Use of dihydropyridine compounds or salts thereof for the preparation of a medicament for the prevention and / or treatment of HPV infection

Abstract

The application relates to 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 pharmaceutical preparations. The application provides application of the dihydropyridine compound or the salt thereof in preparation of the medicine 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 application scheme first reports the application of the dihydropyridine compound or the salt thereof in prevention and / or treatment of HPV, and in-vitro cell experiments are carried out through azelnidipine in the dihydropyridine compound, and further, the inhibiting effect of the dihydropyridine compound on HPV virus is verified through medicine administration of a mouse intradermal virus inoculation model and medicine administration of a mouse vaginal virus inoculation model.

Description

Technical Field

[0001] This application relates to the use of a dihydropyridine compound or its salt in the preparation of a drug for the prevention and / or treatment of HPV infection, and belongs to the field of pharmaceutical and pharmaceutical preparation technology. Background Technology

[0002] Human papillomavirus (HPV) is a small, double-stranded circular DNA virus. Based on its pathogenicity, it is classified into high-risk and low-risk types. High-risk HPV types, such as HPV16, HPV18, HPV31, and HPV45, can cause head and neck cancers in women, including cervical cancer, bladder cancer, oral cancer, and laryngeal cancer. Low-risk HPV types, such as HPV1, HPV5, HPV6, HPV8, and HPV11, are mainly 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 threatening women's health.

[0003] In the medical field, the treatment of HPV infection has always been a hot topic and a challenge. Currently, treatment methods for HPV infection mainly include surgery, physical therapy, and drug therapy. Surgical treatments primarily include cervical conization and loop electrosurgical excision procedure (LEEP). Physical therapy mainly involves photosensitizers combined with photodynamic therapy (PDT). Drug therapy includes interferon therapy, traditional Chinese medicine, and integrated traditional Chinese and Western medicine treatments. However, these methods still have some shortcomings in terms of cost, efficacy, safety, onset time, ease of use, and treatment adherence.

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

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

[0006] Azelnidipine (English name: Azelnidipine; Chinese chemical name: 3-(1-diphenylmethylazacyclobutane-3-yl)5-isopropyl2-amino-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid ester; molecular formula: C2) 33 H 34 N4O6, molecular weight: 582.65, chemical structural formula as follows: Figure 2 (As shown) is a novel dihydropyridine calcium channel blocker that selectively acts on L-type calcium channels. Its chemical structure is similar to that of nifedipine. Clinically, it is used to treat systemic hypertension and angina pectoris, and in a few cases, it is used to treat congestive heart failure, protect the heart and kidneys, fight atherosclerosis, and improve insulin tolerance.

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

[0008] To address the aforementioned issues, this invention provides the application of dihydropyridine compounds or their salts in the preparation of drugs for the prevention and / or treatment of HPV infection. Specifically, it provides a topical formulation of dihydropyridine compounds or their salts for the prevention and / or treatment of high / low-risk HPV infection, along with its preparation method and application. The provided azhedipine formulation has a simple preparation process and is easily industrialized. The azhedipine cream and suppositories exhibit robust performance, good in vitro release, significant therapeutic effect on HPV, and convenient use.

[0009] This application provides the use of dihydropyridine compounds or their salts in the preparation of medicaments for the prevention and / or treatment of HPV infection, wherein the dihydropyridine compounds are selected from one or more of nifedipine, amlodipine, lercanidipine, nimodipine, nifedipine, nisoldipine, felodipine, benidipine, lacidipine, and azaldipine. The core structure of the dihydropyridine compounds is as follows: Figure 1 As shown.

[0010] Optionally, the dihydropyridine compound is azedipine.

[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 may be an ointment, cream, gel, capsule, effervescent tablet, microneedle, suppository, tablet, pill, foam, patch, or lotion.

[0014] Optionally, the drug may also include pharmaceutically acceptable excipients.

[0015] Optionally, the drug may be administered via local administration, transdermal administration, or intravaginal administration.

[0016] Optionally, 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;

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

[0018] This application provides a pharmaceutical composition for the prevention and / or treatment of 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, nifedipine, nisodipine, felodipine, benidipine, lacidipine, and azedidipine.

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

[0021] Optionally, the pharmaceutical composition is a suppository, which comprises, by weight, 0.1-10 parts of azoxydipine, 10-40 parts of oil phase matrix, 0.1-5 parts of absorption enhancer, 0.1-5 parts of surfactant, 0.01-0.05 parts of antioxidant, 0.01-0.05 parts of preservative, and 0.01-0.05 parts of hardening agent.

