Gel for resisting genital tract human papilloma virus infection and preparation method thereof

Abstract

The invention discloses gel for resisting genital tract HPV (human papilloma virus) infection and a preparation method of the gel. The gel is prepared from the following components: 15 to 22 percent of poloxamer 407; 0 to 10 percent of poloxamer 188; 0.01 to 0.25 percent of hydroxypropyl methyl cellulose; 0.5%-5% of an acidic buffer agent (the pH value is 3.5-4.0); 2.5 to 12.5 percent of a wetting agent; 0.01 to 0.2 percent of preservative; and pure water. The anti-genital tract HPV infection gel provided by the invention not only can effectively kill and inhibit genital tract HPV viruses, but also has no irritation and anaphylaxis to genital tract mucous membrane epithelial cells, is good in safety, relatively simple in preparation process and controllable in quality, and can be developed as a medicine or a medical instrument product.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a gel suitable for preventing and treating genital tract HPV infection and a preparation method thereof. Background Art

[0002] Reproductive tract infections have a significant impact on global health. According to the World Health Organization (WHO), there are more than 1 million new sexually transmitted infections (STIs) every day worldwide. Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases in the world.

[0003] HPV is a widely distributed DNA virus with multiple subtypes, some of which are closely related to reproductive tract infections, cervical cancer, and other reproductive system tumors. According to WHO statistics, about 6 million women are newly infected with HPV each year. Cervical cancer, as a direct consequence of HPV infection, is one of the main threats to women's health worldwide, causing the death of about 300,000 women each year. HPV infection may not only cause cervical cancer, but is also related to other reproductive system tumors such as vaginal cancer, vulvar cancer, anal cancer, and oropharyngeal cancer. In addition, HPV infection may also cause sexually transmitted diseases such as genital warts. HPV infection has a serious impact on the patient's quality of life, mental health, and economic burden. Although HPV vaccines are currently available to prevent HPV infections of specific subtypes, the vaccine cannot treat existing infections and cannot cover all high-risk HPV subtypes. In addition, the popularity and vaccination rates of vaccines are still uneven around the world, especially in developing countries.

[0004] HPV infection not only threatens women's health, but also poses a health risk to men. According to a systematic evaluation data, 31% of men are infected with HPV, of which 21% are infected with high-risk HPV. Almost all cervical cancers are caused by high-risk HPV, while 40% of penile cancers and 90% of anal cancers are caused by high-risk HPV. However, due to the incomplete epidemiological data of HPV in men in my country and insufficient clinical attention, there are currently no effective measures for detection and screening, early prevention, and intervention and clearance. Since HPV infection often manifests as asymptomatic or subclinical infection, men can easily become the source of infection for women infected with HPV. Therefore, it is of great significance to the health of both men and women to diagnose and treat HPV-infected men and women together and carry out targeted prevention.

[0005] As people's quality of life improves, many women will choose genital tract gel products to prevent and treat genital tract infections. Genital tract gel preparations have the following characteristics, making them excellent drug carriers for anti-genital tract drugs. First, the active pharmaceutical ingredients in the gel have bactericidal and anti-inflammatory effects on sensitive STIs pathogens; second, the medicinal excipients of genital tract gel are safe and have no side effects, and are non-irritating to the mucosa; and, genital tract gel can maintain the normal physiological pH value of the female genital tract, which is beneficial to the balance of vaginal flora and helps the vagina restore its self-cleaning function. In terms of acceptability, the gel is usually water-soluble, easy to clean, and has a lubricating effect; the gel can be pre-filled in a disposable applicator, with accurate dosage and convenient administration. Therefore, genital tract gel preparation products are expected to become an important intervention technology for preventing genital tract infections.

[0006] In view of the extensiveness and severity of genital HPV infection, the development of new and highly effective genital topical drugs with anti-genital HPV virus effects is of great significance for the effective prevention and treatment of male and female external genital infections. Summary of the invention

[0007] The present invention aims to provide a gel for resisting genital HPV infection and a preparation method thereof. The gel can not only effectively kill genital HPV viruses, but also has no irritation or allergy to genital mucosa, good safety, simple preparation process, and controllable quality, and can be developed and industrialized as a drug or medical device product.

