WO2009088125A1

WO2009088125A1 - A composition for sanitary supplies comprising mucosal immunoadjuvant and a preparation method thereof

Info

Publication number: WO2009088125A1
Application number: PCT/KR2008/002394
Authority: WO
Inventors: Hwan-Joon Jung; Se-Joong Kim; Seung-Hoon Yang
Original assignee: DIDIMBIOTECH CO Ltd
Current assignee: DIDIMBIOTECH CO Ltd
Priority date: 2008-01-08
Filing date: 2008-04-28
Publication date: 2009-07-16
Anticipated expiration: 2010-07-08
Also published as: KR101012507B1; KR20080070525A

Abstract

Disclosed are a composition for sanitary supplies for women (vaginal cleansing solution, vulval cleansing solution, vaginal tablet, etc.) comprising a mucosal immunoadjuvant and a preparation method thereof. Further disclosed are sanitary supplies for women comprising the mucosal immunoadjuvant. The composition of the present invention enhances immunity against infections and diseases by inducing mucosal immunity in the genitourinary system of women, which is susceptible to exposure to infections, thereby further providing an effect of preventing infections and diseases, as well as the intrinsic functions of the sanitary supplies for women.

Description

A COMPOSITION FOR SANITARY SUPPLIES COMPRISING MUCOSAL IMMUNOADJUVANT AND A PREPARATION

METHOD THEREOF

Technical Field

[1] The present invention relates to a composition for sanitary supplies for women (e.g., vaginal cleansing solution, vulval cleansing solution, vaginal tablet, etc.) comprising a mucosal immunoadjuvant, sanitary supplies for women comprising the composition and a preparation method thereof. Background Art

[2] The female genitourinary system including uterus, vagina, vulva, urethra, etc. is susceptible to infection from outside due to its intrinsic anatomical, physiological and functional features. For example, the vagina is located deep in the body and, thus, is warm and moist. Accordingly, bacteria or fungi, which are main causes of vaginitis, may proliferate and lead to diseases. Therefore, it is preferable to always keep the vagina clean to prevent from bacterial infection.

[3] The female reproductive organ may be easily infected by germs through the mucosa during sexual intercourse. It is reported that chronic inflammation may be induced when a contraceptive device is placed inside the uterus for a long period of time (Wagenlehner, et al., Clin. Microbiol. Infect. 9(8), 881-5, 2003). And, during the introduction a medical device into the body or delivery of mammals, the corresponding mucous membrane can be often damaged, which can be at high risk of infection and inflammation.

[4] Inflammation in the genitourinary system is one of the commonest diseases found in women. The inflammation may be caused by bacteria, parasites or viruses. Patients suffer from pruritis, severe inflammation and foul odor. Particularly, the papillomavirus infection is very closely related to cervical cancer. Therefore, it is very important to avoid development into a fatal complication through adequate prevention, diagnosis and treatment. The most common infections of the female genitalia (e.g., vulva, vagina, cervix, etc.) include bacterial vaginosis, trichomonal vaginitis, candidal vulvovaginitis and vaginitis, chronic vulval candidiasis, general inflammatory vaginitis, atrophic vaginitis, cervical vaginitis, and the like. When profuse bleeding occurs during menstruation, the risk of infection increases, in addition to discomfort.

[5] Vaginal cleansing solution (cleanser) or vulval cleansing solution is frequently used to prevent women's diseases and remove foul odors. There are solution or lotion type cleansers used to prevent vaginitis, which comprise sulfonated fatty acid or distilled lauryl alcohol and an emollient, a thickener, a nutrient, a foaming agent, an organic disinfectant, and so forth. Another example is one mainly consisting of a surfactant and/or povidone iodide. The synthetic disinfectant povidone iodide is known to be effective in preventing and treating candidal vaginitis, trichomonal vaginitis and other vaginal infections. But, iodine hypersensitivity, pruritis caused by mucosal damage, burning, skin ulcer, contact dermatitis, etc. may occur, and accumulation of inorganic iodine in the vagina or the increase of blood iodine level may lead to hypothyroidism. In healthy women, the acidity in the vagina is maintained at about pH 3.2 due to the lactic acid secretion by benevolent microorganisms. In such a low acidity, most harmful microorganisms cannot proliferate or die out. Based on this finding, a pH of 3.5 buffer solution comprising lactic acid and lactoserum, which is extracted from milk, has been proposed. However, it is nothing more than an acidity maintaining measure to keep the pH in the vagina within a desirable range and it cannot treat the diseases.

