KR101546802B1 - Antiviral Composition comprising Citric acid, Zinc Boric acid, Albumin and Vitamin C against Papilloma virus - Google Patents

Abstract

The present invention relates to an antiviral composition comprising boric acid, citric acid, zinc, albumin, and vitamin C as active ingredients. In particular, it has an activity of suppressing protein expression of papilloma virus.

Description

An antiviral composition for external use for papilloma virus, which comprises boric acid, citric acid, zinc, albumin and vitamin C,

The present invention relates to an antiviral dermatological composition for papilloma virus comprising boric acid, citric acid, zinc, albumin and vitamin C as active ingredients.

Papilloma viruses are known to infect epithelial cells of various tissues of animals and cause benign tumors, often called warts, on the hands, feet, skin, and larynx. These papilloma viruses have been identified in about 100 genotypes in humans, and several genotypes have been reported to be specific for the infected tissues and cause various diseases (Broker et al., Cold Spraing Harbor Laboratory , 1989, p17.) Most of these genotypes of papillomaviruses were not treated because they were not treated well and caused severe pain in infected patients but did not cause fatal diseases. However, in recent years, certain types of the virus, especially human papilloma viruses 16 and 18, have been associated with malignant tumors in the genitalia, oral cavity, and skin, and are a major factor in causing cervical cancer, accounting for more than 90% In addition, 6b and 11b have been found to induce benign tumors of the male and female genitalia, named as congeria (or genital warts, condyloma acuminata). In addition, epidemiologic studies have suggested that uterine cancer is caused mainly by factors that are transmitted by sexual contact (Durst et al., 1983, Vol 80, p3812), cervical There is much evidence that uterine cancer is caused by infection with papilloma virus, such as 85-100% of all cancerous lesions named intraepithelial neoplasm (CIN) caused by papilloma virus infection (Hansen, H ., Science, 1991, Vol. 254, p. 173).

In addition, the infection rate of human papillomavirus type 16 and 18 in cervical cancer patients is generally reported to be about 50 to 70% in 16 type and 15 to 25% in 18 type. However, in the case of metastatic cancer, 25% of patients with type 16 and 50% of patients with type 18 infection were found to be metastatic (Lorincz et al., Obstetrics and Gynecology, 1992, Vol 79, p328.).

Therefore, it is necessary to develop a preventive and therapeutic agent for various diseases caused by papilloma virus.

As a result of investigating related prior art patents, the prior patent related to papillomavirus treatment is "a composition for prevention or treatment of papilloma virus-inducing diseases including E7 gene having an optimized gene codon and lysosomal targeting signal sequence" Korean Patent Laid-Open Publication No. 2008-0040412), "papillomavirus vaccine" (Korean Patent Laid-Open Publication No. 1997-7003417 ), "Tetrahydrocarbazole derivatives and their pharmaceutical uses" (Korean Patent Laid-Open Publication No. 2006-0017545). However, the antiviral agent described in the aforementioned publications can not be said to provide a complete antiviral agent.

Accordingly, a problem to be solved by the present invention is to provide a new anti-viral external preparation for skin which is more active than the antiviral agent described in the open literature.

In order to attain the above object, the present invention provides a pharmaceutical composition comprising 0.001 to 10% by weight of boric acid, 0.001 to 10% by weight of citric acid, 0.001 to 10% by weight of zinc, 0.01 to 10% by weight of albumin and 0.01 to 10% An antiviral dermatologic composition for papilloma virus is provided.

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In the antiviral composition according to the present invention, the disease caused by papilloma virus may be a wart, a gonad or a uterine cancer. In particular, the uterine cancer may be a cervical cancer, an endometrial cancer or an uterine cancer.

The antiviral composition comprising boric acid, citric acid, zinc, albumin and vitamin C as an active ingredient according to the present invention exhibits an activity of suppressing protein expression of papilloma virus. Accordingly, the antiviral agent composition of the present invention is effective for treating diseases caused by papilloma virus.

