Application of astilbin in preparation of medicine for treating human papilloma virus infection and related diseases

Abstract

The application discloses application of astilbin in preparation of a medicine for treating human papilloma virus (HPV) infection and related diseases, and belongs to the technical field of biological medicine. The in-vivo and in-vitro pharmacodynamic experiments prove that the astilbin can significantly inhibit HPV virus infection on host cells, reduce the virus load of a model animal's vaginal mucosa, and effectively inhibit the proliferation of HPV-positive cervical cancer cells. The application provides an experimental basis for the prevention and treatment of the astilbin for HPV related diseases such as condyloma acuminatum and cervical cancer, has clinical application value, and solves the problem of the lack of safe and effective, small side effect and new anti-HPV treatment drugs which are not easy to produce drug resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of astilbin in the preparation of drugs for treating human papillomavirus infection and related diseases. Background Technology

[0002] Human papillomavirus (HPV) infection is a global public health problem. Globally, approximately 80% of sexually active individuals will be infected with at least one HPV subtype in their lifetime. Low-risk HPV types (primarily HPV 6 and 11) can cause benign proliferative lesions of the skin and mucous membranes. More than 90% of genital warts are caused by HPV 6 / 11 infection. This disease is primarily transmitted through sexual contact, is highly contagious, and has a recurrence rate as high as 30%-70%. The peak recurrence period is 3-6 months after surgery. Repeated treatments not only impose a heavy economic burden on patients but also cause severe psychological stress, significantly impacting their quality of life. Persistent infection with high-risk HPV types (mainly HPV16 and 18) is a necessary cause of cervical cancer. Globally, approximately 99.7% of cervical cancer cases are related to high-risk HPV infection. Every year, there are more than 600,000 new cases of cervical cancer and more than 300,000 deaths worldwide. In my country, the incidence of cervical cancer is increasing year by year and is showing a trend towards affecting younger women. In 2020, there were approximately 110,000 new cases of cervical cancer and approximately 59,000 deaths in my country, making it the second most common malignant tumor among women aged 15-44.

[0003] Currently, clinical treatments for HPV-related diseases have significant limitations: preventative vaccines only cover some HPV subtypes; even the nine-valent HPV vaccine only protects against nine common subtypes, failing to cover all pathogenic subtypes. Furthermore, vaccines only provide preventative protection and have no therapeutic effect on established HPV infections and related lesions. Simultaneously, due to factors such as awareness of vaccination, economic conditions, and the distribution of medical resources, HPV vaccination rates in my country, especially in rural areas, remain low. Physical therapies (laser, cryotherapy, electrocautery, surgical excision, etc.) can only remove visible warts or lesions, failing to eliminate latent viruses under the skin and mucous membranes. Post-operative recurrence rates are high, and treatment of lesions in special locations such as the cervical canal and urethra is challenging, easily causing complications such as mucosal damage and scarring. Chemotherapy also has many drawbacks: immunomodulators such as imiquimod require long-term topical application, which can easily cause local irritation such as erythema, erosion, and itching; cytotoxic drugs such as podophyllotoxin can damage normal tissues, are contraindicated in pregnant women, and have limited effectiveness on large warts; systemic use of drugs such as interferon can easily cause systemic adverse reactions such as fever, fatigue, and bone marrow suppression, and the efficacy varies greatly from person to person. Therefore, the development of safe, effective, and low-side-effect-prone novel anti-HPV drugs has become an urgent clinical need.

[0004] Astilbene (CAS 29838-67-3), chemically named (2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxacyclohexane-2-yl]oxy-2,3-dihydro-4H-chromen-4-one, is a dihydroflavonol glycoside. Modern pharmacological studies have shown that astilbene possesses anti-inflammatory, antioxidant, and immunomodulatory activities, with low toxicity and good safety profile. Currently, no research has been reported on the application of astilbene in the treatment of human papillomavirus infection and related diseases. Summary of the Invention

[0005] To address the aforementioned technical problems, the purpose of this invention is to provide the application of astilbin in the preparation of drugs for treating human papillomavirus infection and related diseases, thereby solving the problem of the lack of safe, effective, minimally invasive, and drug-resistant novel anti-HPV treatment drugs.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the invention provides the use of astilbin in the preparation of medicaments for treating human papillomavirus infection and related diseases.

[0007] Furthermore, human papillomavirus includes low-risk HPV and high-risk HPV; low-risk HPV includes at least one of HPV type 6 and HPV type 11; high-risk HPV includes at least one of HPV type 16 and HPV type 18.