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

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

[0024] 1. Based on the application of dihydropyridine compounds or their salts in the preparation of drugs for the prevention and / or treatment of HPV infection, this application is the first to report the application of dihydropyridine compounds or their salts in the prevention and / or treatment of HPV infection. There are currently no reports of dihydropyridine compounds for the prevention and / or treatment of HPV. This application provides a new approach for the prevention and / or treatment of HPV.

[0025] 2. Based on the application of the dihydropyridine compounds or their salts in the preparation of drugs for the prevention and / or treatment of HPV infection, this application has determined the inhibitory effect of dihydropyridine compounds or their salts on HPV through in vitro cell experiments combined with intradermal and vaginal viral inoculation models in mice, and the effect of treating HPV is significant.

[0026] 3. Based on the application of the dihydropyridine compounds or their salts in the preparation of drugs for the prevention and / or treatment of HPV infection, the preparation process of the azhedipine formulation provided in this application is simple and easy to industrialize. The azhedipine cream and suppositories have robust performance, good in vitro release, significant effect in treating HPV, and are convenient to use. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0028] Figure 1 This is a chemical formula diagram of the core structure of the dihydropyridine compounds involved in this application;

[0029] Figure 2 This is a chemical structural diagram of the azithromycin involved in this application;

[0030] Figure 3 This is a graph showing the inhibition results of different concentrations of azoldipine on HPV16 in the human cervical cancer HeLa cell line involved in Example 5 of this application;

[0031] Figure 4 This is a graph showing the inhibition results of different concentrations of nimodipine on HPV16 in the human cervical cancer HeLa cell line involved in Example 6 of this application;

[0032] Figure 5 This is a graph showing the inhibition results of different concentrations of amlodipine on HPV16 in the human cervical cancer HeLa cell line involved in Example 6 of this application;

[0033] Figure 6 This is a photographic image showing the skin irritation response of azoxyl to guinea pigs as described in Example 7 of this application.

[0034] Figure 7 This is a graph showing the effect of azoxyl cream applied to mice on intradermal HPV16PsV, as described in Example 8 of this application.

[0035] Figure 8 This is a graph showing the results of fluorescein expression in mice after vaginal administration of the azoxydipine suppository involved in Example 9 of this application. Detailed Implementation

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

[0037] Example 1: Cream and its preparation method

[0038] The cream comprises, by weight: 0.1-10% azoxydipine, 0.5-2% skin penetration enhancer, 10-30% oil phase matrix, 0.2-5% emulsifier, 5-30% moisturizer, and may also include 0.1-2% antioxidant, 1-15% emulsifier, 0.1-2% pH adjuster, 0.05-0.2% preservative, with the balance being water.

[0039] The skin penetration enhancer is selected from the group consisting of surfactants, dimethyl sulfoxide and their analogues, 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, anionic surfactant, or a combination thereof; preferably, the emulsifier is selected from the group consisting of: polyoxyethylene sorbitan fatty acids, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, or a combination thereof.

[0041] The antioxidants are selected from the following groups: natural flavonoid antioxidants, phenolic antioxidants, active polysaccharide antioxidants, vitamin antioxidants, saponin and tannin antioxidants, or combinations thereof.

[0042] The emulsifier is selected from the following group: alkanolamide thickeners, fatty alcohol and fatty acid thickeners, ether thickeners, ester thickeners, amine oxide thickeners, cellulose and its modified thickeners, polyacrylamide thickeners, natural gum and its modified thickeners, hydrophobic associative thickeners, or combinations thereof.

[0043] The moisturizers are selected from the following groups: polyol moisturizers, natural moisturizing factors, amino acid moisturizers, high molecular weight biochemical moisturizers, or combinations thereof.

[0044] The preservatives are selected from the following group: parabens, imidazolidinyl ureas, casseroles and hydroxymethyldimethylhydantoin, or combinations thereof.

[0045] In one specific embodiment, the formulation of azoxyl cream is shown in Table 1 below.

