Antibacterial gynecological gel and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine technology. The present invention relates to the use of the polypeptide shown in SEQ ID NO:1 in the preparation of a drug for treating chronic cervicitis combined with HPV infection, and thus obtains an antibacterial gynecological gel. The present invention combines the polypeptide shown in SEQ ID NO:1 with rIFNα‑2b for chronic cervicitis combined with HPV infection, which can effectively improve clinical abnormal symptoms, increase the HPV negative conversion rate and shorten the negative conversion time, and reduce the level of inflammatory cytokines. The effect of using two of them is significantly better than that of using them alone; in addition, the gynecological gel provided by the present invention can improve the condition while promoting the recovery of the patient's vaginal microecological environment, improving the body's immunity and the self-cleaning ability of the vagina.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology. The present invention relates to the use of the polypeptide shown in SEQ ID NO: 1 in preparing a drug for treating chronic cervicitis combined with HPV infection, and thereby obtaining an antibacterial gynecological gel. Background Art

[0002] Chronic cervicitis is a common genital inflammatory disease in clinical practice, often co-occurring with human papillomavirus (HPV) infection. Human papillomavirus (HPV) belongs to the Polyomavirinae subfamily of the Papovaviridae family. It is a double-stranded circular DNA virus that can infect human skin and mucous membranes and cause related clinical symptoms. Currently, a large number of studies have found that persistent high-risk human papillomavirus infection is the direct cause of cervical cancer. Timely and effective precancerous intervention to remove and isolate HPV viruses can effectively reduce the risk of cervical cancer.

[0003] At present, recombinant interferon α2b (rIFNα-2b) is the first choice for the treatment of chronic cervicitis combined with HPV infection. rIFNα-2b can activate antiviral protein genes by binding to target cell receptors to accelerate protein formation and inhibit viral growth. However, this drug is vaginal interferon, which can easily interfere with vaginal microbial flora, resulting in reduced drug efficacy, HPV viral vector inhibition, low HPV negative conversion rate, and poor treatment effect. In addition, most chronic cervicitis combined with HPV infection have serious imbalance of vaginal microecology, and current anti-infective drugs cannot restore the balance of vaginal microecological environment while clearing the virus. Summary of the invention

[0004] We unexpectedly discovered that a peptide with the amino acid sequence of RDLYDDDDKDRMPYSTELIFYIEMDP (hereinafter referred to as R26 peptide) has anti-HPV virus effects, and found that the combination of this peptide and rIFNα-2b for the treatment of chronic cervicitis combined with HPV infection has significantly better efficacy than single use, and this drug can promote the restoration of the imbalanced vaginal microecological environment to normal.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] In one aspect, the present invention provides use of R26 peptide in combination with rIFNα-2b in the preparation of a drug for treating gynecological infections, wherein the amino acid sequence of the R26 peptide is shown in SEQ ID NO:1.

[0007] In a preferred embodiment of the present invention, in the drug, the weight of the polypeptide represented by SEQ ID NO: 1 is 0.1-1.5 wt%.

[0008] In a more preferred embodiment of the present invention, the concentration of the rIFNα-2b in the drug is 10,000 to 20,000 IU / g.

[0009] In a more preferred embodiment of the present invention, the gynecological infection is chronic cervicitis combined with HPV infection.

[0010] In another aspect, the present invention provides an antibacterial gynecological gel, the gel comprising the following components:

[0011] 0.1-1.5 wt% of the polypeptide shown in SEQ ID NO: 1;

[0012] rIFNα-2b 10,000 to 20,000 IU / g;

[0013] Thickener 1-4wt%;

[0014] Moisturizing agent 2-8wt%;

[0015] Water balance.

[0016] In a preferred embodiment of the present invention, the thickener is one or more of carbomer, poloxamer, chitosan, methyl cellulose, and sodium carboxymethyl cellulose.

[0017] In a more preferred embodiment of the present invention, the moisturizing agent is one or two of hyaluronic acid, trehalose, propylene glycol and glycerin.