[0008] Furthermore, the associated diseases include at least one of the following: condyloma acuminata, warts, and flat warts caused by low-risk HPV infection, and cervical intraepithelial neoplasia and cervical cancer caused by high-risk HPV infection.

[0009] In a second aspect, the present invention provides a medicament for treating human papillomavirus infection and related diseases, wherein the active ingredient comprises the aforementioned astilbin.

[0010] Furthermore, the drug consists of astilbene and a pharmaceutically acceptable carrier.

[0011] Furthermore, the dosage form of the drug is a topical preparation, an oral preparation, a cavity preparation, or an injectable preparation.

[0012] Furthermore, topical preparations include any one of gels, lotions, suppositories, ointments, powders, creams, patches, films, and sprays; oral preparations include any one of tablets, capsules, granules, powders, and pills; cavity preparations include any one of suppositories, vaginal tablets, vaginal capsules, enemas, rectal ointments, and creams; and injectable preparations include any one of injection solutions, lyophilized powder for injection, liposomes for injection, and microspheres.

[0013] Furthermore, the drug can be administered either locally or systemically.

[0014] Furthermore, local administration includes at least one of vaginal administration, transdermal administration, and intratumoral injection; systemic administration includes at least one of oral administration, intravenous injection, and intraperitoneal injection.

[0015] The present invention has the following beneficial effects: This invention discovered the anti-HPV and HPV-positive cervical cancer activity of astilbene through in vitro HPV infection experiments, HPV-positive cell proliferation inhibition experiments, and HPV-positive cervical cancer cell xenograft experiments. In vivo and in vitro pharmacodynamic experiments confirmed that astilbene can significantly inhibit HPV infection of host cells, reduce viral load in the vaginal mucosa of model animals, and effectively inhibit the proliferation of HPV-positive cervical cancer cells. The therapeutic effects are significantly better than those of epigallocatechin gallate (EGCG). Attached Figure Description

[0016] Figure 1 The image shows the results of fluorescence staining in the in vitro inhibitory effect of astilbene and EGCG on HPV6 pseudovirus infection. Figure 2 Figure 1 shows the experimental results of the inhibitory effects of different concentrations of astilbene and EGCG on the proliferation of HeLa cells. ±s, n=6); Figure 3 The figure shows the experimental results of the inhibitory effects of astilbene and EGCG on HeLa subcutaneous xenografts in nude mice. In the figure, A is a representative image of the excised tumor, and B is the tumor growth curve of the subcutaneous tumor in nude mice (n=6). Detailed Implementation

[0017] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0018] Example 1: Preparation of astilbene topical gel Prescription composition (per 100 g): astilbin 1 g, carbomer 940 0.8 g, glycerol 8 g, triethanolamine 0.3 g and ethylparaben 0.08 g, purified water added to 100 g.

[0019] The preparation method includes the following steps: S1. Take Carbomer 940, add 80 mL of purified water, stir well, and let it stand for 24 h to swell, and obtain the gel matrix. S2. Take omeprazole, glycerol and ethylparaben, add 10 mL of purified water, and stir until completely dissolved to obtain a completely dissolved solution; S3. Slowly add the completely dissolved solution obtained in S2 to the gel matrix obtained in S1 while stirring, and mix evenly to obtain a mixed gel. S4. Add triethanolamine dropwise to the mixed gel obtained in S3 to adjust the pH value to 5.5-6.5, add purified water to the total volume, stir evenly, and obtain 1 wt% astilbin external gel.

[0020] Example 2: Preparation of astilbene lyophilized powder for injection Prescription composition (per 1000 vials): 50 g astilcin and 80 g mannitol, with water for injection added to 2000 mL.

[0021] The preparation method includes the following steps: S1. Take the prescribed amounts of astilbene and mannitol, add 1600 mL of water for injection, and stir until completely dissolved to obtain a completely dissolved solution; S2. Add 0.1% (w / v) activated carbon to the completely dissolved solution obtained in S1, stir for 15 min, filter to remove carbon, and obtain the decarbonized solution. S3. Add water for injection to the decarbonized solution obtained in S2 until the volume is full, filter it through a 0.22 μm microporous membrane to remove bacteria, and obtain the filtrate. S4. Dispense the filtrate obtained in S3 into vials, 2 mL per vial, freeze-dry, and then stopper and cap to prepare astilbene lyophilized powder for injection, with each vial containing 50 mg of astilbene lyophilized powder.