[0008] The present invention provides a gel for preventing genital HPV infection, characterized in that it is prepared from the following components in percentage by mass:

[0009] 15-32% gel matrix excipient;

[0010] 0.01-0.25% thickener;

[0011] 0.5-5% acid buffer;

[0012] 2.5-12.5% ​​wetting agent;

[0013] 0.01-0.2% preservative;

[0014] The balance is pure water;

[0015] The pH of the gel is adjusted to 3.5 to 4.0 using the acidic buffer.

[0016] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0017] The dosage of the above acidic buffer is 0.5-2.0%.

[0018] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0019] The acidic buffer mentioned above can be any acidic buffer commonly used in the art, including but not limited to: acetate buffer, phosphate buffer, carbonate buffer, preferably acetate buffer. Preferably, it contains 1.0-2.0 wt% acetate buffer.

[0020] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0021] The gel matrix excipients can be selected from nonionic surfactants, such as block polymers of polyoxyethylene-polyoxypropylene, alginic acid, chitosan, carbomer, tragacanth gum, gelatin, polycarbophil, carboxymethyl cellulose and the like; preferably poloxamer 407 and / or poloxamer 188.

[0022] The gel matrix excipients include poloxamer 407 and poloxamer 188;

[0023] Wherein, the usage of the above-mentioned poloxamer 407 is 15-18%.

[0024] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0025] The thickener mentioned above can be any thickener commonly used in the art, including but not limited to: one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, sodium polyacrylate, xanthan gum, etc. Preferably, it contains 0.01-0.1wt% hydroxypropyl methylcellulose;

[0026] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0027] The above-mentioned moisturizing agent can be selected from any moisturizing ingredients commonly used in beauty products, daily chemicals, and body care products, such as: one or more of glycerol, polyethanol, propylene glycol, sorbitol, sodium lauryl sulfate, sodium dicaprylate sulfonate, sulfonated castor oil, triacetin, octyl palmitate coconut oil glyceride, and stearyl alcohol; preferably contains 5-12.5wt% glycerol.

[0028] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0029] The preservative can be selected from any preservative ingredients commonly used in cosmetics, daily chemicals, and body care products, such as propolis, benzoic acid, sodium benzoate, parabens (parabens), ethanol, quaternary ammonium salts, sorbic acid, 30% or more glycerol solution Germal IS45, Glydant, etc. Preferably, 0.02-0.1wt% of parabens is contained.

[0030] Furthermore, the present invention also provides a method for preparing the above-mentioned gel for resisting genital tract HPV infection, which is characterized by comprising the following steps:

[0031] S1. Soak poloxamer in pure water, let stand, stir, fully hydrate and disperse evenly to obtain a poloxamer solution for later use;

[0032] S2. Slowly add the thickener, wetting agent, acidic buffer and preservative into S1 in order, and finally add pure water, stir evenly and adjust the volume to obtain a gel for preventing genital HPV infection.

[0033] Based on the difference in poloxamer composition, the preparation method can include the following two schemes:

[0034] The present invention provides a method for preparing an anti-genital HPV infection gel, comprising the following steps: step 1, taking 15-22% of poloxamer 407 by mass percentage, soaking it in pure water, standing, stirring, fully hydrating and evenly dispersing it, to obtain a poloxamer 407 solution for standby use; step 2, taking 0-10% of poloxamer 188 by mass percentage, soaking it in pure water, standing, stirring, fully hydrating and evenly dispersing it, to obtain a poloxamer 188 solution for standby use; step 3, slowly adding the poloxamer 188 solution obtained in step 2 to the poloxamer 407 solution in step 1, and stirring it evenly; step 4, taking 15-22% of poloxamer 407 by mass percentage, soaking it in pure water, standing, stirring, fully hydrating and evenly dispersing it, to obtain a poloxamer 188 solution for standby use; step 5, slowly adding the poloxamer 188 solution obtained in step 5 to the poloxamer 407 solution in step 1, and stirring it evenly; step 6, Take 0.01-0.25% of hydroxypropyl methylcellulose by mass percentage, soak it in pure water, stir it, fully dissolve it, and obtain a hydroxypropyl methylcellulose solution for later use; step five, take 2.5-12.5% ​​of a wetting agent by mass percentage, and set it aside; step six, take 0.5-5% of an acid buffer solution by mass percentage, and set it aside; step seven, take 0.01-0.2% of a preservative solution by mass percentage, and set it aside; the solutions obtained in steps four, five, six and seven are slowly added to the solution in step three in sequence, and finally pure water is added, stirred evenly, and fixed to volume to obtain a gel for resisting genital tract HPV infection.