[0018] In another aspect, the present invention provides an antibacterial gynecological gel, the gel comprising the following components:

[0019] 0.1-1.5 wt% of the polypeptide shown in SEQ ID NO: 1;

[0020] rIFNα-2b 10,000 to 20,000 IU / g;

[0021] Carbomer 1-4wt%;

[0022] Hyaluronic acid 2-8wt%;

[0023] Water balance.

[0024] In another aspect, the present invention provides a method for preparing the antibacterial gynecological gel, comprising the following steps:

[0025] Weigh hyaluronic acid according to the weight ratio, add water, and stir evenly; add carbomer, stir, and fully swell; then add the polypeptide shown in SEQ ID NO: 1 and rIFNα-2b, stir and dissolve completely, then discharge and fill to obtain the product.

[0026] The present invention has the following beneficial effects:

[0027] 1. The present invention uses R26 peptide and rIFNα-2b in combination for chronic cervicitis complicated with HPV infection, which can effectively improve clinical abnormal symptoms, increase the HPV negative conversion rate and shorten the negative conversion time, and reduce the level of inflammatory cytokines. The effect of using both is significantly better than that of using either alone.

[0028] 2. The gynecological gel provided by the present invention can improve the condition and promote the recovery of the patient's vaginal microecological environment, thereby improving the body's immunity and the self-cleaning ability of the vagina. DETAILED DESCRIPTION

[0029] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0030] Examples 1 to 4, antibacterial gynecological gel (unit: g, per 100 g of drug)

[0031] Components Embodiment 1 Embodiment 2 Embodiment 3 Embodiment 4 R26 peptide 1.0 0.1 0.5 1.5 rIFNα-2b 2 million IU 2 million IU 2 million IU 1 million IU Carbomer 3.0 3.0 3.0 3.0 Hyaluronic acid 5.0 2.0 5.0 8.0 water margin margin margin margin

[0032] Preparation process: weigh hyaluronic acid according to the weight ratio, add water, and stir evenly; add carbomer, stir, and fully swell; then add R26 peptide and rIFNα-2b, stir and dissolve completely, then discharge the material and fill it according to the specification of 5g / pill.

[0033] The difference between the comparative example group and the example one is that the components do not contain the R26 peptide, and the other parameters are the same as those of the example one.

[0034] The difference between the comparative example 2 and the example 1 is that the components do not contain rIFNα-2b, and the other parameters are the same as those of the example 1.

[0035] Test Example 1: Irritation test on vaginal mucosa

[0036] Fifteen healthy ICR mice were randomly divided into a blank control group and groups 1 to 4 of Examples, with 3 mice in each group. 1 g of the gel from groups 1 to 4 of Examples was taken respectively and delivered into the vagina of the mice using a vaginal syringe. The blank control group was treated with normal saline. The drug was allowed to act on the vaginal mucosa for 12 h, during which the diet, mental state and excretion of the mice in each group were recorded. The drugs were administered for 7 consecutive days, and the mice were killed on the 8th day. The swelling of the administration site was observed with the naked eye, and the vaginal tissue was sliced ​​and stained with HE. The erythema and edema were observed under an optical microscope.

[0037] Results: The diet, spirit and excretion of mice in each drug administration group were normal, and no abnormalities such as swelling were observed at the administration site. Under an optical microscope, it was observed that the vaginal tissue sections of mice in each group had no erythema and edema, and no inflammatory cell infiltration was observed, indicating that the drugs in each group of Examples 1 to 4 had less irritation to the vaginal mucosa.