FIG. 1 shows that the expression of human papilloma virus type 16 or 18 E6 protein was reduced by treating the cervical cancer cell lines Ca Ski, HeLa and C-33 A with the antiviral composition of the present invention.
FIGS. 2A to 2C show the effect of the antiviral composition of the present invention on the cervical cancer cell lines Ca Ski (), HeLa () and C-33 A () in terms of cell viability through MTT assay.
FIG. 3 shows the antiviral composition of the present invention applied to the vulva of female patients suffering from papules to observe the treatment of the papules.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing an antiviral composition comprising as active ingredients boric acid, citric acid and zinc in a therapeutically effective amount together with a pharmaceutically acceptable carrier.

The antiviral composition comprises 0.001 to 10% by weight, preferably 2 to 6% by weight, of boric acid per total weight of the composition and 0.001 to 10% by weight, preferably 0.01 to 5% by weight, By weight of the composition, zinc is contained in an amount of 0.001 to 10% by weight, preferably 0.01 to 5% by weight, based on the total weight of the composition, and albumin is added as an additional active ingredient in an amount of 0.01 to 10% by weight, preferably 3 to 6% %, And vitamin C in an amount of 0.01 to 10% by weight, preferably 3 to 6% by weight.

The composition also includes a pharmaceutically acceptable carrier. Carriers include carriers, adjuvants or vehicles commonly used in the pharmaceutical field.

The citric acid used in the present invention can be extracted by extracting citric acid present in a free state in seeds or juices of various plants. In addition, citric acid is also produced by a surface fermentation process or a submerged fermentation process using Aspergillus niger, which is a fungus, which can also be used in the composition of the present invention. In the production process of citric acid, molasses is used as a source of sugar-containing raw materials, and various centrifuges such as a nozzle type centrifuge, an automatic discharge type centrifuge, and a decanter are utilized.

The zinc used in the present invention can be extracted and used in an animal food such as oysters, crustaceans, fish, red meat, and a variety of plant foods such as grain, legumes, nuts, and seeds. The zinc forms that can be contained in the compositions of the present invention are very diverse, and most clinical studies have used zinc sulfate, but the body absorbs and exploits other forms as well. Examples include picolinate and acetate, citrate, glycerate, or a combination of monomethionine and zinc. The albumin used in the present invention may be serum albumin, and the vitamin C may be L-ascorbic acid.

The antiviral composition can prevent or treat various diseases caused by the infection with viruses, and can preferably prevent or treat warts, pruritus, or uterine cancer caused by infection with papilloma virus.

The activity of the antiviral composition of the present invention can be demonstrated by analyzing the protein of papilloma virus expressed in a cervical cancer cell line. That is, after the composition of the present invention is treated in a cervical cancer cell line, the protein expressed in the cancer cells can be extracted and analyzed by a molecular biological method commonly known in the art. The papillomavirus protein to be analyzed in the present invention is known as an antigen protein that induces uterine cancer as human papillomavirus type 16 or 18 type E6. Thus, the present inventors have proved that the mechanism by which the protein is reduced in the uterine cancer cell line is due to the treatment of the antiviral composition according to the present invention (see FIG. 1).

In addition, the activity of the antiviral composition of the present invention can be proved by treating the composition of the present invention in a female patient who is infected with papilloma virus and is caused by a ponytail around the penis to treat the papilloma (see FIG. 3).

Accordingly, diseases that can be treated or prevented by the composition of the present invention include, but are not limited to, the following: locally infected epidermis caused by papillomavirus, which is a hyperkeratotic tumor; Congestion caused by penis or viruses infected by papilloma virus; And uterine cervical cancer, endometrial cancer or uterine cancer, which are induced in uterine epithelial cells infected by papilloma virus.

The antiviral composition of the present invention may be formulated with a therapeutically effective amount of the active ingredient together with a pharmaceutically acceptable carrier.