[0035] As another scheme, a method for preparing a gel for resisting genital tract HPV infection includes the following steps: step 1, taking 15-18% of poloxamer 407 by mass percentage, soaking it in pure water, letting it stand, stirring, and fully dissolving it to obtain a poloxamer 407 solution for standby use; step 2, taking 0.01-0.1% of hydroxypropyl methylcellulose by mass percentage, soaking it in pure water, stirring, and fully dissolving it to obtain a hydroxypropyl methylcellulose solution for standby use; step 3, taking 5-12.5% ​​of a wetting agent by mass percentage for standby use; step 4, taking 0.5-2.0% of an acetate buffer solution by mass percentage for standby use; step 5, taking 0.01-0.1% of a preservative solution by mass percentage for standby use; the solutions obtained in steps 2, 3, 4, and 5 are slowly added to the solution in step 1 in sequence, and finally pure water is added, stirred evenly, and fixed to volume to obtain a gel for resisting genital tract HPV infection.

[0036] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0037] The above gel is a colorless clear liquid at <20°C with a viscosity of less than 1.0 Pa.s; and a colorless clear gel at ≥29°C with a viscosity of more than 50.0 Pa.s.

[0038] Furthermore, the present invention provides a gel for preventing genital HPV infection, which is also characterized by:

[0039] During use, apply to the female vagina and / or male genitalia.

[0040] Furthermore, the drug delivery product is a gel or cream (ointment) for condoms, or for vagina and / or male external genitalia.

[0041] After research, the inventors found that anti-genital HPV gel can effectively inactivate or inhibit the activity of HPV virus.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The anti-genital HPV infection gel of the present invention can effectively kill and inhibit genital HPV viruses. The gel can not only effectively kill and inhibit genital HPV viruses, but also has no irritation and allergy to genital mucosal epithelial cells, good safety, relatively simple preparation process, and controllable quality, and can be developed as a drug or medical device. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Thermosensitive phase transition curve of gel against HPV infection in reproductive tract

[0045] Figure 2Cytostatic activity of anti-genital HPV infection gel against HPV16 PsV

[0046] Figure 3 The inhibitory effect of anti-genital HPV infection gel on HPV pseudovirus infection in mice. Fluorescence imaging (A) and fluorescence intensity statistics (B) of the vaginal area of ​​mice infected with HPV16 pseudovirus after vaginal injection of anti-genital HPV infection gel or control (PBS).

[0047] Figure 4 Irritation test of anti-genital HPV infection gel on rabbit vaginal mucosa

[0048] Figure 5 Cytotoxicity test of anti-genital HPV infection gel on VK2 cells (HE staining, 20×), A matrix control group; B anti-genital HPV infection gel group; C N-9 gel group DETAILED DESCRIPTION

[0049] The present embodiment provides a gel for preventing genital HPV infection, which is prepared from the following components in percentage by mass: 15-32% gel matrix excipient; 0.01-0.25% thickener; 0.5-5% acidic buffer; 2.5-12.5% ​​wetting agent; 0.01-0.2% preservative; and the balance pure water; wherein the pH of the gel is adjusted to 3.5-4.0 by the acidic buffer.

[0050] The acidic buffer is selected from acetate buffer solution, phosphate buffer solution, and carbonate buffer solution, and its usage is preferably 0.5-2.0%, and most preferably 1-2%.

[0051] The gel matrix excipient comprises poloxamer 407 and / or poloxamer 188; wherein the amount of poloxamer 407 is 15-18% of the total weight.

[0052] The thickener is selected from one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, sodium polyacrylate and xanthan gum.

[0053] The wetting agent is selected from one or more of glycerol, polyethanol, propylene glycol, sorbitol, sodium lauryl sulfate, sodium dicaprylate sulfonate, sulfonated castor oil, triacetin, octyl palmitate coconut oil glyceride, and stearyl alcohol.

[0054] The preservative is selected from one or more of propolis, benzoic acid, sodium benzoate, parabens, ethanol, quaternary ammonium salts, sorbic acid, 30% or more glycerol solution Germal IS45, and Glydant.

