A gel preparation for gynecological conditioning and prevention of cervical HPV and its preparation process

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a gel preparation for gynecological conditioning and prevention of cervical HPV and its preparation process. The gel preparation of the present invention uses a fusion protein of human interferon α-2b and antimicrobial peptide as an active ingredient, and has a good effect in inhibiting the cell proliferation rate of Hela cells and reducing the HPV18 viral load in Hela cells. Clinical experimental data show that the gel preparation of the present invention for gynecological conditioning and prevention of cervical HPV has a high efficiency and has produced significant progress.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a gel preparation for gynecological conditioning and prevention of cervical HPV and a preparation process thereof. Background Art

[0002] Human papillomavirus (HPV) is a spherical DNA virus. Currently, more than 150 HPV subtypes have been isolated and identified. HPV can be divided into low-risk and high-risk types according to the degree of pathogenicity or carcinogenic risk. It can be divided into four types according to the different tissue sites invaded: low-risk skin type: related to common warts, flat warts, plantar warts, etc., including HPV 1, 2, 3, 4, 7, 10, 12, 15, etc. High-risk skin type: related to verrucous epidermodysplasia, and others may also include vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer, etc., including HPV 5, 8, 14, 17, 20, 36, 38. Low-risk mucosal type: mainly infects the genitals, anus, oropharynx, and esophageal mucosa, including HPV 6, 11, 13, 32, 34, 40, 42, 43, 44, 53, 54, etc. High-risk mucosal types: mainly cause cervical cancer, rectal cancer, oral cancer, tonsil cancer, etc., including human papillomavirus types 16, 18, 30, 31, 33, 35, and 39. In addition, high-risk HPV can cause a variety of tumors, such as cervical cancer, anal cancer, and penile cancer. The main transmission routes include contact transmission, sexual transmission, and mother-to-child transmission, among which sexual contact is the most common route of infection. People with factors such as premature sexual intercourse, disordered sex hormone levels, low immunity, multiple sexual partners, and smoking are also more likely to be infected with HPV.

[0003] Cervical cancer is one of the most common malignant tumors in gynecology. In recent years, the age of onset has tended to be younger, and the incidence rate is second only to breast cancer, which has attracted widespread attention from all over the world. Studies have found that persistent high-risk HPV infection is a necessary condition for the occurrence of cervical precancerous lesions and cervical cancer. Therefore, cervical cancer has become the only malignant tumor that can be detected, prevented and treated early.

[0004] At present, there are many ways to prevent and treat HPV infection in clinical practice, including physical therapy, surgical treatment, drug therapy, vaccine treatment and traditional Chinese medicine treatment. Physical therapy includes laser, cryotherapy, focused ultrasound, photodynamic therapy, etc., which are more effective for the early stage of infection. Surgical treatment is generally for the treatment of cervical intraepithelial neoplasia. Although vaccine treatment has achieved good results in clinical practice, the high price of vaccines makes it difficult to be widely used. Traditional Chinese medicine treatment mostly works by reducing the viral load, changing the local immune microenvironment of the cervix or the systemic immune function, but due to the large individual differences in immune function, there are still doubts whether the treatment can be applied to all infected people and achieve the expected effect. Therefore, the most commonly used HPV prevention and treatment method in clinical practice is drug therapy, and the most commonly used drug is human recombinant interferon. Commonly used drug forms include effervescent and suppositories. Human recombinant interferon is a broad-spectrum antiviral drug that can inhibit the proliferation of abnormal cells on the one hand and improve the body's immunity on the other.

