CN114652741A

CN114652741A - Composition and preparation for inhibiting HPV and application thereof

Info

Publication number: CN114652741A
Application number: CN202210059538.4A
Authority: CN
Inventors: 张磊; 陆可望; 魏洁; 朱玲; 周磊
Original assignee: Kunming Yeshui Biotechnology Co ltd
Current assignee: Kunming Yeshui Biotechnology Co ltd
Priority date: 2022-01-19
Filing date: 2022-01-19
Publication date: 2022-06-24
Anticipated expiration: 2042-01-19
Also published as: CN114652741B

Abstract

The invention discloses a composition for inhibiting HPV, a preparation and application thereof. The composition can effectively inhibit the activity of HPV, can continuously act on a lesion part, almost has no stimulation and toxic or side effect on the lesion part, has high activity utilization rate and obvious treatment effect, has uncomplicated preparation process and low material cost, can be used by being matched with other conventional treatment strategies, can be used for treating HPV infection and preventing cervical or other tissue lesions caused by HPV infection, and provides a new way for clinically treating HPV infection and preventing HPV infection.

Description

Composition and preparation for inhibiting HPV and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a composition and a preparation for inhibiting HPV, and application thereof.

Background

Human Papillomaviruses (HPV) are a group of small DNA viruses without envelopes, belong to the family of papillomaviruses, can infect the skin and mucosal epithelial tissues of human beings, induce the formation of verrucous hyperplasia and even cause benign and malignant tumors. More than 100 HPV virus subtypes are separated and identified at present, and HPV can be divided into two main types, namely a skin-philic group and a mucosa-philic group according to different infection parts. The mucosal group is divided into firstly, high-risk HPV for inducing epithelial tissue malignant hyperplasia according to different benign and malignant diseases of the induced lesion after infection, and the high-risk HPV infection is closely related to the occurrence of cervical cancer and precancerous lesion; low risk type inducing benign hyperplasia of epithelial tissue, wherein the low risk type HPV infection is closely related to the occurrence of more than 90 percent of genital warts and cervical low-grade squamous hyperplasia lesions.

Cervical cancer is the second most common malignancy in women except breast cancer, is the first malignancy of genital tract, and accounts for the first malignancy in developing countries, and in recent years, with the popularization of publicity, more and more people begin to know that cervical cancer is related to a virus called HPV (human papilloma virus). Persistent infection with HPV means a higher risk of developing cervical cancer. In 1974, Harald Zur Hausen proposed a hypothesis that HPV is involved in the onset of cervical cancer; in 1977, Laverty observed the presence of HPV particles in cervical cancer biopsies in electron microscopy; keerti Shah in 1989 demonstrated that cervical cancer is directly associated with HPV infection; in 1995, the international association for cancer research considered persistent infection with high-risk HPV types to be the major cause of cervical cancer. Hausen shared a 2008 nobel physiological or medical prize for finding Human Papillomavirus (HPV) causing cervical cancer with two other scientists. More than 35 of the 100 HPV subtypes which are found at present can infect the reproductive organs of human, about 30 are related to tumors, wherein the low-risk type is not directly related to the occurrence of cervical cancer, and can cause verruca vulgaris, condyloma acuminatum, papilloma on mucosa and the like, such as type 6, type 11 and the like; the high-risk type is directly related to the occurrence of cervical cancer, such as 16 type, 18 type, 45 type and the like.

Today, treatment for HPV is mainly achieved by HPV therapeutic vaccines, which are mostly rationally designed against the antigens E6/E7, aiming at activating cell-mediated immune responses. The therapeutic HPV vaccine is used in treating or preventing disease aggravation by re-exciting specific T cell mediated immune reaction in body with infected HPV virus disease to form cancer transformation. However, due to the problems of effectiveness and safety, the therapeutic vaccine needs to be further researched and verified, and meanwhile, the preparation process of the therapeutic vaccine is complex and high in cost, so that the therapeutic vaccine is difficult to be widely applied to the population in developing countries.

Disclosure of Invention

The invention aims to: aiming at the existing problems, the composition and the preparation for inhibiting HPV and the application thereof are provided, are used for treating HPV infection and preventing cervical or other tissue lesions caused by HPV infection, and provide a new way for clinically treating HPV infection and preventing HPV infection.