[0022] Example 3: Preparation of astilbene cream Prescription composition (per 100 g): astilbene 2 g, stearic acid 12 g, glyceryl monostearate 4 g, white petrolatum 8 g, liquid paraffin 10 g, glycerin 10 g, triethanolamine 1 g and ethylparaben 0.1 g, purified water to 100 g.

[0023] The preparation method includes the following steps: S1. Stearic acid, glyceryl monostearate, white petrolatum and liquid paraffin are mixed and heated to 80°C, and stirred until completely melted to obtain the oil phase; S2. Add glycerol, triethanolamine and ethylparaben to purified water, heat to 80°C, and stir until completely dissolved to obtain the aqueous phase; S3. Slowly add the oil phase obtained in S1 to the aqueous phase obtained in S2 while stirring, and emulsify for 30 min to obtain the cream base. S4. After the cream base obtained in S3 has cooled to below 40°C, add the prescribed amount of astilbene and stir evenly to obtain a 2wt% astilbene cream.

[0024] Example 4: Preparation of astilbene suppositories Prescription composition (per 100 tablets): astilbin 50 g, semi-synthetic fatty acid glycerides 300 g and polysorbate 8010 g.

[0025] The preparation method includes the following steps: S1. Take semi-synthetic fatty acid glycerides, heat them in a water bath to 40°C to melt them, add polysorbate 80, stir evenly, then add the prescribed amount of astilbene, grind until evenly dispersed, and obtain a mixture; S2. Pour the mixture into a preheated suppository mold, cool to room temperature, and demold to obtain astilbestidine suppositories containing 500mg of astilbestidine per suppository.

[0026] Experimental Example 1: Study on the in vitro anti-HPV6 pseudovirus activity of astilbin I. Experimental Materials Human embryonic kidney epithelial cells 293FT and human immortalized epidermal cells HaCaT were purchased from the cell bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; HPV6 structural gene plasmid p6sheLLr and luciferase reporter gene plasmid pCLucf were purchased from Addgene, USA; Lipofiter TM Transfection reagents were purchased from Hanheng Biotechnology Co., Ltd.; astilbin (purity ≥98%) was purchased from Chengdu Mansite Biotechnology Co., Ltd.; epigallocatechin gallate (EGCG, purity ≥98%) was purchased from Sigma-Aldrich, USA; and firefly luciferase assay kit was purchased from Beyotime Biotechnology Co., Ltd.

[0027] II. Preparation and Titer Determination of HPV6 Pseudovirus Take 293FT cells in the logarithmic growth phase and administer at a dose of 2×10⁻⁶. 6 Seed each cell in a 10 cm cell culture dish and culture for 24 h until the cells reach 70%-80% confluence. Transfect the cells by dissolving 10 μg of p6sheLLr plasmid and 10 μg of pCLucf plasmid in 500 μL of serum-free DMEM medium and gently mixing. Separately, add 30 μL of Lipofiter to 500 μL of serum-free DMEM medium. TM Mix the transfection reagent gently and let stand at room temperature for 5 minutes. Mix the two solutions thoroughly and incubate at room temperature for 20 minutes to form the transfection complex. Add the complex dropwise to the culture dish and gently shake the dish to distribute the complex evenly.

[0028] Forty-eight hours after transfection, cells and supernatant were collected, subjected to three freeze-thaw cycles, and centrifuged at 3000×g for 10 min at 4°C. The supernatant was collected and filtered through a 0.45 μm sterile filter to obtain the HPV6 pseudovirus stock solution. The viral titer was determined using the endpoint dilution method, and the results showed that the HPV6 pseudovirus titer prepared in this experiment was 8.1×10⁻⁶. 7 TU / mL.

[0029] III. In vitro inhibition test HaCaT cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension, and then... 4 The cells were seeded at a density of 100 μL per well in 96-well plates and cultured overnight in a cell culture incubator at 37°C, 5% CO2, and saturated humidity until the cell confluence reached approximately 70%.

[0030] The original culture medium was discarded. To investigate the direct inactivation effect of astilbene on HPV6 pseudovirus, HPV6 pseudovirus was mixed with astilbene at final concentrations of 25 mg / L and 50 mg / L (all drugs were prepared in serum-free MEM medium containing 0.1% DMSO to ensure that the final solvent concentration was completely consistent in each group). After pre-incubation at 37°C for 1 h, the mixture was added to cell wells. Simultaneously, the following control groups were set up in parallel: 50 mg / L EGCG positive control group (prepared in the same manner), solvent control group (serum-free MEM medium containing 0.1% DMSO + an equal amount of virus), normal control group (serum-free MEM medium containing 0.1% DMSO, without virus or drug), and zeroing wells (containing only serum-free MEM medium and cell lysis buffer, without cells or virus).