[0005] Gel preparations refer to thick liquid or semi-solid preparations in the form of solutions, suspensions or emulsions made of drugs and excipients that can form gels. Usually, gels are limited to local use on the skin and body cavities (such as the nasal cavity, vagina and rectum). Common gynecological gel preparations include aqueous gels and oily gels. The common effects of gynecological gel preparations include antibacterial and anti-inflammatory, cleaning and care, lubrication and moisturizing, symptom relief, and restoration of vaginal pH balance. Gel preparations are often used in the prevention or treatment of gynecological diseases for the following reasons: Gel preparations can directly act on gynecological parts such as the vagina or cervix, allowing the drug to directly contact the affected area and improve the therapeutic effect. Gel preparations usually have good biocompatibility, are not greasy when used, are easy to spread and wash off, can absorb tissue exudate, do not interfere with the normal function of the skin, and have a certain protective effect on the vaginal mucosa, which can reduce irritation and discomfort. Gel preparations can provide a certain period of drug release, reduce the number of dosing times, and improve patient compliance. Summary of the invention

[0006] Based on the prior art, the present invention aims to provide a gel preparation for gynecological conditioning and prevention of cervical HPV and its preparation process, wherein the fusion protein of human interferon α-2b and antimicrobial peptide is used as an active ingredient and is configured as a gel preparation to improve medication compliance. The gel preparation of the present invention aims to improve the effect of preventing and treating HPV infection, reduce the economic burden of consumers, facilitate clinical promotion and application, and provide a new method for gynecological conditioning and prevention of cervical HPV.

[0007] Specifically, the technical solution of the present invention is achieved as follows:

[0008] In a first aspect, the present invention provides a fusion protein of human interferon α-2b and antimicrobial peptide.

[0009] The fusion protein of human interferon alpha-2b and antimicrobial peptide is formed by fusing human interferon alpha-2b and antimicrobial peptide via a linker.

[0010] The nucleotide sequence encoding the human interferon α-2b is shown in SEQ ID NO.1.

[0011] The amino acid sequence of human interferon α-2b is shown in SEQ ID NO.2.

[0012] The nucleotide sequence encoding the antimicrobial peptide is shown in SEQ ID NO.3.

[0013] The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.4.

[0014] The linker is GGGGSGGGGS, and the sequence is shown in SEQ ID NO.5.

[0015] The amino acid sequence of the fusion protein of human interferon α-2b and antimicrobial peptide is shown in SEQ ID NO.6.

[0016] In a second aspect, the present invention provides a gel preparation for gynecological conditioning and prevention of cervical HPV, comprising the following ingredients: a fusion protein of human interferon α-2b and antimicrobial peptide, and an aqueous gel matrix.

[0017] The fusion protein of human interferon alpha-2b and antimicrobial peptide is formed by fusing human interferon alpha-2b and antimicrobial peptide via a linker.

[0018] The nucleotide sequence encoding the human interferon α-2b is shown in SEQ ID NO.1.

[0019] The amino acid sequence of human interferon α-2b is shown in SEQ ID NO.2.

[0020] The nucleotide sequence encoding the antimicrobial peptide is shown in SEQ ID NO.3.

[0021] The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.4.

[0022] The linker is GGGGSGGGGS, and the sequence is shown in SEQ ID NO.5.

[0023] The fusion protein of human interferon α-2b and antimicrobial peptide is formed by fusion of human interferon α-2b and antimicrobial peptide via a linker, and the amino acid sequence is shown in SEQ ID NO.6.

[0024] The aqueous gel matrix comprises glycerol, carbomer, sodium alginate, PEG400, Tween 80, a preservative, a pH regulator and sterile water.

[0025] Preferably, the present invention provides a gel preparation for gynecological conditioning and prevention of cervical HPV, which is composed of the following components by mass percentage: 0.01% to 0.15% of a fusion protein of human interferon α-2b and an antimicrobial peptide, and the remainder of an aqueous gel matrix;

[0026] Each kilogram of the aqueous gel matrix includes the following components: 30-100 g of glycerol, 10-35 g of carbomer, 25-50 g of sodium alginate, 10-25 g of PEG400, 1-5 g of Tween 80, 0.1-1 g of a preservative, 0.1-1 g of a pH regulator, and the balance of sterile water.

[0027] The preservative is selected from any one of chlorobutanol, benzyl alcohol and p-hydroxybenzoate.