[0038] Test Example 2: In vitro anti-HPV effect

[0039] Human cervical cancer Hela cells (purchased from Kunming Cell Bank, with a collection number of KCB 86019YJ, expressing HPV18E7) ​​and caski cells (expressing HPV16E7) were inoculated into 96-well plates, respectively. 1.0 g of the gel described in Example 1 (rIFNα-2b+R26 peptide group), Comparative Example 1 (rIFNα-2b group) and Comparative Example 2 (R26 peptide group) was diluted to 500 μg / ml with DMEM culture medium containing 10% FBS and added to a 96-well plate at 2 ml / well. Six replicate wells were set for each group. The blank control group was a matrix group without rIFNα-2b and R-26 polypeptides. Each group was cultured in an incubator at 37° C., 5% CO2 for 24 h. The cells in each group were collected and resuspended in PBS buffer. The total RNA of each group of cells was extracted according to the instructions of the Trizol kit for RT-PCR detection. The changes in the mRNA expression levels of HPV18E7 and HPV16E7 in each group of cells were compared to determine the inhibitory effect of each group of gel on HPV18 and HPV16 viruses. The primer sequences are shown in Table 1 below, and the results are shown in Table 2.

[0040] Table 1: Primer sequences

[0041]

[0042] PCR reaction conditions: 94°C, 5 min pre-denaturation, 94°C for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles, and 72°C extension for 10 min.

[0043] Table 2: Comparison of relative expression levels of target gene mRNA in each group ( n=6)

[0044]

[0045]

[0046] Note: Compared with the blank control group, ** P<0.01, *** P<0.001, compared with the embodiment group, ## P<0.01.

[0047] Compared with the blank control group, both Example 1 and Comparative Example 1 can significantly reduce the mRNA expression levels of HPV16 E7 and HPV18 E7, especially Example 1, the difference between which was also statistically significant (P < 0.01). There was no statistically significant difference in the mRNA expression levels of the two viruses in Comparative Example 2 compared with the blank control group. This shows that the effect of the combined use of rIFNα-2b and R-26 polypeptide is far greater than the effect of single use, and a synergistic effect is produced between the two. Test Example 3: Therapeutic effect on chronic cervicitis combined with HPV infection

[0048] 1 Materials and methods

[0049] 1.1 General Information

[0050] 90 patients with chronic cervicitis complicated with HPV infection were selected, and HPV typing was: 48 cases of HPV16 / 18, 22 cases of HPV52 / 56, 15 cases of 31 / 33, and 5 cases of HPV58. The patients were divided into Example 1, Comparative Example 1 and 2 according to the random number table method, with 30 cases in each group. Inclusion criteria for chronic cervicitis complicated with HPV infection: ① Chronic cervicitis complicated with HPV infection was confirmed by gynecological examination and pathological results; ② The patient gave informed consent to the study. Exclusion criteria: ① The patient had severe organ disease; ② Gynecological malignant tumors; ③ Allergic to the study drugs. The general data of the patients in each group were compared, and there was no statistically significant difference (P>0.05), which was comparable.

[0051] 1.2 Methods

[0052] Three days after the end of menstruation, the patients in each group cleaned the vulva and placed the drug in the posterior fornix of the vagina using a pusher. The drug was taken once every other day, 1 tablet (5 g / tablet) each time, 10 times as a course of treatment, and the total course of treatment lasted for 3 menstrual cycles.

[0053] 1.3 Observation indicators and evaluation criteria

[0054] (1) Effectiveness: Ineffectiveness is defined as no change or increase in the HPV-DNA viral load and no significant improvement in clinical symptoms. Effectiveness is defined as a decrease in the HPV-DNA viral load by at least one order of magnitude, or a change from high-risk to low-risk type, and alleviation of clinical symptoms. Cure is defined as disappearance of clinical symptoms and a negative HPV-DNA test result. Total effective rate = cure rate + effective rate.

[0055] (2) Negative conversion rate: At the end of the first and second menstrual cycles and after the end of treatment, epithelial cells were collected from the cervical transition zone using cervical swabs. The HPV-DNA viral load was detected using a LightCycler Nano 32-well fluorescence quantitative PCR instrument, and the negative conversion rate of each group was recorded.