[6] Also, tablet, lotion, cream, ointment or solution type vaginal products comprising antibiotics as active ingredient to prevent or treat the diseases are used.

[7] The use of bactericide, disinfectant, antibiotics and the like not only increases the possibility of antibiotic resistance but also tends to indiscriminately attack even the benevolent microorganisms that maintain the acidity in the vagina and perform self- protection, replacing them with pathogenic anaerobic bacteria.

[8] The risk of infection by pathogenic bacteria and inflammation increases when the body's immune system is weakened. Accordingly, in order to prevent or treat the pathogen infection, it is necessary to enhance resistance against pathogens, microorganisms, viruses or cancer cells by activating the immunoregulatory system of the body. One way of activating the immunoregulatory system is immuno- augmentation through the mucous membrane. The mucous membranes are originated from the endoderm and are distributed over the organs involved in absorption and secretion. They line various body cavities that are exposed to the external environment and internal organs: respiratory organs including the nostrils, the airway and the tonsil; digestive organs including the small intestine and the rectum; and reproductive organs. The mucous cells perform the primary defensive function against pathogens from external environment at the very forefront. As such, the immunity through mucous cells is very important for the defense of the body.

[9] Examples of mucosal immunoadjuvants include β -glucan, CT (cholera toxin) B- subunit, E. coli LT (heat-labile toxin), LPS (lipopoly saccharide) including pertussis toxin, MPL (monophosphoryl lipid A), CgP ODN (oligodeoxynucleotides containing unmethylated dinucleotides), an aluminum salt, elethero (Acanthopanax Senticosus) extract, a mistletoe extract and alum. [10] The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention. Disclosure of Invention Technical Problem

[11] The existing sanitary supplies used to keep the female genitourinary system clean and prevent and/or treat infection thereof contain bactericides and disinfectants which indiscriminately attack even the benevolent microorganisms in the vagina, leading to the loss of the body's self -protection. Recently, the human body's resistance and response to pathogens is decreasing gradually due to pollution, intake of processed foods, and so forth. Accordingly, an object of the present invention is to provide sanitary supplies for women which, in addition to their inherent function of cleansing, enhance the body's immunity, thereby providing superior stability and enabling the body to protect itself from harmful microorganisms in the vagina. Technical Solution

[12] The present inventors have achieved the aforesaid object by providing a composition for sanitary supplies for women, such as vaginal cleansing solution, comprising a mucosal immunoadjuvant. Introduction of immunoadjuvants to sanitary supplies for women such as cleanser, vaginal tablet, etc. has never been attempted before. The present invention provides a safer and more convenient way of enhancing basic immunity, while providing the intrinsic functions of sanitary supplies such as cleansing, sterilization, and the like.

[13] The present invention provides a composition for sanitary supplies for women comprising a mucosal immunoadjuvant and a preparation method thereof. The present invention also provides sanitary supplies for women prepared from a composition comprising a mucosal immunoadjuvant.

[14] More specifically, the present invention provides a cleansing solution for the urinogenital organs of women in which a suitable amount of an immunoadjuvant that induces mucosal immunity is mixed with a vaginal cleansing solution or a vulval cleansing solution. Such a cleansing solution provides the effect of preventing venereal diseases, infections and various diseases by enhancing basic immunity through inducing mucosal immunity, in addition to its intrinsic functions as cleanser. By including an immunoadjuvant in the vaginal tablet, lotion, cream, ointment or solution applied to prevent or treat infection in the vagina, the stimulation to the mucous membrane is maximized and the mucosal immunity and the systemic immunity are enhanced.