[0028] In a third aspect, the present invention provides a method for preparing a gel preparation for gynecological conditioning and prevention of cervical HPV, the steps comprising:

[0029] Weigh the fusion protein of human interferon α-2b and antimicrobial peptide, glycerol, carbomer, sodium alginate, PEG400, Tween 80, preservative and sterile water, and mix them evenly at 30-45°C;

[0030] Slowly add the pH adjuster and continue mixing at 30-45°C;

[0031] The uniformly mixed materials are mechanically stirred and emulsified to obtain the product.

[0032] The preservative is selected from any one of chlorobutanol, benzyl alcohol and p-hydroxybenzoate.

[0033] The pH adjuster is selected from any one of lactic acid, tartaric acid and citric acid.

[0034] Preferably, the gel preparation for gynecological conditioning and prevention of cervical HPV is composed of the following components by mass percentage: 0.01% to 0.15% of a fusion protein of human interferon α-2b and an antimicrobial peptide, and the remainder of an aqueous gel matrix;

[0035] Each kilogram of the aqueous gel matrix includes the following components: 30-100 g of glycerol, 10-35 g of carbomer, 25-50 g of sodium alginate, 10-25 g of PEG400, 1-5 g of Tween 80, 0.1-1 g of a preservative, 0.1-1 g of a pH regulator, and the balance of sterile water.

[0036] The preservative is selected from any one of chlorobutanol, benzyl alcohol and p-hydroxybenzoate.

[0037] The pH adjuster is selected from any one of lactic acid, tartaric acid and citric acid.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention uses a fusion protein of human interferon α-2b and an antimicrobial peptide as an active ingredient. In a cell model, the fusion protein of the present invention is significantly superior to the combined administration of human interferon α-2b and an antimicrobial peptide or the single administration of human interferon α-2b in inhibiting the cell proliferation rate of Hela cells and reducing the HPV18 viral load in Hela cells. In terms of clinical treatment, the gel preparation of the present invention for gynecological conditioning and prevention of cervical HPV has also been shown to have a high efficacy, which is significantly superior to the existing human interferon α-2b gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Statistical graph of cell proliferation rate of Hela cells;

[0041] Figure 2 Statistical graph of HPV18 viral load in Hela cells; DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0043] The antimicrobial peptide of the present invention is independently developed, and the proteins or polypeptides involved in the technical solution are commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis.

[0044] The aqueous gel matrix raw materials in the gel preparation for gynecological conditioning and prevention of cervical HPV of the present invention are all commercial reagents.

[0045] Preparation Example

[0046] A gel preparation for gynecological conditioning and prevention of cervical HPV, comprising the following components by mass percentage: 0.10% of a fusion protein of human interferon α-2b and an antimicrobial peptide, and the remainder of an aqueous gel matrix;

[0047] Each kilogram of the aqueous gel matrix includes the following components: 80 g glycerol, 25 g carbomer, 45 g sodium alginate, 15 g PEG400, 3 g Tween 80, 0.3 g benzyl alcohol, 0.2 g citric acid and the balance sterile water;

[0048] The preparation method of the gel preparation for gynecological conditioning and prevention of cervical HPV comprises:

[0049] Weigh the raw materials according to the amount;

[0050] The fusion protein of human interferon α-2b and antimicrobial peptide, glycerol, carbomer, sodium alginate, PEG400, Tween 80, benzyl alcohol and sterile water were mixed uniformly at 35°C;

[0051] Slowly add citric acid and continue mixing at 35°C;

[0052] The mixed materials are mechanically stirred and emulsified to obtain the product.