[0055] By configuring the above components and contents, a product can be obtained which is a colorless clear liquid at <20℃ and has a viscosity of less than 1.0Pa.s; and a colorless clear gel at ≥29℃ and has a viscosity of more than 50.0Pa.s.

[0056] 1. Best Experimental Example

[0057] Example 1: Preparation of Anti-genital HPV Infection Gel 1

[0058] The present invention provides a method for preparing an anti-genital HPV infection gel, comprising the following steps: step 1, taking 18% of poloxamer 407 by mass percentage and soaking it in pure water, standing, stirring, fully hydrating and evenly dispersing it to obtain a poloxamer 407 solution for standby use; step 2, taking 0.5% of poloxamer 188 by mass percentage and soaking it in pure water, standing, stirring, fully hydrating and evenly dispersing it to obtain a poloxamer 188 solution for standby use; step 3, slowly adding the poloxamer 188 solution obtained in step 2 to the poloxamer 407 solution in step 1, stirring and evenly dispersing it uniform; step 4, taking 0.10% hydroxypropyl methylcellulose by mass percentage, soaking it in pure water, stirring, fully dissolving, obtaining a hydroxypropyl methylcellulose solution for standby use; step 5, taking 10% wetting agent by mass percentage for standby use; step 6, taking 1% acid buffer solution by mass percentage for standby use; step 7, taking 0.1% preservative solution by mass percentage for standby use; the solutions obtained in steps 4, 5, 6 and 7 are slowly added to the solution in step 3 in sequence, and finally pure water is added, stirred evenly, and fixed to volume to obtain a gel for resisting genital HPV infection.

[0059] Test results:

[0060] It can be seen that the above anti-genital HPV infection gel is a temperature-sensitive water-soluble colorless clear gel with a pH value of 3.9±0.2. At 20°C, the gel is in liquid state with a viscosity of less than 1.0 Pa.s; at 29°C, the gel is in gel state with a viscosity greater than 50.0 Pa.s. The results of the 3-month stability test showed that the gel was stable in all properties under the conditions of 25°C±2°C and RH60%±5%.

[0061] Example 2: Preparation of Anti-genital HPV Infection Gel 2

[0062] As a preferred solution, a method for preparing a gel for resisting HPV infection in the reproductive tract includes the following steps: step 1, taking 17% of poloxamer 407 by mass percentage, soaking it in pure water, standing, stirring, fully dissolving, obtaining a poloxamer 407 solution for standby use; step 2, taking 0.05% of hydroxypropyl methylcellulose by mass percentage, soaking it in pure water, stirring, fully dissolving, obtaining a hydroxypropyl methylcellulose solution for standby use; step 3, taking 8% of a wetting agent by mass percentage for standby use; step 4, taking 1.5% of an acetic acid buffer solution by mass percentage for standby use; step 5, taking 0.05% of a preservative solution by mass percentage for standby use; the solutions obtained in steps 2, 3, 4 and 5 are slowly added to the solution in step 1 in sequence, and finally pure water is added, stirred evenly, and the volume is fixed to obtain a gel for resisting HPV infection in the reproductive tract. The properties, pH value, viscosity, phase transition temperature and stability of the gel are checked.

[0063] Test results:

[0064] It can be seen that the anti-genital HPV infection gel of this product is a temperature-sensitive water-soluble colorless clear gel with a pH value of 3.8±0.2. The gel is in liquid state at 20°C with a viscosity of less than 1.0 Pa.s; it is in gel state at 29°C with a viscosity greater than 50.0 Pa.s. The results of the 3-month stability test show that the gel is stable in all properties under the conditions of 25°C±2°C and RH60%±5%. Compared with the anti-genital HPV infection gel of Example 1, the pH range of this product is more suitable for maintaining the normal vaginal microenvironment.

[0065] 2. Effect Verification Experiment

[0066] Experiment 1: Dynamic temperature scanning and phase transition temperature comparison of anti-genital HPV infection gel

[0067] Dynamic strain scanning was performed under constant shear rate conditions, and a rotational rheometer was used to measure the dynamic viscosity change of the anti-genital HPV infection gel of the above embodiment during the temperature increase from 20°C to 40°C. The measuring fixture used a flat plate with a diameter of 40 mm and a shear rate of 1 s -1 , heating rate 3.33℃·min -1 .