[15] The present invention is based on the experimental results that, in the case of applying a vaginal cleansing solution prepared by adding a substance known to have an effect of enhancing the stimulation of mucosal immunity to the vaginas of mice, the ratio of B -/T -cell differentiation and the differentiation potential of T cells increase in splenocytes, and epithelial cells in the urinogenital organs are activated to produce a large amount of immunoglobulins and enhance the secretion of ThI type cytokines, thereby enhancing immunity, as compared to the control group without the substance that enhances mucosal immunity.

[16] The present inventors added mucosal immunoadjuvants to the already known vaginal cleansing solutions and injected each of them into the vaginas of female BALB/C mice. The mucosal immunoadjuvants were β -glucan, CT B-subunit, E. coli LT, LPS including pertussis toxin, CgP ODN, an aluminum salt, an eleuthero extract, mistletoe lectin, mistletoe lectin B-chain and cocktails thereof. The mucosal immunoadjuvants were added to commercially available vaginal cleansing solution and vulval cleansing solution (e.g., Cepee®, Ladies Cleanser®).

[17] The test groups treated with the cleansing solutions including the mucosal immuno- adjuvant exhibited twice or higher B-cell and T-cell activity, as compared to the control group treated with the cleansing solution including a phosphate buffer instead of mucosal immunoadjuvant.

[18] The test groups treated with the cleansing solution including the mucosal immunoadjuvants or cocktails thereof showed overall enhancement in immunity with at least 1:64,000 of IgG titration in serum, at least 1: 16,000 of IgG titration in mucosal cleansing solution samples, and at least 1:8,000 of IgA titration in mucosal cleansing solution samples of the urinary organ, as compared to the control group treated with the cleansing solution including a phosphate buffer instead of mucosal immunoadjuvant.

[19] Further, from the concentrations of ThI type cytokine such as TNF- α , IL-2 and

INF- γ in serum, we confirmed that immunity is outstandingly enhanced in the immunoadjuvant treated groups than the control group.

[20] Based on these experimental results, the present invention provides the followings.

[21] [I] A vaginal or vulval cleansing composition comprising a mucosal immunoadjuvant.

[22] [2] The cleansing composition according to [1], wherein the immunoadjuvant is selected from the group consisting of β -glucan, CT B-subunit, E. coli LT, pertussis toxin, LPS, MPL, CgP ODN, an aluminum salt, an eleuthero extract, European mistletoe lectin, European mistletoe lectin B-chain, a Korean mistletoe extract, Korean mistletoe lectin, Korean mistletoe lectin B-chain and cocktails thereof.

[23] [3] The cleansing composition according to [1], wherein the immunoadjuvant is selected from the group consisting of CT B-subunit, European mistletoe lectin, Korean mistletoe lectin, European mistletoe lectin and CpG ODN, Korean mistletoe lectin and CpG ODN, LPS and Korean mistletoe lectin, CT B-subunit and Korean mistletoe lectin, and β -glucan and Korean mistletoe lectin.

[24] [4] A preparation method of a vaginal or vulval cleansing composition comprising adding a mucosal immunoadjuvant.

[25] [5] A vaginal or vulval cleansing solution comprising a composition according to any of [1] to [3].

[26] [6] A vaginal product or sanitary supply for women comprising an immunoadjuvant capable of inducing mucosal immunity.

[27] [7] The vaginal product or sanitary supply for women according to [6], wherein the vaginal product or sanitary supply for women is in the form selected from the group consisting of vaginal tablet, lotion, cream, ointment and solution.