[0046] Table 1. Formulation of Azaldipine Cream

[0047]

[0048] In one specific embodiment, the preparation method of azoxystrobin cream includes the following steps:

[0049] 1) Preparation of oil phase: Take 10g of white petrolatum, 4g of stearic acid, 3g of glyceryl monostearate, 18g of silicone oil, 1g of vitamin E, and 2g of laurocapram and put them into a beaker. Mix the reagents evenly under stirring and temperature of 80℃ to prepare the oil phase.

[0050] 2) Aqueous phase preparation: Take 2g of emulsifier sodium fatty alcohol polyoxyethylene ether sulfate, 1g of Tween 80, 5g of active ingredient, and 10g of glycerin, add them to water, and mix the reagents evenly under stirring and a water temperature of 80℃ to prepare the aqueous phase. Add an appropriate amount of 0.1% methylparaben dissolved in anhydrous ethanol to the aqueous phase, and add triethanolamine dropwise until the pH reaches 7.

[0051] 3) Cream preparation: Mix the prepared oil phase and water phase, keep the oil and water phases at 80°C and slowly add the water phase to the oil phase while continuously stirring to fully emulsify the oil and water phases; after stirring evenly, allow the cream to cool naturally to room temperature, and continue stirring during the cooling process to ensure the uniformity of the cream.

[0052] 4) Packaging and storage: After cooling, the cream is inspected to ensure that its appearance, texture, pH value, etc. meet the requirements. Then it is dispensed into suitable packaging containers and sealed for storage.

[0053] Example 2 Suppositories and their preparation method

[0054] The suppository comprises, by weight: 0.1-10 parts of azoxystrobin, 10-40 parts of oil phase matrix, 0.1-5 parts of absorption promoter, 0.1-5 parts of surfactant, 0.01-0.05 parts of antioxidant, 0.01-0.05 parts of preservative, and 0.01-0.05 parts of hardener.

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

[0056] The absorption enhancers are selected from the following group: laurocapram, isopropyl myristate, taurine, glycine, menthol, or combinations thereof.

[0057] The surfactant is selected from the following group: glyceryl monostearate, polysorbate 80, Span 80, sodium dodecyl sulfate, or combinations thereof.

[0058] The antioxidants include vitamin E, sodium sulfite, sodium thiosulfate, or combinations thereof.

[0059] The preservatives are selected from the following group: methylparaben, sodium benzoate, potassium sorbate, benzalkonium chloride, or combinations thereof.

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

[0061] In one specific embodiment, the formulation of azoxyl suppositories is shown in Table 2 below.

[0062] Table 2. Composition of Azuldipine Suppository Formulation

[0063]

[0064] In one specific embodiment, the preparation method of azoxyl suppositories includes the following steps:

[0065] 1) Preparation of oil phase matrix: Place the prescribed amount of cocoa butter in a beaker and melt it under a 50°C water bath. Stir continuously during heating until it is completely melted and evenly mixed. In the melted oil phase matrix, add the prescribed amounts of azithromycin, laurocapram, glyceryl monostearate, Span 80, vitamin E, methylparaben, benzalkonium chloride, and carnauba wax in sequence. Stir continuously at 50°C to ensure that each component is fully dissolved and evenly dispersed in the oil phase matrix.

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

[0067] 3) Demolding and Packaging: After the suppository has completely solidified, carefully demold it and inspect its appearance to ensure that the suppository surface is smooth, without cracks, bubbles or other defects; package the qualified suppositories and seal them for storage.

[0068] Example 3: Microneedle Formulation and its Preparation Method

[0069] The microneedle formulation comprises, by weight: 1-24 parts of azoxydipine, 20-40 parts of biodegradable polymer, 1-15 parts of plasticizer, 10-50 parts of excipient, 0.1-5 parts of surfactant, 0.1-2 parts of antioxidant, and 0.01-0.05 parts of preservative.

[0070] The biodegradable polymers are selected from the following group: polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), chitosan, hyaluronic acid, or combinations thereof.

[0071] The plasticizer is selected from the following group: glycerin, polyethylene glycol, propylene glycol, triethyl citrate (TEC), or combinations thereof.

[0072] The excipients are selected from the following group: lactose, mannitol, sodium chloride, sodium carboxymethyl cellulose, polyvinylpyrrolidone (PVP), or combinations thereof.

[0073] The surfactant is selected from the following group: Tween-80, Span-80, poloxamer, or combinations thereof.

[0074] The antioxidants are selected from the following group: vitamin E, vitamin C, propyl gallate, or combinations thereof.