The technical scheme adopted by the invention is as follows: a composition for inhibiting HPV, wherein the effective component in the composition is a cationic polymer, and the cationic polymer is a quaternary aminated polysaccharide or/and a quaternary aminated acrylamide copolymer.

In the invention, the inventor firstly finds that the cationic polymer has the effect of inhibiting HPV and can be used for treating or preventing HPV infection, and the cationic polymer can effectively inhibit the splitting function of the virus through the charge activity of the cationic polymer so as to make the virus lose the reproductive capacity. The inventor discovers that the cationic polymer has lower charge activity compared with a cationic bactericide used as a disinfectant, and can continuously act on a pathological change part because the cationic polymer has lower activity and is matched with the characteristic that a gel state with stronger adhesiveness can be formed after the cationic polymer absorbs water, the lower activity is beneficial to relieving the stimulation and other side effects on the pathological change part, the reduction of the utilization rate due to the action with other non-target substances (such as various bacteria) can be effectively avoided, and the aim of inhibiting HPV for a long time is fulfilled.

Compared with the strong cationic bactericide which inhibits the growth of viruses by the charge property, the cationic polymer of the invention has the advantages that the pathological change part is positioned in the female genital tract, the problem of application needs to be considered firstly, the strong cationic bactericide has no adhesiveness, and a long-acting action area is difficult to form at the pathological change part, if the application form is adjusted to be the gel state, a gel carrier needs to be matched, the adhesive property of the gel carrier, the influence on the activity of the strong cationic bactericide and the side effect on the female genital tract are also considered, and the cationic polymer of the invention can directly form gel after absorbing water, has strong adhesiveness, has no stimulation and toxic or side effect on the female genital tract, and shows obvious technical advantages in the aspect of application. Secondly, because the strong cationic bactericide has high activity, when the strong cationic bactericide acts on a lesion part, although the inhibition effect on HPV is better than that of the application, the strong cationic bactericide has high toxicity on normal cells; the cationic polymer has mild performance, can continuously act on a lesion part, has low toxicity to normal cells, and can achieve the aim of inhibiting HPV for a long time.

Further, the quaternary aminated polysaccharide is chlorinated-2-hydroxy-3- (trimethylamino) propyl polyethylene oxide cellulose ether or/and cationic guar gum. Further, the quaternized acrylamide copolymer is an N, N-trimethyl-2- (2-methyl-1-oxo-2-propenyloxy) ethylammonium chloride-acrylamide copolymer. These materials are conventionally used as thickeners with good thickening effect in liquids, and in the present invention, they are used for inhibiting HPV, thereby achieving the treatment and prevention of HPV infection.

Further, the mass ratio of the active ingredient in the composition is 0.01 to 100%, which is preferably 0.01 to 30%, more preferably 0.01 to 20%, and further preferably 0.1 to 2.0%.

Further, when the composition contains purified water, the mass ratio of the effective components in the composition is 0.01-30%.

The invention also comprises a preparation for inhibiting HPV, which comprises an effective component and medically acceptable auxiliary materials, wherein the effective component is the composition, and the medically acceptable auxiliary materials generally refer to excipients and additives used in medicine production or prescription preparation, can be selected in a matching way according to the auxiliary materials listed in Chinese pharmacopoeia, belong to the conventional technical means in the technical field, and are not repeated herein. The composition is mixed with auxiliary materials and then prepared into paste, gel, suppository, powder or tablets, preferably the gel, and can also be prepared into other preparations, for the powder and the tablets, the solid physical form of the composition is generally used, so that the storage and transportation process of the medicine is facilitated, when the composition is used, water can be added to prepare the paste or the gel, the powder can be directly sprayed on a wet pathological change part by using a spray pipe, and the powder composition absorbs water at the pathological change part to form paste or gel, so that the effect of inhibiting HPV can be achieved. Of course, the preparation of the invention can also be used together with other treatment factors, for example, the preparation of the invention can be added with proper amount of cationic bactericide such as PHMG, PHMB and the like to strengthen the curative effect of the initial administration and improve the effect of the initial treatment.