[28] The cleansing solution of the present invention induces mucosal immunity by stimulating the female genitalia, thereby resulting in nonspecific immunity, which is spread to all the mucosal area in the body, including serum, oral mucous membranes, ocular mucous membranes, nasal mucous membranes, gastrointestinal mucous membranes, respiratory mucous membranes, urinary mucous membranes, and genital mucous membranes. Accordingly, the resistance to initial infection by pathogens is enhanced and the initial immunity against respiratory infectious diseases, food-borne infectious diseases, anal and rectal infectious diseases, and urinary infectious diseases is enhanced. In particular, through activation of nonspecific immunity, the present invention is effective in initially blocking infectious diseases caused by most microorganisms and a few viruses and, thus, is valuable for non-drug products. Further, the present invention can provide prevention against the pandemic infectious diseases, including AIDS, SARS and aviation flu, and keep the genitalia healthy.

[29] The mucosal immunoadjuvant added to the sanitary supplies for women and vaginal products including vaginal cleansing solution, vulval cleansing solution, vaginal tablet, etc. may be any one known in the related art, as long as the advantageous effect provided by the present invention is attained. Examples include β -glucan, CT B- subunit, E. coli LT, LPS including pertussis toxin, MPL, CgP ODN, an aluminum salt, an eleuthero extract, European mistletoe lectin, European mistletoe lectin B-chain, a Korean mistletoe extract, Korean mistletoe lectin, Korean mistletoe lectin B-chain and cocktails thereof. A mistletoe extract, a mistletoe lectin or a cocktail thereof with other immunoadjuvants are preferred.

[30] The composition provided by the present invention may be included in any known or commercially available sanitary supplies for women, as long as the advantageous effect provided by the present invention is attained.

[31] The vaginal cleansing composition of the present invention may be in various forms.

For example, the composition may be in liquid form, such as solution, suspension, emulsion, etc., and may be prepared by adding a solubilizer, an emulsifier, a buffer for pH control, etc. to sterilized water. The composition may further comprise an excipient in addition to the active ingredient. The excipient may be a binder, an anticaking agent, a disintegrant, a vehicle, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a pigment, a fragrance, a buffer, a preservative, a solubilizer, a tonifier, a matrix, a filler, a lubricant, a surfactant, etc. Further, polysaccharide, oligosaccharide, oil and fat, protein, vitamin, steroid, hormone, low alcohol, polyalcohol, vasodilator, spermicide, bactericide, antiviral agent, etc. may be included. These may be included in an amount which is normally used in the pharmaceutical field.

[32] The composition according to the present invention may comprise the mucosal im- munoadjuvant in an amount from 0.1 % by weight to 15 % by weight, preferably from 3 % by weight to 10 % by weight, based on the total weight of the composition. The composition of the present invention is administered at a pharmaceutically effective dosage, which may be determined depending on the disease and severity thereof, age and sex of the patient, activity of the drug, sensitivity to the drug, time and route of administration, discharge ratio, duration of treatment, concomitant use of other drugs, and other well-known factors. It is important to determine the dosage that can lead to the best effect with the smallest amount, which can be readily performed by those skilled in the art.

[33] Another object of the present invention is to provide a preparation method of sanitary supplies for women and vaginal medications including vaginal cleansing solution, vulval cleansing solution, vaginal tablet, etc. by adding an immunoadjuvant capable of inducing mucosal immunity. The sanitary supplies for women and vaginal medications used in the present invention may be already known ones and the mucosal immunoadjuvant is added in an appropriate manner according to the product type. Advantageous Effects

[34] By inducing mucosal immunity and enhancing basic immunity against venereal diseases, infections and other diseases, the composition of the present invention and the sanitary supplies for women comprising the same provide the effect of preventing and treating vaginal infection, as well as providing the intrinsic functions of sanitary supplies.

[35] Through enhanced basic immunity, various infections can be prevented at early stage, pandemic infectious diseases such as AIDS, SARS, avian flu, etc. may be prevented, or the genitalia can be maintained healthy.