[0053] Experimental Example 1

[0054] Stably growing Hela cells were taken and cultured at 5×10 4 The cells were inoculated in a 96-well plate at a density of 10 cells / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. Then the old culture medium was removed and the culture was continued for 48 hours after treatment according to the following dosing regimen. The old culture medium was removed and replaced with an equal amount of fresh culture medium containing 10% CCK8 reagent and continued to incubate in the incubator for 120 minutes. The culture medium was RPMI-1640, containing 10% FBS and 1% penicillin-streptomycin. The OD value at 450nm was measured with an enzyme-labeled instrument. Six replicate wells were set for each group, and the cell proliferation rate (%) was calculated based on the average value.

[0055] The dosing regimen is as follows:

[0056] Blank group: fresh culture medium only;

[0057] Fusion group: fresh culture medium containing the fusion protein of human interferon α-2b and antimicrobial peptide at a final concentration of 0.10%;

[0058] Combined group: fresh culture medium containing final concentration of 0.10% human interferon α-2b and 0.10% antimicrobial peptide;

[0059] Human interferon α-2b group: fresh culture medium containing human interferon α-2b at a final concentration of 0.10%.

[0060] Table 1 Cell proliferation rate of Hela cells (%)

[0061]

[0062] *: compared with the blank group, p<0.001; #: compared with the fusion group, p<0.05.

[0063] The OD value of the 48h drug administration treatment was measured. The cell proliferation rate (%) of the fusion group was 51.65±4.11, the cell proliferation rate (%) of the combination group was 60.93±2.24, and the cell proliferation rate (%) of the human interferon α-2b group was 63.47±3.06. The results of the fusion group were statistically significantly different from those of the combination group or the human interferon α-2b group. The above experimental results show that the fusion protein of human interferon α-2b and antimicrobial peptide has a better inhibitory effect on the proliferation of Hela cells than the combined administration of human interferon α-2b and antimicrobial peptide, and also produces a more significant inhibitory effect than the administration of human interferon α-2b alone.

[0064] Experimental Example 2

[0065] HPV18 was detected in Hela cells by PCR amplification. Stably growing Hela cells were taken and 1×10 5 The cells were inoculated at a density of 10 cells / mL in a 24-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. The old culture medium was then removed and the cells were cultured for 48 hours after treatment according to the following dosing regimen. After the culture medium was removed, the cells were collected and the HPV18 viral load of Hela cells was measured using a human papillomavirus (HPV) nucleic acid detection kit (hybridization capture-chemiluminescence method) (purchased from Hangzhou Detong Biotechnology Co., Ltd.).

[0066] The dosing schedule is as follows (three replicate wells are set for each group):

[0067] Blank group: fresh culture medium only;

[0068] Fusion group: fresh culture medium containing the fusion protein of human interferon α-2b and antimicrobial peptide at a final concentration of 0.10%;

[0069] Combined group: fresh culture medium containing final concentration of 0.10% human interferon α-2b and 0.10% antimicrobial peptide;

[0070] Human interferon α-2b group: fresh culture medium containing human interferon α-2b at a final concentration of 0.10%.

[0071] Table 2 HPV18 viral load in Hela cells (pg / mL)

[0072]

[0073] *: compared with the blank group, p<0.01; #: compared with the fusion group, p<0.05.

[0074] After testing, the HPV18 viral load of the blank group was 21.63±0.77pg / mL, the HPV18 viral load of the fusion group was 10.24±1.02pg / mL, the HPV18 viral load of the combination group was 14.37±0.86pg / mL, and the HPV18 viral load of the human interferon α-2b group was 15.28±0.92pg / mL. Compared with the blank group, the fusion group, the combination group and the human interferon α-2b group all had statistically significant differences (p<0.01); compared with the fusion group, the combination group and the human interferon α-2b group all had statistically significant differences (p<0.05). The experimental results are as follows Figure 2 shown.

[0075] The above experimental results show that the fusion protein of human interferon α-2b and antimicrobial peptide is more effective in reducing the HPV18 viral load in Hela cells, which is significantly better than the combined administration of human interferon α-2b and antimicrobial peptide or the administration of human interferon α-2b alone.