In the invention, the composition has better charge performance under acidic or weakly acidic conditions, if the pH value is higher, the activity performance is poorer, the effect of inhibiting HPV is difficult to achieve, and the pH value of the preparation is preferably not more than 6.5 as summarized by experiments.

Further, when the preparation contains purified water, the composition is mixed with purified water to prepare a paste, a gel or a suppository.

The preparation of the invention is mainly used in anti-HPV virus drugs, and the using method is as follows: directly smeared or coated or covered on the tissue lesion caused by HPV infection.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the inventor discovers for the first time that the cationic polymer has the effect of inhibiting HPV and can be used for treating or preventing HPV infection, and the cationic polymer can effectively inhibit the splitting function of the virus through the charge activity of the cationic polymer so as to make the virus lose the reproductive capacity;

2. the cationic polymer is prepared into a preparation, and the preparation can form a gel state with strong adhesion after water absorption, can continuously act on a pathological change part by combining drug properties with moderate activity, almost has no stimulation and toxic or side effect on the pathological change part, has high activity utilization rate, and can achieve the purpose of inhibiting HPV for a long time;

3. the preparation can be effectively adhered to a lesion part, has long action time and obvious treatment effect, has uncomplicated preparation process and low material cost, can be used in combination with other conventional treatment strategies, such as therapeutic vaccines, chemotherapy, radiation, biological agents and other immunotherapy methods, improves the treatment effect of HPV, and provides a new way for clinically treating HPV infection and preventing HPV infection.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Example 1

The liquid product is prepared from the following raw materials in percentage by weight: 0.5% of chlorinated-2-hydroxy-3- (trimethylamino) propylpolyethylene oxide cellulose ether, and the balance of purified water.

Preparation: firstly, taking the raw materials according to the mass ratio;

pre-dissolving chlorinated-2-hydroxy-3- (trimethylamino) propyl polyethylene oxide cellulose ether in purified water to prepare homogeneous solution;

and thirdly, subpackaging the mixture into proper containers by using a filling machine to obtain finished products.

Example 2

The following table 1 lists the raw materials of 8 formulas and the mass ratios thereof, and the raw materials are taken according to the mass ratio for each formula.

Table 1 raw materials of formulas 1 to 8 and mass percent (%) ratios

S1: chlorinated-2-hydroxy-3- (trimethylamino) propyl polyethylene oxide cellulose ether; s2: a cationic guar gum; s3: n, N, N-trimethyl-2- (2-methyl-1-oxo-2-propenyloxy) ethylammonium chloride-acrylamide copolymer.

HPV gene inhibition test

1. Test materials and instruments

1) Experimental group cells: SiHa (HPV16 positive cervical carcinoma cell)

Control cells: HaCaT (human immortalized epidermal cells) was used as a control to test the killing ability of the preparation against cells.

2) Test samples: formulation 1 to formulation 8

3) Cell culture solution: DMEM culture solution containing 10% fetal calf serum

4) PBS phosphate buffer, pancreatin

5) 12-hole cell culture plate

2. Laboratory apparatus

1) Cell culture box

2) qPCR instrument (using conventional real-time fluorescent quantitative PCR detection method (qRT-PCR) detection)

3) Phase contrast microscope

4) RNA extraction reagent

5) Reverse transcription kit

6) Primers and sequences:

E6(F:CAGCAATACAACAAACCG，R：GCAACAAGACATACATCG)

E7(F:CAGAGGAGGAGGATGAAATAG，R：AGGTCTTCCAAAGTACGAATG)，β-actin(F： CCATCGTCCACCGCAAAT,R：GCTGTCACCTTCACCGTTCC)

3. method of producing a composite material

1) Plating SiHa and HaCaT cells with good growth, wherein the final volume of each hole is 1 mL; each cell is provided with 2 groups (1 blank control group and 1 experimental group), each group is provided with 3 compound holes, and the drug adding treatment is carried out when the cell confluence reaches 80%. Respectively adding formula 1-formula 8 and 10uL into the cells of the experimental group, and culturing for 48 hours;

2) observing the growth conditions of SiHa and HaCaT cells after adding the formula 1-the formula 8 by a microscope; then SiHa cells are collected, total RNA is extracted, and reverse transcription is carried out to obtain cDNA;

3) qPCR detected changes in HPV 16E 6/E7 mRNA expression in SiHa cells under the action of formulations 1-8.