[36] The mucosal immunity induced in the genital or urinary organ enhances mucosal immunity of other organs in the body. Therefore, not only the infection at the genital or urinary organ, immunity against infectious diseases at other organs, including respiratory and gastrointestinal organs, may be augmented. Description of Drawings

[37] FIG. 1 is a drawing illustrating the ratio of B-/T-cell differentiation in the splenocytes of mice, vaginas of which were stimulated with the vaginal cleansing composition of the present invention. [38] FIG. 2 is a drawing illustrating the differentiation potential of T-cells in the splenocytes of mice, vaginas of which were stimulated with the vaginal cleansing composition of the present invention. [39] FIG. 3 is a drawing illustrating the concentration of IgG and IgA in sera of mice, vaginas of which were stimulated with the vaginal cleansing composition of the present invention. [40] FIG. 4 is a drawing illustrating the concentration of IgG and IgA in wash samples of urinary and reproductive organs of mice, vaginas of which were stimulated with the vaginal cleansing composition of the present invention. [41] FIG. 5 is a drawing illustrating the concentration of TNF- α and IL-2 in sera of mice, vaginas of which were stimulated with the vaginal cleansing composition of the present invention. [42] FIG. 6 is a drawing illustrating the concentration of INF- γ in sera of mice, vaginas of which were stimulated with the vaginal cleansing composition of the present invention.

Mode for Invention [43] Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for the illustrative purpose only, and the invention is not intended to be limited by the examples.

[44] Example 1: Preparation of cleansing solution comprising mucosal immunoadjuvant

[45] Using a commercially available cleansing composition comprising purified water, ethanol, fragrance, sodium benzoate, whey and lactic acid, vaginal cleansing solutions including a mucosal immunoadjuvant were prepared as in Table 1. [46] [ Table 1 ]

[47] Compositions of vaginal cleansing composition

[48] [Table 1] [Table ]

[49] Also, cocktail compositions comprising more than one mucosal immunoadjuvant were prepared. The compositions are given in Table 2. [50] The single immunoadjuvants or cocktail mixtures listed in Table 2 were directly mixed with the commercially available cleansing solutions (Cepee®, Ladies Cleanser®), or the mucosal immunoadjuvants were dissolved in purified water and then mixed with the cleansing compositions.

[51] The mucosal immunoadjuvants were either commercially purchased or prepared according to the method described in Korean Patent Application Nos. 10-1996-5732, 10-2004-31109 or 10-1999-41288.

[52] The resultant cleansing solutions containing a single mucosal immunoadjuvant or a cocktail of mucosal immunoadjuvants induced mucosal immunity and, as a result, the overall systemic immunity was enhanced.

[53] [ Table 2 ] [54] Compositions of mucosal immunoadjuvants [55]

[Table 2] [Table ]

[56] Example 2: Examination of differentiation potential of B-/T-cells of mouse splenocytes using FACScan

[57] The vaginas of mice were stimulated by washing with each of 500 μ L of the cleansing solutions comprising 0.01 % of each β -glucan, CT B-subunit, CpG ODN, LPS, aluminum salt, mistletoe lectin, and mistletoe lectin B-chain using a sterilized pipette. Such treatment was carried out for 15 days (once a day). The control group was treated similarly using a phosphate buffer (PBS) instead of the mucosal immuno- adjuvant. On day 16, the mice were sacrificed, and splenocytes were isolated from the spleens. Then, the ration of B-/T-cell differentiation was observed using antibodies by FACScan. The test groups treated with the cleansing solutions containing the mucosal immunoadjuvants showed twice or higher ratio than the control group (see FIG. 1).

[58] Example 3: Examination of differentiation potential of T-cells from mouse splenocytes using FACScan

[59] For the mouse splenocytes obtained from the test groups and the control group in

Example 2, differentiation potential of T-cells was examined. More CD3 was observed since the response was a nonspecific immune response (see FIG. 2). If antigens had been administered together, more CD4 or CD8 would have been observed than CD3. To conclude, the treatment of the vaginal mucosal membranes with the cleansing solutions comprising the immunoadjuvants enhanced nonspecific immunity, and the differentiation potential of T-cells increased on the whole. Accordingly, it is thought that immunity against various antigens or pathogens was activated.