Carriers contained in the present invention include carriers, adjuvants and vehicles commonly used in the pharmaceutical field and are collectively referred to as "pharmaceutically acceptable carriers ". Pharmaceutically acceptable carriers that may be used in the antiviral compositions of the present invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins, buffer substances (e.g., various phosphates, glycine, sorbic acid , Potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, poly Polyvinyl pyrrolidone, cellulose-based substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-terminated polymers, polyethylene glycols and wool.

The antiviral composition of the present invention can be administered via any conventional route so long as it can reach the desired tissue. Accordingly, the antiviral agent composition of the present invention can be administered locally, orally, parenterally, transdermally, rectally or intestinally. The term "parenteral" as used herein includes subcutaneous, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intrasynovial, intrasternal, intracardiac, intrathecal, intralesional and intracranial injection or infusion techniques .

Meanwhile, the antiviral agent composition of the present invention can be formulated as a solution, a suspension, a tablet, a granule, a powder, a capsule, an ointment or a cream.

In one embodiment, when formulating the antiviral composition of the present invention with an oral solid, the active ingredient may be formulated with a diluent (for example, lactose, dextrose, sucrose, cellulose, corn starch or potato starch) Such as, for example, silica, talc, stearic acid, magnesium or calcium stearate and / or polyethylene glycol, binders such as starch, arabic gum, gelatin methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone, (For example, starch, alginic acid, alginate or sodium starch glycolate), formamide mixtures, dyes, sweeteners, humectants (for example lecithin, polysorbate, lauryl sulfate) Inactive materials. These medicaments can be prepared by known methods, for example by means of mixing, granulating, tabletting, sugar or film-coating processes.

In another embodiment, the antiviral composition of the present invention comprises a commonly used inert diluent (e.g., purified water, ethanol) when formulated into liquid compositions for oral administration such as solutions, emulsions, suspensions, syrups and elixirs . If desired, the liquid composition may further comprise adjuvants such as wetting and suspending agents, sweetening agents, perfume fragrances and preservatives.

In another embodiment, the antiviral composition of the present invention may be prepared as an aqueous solution for parenteral administration. Preferably, a suitable buffer solution such as Hank's solution, Ringer's solution or physically buffered saline can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, suspensions of the active ingredient are suitable lipophilic solvents or carriers suitable for oily injection suspensions, including fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension may employ a suitable stabilizing agent or agent to increase the solubility of the compound and to produce a high concentration of solution.

In another embodiment, the antiviral composition of the present invention may be in the form of a sterile injectable preparation as a sterile injectable aqueous or oily suspension. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e. G., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. For this purpose, any non-volatile oil with low irritation, including synthetic mono- or diglycerides, may be used. Fatty acids, such as oleic acid and glyceride derivatives thereof, are useful in the form of pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially as injectables, as are their polyoxyethylated versions.

The previously prepared liquid composition is usually sterilized by filtration through a bacterial capture filter or the like, incorporating a sterilizing agent or radiation. The sterilized composition can be solidified, for example, by lyophilization to obtain a solid composition, which is dissolved in sterile water or sterile diluent at the time of use.

In another aspect, the antiviral composition of the present invention may be formulated for transdermal administration together with a pharmaceutically acceptable carrier in a therapeutically effective amount of the active ingredient. The term "transdermal administration" means that a pharmaceutical composition is topically administered to the skin such that a therapeutically effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

The transdermal preparation according to the present invention includes a cream preparation, an ointment preparation, a lotion preparation, a gel preparation, a solution for external use, a pasta preparation, a liniment preparation, an external preparation, an air- These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania, 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