[0031] Each group was configured with 8 replicates. 200 μL of sterile PBS was added to the edge wells of the 96-well plate to prevent liquid evaporation. The culture plates were incubated at 37°C in a 5% CO2 incubator for 48 h. The culture medium was then discarded, and 50 μL of cell lysis buffer was added to each well. The plates were incubated on ice for 10 min. The luciferase activity of each well was detected using a multi-functional microplate reader (detection wavelength 560 nm, integration time 1 s) following the instructions of the firefly luciferase assay kit.

[0032] The formula for calculating the viral infection inhibition rate is as follows: Inhibition rate (%) = [1 - (average fluorescence intensity of the drug-treated group - average fluorescence intensity of the zeroing well) / (average fluorescence intensity of the solvent control group - average fluorescence intensity of the zeroing well)] × 100%.

[0033] IV. Experimental Results and Analysis Experimental results are as follows Figure 1 As shown in Table 1.

[0034] Table 1. In vitro inhibitory effect of astilbin on HPV6 pseudovirus infection ( ±s, n=8)

[0035] Note: Compared with the virus control group, ** P <0.01.

[0036] The results showed that astilbin significantly inhibited HPV6 pseudovirus infection of HaCaT cells, and the inhibitory effect was clearly dose-dependent. Furthermore, the in vitro anti-HPV6 activity of astilbin was significantly superior to that of EGCG.

[0037] Experimental Example 2: Inhibitory effect of astilbin on the in vitro proliferation of HPV18-positive HeLa cells I. Experimental Materials Human cervical cancer cells (HeLa) were purchased from the cell bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the CCK-8 reagent kit was purchased from Beyotime Biotechnology Co., Ltd.; astilbin and EGCG were used in the same test case 1.

[0038] II. Experimental Methods Persistent HPV infection is a necessary prerequisite for the development of cervical cancer. Among them, HPV types 16 and 18 are the most common carcinogenic subtypes, accounting for approximately 70% of cervical cancer cases worldwide. HeLa cells are immortalized cell lines derived from human cervical adenocarcinoma, and their genomes stably integrate the HPV type 18 viral genome. Therefore, they are widely used as a classic cell model for the development of drugs to treat HPV-related cervical cancer.

[0039] In this experimental example, HeLa cells in the logarithmic growth phase were first digested with trypsin to prepare a single-cell suspension, and then... 3 The cells were seeded at a density of 100 μL per well in 96-well plates and cultured for 24 h in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0040] Then, the original culture medium was discarded, and fresh MEM medium containing astilbin at final concentrations of 6.25, 12.5, 25, 50, and 100 mg / L was added, respectively. Simultaneously, EGCG positive control, 0.1% DMSO solvent control (cells and medium containing 0.1% DMSO only), and zeroing wells (culture medium and CCK-8 solution only, no cells) were set up with the same concentration gradient. Each group had 6 replicates, and 200 μL of sterile PBS was added to the edge wells to prevent evaporation.

[0041] Finally, the culture was continued for 48 h, then 10 μL of CCK-8 solution was added to each well, the culture plate was gently shaken to mix well, the mixture was incubated at 37°C in the dark for 2 h, and the absorbance (OD value) at a wavelength of 450 nm was detected using a multi-function microplate reader.

[0042] Calculation formula for cell proliferation inhibition rate: Inhibition rate (%) = [1 - (average OD value of the administration group - average OD value of the zero-adjustment well) / (average OD value of the solvent control group - average OD value of the zero-adjustment well)] × 100%.

[0043] Nonlinear regression fitting was performed using GraphPad Prism 8.0 software to calculate the half maximal inhibitory concentration (IC 50 ) of astilbin and EGCG.

[0044] III. Experimental Results and Analysis The experimental results are shown in Figure 2 . The results show that astilbin can significantly inhibit the in vitro proliferation of HeLa cells, and the inhibitory effect presents a good dose-dependent relationship. After nonlinear regression fitting, the IC 50 of astilbin for inhibiting HeLa cell proliferation is 74.8 mg / L, which is significantly lower than that of the positive control drug EGCG (IC 50 =117.2 mg / L), indicating that astilbin has better in vitro inhibitory activity against HPV18-positive cervical cancer cells than EGCG.