[0075] The preservatives are selected from the following group: methylparaben, sodium benzoate, benzalkonium chloride, or combinations thereof.

[0076] In one specific embodiment, the formulation of the azoxyl microneedle preparation is shown in Table 3 below.

[0077] Table 3. Composition of Azaldipine Microneedle Formulation

[0078]

[0079] In one specific embodiment, the preparation method of azoxyl microneedle formulation includes the following steps:

[0080] 1) Polymer solution preparation: Weigh the prescribed amount of PLGA and add it to an appropriate amount of dichloromethane, stirring until fully dissolved. Add the prescribed amounts of aztredipine, glycerin, lactose, mannitol, Tween-80, vitamin E, propyl gallate, and methylparaben sequentially to the polymer solution. Stir continuously during the addition process to ensure that all components are evenly dispersed in the solution.

[0081] 2) 3D Printing Model Design: Using computer-aided design (CAD) software, design the 3D model of the azurh microneedles. Determine the shape, length, diameter, array layout, and other parameters of the microneedles.

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

[0083] 4) Microneedle post-processing: After printing, carefully remove the microneedles from the printing platform and remove excess solution or impurities from the surface. Use vacuum drying to remove organic solvents from the microneedles, allowing them to solidify and take shape. Package and store the qualified azuril microneedles.

[0084] Example 4: Cytotoxicity evaluation of azoxystrobin

[0085] Human cervical cancer cells (HeLa) and human skin fibroblast-like cells (HSF) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin. Human immortalized epidermal cells (HaCat) were also cultured in MEM high-glucose medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells in logarithmic growth phase were seeded at 7000 cells / well (180 μl) in 96-well plates and cultured for 24 h. Then, different concentrations of azledipine (2.5, 5, 10, 20, 50, 100 μM) were added, and an equal volume of culture medium was added to the blank control group. Each concentration was used in quadruplicate wells, and the cells were cultured for another 72 h.

[0086] After 72 hours of treatment with azhedipine, cells were fixed in each well with 50% (m / v) cold trichloroacetic acid (TCA), stained with SRB, and then 150 μl of Tris solution was added to each well. The OD value at 540 nm was measured using a microplate reader.

[0087] The cell growth inhibition rate is calculated using 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 3-5 below.

[0090] Table 3. Effects of different concentrations of azordipine on cell proliferation after direct in vitro contact with HeLa cells for 72 h.

[0091]

[0092] Table 4. Effects of different concentrations of azordipine on HaCat cell proliferation after 72 hours of direct in vitro contact with HaCat cells.

[0093]

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

[0095]

[0096] According to the results in the table above, azithromycin did not show significant cytotoxicity against 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 azoxystrobin

[0098] Cervical cancer cells (HeLa) and human skin fibroblast-like cells (HSF) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin. Human immortalized epidermal cells (HaCat) were also cultured in MEM high-glucose medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells in logarithmic growth phase were then cultured at a density of 1 × 10⁻⁶ cells / mL. 4 85 μl of virus per well was seeded into a 96-well plate. When the cell confluence reached approximately 50%, 5 μl of different HPV genotypes (HPV16, HPV5, HPV6, HPV8, HPV11, HPV31, HPV45) and 10 μl of azledipine solutions at different concentrations (0.625, 1.25, 2.5, 5, 10 μM) were added. An equal volume of virus and culture medium was added to the blank control group. Each concentration was used in triplicate, and the cells were cultured for another 48 hours.

[0099] After 48 hours, the culture medium was discarded, and 100 μl of lysis buffer was added to each well. The plate was placed on ice and shaken 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. The relative light unit (RLU) was measured using a microplate reader.

[0100] Inhibition rate (%) = (Experimental group RLU - Blank group RLU) / (Virus group RLU - Blank group RLU) × 100%, and IC50 is calculated based on the inhibition rate of different concentrations.

[0101] The inhibitory effects of different concentrations of azoldipine on HPV16 in the human cervical cancer HeLa cell line are shown in Table 6. Figure 3 As shown.

[0102] Table 6. Anti-HPV16 activity of HeLa cells

[0103]

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

[0105] Table 7 IC50 values ​​of azithromycin against different HPV genotypes in different cell lines.