Typically, the cream preparations of the present invention are applied as an o / w-based (o / w) system containing a cold cream, an emollient cream, a shaving cream, a vanishing cream and a hand cream and generally containing stearic acid and water Varnish cream is preferably used which evaporates water and leaves a film of stearic acid. In addition, cold creams may be suitably used. These preparations are semi-solid white, water-in-oil (w / o) formulations prepared with cetyl, alcohol, beeswax, white spirit, circulating paraffin, sodium borate and purified water. The ointments of the present invention can be classified into a hydrocarbon group, an absorbent group, a water-soluble group and a water-soluble group depending on the kind of the base used therein, all of which are well known to experts in the field of formulation. The lotion agent of the present invention is classified into a suspension and an emulsion, usually prepared using emulsifiers or other suitable stabilizers. All of which are included in the present invention as are well known to those skilled in the art of formulation.

Hereinafter, a method of manufacturing a transdermal patch according to the present invention will be described with reference to the above description with reference to a cream, an ointment, a gel, and a pasta.

As a typical example of a dermal agent, a cream agent is used. The cream agent includes an oil phase and a sorbitol solution such as alcohol, beeswax, and sorbitan monooleate, polysorbate, methylparaben, propylparaben, glycerin, The aqueous phase consisting of a mixed solution of propylparaben, potassium hydroxide and purified water is separately heated to 65-75 占 폚, and the oil phase is slowly added to the aqueous phase while stirring to form a crude emulsion. The thus-formed emulsion can be obtained by cooling to a temperature at which volatilization and decomposition do not occur, homogenizing and stirring at a constant rate until solidification.

Ointment can be manufactured on a large scale or on a small scale, and when a special preparation is taken, it depends heavily on the nature of the ingredient. In small scale manufacture of ointments, incorporation may be used in which ointment ingredients are mixed using various methods until a uniform formulation is obtained. In large scale manufacture, all or part of the ointment is melted And then the mixture is cooled and stirred while stirring at a constant speed until solidification. At this time, the components that are not melted are usually cooled and mixed. In general, heat-labile substances or volatile substances will produce ointments at low temperatures where the temperature of the mixture does not degrade or volatilize the ingredients.

The gel is prepared by dispersing metatocell in purified water heated to 80 to 90 캜, allowing it to stand for about 12 hours, and dispersing the gel composition in the settled purified water to prepare a dispersion. The sodium hydroxide solution is then added to the dispersion to adjust to neutral pH. The dispersion thus obtained is mixed with methylparaben, methocel, carbo- bole, propylene glycol or the like to obtain a gel.

The pasta preparation can be prepared by mixing the composition of pasta into a predetermined container such as a mortar, powdering it, and uniformly mixing it with vaseline or the like.

The active ingredients citric acid, zinc and L-arginine in the transdermal formulations of the present invention can promote absorption into the skin by known physical and chemical methods. A physical method using mainly heat, electricity and ultrasonic energy may be suitable, but preferably a chemical method can be used. The transdermal absorption enhancers that can be used in the chemical method according to the present invention include those listed in Table 1 below.

representative Percutaneous  Types and types of absorption promoters Chemical absorption promoter
Clan Usage example Surfactants



Nonionic Twins, Brij, Spans, Triton X-100, Polozyme Anionic Sodium lauryl sulfide Cationic N, N-bis (2-hydroxyethyl) oleylamine Sulfoxide DMSO, dodecyl methyl sulfoxide (DCMS), DMF Atsushi Shirate Dodecanol, hexanesysylate Solvents

Alcohol ethanol Polyol Propylene glycol, polyethylene glycol, glycerin Pyrrolidone PVP, 2-pyrrolidone, N-methyl-2-pyrrolidone Fatty
Fatty acids Oleic acid, linolenic acid, lauric acid, myristic acid, stearic acid, IPM, IPP, caric acid, Fatty alcohols Olyl, lauryl, stearyl Position Dodecyldimethylaminopropane sulfate amides n, diethyl-m-tau amide (DEET) Elements 1-dodecyl urea, 1,3-didodecyl urea, 1,3-diphenylurea Terpen Eucalyptol, menthol, limonene oxide Lactams Lauro Kappram (Azone) Cheratos Disodium EDTA Bile salt Glycollate, calcium-thioglycolate Macrosalicinic Macrosalicinic ketone / lactone

The term "therapeutically effective amount " as used in connection with the antiviral composition according to the present invention means the amount of active ingredient which exhibits a preventive or therapeutic effect on a disease to which the antiviral composition of the present invention is applied.