4. Results

1) The results of the effect of formula 1-formula 8 on cell growth when added to SiHa and HaCaT cells show that: the results show that after the formula 1-the formula 8 are added into the cells, the cells grow well, and no obvious difference exists between the blank control group and the test group.

2) The effect on the expression of HPV 16E 6/E7 mRNA in SiHa cell lines is shown in Table 1 (2)^–ΔΔCTValue):

TABLE 2 Gene expression profiles

5. Conclusion

The formula 1-formula 8 have down-regulation effect on mRNA expression of HPV 16E 6/E7 genes in SiHa cells, 3 kinds of cationic polymers with the concentration of 0.1% and 0.5% respectively have approximate inhibition effect on mRNA expression of E6/E7 genes, and the inhibition effect of the formula 6 and the formula 8 is relatively good.

As can be seen from Table 2, the inhibition effect of the 3 cationic polymers on the mRNA expression of the E6/E7 gene was higher with the concentration.

Second, gel formulation adhesion test

1. Test materials

1) Chlorinated-2-hydroxy-3- (trimethylamino) propyl polyethylene oxide cellulose ether; (S1)

2) A cationic guar gum; (S2)

3) N, N-trimethyl-2- (2-methyl-1-oxo-2-propenyloxy) ethylammonium chloride-acrylamide copolymer; (S3)

4) Hydroxypropyl methylcellulose;

5) a standard glass slide;

6) pig large intestine.

2. Method of producing a composite material

1) Preparing 3% hydrogel from the polymers of items 1-4 above;

2) stretching the inner membrane of the large intestine of the pig outwards on a glass slide to prepare 4 pieces;

3) respectively dripping 0.5 g of 4 different gels at the rightmost end of 4 glass slides stretched with the pig large intestine;

4) the slide was inclined at an angle of 45 ℃ and the results were observed after 6 hours.

3. Results

Hydroxypropyl methylcellulose gel has slipped to the bottom of the slide; the remaining S1, S2, S3 gels were also located approximately at a distance from the bottom 1/3 of the slide. The adhesion force of the gel of S1, S2 and S3 on the mucous membrane is better than that of hydroxypropyl methyl cellulose, and the adhesion effect is better.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A composition for inhibiting HPV, wherein the effective component in the composition is a cationic polymer, and the cationic polymer is a quaternary aminated polysaccharide or/and a quaternary aminated acrylamide copolymer.

2. The composition for inhibiting HPV according to claim 1, wherein said quaternary aminated polysaccharide is a chlorinated-2-hydroxy-3- (trimethylamino) propyl polyethylene oxide cellulose ether or/and a cationic guar gum.

3. The composition for inhibiting HPV according to claim 1, wherein the quaternary aminated acrylamide copolymer is N, N-trimethyl-2- (2-methyl-1-oxo-2-propenyloxy) ethylammonium chloride-acrylamide copolymer.

4. The composition for inhibiting HPV according to claim 1, wherein the effective ingredient in the composition is 0.01-100% by mass.

5. The composition for inhibiting HPV according to claim 4, wherein the composition comprises 0.01-30% by mass of the active ingredient when purified water is present in the composition.

6. A preparation for inhibiting HPV, which comprises an effective component and medically acceptable auxiliary materials, wherein the effective component is the composition as claimed in any one of the claims 1-5, and the composition is prepared into paste, gel, suppository, powder or tablet after being mixed with the auxiliary materials.

7. The formulation for inhibiting HPV according to claim 6, wherein the formulation has a pH of no more than 6.5.

8. The formulation for inhibiting HPV according to claim 6, wherein, when the formulation contains purified water, the composition is mixed with purified water to prepare a paste, gel or suppository.

9. The use of an agent for inhibiting HPV according to claim 6 in an anti-HPV medicament.

10. Use according to claim 9, wherein the formulation is for application or coating or covering of tissue lesions caused by HPV infection.