[0068] Experimental results:

[0069] The results show that Figure 1), the viscosity of the anti-genital HPV infection gel of Example 1-2 increases with the increase of temperature in an S-shaped curve. When it is less than 29°C, the viscosity of the sample is very low; when the temperature rises to 28-29°C, the viscosity suddenly increases, and an exponential increase stage appears. The preparation changes from a solution state to a semi-solid gel state. The entire phase change process is completed within about 3-4 temperature units, and then enters a plateau phase, at which time the viscosity reaches the highest value. And the viscosity of the three batches of anti-genital HPV infection gel samples with optimized formulas changes with temperature in a consistent curve, indicating that the preparation process has good reproducibility and controllable quality.

[0070] Compared with the anti-genital HPV infection gel of Example 1, the anti-genital HPV infection gel of Example 2 has a phase transition temperature that is more consistent with the temperature in the vagina, has better viscosity stability as the temperature rises, and has rheological properties that are more suitable for vaginal administration. Subsequent experiments were all conducted using the anti-genital HPV infection gel of Example 2.

[0071] Experiment 2. Inhibitory activity of the anti-genital HPV infection gel of Example 2 on HPV16 PsV in cells

[0072] The anti-genital HPV infection gel was diluted in a gradient manner, with 3 replicates for each concentration, and mixed with HPV16 pseudovirus in equal volumes, and incubated at room temperature for 30 minutes; added to a 96-well culture plate pre-plated with Hela cells, and the cell supernatant was replaced with DMEM medium containing 2% FBS after 12 hours of infection, and the expression of the luciferase reporter gene and the enzyme activity of each well were detected after 48 hours of continuous culture; the cell wells (not infected with viruses) were blank controls, and the cell wells infected with pseudoviruses alone were positive controls. The pseudovirus inhibition rate of each sample can be calculated by the following formula:

[0073] Pseudovirus inhibition rate (%) = [(average RLU of positive control - average RLU of sample) / (average RLU of positive control - average RLU of blank control)] × 100

[0074] CalcuSyn software was used to calculate the half-effective (entry inhibition) concentration (IC) of each modified compound sample against each pseudovirus. 50 ).

[0075] Experimental results:

[0076] like Figure 2 As shown in Figure 2, in Hela cells, the anti-genital HPV infection gel can effectively inhibit the infection of HPV16 pseudovirus, and its IC 50 The value is 7.56mg / mL.

[0077] Experiment 3. Inhibitory effect of the anti-genital HPV infection gel of Example 2 on HPV pseudovirus-infected mice

[0078] On the first day of implementation, mice were intraperitoneally injected with medroxyprogesterone; on the fourth day of implementation, nonoxynol 9 (N-9) was injected vaginally, and mice were anesthetized 6 hours later. After destroying the integrity of the vaginal epithelium with a cell brush, premixed anti-genital HPV infection gel and HPV16 pseudovirus were injected vaginally. The control group used PBS instead of anti-genital HPV infection gel. On the seventh day of implementation, firefly luciferase (Promega product P1041) was injected intraperitoneally to detect luciferase expression in mice, and imaging was performed using the IVIS spectral imaging system.

[0079] Experimental results:

[0080] like Figure 3 As shown, the average fluorescence intensity in the vagina of mice injected with PBS reached 1.2×10 4 p / s / cm2 / sr, while the average fluorescence intensity in the vagina of mice injected with anti-genital HPV infection gel was only 2.7×10 3 p / s / cm2 / sr. This shows that compared with PBS, the anti-genital HPV infection gel can effectively prevent vaginal infection of HPV16 pseudovirus.

[0081] Experiment 4: Cytotoxicity test of the anti-genital HPV infection gel of Example 2 on vaginal mucosal cells VK2 / E6E7

[0082] VK2 / E6E7 cells in the logarithmic growth phase were inoculated into 96-well culture plates at a concentration of 0.5-1×10 4 Cells / well were pre-cultured overnight in a CO2 cell culture incubator at 37°C;

[0083] An equal volume of matrix control and the anti-genital HPV infection gel of Example 2 were added to each well, and Defined Keratinocyte-SFM was used as a blank control. After 4 hours of action, each well was replaced with fresh culture medium, and 10 μL MTT solution (5 mg / mL) was added and placed in a CO2 cell culture incubator at 37°C for another 4 hours;