Therefore, the therapeutically effective amount of the antiviral agent composition according to the present invention may vary depending on the age, sex, application site, administration time, administration time, formulation, kind of adjuvant, etc., Preferably 100 to 3000 mg per day, and 1 to 2,000 mg, preferably 250 to 1,000 mg, most preferably 300 to 500 mg per day for oral administration, per day It may be administered 1 to 5 times. In addition, in the case of a transdermal preparation, it is generally possible to exhibit the effect of prevention or treatment by applying 500 to 2000 mg once or twice a day for about 2 to 3 weeks.

The present invention will be more specifically illustrated by the following examples. However, these embodiments are merely examples of the present invention and do not limit the scope of the present invention.

1. Injection preparation

3% by weight of boric acid (Sigma, USA)

Citric acid (Sigma, U. S. A.) 2%

0.1% by weight of zinc (Sigma, U.S.A.)

Sterile water 100% Composition

The appropriate amount of boric acid, citric acid, and zinc in the above-described amounts was mixed with a 100% composition amount of sterilized water in a suitable container, and then filled in a vial (1,000 mg) to prepare an antiviral composition of the present invention.

2. Manufacture of tablets

5% by weight of boric acid (Sigma, USA)

Citric acid (Sigma, U. S. A.) 2%

0.1% by weight of zinc (Sigma, U.S.A.)

Lactose 30 wt%

Magnesium stearate 5 wt%

Sodium starch glycolate 10 wt%

Sterile water 100% Composition

In a suitable container, 30 wt% of lactose, 5 wt% of magnesium stearate, 10 wt% of sodium starch glycolate, and 100 wt% of sterilized water are mixed with boric acid, citric acid and zinc in the amounts described above, Lt; 0 > C for 1 hour, cooled to room temperature, and then tableted according to a conventional method for preparing tablets to prepare tablets containing 350 mg of the composition.

3. Percutaneous  Produce

To prepare the antiviral composition of the present invention as a transdermal preparation, 5% by weight of boric acid, 2% by weight of citric acid and 0.1% by weight of zinc (pH buffering effect) were dissolved in 1 liter of ethanol-filled beaker, And stored frozen. The sediment was then collected from the beaker and used as the active ingredient in the transdermal formulation. The sediment has a concentration ratio of about 10 times.

(One) Cream

<Remission>

Stearic acid 13 wt%

1% by weight of stearyl alcohol

1% by weight of ethyl alcohol

The sediment 5 wt%

<Awards>

10% by weight of glycerin

Methyl paraben 0.1 wt%

0.05% by weight of propylparaben

Potassium hydroxide 0.9 wt%

Purified water 100% Composition

The oil phase and water phase were separately heated to 65 DEG C and the oil phase was slowly added to the water phase while stirring to form a crude emulsion. The formed emulsion was cooled to about 50 캜 and homogenized. The homogeneous cargo was cooled while shaking until condensation, yielding a cream.

(2) Ointment system

Vaseline 80 wt%

3% by weight of stearyl alcohol

9 wt%

3% by weight cholesterol

The sediment 5 wt%

Stearyl alcohol, pewter, cholesterol and the precipitate were dissolved in a steam bath, and vaseline was added, followed by gradually heating until a liquid was formed. The ointment agent was obtained by stirring and cooling to thereby obtain an ointment agent.