[0056] (3) Analysis of vaginal microecological recovery: An appropriate amount of vaginal secretions were collected from patients before and after treatment, and their pH values ​​were tested. Subsequently, transmission electron microscopy was used to observe the vaginal microecological status. Vaginal microecological status evaluation criteria: ① Normal: pH of vaginal secretions ≤ 4.5, bacterial density and diversity of 10-999 bacteria / oil immersion mirror, catalase and sialidyl anhydrase were both negative, and Nugent score ≤ 3 points; ② Imbalance: Gram-negative short rods, cocci or miscellaneous bacteria were the dominant flora, bacterial density and diversity were 1-9 or ≥ 1000 bacteria / oil immersion mirror, vaginal secretion pH > 4.5, Nugent score > 3 points, sialidyl anhydrase and catalase were positive. Nugent score: 0-3 points are normal, 4-6 points indicate intermediate, and ≥ 7 points indicate BV.

[0057] (4) Observation of changes in inflammatory factors: Fasting venous blood was drawn from patients in the morning before and after treatment and centrifuged at 3600 r / min for 12 min. The supernatant was collected and the ELISA kit was used in strict accordance with the instructions to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and C-reactive protein (CRP) in the serum of patients in each group.

[0058] 1.4 Statistical methods

[0059] SPSS 26.20 statistical software was used to analyze the data and the data were expressed as mean ± standard error. Represents measurement data.

[0060] 2 Results

[0061] 2.1 Comparison of therapeutic effects: The results in Table 3 show that the total effective rate of the Example group is 96.66%, which is higher than the 76.66% of the recombinant human interferon α-2b group (Comparative Example group).

[0062] Table 3: Comparison of efficacy (cases / %)

[0063]

[0064] 2.2 Negative conversion rate: The results in Table 4 show that the negative conversion rate of HPV-DNA in Example 1 was the highest (83.33%), which was significantly different from that in the other groups. This indicates that the combined use of rIFNα-2b and R26 polypeptide can increase the negative conversion rate of HPV and shorten the negative conversion time, and the effect is far better than when rIFNα-2b or R26 polypeptide is used alone.

[0065] Table 4: Comparison of HPV negative conversion rate in each group (cases / %)

[0066]

[0067] 2.3 Analysis of vaginal microecological recovery: The results in Table 5 show that after treatment in the embodiment group, the number of patients with vaginal microecological imbalance decreased significantly, and the number of normal patients increased, while the control group had the opposite effect after treatment, with 2 more patients with imbalance. This shows that compared with the use of recombinant human interferon α-2b, the combined use of rIFNα-2b and R-26 polypeptide can adjust the vaginal microecological environment and promote the restoration of the imbalanced microecology to normal, and the above effects are not achieved when the two are used alone.

[0068] Table 5: Comparison of vaginal microecological recovery

[0069]

[0070] 2.4 Changes in inflammatory factors: The results in Table 6 show that after the treatment, the levels of TNF-α, IL-1β and CRP in the serum of the Example group and the Comparative Example group were significantly decreased compared with those before treatment, especially the Example group, which showed extremely significant differences compared with those before treatment (P < 0.01).

[0071] Table 6: Comparison of changes in serum inflammatory factors

[0072]

[0073]

[0074] Compared with before treatment, * P<0.05, ** P<0.01.

[0075] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a polypeptide combined with rIFNα-2b in the preparation of a drug for treating chronic cervicitis combined with HPV infection, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1. The drug is an antibacterial gynecological gel. 100 g of the gel contains the following components: 1.0 g of the polypeptide shown in SEQ ID NO: 1; rIFNα-2b 2 million IU; Carbomer 3.0g; Hyaluronic acid 5.0g; Water balance.

2. An antibacterial gynecological gel, characterized in that: The gel 100g contains the following components: 1.0 g of the polypeptide shown in SEQ ID NO: 1; rIFNα-2b 2 million IU; Carbomer 3.0g; Hyaluronic acid 5.0g; Water balance.

3. A method for preparing the antibacterial gynecological gel as claimed in claim 2, characterized in that: The following steps are involved: Weigh hyaluronic acid according to the weight ratio, add water, and stir evenly; add carbomer, stir, and fully swell; then add the polypeptide shown in SEQ ID NO: 1 and rIFNα-2b, stir and dissolve completely, then discharge and fill to obtain the product.

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