[0084] Remove the culture medium, add 100 μL DMSO to each well to lyse the cells, measure the optical density of each well at a wavelength of 570 nm using an ELISA reader, and calculate the cell viability:

[0085]

[0086] Experimental results:

[0087] The experimental results show that ( Figure 4), after 4 hours of treatment with the matrix control and the anti-genital HPV infection gel of Example 2, the cell proliferation rates of VK2 / E6E7 cells were 98.47±2.95% and 95.88±3.67%, respectively. The results of the study suggest that compared with the matrix control, the anti-genital HPV infection gel of Example 2 has no significant difference in effect on vaginal mucosal epithelial cells (P>0.05), and has good safety.

[0088] Experiment 5: Rabbit vaginal mucosal irritation test of the anti-genital HPV infection gel of Example 2

[0089] According to the Eckstein experimental method and evaluation criteria, 18 rabbits were divided into 3 groups, 6 in each group, and 2.5 mL of gel was administered vaginally to different treatment groups once a day for 10 consecutive days. The animals were killed on the 11th day, the vagina was cut open for naked eye observation, and then the vaginal tissue was separated, fixed with 10% formalin solution, embedded, sliced, and pathological histological examination was performed. The four lesions of vaginal mucosal tissue, mucosal edema, vascular congestion, leukocyte infiltration, and epithelial ulcer, were scored according to the different degrees, each item was recorded with 1 to 4 points, a total of 16 points. If the total score is between 0 and 8 points, it indicates that the mucosal irritation of the drug is acceptable.

[0090] from Figure 5 It can be seen that the vaginal mucosal epithelium of rabbits in the matrix control group was intact, with some epithelial cells slightly swollen, and occasional mild congestion and inflammatory cell infiltration of submucosal capillaries. The vaginal mucosal epithelium of rabbits in the commercially available N-9Gel group showed local necrosis and shedding, and the mucosal epithelial damage was more obvious, accompanied by moderate mucosal irritation. The congestion and inflammatory cell infiltration of submucosal capillaries in the anti-genital HPV infection gel group were mild, and no mucosal epithelial shedding or necrosis was observed.

[0091] The scores of vaginal mucosal irritation lesions were calculated, and the matrix control group was 1.33, the commercially available N-9Gel group was 7.93, and the anti-genital HPV infection gel group was 2.86. It is suggested that the irritation of the anti-genital HPV infection gel to the rabbit vaginal mucosa is slightly higher than that of the matrix control group, with no statistical difference (P>0.05); it is significantly lower than the positive control commercially available 4% N-9Gel, and the difference is statistically significant (P<0.05). The total scores of the lesions caused by the above-mentioned groups of gels to the rabbit vaginal mucosa are all <8 points, which are within the clinically acceptable range according to the rabbit vaginal mucosal irritation standards stipulated by the WHO. 3. Scheme comparison test:

[0092] Comparative Example 1: Effects of Anti-genital HPV Infection Gels with Different pH Values ​​on HPV Virus Activity

[0093] Experimental method: Other conditions remained unchanged, and three anti-genital HPV infection gels with different pH values ​​were prepared. The pH values ​​of the gel preparations were adjusted to 3.8, 4.5 and 6.0 with acetate buffer solution. The gel samples were gradient diluted, with 3 replicate wells for each concentration, mixed with equal volumes of HPV16 pseudovirus, and incubated at room temperature for 30 minutes; added to a 96-well culture plate pre-plated with Hela cells, and after 12 hours of infection, the cell supernatant was replaced with DMEM medium containing 2% FBS, and the expression of the luciferase reporter gene and enzyme activity in each well were detected after 48 hours of continued culture; the cell wells (not infected with virus) were blank controls, and the cell wells infected only with pseudovirus were positive controls. Calculate the pseudovirus inhibition rate of gel samples with different pH values. CalcuSyn software was used to calculate the IC values ​​of samples with different pH values ​​for HPV16 pseudovirus. 50 .

[0094] Experimental results: In Hela cells, the anti-genital HPV infection gel with a pH value of 3.8, 4.5 and 6.0 adjusted with acetate buffer solution inhibited the infection of HPV16 pseudovirus with IC 50 The values ​​were 7.56 mg / mL, 15.36 mg / mL and 58.62 mg / mL, respectively, indicating that under the experimental conditions, the anti-genital HPV infection gel inhibited the IC of HPV at a pH of about 3.8. 50 The lowest value means the best suppression effect.