(3) Gelse

Methocel 90 H.C. 4000 0.8 wt%

Carbocall 934 0.24 wt%

Propylene glycol 16.7 wt%

Methyl paraben 0.015 wt%

The sediment 5 wt%

Sodium hydroxide pH7 Adjusted amount

Purified water 100% Composition

The metocell was dispersed in hot (80-90 ° C) water and cooled overnight in the refrigerator to make a solution. Carbocol 934, the precipitate was dispersed in water and a sufficient amount of sodium hydroxide solution was added to the dispersion to adjust the pH to 7.0. Purified water was added to make the volume 40 ml. Methyl paraben was dissolved in propylene glycol. Methocel, Carbocol 934 and propylene glycol fraction were mixed to obtain a gel.

(4) Pasta

Zinc oxide 25 wt%

Starch 25 wt%

Calamine 5 wt%

The sediment 5 wt%

Vaseline 100% Composition

The calamine was titrated with zinc oxide, the precipitate and the starch, and powdered in a mortar and uniformly mixed with vaseline to obtain a pasta.

4. Culture of uterine cancer cell lines

To investigate the effect of the antiviral composition of the present invention on the papilloma virus protein expressed in the cervical cancer cell line and the effect on the growth and survival of the cancer cell, the cervical cancer cell line Ca Ski (human cervical cancer cell line, ATCC number: CRL- 1550), HeLa (human cervical cancer cell line, ATCC number: CCL-2) and C-33A (human cervical cancer cell line, ATCC number: HTB-31). Among these cell lines, Ca Ski is a cervical cancer cell line infected with human papilloma virus type 16, HeLa is a cervical cancer cell line infected with human papilloma virus type 18, and C-33 A is a cervical cancer cell line negative to human papilloma virus.

Each of the above cell lines was put into 96-well plates so as to be 3 x 103 cells / well and then cultured for 12 hours. The culture medium was DMEM supplemented with 10% (v / v) fetal bovine serum (FBS), 100 μg / ml streptomycin, 100 U / ml penicillin and 100 μg / ml L-glutamine Eagle's medium, Life Technology, Inc., USA).

5. Protein analysis of papilloma virus expressed in uterine cancer cells

To investigate whether the antiviral composition of the present invention reduces the protein of papilloma virus expressed in a cervical cancer cell line, the expression of human papilloma virus type 16 or 18 E6 was analyzed.

The Ca-Ski and HeLa cell lines cultured in Example 1 were placed in 96-well plates at 2 x 106 cells / well, and 100 ml of the antiviral composition of the present invention prepared in Example 1 After the treatment, cells cultured for 6 hours, 12 hours, 24 hours, and 48 hours were collected at 37 DEG C under the condition of supplying 5% carbon dioxide and 95% oxygen. Cells recovered according to the elapsed time were resolved by dissolving the cells using a lysis buffer (10 mM Tris, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, protease inhibitor) to dissolve the sample, and the sample was subjected to SDS-electrophoresis Proteins were transferred to an ECL (Electrochemiluminescence) nitrocellulose membrane (Amersham Life Science, UK). The membrane was treated with a blocking solution (5% skin milk in TBST 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and allowed to react overnight at 4 ° C.

Thereafter, Immunoblotting method was used for detection of human papillomavirus type 16 or 18 type E6 protein. That is, primary antibodies (Santa Cruz Biotechnology, USA) binding to human papillomavirus type 16 E6 and human papillomavirus type 18 E6 were diluted 1: 500 in blocking solution and reacted overnight at 4 ° C. And the secondary antibody (goat anti-rabbit IgG-HRP Santa Cruz Biotechnology, USA) was diluted 1: 5000 in the blocking solution and reacted at room temperature for 1 hour. Proteins reacted with the primary and secondary antibodies were washed three times for 15 minutes and checked by exposure to ECL hyperfilm (Amersham Life Science, U.K.). This is shown in FIG. The composition of the active ingredient used herein was 5% by weight of boric acid (Sigma, USA), 2% by weight of citric acid (U.S.A.) and 0.1% by weight of zinc (Sigma, USA).