[0045] Test Example 3: Inhibitory Effect of Astilbin on HPV18-Positive HeLa Cell Subcutaneous Xenograft Tumors in Nude Mice I. Experimental Animals SPF-grade female BALB / c nude mice, 4-5 weeks old, weighing 16-18 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. [Experimental Animal Production License No.: SCXK (Beijing) 2024-0001]. The animals were reared in an SPF-grade barrier system at a rearing environment temperature of 22±2°C, a relative humidity of 50%-60%, with a 12 h / 12 h light-dark cycle, free access to drinking water and food, and were used for the experiment after 1 week of adaptive rearing.

[0046] II. Experimental Methods (1) Animal Model Establishment HeLa cells in the logarithmic growth phase were collected, and pre-cooled sterile PBS buffer was used to adjust the cell concentration to 1×10 7 cells / mL, and 0.1 mL of the cell suspension (containing 1×10 6 HeLa cells) was subcutaneously inoculated into the right axilla of each nude mouse. The state of the nude mice and tumor growth were observed daily. When the tumor volume grew to 40-100 mm 3 , the model establishment was completed, and subsequent grouping experiments were carried out.

[0047] (2) Grouping and processing methods Nude mice that successfully developed the model were randomly divided into four groups of six mice each: a model control group (injected with an equal volume of physiological saline into the tumor), an EGCG positive control group (30 mg / kg), a low-dose astilbene group (10 mg / kg), and a high-dose astilbene group (30 mg / kg). The mice were administered the medication once every two days for a total of 10 doses.

[0048] (3) Indicator detection During the course of treatment, the long and short diameters of the tumor were measured every two days using vernier calipers, and the tumor volume (V, mm) was calculated according to the formula. 3 ): ; In the formula, a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm).

[0049] After the experiment, nude mice were euthanized by cervical dislocation, and tumor tissue was completely removed to calculate the tumor inhibition rate.

[0050] III. Experimental Results and Analysis Experimental results are as follows Figure 3 As shown in Table 2.

[0051] Table 2. Inhibitory effect of astilbin on HeLa subcutaneous xenograft tumors in nude mice ( ±s, n=6)

[0052] Note: Compared with the model group, ** P <0.01.

[0053] like Figure 3 As shown in Figure A, tumor growth was rapid in the model control group during the drug administration period, while tumor growth was significantly inhibited in all astilbestidine administration groups in a dose-dependent manner. The tumor volume in the high-dose astilbestidine group was significantly smaller than that in the EGCG group, indicating that astilbestidine can significantly inhibit the growth of HeLa cell subcutaneous xenografts in nude mice, and the inhibitory effect is dose-dependent. The growth curves of the nude mice in each group are shown in Figure A. Figure 3 Figure B shows the tumor inhibition rate, as shown in Table 2. At a dose of 30 mg / kg, ascorbic acid showed a tumor inhibition rate of 65.8%, significantly higher than that of the EGCG group at the same dose (54.2%). P The value <0.01 indicates that astilbene has superior activity against HPV-positive cervical cancer in vivo compared to EGCG.

[0054] 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. Application of astilbin in the preparation of drugs for treating human papillomavirus infection and related diseases.

2. The application according to claim 1, characterized in that, The human papillomavirus includes low-risk HPV and high-risk HPV; the low-risk HPV includes at least one of HPV6 and HPV11; the high-risk HPV includes at least one of HPV16 and HPV18.

3. The application according to claim 1, characterized in that, The related diseases include at least one of the following: condyloma acuminata, warts, and flat warts caused by low-risk HPV infection, and cervical intraepithelial neoplasia and cervical cancer caused by high-risk HPV infection.

4. A drug for treating human papillomavirus infection and related diseases, characterized in that, The active ingredient includes astilbin as described in claim 1.

5. The drug according to claim 4, characterized in that, The drug consists of astilbin and a pharmaceutically acceptable carrier.

6. The drug according to claim 4, characterized in that, The dosage form of the drug is a topical preparation, an oral preparation, a cavity preparation, or an injectable preparation.

7. The drug according to claim 6, characterized in that, The external preparations include any one of gels, lotions, suppositories, ointments, powders, creams, patches, films, and sprays; the oral preparations include any one of tablets, capsules, granules, powders, and pills; the cavity preparations include any one of suppositories, vaginal tablets, vaginal capsules, enemas, rectal ointments, and creams; and the injectable preparations include any one of injection solutions, lyophilized powder for injection, liposomes for injection, and microspheres.

8. The drug according to claim 4, characterized in that, The drug can be administered locally or systemically.

9. The medicament according to claim 8, characterized in that, The local administration includes at least one of vaginal administration, skin administration, and intratumoral injection; the systemic administration includes at least one of oral administration, intravenous injection, and intraperitoneal injection.

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