[0095] In addition, when the pH value is too low, less than 3.6-3.8, it may affect the local microecological balance, cause irritation and inflammation of the reproductive tract mucosa, and does not meet the requirements of use.

[0096] Comparative Example 2: Rabbit vaginal mucosal irritation test of the anti-genital HPV infection gel of Example 2 containing different acetic acid concentrations

[0097] Experimental method: Other conditions remained unchanged, and 4 anti-genital HPV infection gels containing different acetic acid concentrations were prepared, with acetic acid concentrations of 0.5%, 1%, 2%, and 3%, respectively. The rabbit vaginal mucosal irritation test was carried out according to the Eckstein method approved by WHO. Specifically, 30 New Zealand rabbits were randomly divided into 5 groups, with 6 rabbits in each group. Different treatment groups were given 2.5 ml of gel once a day for 10 consecutive days. The animals were killed on the 11th day, and the vaginal tissue was separated for pathological histological examination. Evaluation criteria: According to the different degrees of mucosal edema, vascular congestion, leukocyte infiltration, and epithelial ulcers in the vaginal mucosal tissue, each item was recorded with 1 to 4 points, for a total of 16 points. The higher the total score of the pathological score, the stronger the irritation of the sample to the vaginal mucosa.

[0098] Experimental results: As shown in the table below, the total score of vaginal mucosal irritation lesions in the matrix control group was the lowest at 1.41, and the total pathological scores of the anti-HPV gel groups containing 0.5%, 1% and 2% acetic acid were 1.70, 2.53 and 3.42 respectively, while the pathological score of the gel group containing 3% acetic acid was higher at 6.31, which was significantly higher than that of the acetic acid gel groups with other doses. The experimental results suggest that under the experimental conditions, the gel containing 0.5-2% acetic acid concentration has low irritation to the vaginal mucosa, good safety and high acceptability.

[0099]

[0100] Comparative Example 3: Effects of different components of anti-genital HPV infection gel on HPV virus activity

[0101] Experimental method: Other conditions remained unchanged, and purslane, frankincense, purslane + frankincense, ethanol, and salicylic acid were used to prepare the gel instead of the acetic acid buffer solution. The gel samples were diluted in a gradient manner, with 3 replicate wells for each concentration, mixed with equal volumes of HPV16 pseudovirus, and incubated at room temperature for 30 minutes; added to a 96-well culture plate pre-plated with Hela cells, and after 12 hours of infection, the cell supernatant was replaced with DMEM medium containing 2% FBS, and the expression of the luciferase reporter gene and the enzyme activity of each well were detected after 48 hours of continued culture; the cell wells (not infected with virus) were blank controls, and the cell wells infected only with pseudovirus were positive controls. Calculate the pseudovirus inhibition rate of gel samples with different pH values. Use CalcuSyn software to calculate the IC of HPV16 pseudovirus respectively. 50 .

[0102] Experimental results: In Hela cells, the anti-genital HPV infection gel of purslane, frankincense, purslane + frankincense, ethanol, and salicylic acid inhibited the infection of HPV16 pseudovirus with IC 50 The values ​​were 15.22 mg / mL, 18.31 mg / mL, 16.99 mg / mL, 21.78 mg / mL, and 18.91 mg / mL respectively.

[0103] Comparative Example 4: Effects of different components of anti-genital HPV infection gel on HPV virus activity

[0104] Experimental method: Other conditions remain unchanged, and 0.2% purslane, 0.2% frankincense, and 0.1% purslane + 0.1% frankincense are added to the anti-genital HPV infection gel, and the pH value of the gel preparation is adjusted to 3.8 with acetic acid buffer solution. The gel sample is diluted in a gradient manner, and 3 replicate wells are set for each concentration. It is mixed with HPV16 pseudovirus in equal volumes and incubated at room temperature for 30 minutes; it is added to a 96-well culture plate pre-plated with Hela cells, and the cell supernatant is replaced with DMEM medium containing 2% FBS after 12 hours of infection. After continuing to culture for 48 hours, the expression of the luciferase reporter gene and the enzyme activity of each well are detected; the cell wells (not infected with viruses) are blank controls, and the cell wells infected only with pseudoviruses are positive controls. Calculate the pseudovirus inhibition rate of gel samples with different pH values. CalcuSyn software was used to calculate the IC values ​​of samples with different pH values ​​for HPV16 pseudovirus. 50 .

