt thereof has the effect of inhibiting HPV activity.

5. The use according to claim 1, characterized in that The medicine is an ointment, cream, gel, capsule, effervescent tablet, microneedle, suppository, tablet, pill, foam, patch or lotion.

6. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 1, characterized in that The administration methods of the drug include topical administration, transdermal administration, and vaginal administration.

8. The use according to claim 1, characterized in that The drug is used to prevent and / or treat skin warts, vaginitis, cervical erosion, cervical cancer, cervical and / or vaginal intraepithelial neoplasia, polyps and warts caused by HPV infection; The skin warts include common warts, flat warts, plantar warts, condyloma acuminata, epidermodysplasia verruciformis, anal and / or genital warts.

9. A pharmaceutical composition for preventing and / or treating HPV infection, characterized in that: The pharmaceutical composition includes a dihydropyridine compound or a salt thereof; The dihydropyridine compound is selected from one or more of nifedipine, amlodipine, lercanidipine, nimodipine, nitrendipine, nisoldipine, felodipine, benidipine, lacidipine and azelnidipine.

10. The pharmaceutical composition for preventing and / or treating HPV infection according to claim 9, characterized in that: The pharmaceutical composition is a cream, which comprises, by weight: 0.1-10 parts of azelnidipine, 0.5-2 parts of a skin permeation enhancer, 10-30 parts of an oily base, 0.2-5 parts of an emulsifier, 5-30 parts of a moisturizer, 0.1-2 parts of an antioxidant, 1-15 parts of an emulsifier, 0.1-2 parts of a pH regulator, and 0.05-0.2 parts of a preservative; or The pharmaceutical composition is a suppository, which comprises, by weight: 0.1-10 parts of azelnidipine, 10-40 parts of an oily base, 0.1-5 parts of an absorption enhancer, 0.1-5 parts of a surfactant, 0.01-0.05 parts of an antioxidant, 0.01-0.05 parts of a preservative, and 0.01-0.05 parts of a hardener; or The pharmaceutical composition is a microneedle, which comprises, by weight, 1 to 24 parts of azelnidipine, 20 to 40 parts of a biodegradable polymer, 1 to 15 parts of a plasticizer, 10 to 50 parts of an excipient, 0.1 to 5 parts of a surfactant, 0.1 to 2 parts of an antioxidant, and 0.01 to 0.05 parts of a preservative.

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