As shown in FIG. 1, E6 of human papillomavirus type 16, which is a carcinogenic protein, can be observed to be gradually decreased in the expression of the Cask and HeLa cell lines treated with the antiviral composition according to the present invention,

That is, the results shown in FIG. 1 indicate that when the antiviral composition of the present invention is treated with uterine cancer cells, the expression of human papillomavirus type 16 or 18 E6 is suppressed.

The Antiviral agent  Analysis of cell viability by composition treatment

In order to investigate the effect of the antiviral composition of the present invention on the growth and survival of the uterine cancer cell line, the cell survival rate of the uterine cancer cell line was measured by treating the antiviral composition of the present invention. The composition of the active ingredient used herein was 5% by weight of boric acid (Sigma, USA), 2% by weight of citric acid (U.S.A.) and 0.1% by weight of zinc (Sigma, USA).

The Ca-Ski, HeLa and C-33 A cell lines cultured in Example 1 were treated with the antiviral composition of the present invention prepared in Example 1, respectively, and then 5% carbon dioxide and 95% oxygen were supplied The cells were cultured for 6 hours, 12 hours, 24 hours, and 48 hours in a state where the cell viability was measured.

Cell viability was determined by the well known MTT (3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl-2H-tetrazolium bromide) assay (Hansen et al. J. Immunol. Methods, 1989, Vol.172, p203). 20 [mu] l MTT solution (MTT [10 mg / ml in phosphate buffered saline (phosphate buffered saline)] was added to each well and the plate was incubated at 37 [deg.] C for 4 hours. After the medium was removed, formazan crystals were dissolved in 200 μl of DMSO (dimethyl sulfoxide, dimethylsulfoxide) and added to each well. This was mixed by shaking at room temperature for 10 minutes and measured at 540 nm using a Bio-Rad model 3550 microplate reader (Microplate Reader, Richmond, Calif.). At this time, DMEM-FBS and MTT were added, and wells to which no composition was added were used as a control.

In addition, an experiment was conducted in which 3% by weight of albumin was further added to the antiviral agent under the same conditions as described above. The results are shown in FIG. 2B. Experiments with addition of 3% by weight of albumin and 5% The results are shown in FIG. 2C.

As shown in FIGS. 2A to 2C, the cell survival rate of cervical cancer cell lines treated with the antiviral composition of the present invention was found to be significantly decreased with the lapse of time. In addition, it can be seen that the addition of albumin and the addition of albumin and vitamin C further lower the cell survival rate than those of the three active ingredients (boric acid, citric acid and zinc). These results indicate that the antiviral composition specifically acts on human uterine cancer cells induced by human papillomavirus, thereby killing the cells.

Papilloma virus-induced Gnarled  cure.

Six female patients suffering from papules on the vulva were treated with 1 g of the ointment preparation prepared in Example 1 on each affected area. As a result, these patients were treated within 2 to 12 weeks depending on the size of the lesion twice a day. This is shown in FIG. 3, and an excellent therapeutic effect can be seen from FIG.

Claims (7)

Antiviral skin externally to papilloma virus comprising 0.001 to 10% by weight of boric acid, 0.001 to 10% by weight of citric acid, 0.001 to 10% by weight of zinc, 0.01 to 10% by weight of albumin and 0.01 to 10% A pharmaceutical composition.
The antiviral agent for papilloma virus according to claim 1, which comprises 5% by weight of boric acid, 2% by weight of citric acid, 0.1% by weight of zinc, 3% by weight of albumin and 5% A dermatological pharmaceutical composition.
The method according to claim 1,
An antiviral dermatological pharmaceutical composition for papillomavirus wherein the disease caused by papilloma virus is wart, gonad, or uterine cancer.
The method of claim 3,
Wherein said cervical cancer is cervical cancer, endometrial cancer or uterine cancer. delete delete delete

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