[0105] Experimental results: In Hela cells, the anti-genital HPV infection gel of purslane, frankincense, and purslane + frankincense inhibited the infection of HPV16 pseudovirus with IC 50 The values ​​were 10.96 mg / mL, 12.86 mg / mL and 12.69 mg / mL, respectively, indicating that under the conditions of this experiment, the anti-genital HPV infection gel did not achieve better effect after adding purslane and frankincense.

[0106] In summary, the above-mentioned gel for resisting genital HPV infection of the present invention has a relatively simple preparation process, stable and controllable quality, and is a temperature-sensitive water-soluble colorless clear gel with a pH value of 3.5-4.0. The gel is in liquid state at 20°C, and the viscosity is less than 1.0 Pa.s; it is in gel state at 29°C, and the viscosity is greater than 50.0 Pa.s. The results of a 3-month stability test showed that the various properties of the gel were stable under the conditions of 25±2°C and RH60±5%. Compared with the gel for resisting genital HPV infection of Example 1, the pH value range of the gel for resisting genital HPV infection of Example 2 is more suitable for maintaining a normal vaginal microenvironment. The research results show that the gel for resisting genital HPV infection of the present invention can effectively kill and inhibit genital HPV viruses, and the median lethal concentration (IC 50 ) is 7.56 mg / mL; and the gel has no obvious irritation to the reproductive tract mucosal epithelial cells, has good safety, and has the prospect of being developed as a drug or medical device product.

Claims

1. A gel for preventing genital tract HPV infection, characterized in that: It is prepared from the following components in percentage by weight: 15-32% gel matrix excipient; 0.01-0.25% thickener; 0.5-5% acid buffer; 2.5-12.5% ​​wetting agent; 0.01-0.2% preservative; The balance is pure water; Wherein, the pH of the gel is adjusted to 3.5-4.0 by using the acidic buffer.

2. The gel for preventing genital tract HPV infection according to claim 1, characterized in that: The dosage of the acidic buffer is 0.5-2.0%.

3. The gel for preventing genital tract HPV infection according to claim 1, characterized in that: The acidic buffer is selected from acetate buffer solution, phosphate buffer solution and carbonate buffer solution.

4. The gel for preventing genital tract HPV infection according to claim 1, characterized in that: The gel matrix excipient comprises poloxamer 407 and / or poloxamer 188; Wherein, the dosage of Poloxamer 407 is 15-18%.

5. The gel for preventing genital tract HPV infection according to claim 1, characterized in that: The thickener is selected from one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, sodium polyacrylate and xanthan gum.

6. The gel for preventing genital tract HPV infection according to claim 1, characterized in that: The wetting agent is selected from one or more of glycerol, polyethanol, propylene glycol, sorbitol, sodium lauryl sulfate, sodium dicaprylate sulfonate, sulfonated castor oil, triacetin, octyl palmitate coconut oil glyceride, and stearyl alcohol.

7. The gel for preventing genital tract HPV infection according to claim 1, characterized in that: The preservative is selected from one or more of propolis, benzoic acid, sodium benzoate, parabens, ethanol, quaternary ammonium salts, sorbic acid, 30% or more glycerol solution Germal IS45, and Glydant.

8. The method for preparing a gel for preventing genital tract HPV infection according to claim 1, characterized in that: The following steps are involved: S1. Soak the gel matrix excipient in pure water, let stand, stir, fully hydrate and disperse evenly to obtain a gel matrix excipient solution for later use; S2. Slowly add the thickener, wetting agent, acidic buffer and preservative into S1 in order, and finally add pure water, stir evenly and adjust the volume to obtain a gel for preventing genital HPV infection.

9. The gel for preventing genital tract HPV infection according to claim 1, characterized in that: The gel is a colorless clear liquid at a temperature below 20°C, with a viscosity of less than 1.0 Pa.s; and a colorless clear gel at a temperature above 29°C, with a viscosity of more than 50.0 Pa.s.

10. A gel for preventing genital tract HPV infection according to any one of claims 1 to 9, characterized in that: During use, apply to the female vagina and / or male genitalia.

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