[0106]

[0107] As shown in the table above, arzedipine exhibits 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] HeLa cervical cancer cells were used and 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 stocked at a density of 1 × 10⁻⁶ cells / mL. 4 85 μl of virus per well was seeded into a 96-well plate. When the cell confluence reached approximately 50%, 5 μl of HPV16 and 10 μl of nimodipine / amlodipine solutions of different concentrations (5 μM and 10 μM) were added. The blank control group was treated with an equal volume of virus and culture medium. Each concentration was used in quadruplicate wells, and the cells were cultured for another 48 hours.

[0110] After 48 hours, the culture medium was discarded, and 100 μl of lysis buffer was added to each well. The plate was placed on ice and shaken 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. The relative light unit (RLU) was measured using a microplate reader.

[0111] Inhibition rate (%) = (Experimental group RLU - Blank group RLU) / (Virus group RLU - Blank group RLU) × 100%, and IC50 is 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 lines, such as Figure 4 and Figure 5 As 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 azoxystrobin cream

[0115] Four guinea pigs, aged 4–8 weeks, half male and half female, weighing 200–300g, were used as experimental mice. Hair was removed from both sides of the spine on the back of the guinea pigs, with the hair removal area exceeding 3*3cm. The day after hair removal, 0.5mL (g) of the test sample was directly applied to the skin of the same animal on a symmetrical 2.5*2.5cm area. Azuldipine cream was applied to one side as a blockade, while a blank cream was applied to the other side, leaving a blank area as a control. The drug was removed 4 hours after administration. Photographs were taken at 1, 24, 48, and 72 hours after drug removal to evaluate the skin irritation of a single dose. Skin erythema and edema reactions after a single dose were scored according to the "Technical Guidelines for Drug Irritation, Allergy, and Hemolysis Studies 2014".

[0116] Table 8. Scoring Criteria for Skin Irritation Response

[0117] Stimulus response Score erythema No erythema 0 Mild erythema (barely visible) 1 Moderate erythema (clearly visible) 2 Severe erythema 3 From purplish-red erythema to mild eschar formation 4 edema No edema 0 Mild edema (barely visible) 1 Moderate edema (obvious swelling) 2 Severe edema (skin bulges 1mm, with clear contours) 3 Severe edema (skin bulges greater than 1 mm and enlargement). 4 Highest total score 8

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

[0119] Table 9. Skin irritation response scores of azithromycin in guinea pigs.

[0120]

[0121] Based on the results in the images and tables, it can be seen that, in guinea pig model testing, a single application of azaldipine cream to the skin did not cause symptoms such as edema, crusting, or erythema, indicating that a single application of azaldipine did not irritate the guinea pig skin. Therefore, it can be concluded that a single application of azaldipine did not show irritation to the guinea pig skin, and the drug has good safety.

[0122] Example 8: Inhibitory activity of azoxystrobin cream against intradermal inoculation of HPV16 PsV virus in mice.

[0123] Balb / c nude mice, 6–8 weeks old, female, weighing 18–20 g, were used as experimental mice. On day 0, nude mice were infected by intradermal injection of 100 μL of HPV1 6PsV. Each mouse was also injected with 50 μL of pseudovirus solution (2 cm * 2 cm) at a fixed area, with a viral titer of 500,000. On day 2, observation was performed using a small animal imaging system. Based on the semi-quantitative analysis of in vivo fluorescence expression, mice were grouped and administered drugs accordingly. Twelve nude mice were randomly divided into two groups: a model group and a drug treatment group, with six mice in each group. The model group was only infected with the pseudovirus. The drug treatment group was administered the drug topically 0.5 h after viral infection, with low (2 mg / kg), medium (5 mg / kg), and high (10 mg / kg) doses. On day 5, the expression level of luciferase in the mice of different groups was detected. The antiviral effect of azoxylin cream was recorded using small animal in vivo imaging.

[0124] The luciferase substrate (XenoLight D-Luciferin) was dissolved in physiological saline to a concentration of 15 mg / ml. 25 μL of the substrate was injected intraperitoneally into each mouse using a volumetric pipette. After injection, the mice were placed in an isoflurane anesthesia chamber for induction, with anesthesia reaching stability in approximately 3 minutes. The animals were then transferred to the detection chamber of the Xenogen In Vivo Imaging System (IVIS) and anesthetized. Images were acquired using livingimage 2.0 software with the following parameters: Bin = 8, f / stop = 1, Exposure time = 60 s. Strong signal areas were selected within the detected images, and the signal values ​​within the selected areas were acquired and recorded.