[0076] Experimental Example 3

[0077] In order to evaluate the efficacy of the gel preparation for gynecological conditioning and prevention of cervical HPV of the present invention, HPV-infected patients were jointly recruited as test volunteers with local tertiary hospitals. 40 HPV-infected patients aged 25-50 years were selected from the hospital outpatient clinic. Exclusion criteria: patients with transient infection, persistent or repeated infection, including some patients with mild cytological changes (below CIN grade Ⅰ), were selected, and patients with CIN Ⅱ and above, cervical cancer and cervical cancer surgery were excluded. The patients were randomly divided into 3 groups, namely, fusion protein group, combined administration group, and human interferon α-2b group. The fusion protein group used the gel preparation for gynecological conditioning and prevention of cervical HPV of the present invention. The difference between the gel preparation used in the combined administration group and the fusion protein group was only that the fusion protein of human interferon α-2b and antimicrobial peptide was replaced with an equal amount of human interferon α-2b and antimicrobial peptide. The difference between the gel preparation used in the human interferon α-2b group and the fusion protein group was only that the fusion protein of human interferon α-2b and antimicrobial peptide was replaced with an equal amount of human interferon α-2b.

[0078] Usage: Clean the outside of the vagina and insert about 3g of gel deep into the vagina. Use once a day for 3 months. Stop using during menstruation. Check again after 4 weeks of discontinuation.

[0079] Table 3 Treatment effect

[0080]

[0081] *: compared with the fusion protein group, p < 0.05.

[0082] Clinical treatment results show that the gel preparation for gynecological conditioning and prevention of cervical HPV of the present invention is significantly more effective in HPV-infected patients than gel preparations with human interferon α-2b and antimicrobial peptide as active ingredients or with human interferon α-2b as active ingredient, and has more significant therapeutic effect.

[0083] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the description only describe the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A fusion protein of human interferon α-2b and antimicrobial peptide, characterized in that: The fusion protein of human interferon α-2b and antimicrobial peptide is formed by fusing human interferon α-2b and antimicrobial peptide via a linker; the amino acid sequence of the fusion protein of human interferon α-2b and antimicrobial peptide is shown in SEQ ID NO.

6.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

4.

3. The fusion protein according to claim 1, characterized in that The amino acid sequence of human interferon α-2b is shown in SEQ ID NO.

2.

4. A gel preparation for gynecological conditioning and prevention of cervical HPV, comprising the following ingredients: a fusion protein of human interferon α-2b and antimicrobial peptide, and an aqueous gel matrix; The amino acid sequence of the fusion protein of human interferon α-2b and antimicrobial peptide is shown in SEQ ID NO.

6.

5. The gel preparation according to claim 4, characterized in that The amino acid sequence of human interferon α-2b is shown in SEQ ID NO.

2.

6. The gel preparation according to claim 4, characterized in that The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

4.

7. The gel preparation according to claim 4, characterized in that The aqueous gel matrix comprises glycerol, carbomer, sodium alginate, PEG400, Tween 80, a preservative, a pH regulator and sterile water.

8. The gel preparation according to claim 7, characterized in that Each kilogram of the aqueous gel matrix includes the following components: 30-100 g of glycerol, 10-35 g of carbomer, 25-50 g of sodium alginate, 10-25 g of PEG400, 1-5 g of Tween 80, 0.1-1 g of a preservative, 0.1-1 g of a pH adjuster and the balance of sterile water.

9. The gel preparation according to claim 8, characterized in that The preservative is selected from any one of chlorobutanol, benzyl alcohol and p-hydroxybenzoate.

10. The method for preparing the gel preparation for gynecological conditioning and prevention of cervical HPV according to any one of claims 4 to 9, comprising the steps of: Mix human interferon α-2b and antimicrobial peptide fusion protein, glycerol, carbomer, sodium alginate, PEG400, Tween 80, preservatives and sterile water at 30-45°C. Slowly add the pH adjuster and continue mixing at 30-45°C; The uniformly mixed materials are mechanically stirred and emulsified to obtain the product.

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