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

[0126] Table 10 Semi-quantitative analysis of fluorescein expression in mice after administration of azaldipine 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 images and tables show that, on day 5 after administration in the animal group, the expression of luciferin in mice was significantly lower (P<0.0001) compared to the model group, indicating a significant decrease in viral expression. This suggests that azordipine cream has a significant inhibitory effect on HPV16PsV. Therefore, it can be concluded that azordipine has an inhibitory effect on HPV16PsV induced intradermally inoculated in nude mice.

[0129] Example 9: Inhibitory activity of azoxyl suppositories against vaginal inoculation of HPV16 PsV virus in mice.

[0130] Balb / c nude mice, 6–8 weeks old, female, weighing 18–20 g, were used as experimental mice to establish a mouse vaginal model of HPV 16PsV infection. Hormone induction was achieved by subcutaneous injection of 30 mg / ml Depo-Provera (medroxyprogesterone acetate). Three days later, 20 μl of N-9 (final N-9 concentration 4%) was injected into the mouse vagina to induce chemical damage. Six hours later, a cell brush was inserted into the vagina, rotated clockwise 10 times, and then counterclockwise 10 times to induce vaginal bleeding. Twelve nude mice were randomly divided into two groups: a model group and a treatment group, with six mice in each group. The model group was infected with only pseudovirus, while the treatment group received HPV 16PsV pseudovirus inoculation solution and azoxylin suppositories injected into the vagina, divided into low (2 mg / kg), medium (5 mg / kg), and high-dose (10 mg / kg) groups. The expression level of luciferase in the mice was measured on day 4. Small animal in vivo imaging was used to record the anti-HPV16 virus infection ability of azoxystrobin suppositories in mice after vaginal administration.

[0131] The luciferase substrate (XenoLight D-Luciferin) was dissolved in physiological saline to a concentration of 15 mg / ml. 25 μL of the substrate was injected intraperitoneally into each mouse using a volumetric pipette. After injection, the mice were placed in an isoflurane anesthesia chamber for induction, with anesthesia reaching stability in approximately 3 minutes. The animals were then transferred to the detection chamber of the Xenogen In Vivo Imaging System (IVIS) and anesthetized. Images were acquired using livingimage 2.0 software with the following parameters: Bin = 8, f / stop = 1, Exposure time = 60 s. Strong signal areas were selected within the detected images, and the signal values ​​within the selected areas were acquired and recorded.

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

[0133] Table 11 Semi-quantitative analysis of fluorescein expression in mice after administration of azaldipine 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] The results in the images and tables show that, on day 4 after administration in animals, the expression of luciferin in mice in the high-dose group was significantly lower (P<0.05) compared to the model group, indicating a significant decrease in viral expression. This suggests that azoxybipine suppositories have a significant inhibitory effect on HPV16 PsV. Therefore, it can be concluded that azoxybipine has an inhibitory effect on HPV16 PsV induced by vaginal inoculation in nude mice.

[0136] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. The use of dihydropyridine compounds or their salts in the preparation of HPV-inhibiting drugs for the prevention and / or treatment of HPV infection, characterized in that, The dihydropyridine compound is one or both of amlodipine and nimodipine; The HPV referred to is HPV16.

2. The application according to claim 1, characterized in that, The drug is used to prevent and / or treat cervical cancer, cervical and / or vaginal intraepithelial neoplasia caused by HPV infection.

3. The use of dihydropyridine compounds or their salts in the preparation of HPV-inhibiting drugs for the prevention and / or treatment of HPV infection, characterized in that, The dihydropyridine compound is azuldipine; The HPV is one or more of HPV16, HPV45, HPV6, HPV5, HPV8, HPV11, and HPV31.

4. The application according to claim 3, characterized in that, The drug is used to prevent and / or treat cervical cancer, cervical and / or vaginal intraepithelial neoplasia, and anal and / or genital warts caused by HPV infection.

5. The application according to claim 3, characterized in that, The drug is used to prevent and / or treat genital warts caused by HPV infection.

6. The application according to claim 1 or 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application according to claim 1 or 3, characterized in that, The drug is an ointment, cream, gel, capsule, microneedle, suppository, tablet, pill, patch, or lotion.

8. The application according to claim 1 or 3, characterized in that, The drug can be administered via local or transdermal methods.

9. The application according to claim 8, characterized in that, The medication is administered vaginally.

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