[60] Example 4: Examination of antibody titration in mucosal wash samples of mice

[61] From the test groups and the control group of Example 2, serum and wash samples of the urinary and genital organs were taken. The serum sample was taken by drawing blood from below eyes. The wash samples were obtained by adding each 500 μ L of PBS to the urinary and genital tissues, washing 20 times, and then placing in sterilized E-tubes. Such operation was repeated 10 times to take each 5 mL of wash samples. Centrifuge was performed at 6,000 rpm, and only the supernatant was taken and stored at 4 ° C for 24 hours. A protein G column chromatography was performed on the wash samples to isolate IgG, and fractionated IgA was taken separately.

[62] Each of the two samples obtained above was subjected to time-resolved fluorescence

Immunoassay (TR-FIA), which was performed according to the method by Kensner, et al. {Steroids, 59, 1994, 206). The method will be put briefly in the following. Isolated IgG antibodies are diluted starting from 1,000 times to 128,000 times by 2-fold serial dilution, and each 0.1 mL of eight diluted samples are applied to 96-well plates and left at 37 ° C for 2 hours. Then, the plates are repeatedly washed 5 to 6 times using an automatic washer, and goat IgG anti-mouse IgG-Eu particle conjugates were added thereto at a diluted concentration of 1:5000, and reacted at 37 ° C for 2 hours. Subsequently, the plates are repeatedly washed 5 to 6 times using an automatic washer. Then, after adding 200 μ L of enhancement solution and further reacting for about 5 minutes, measurement is made using Delfia (Wallac, Model 1420).

[63] Titration of fractionated IgA was performed in the same manner as mentioned above, except for using goat IgG anti-mouse IgA-Eu particle conjugates.

[64] The mice vaginas of which were stimulated with the cleansing solutions comprising the immunoadjuvants showed at least 1:64,000 of IgG titration in serum, and at least 1: 16,000 of IgG titration in the mucosal cleansing solution samples, as compared to the control group. IgA was not observed in the serum, and IgA titration in the mucosal cleansing solution samples excluding serum was at least 1:8,000 (see FIG. 3 and FIG. 4).

[65] Example 5: Examination of cytokine secretion in mouse blood

[66] Blood level of TNF- α , IL-2 and INF- γ was measured for the test groups and the control group of Example 2. The mice vaginas of which were stimulated with the cleansing solutions comprising the immunoadjuvants showed 10 to 25 times increased cytokine level, as compared to the control group. This confirms that the treatment with the cleansing composition of the present invention enhanced ThI type immunity (see FIG. 5 and FIG. 6).

[67] While the present invention has been described with an emphasis upon preferred embodiments, it will be obvious to those skilled in the art that variations in the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the sprit and scope of the invention as defined by the claims that follow.

Claims

[1] A vaginal or vulval cleansing composition comprising one or more mucosal im- munoadjuvants .

[2] The cleansing composition according to claim 1, wherein one or more immuno- adjuvants are selected from the group consisting of β -glucan, CT (cholera toxin) B-subunit, E. coli LT (heat-labile toxin), pertussis toxin, LPS (lipopolysaccharide), MPL (monophosphoryl lipid A), CgP ODN (oligodeoxynucleotides containing unmethylated dinucleotides), an aluminum salt, an eleuthero extract, European mistletoe lectin, European mistletoe lectin B- chain, a Korean mistletoe extract, Korean mistletoe lectin, Korean mistletoe lectin B-chain and cocktails thereof.

[3] The cleansing composition according to claim 1, wherein one or more immuno- adjuvants are selected from the group consisting of CT B-subunit, European mistletoe lectin, Korean mistletoe lectin, European mistletoe lectin and CpG ODN, Korean mistletoe lectin and CpG ODN, LPS and Korean mistletoe lectin, CT B-subunit and Korean mistletoe lectin, and β -glucan and Korean mistletoe lectin.

[4] A preparation method of a vaginal or vulval cleansing composition comprising adding one or more mucosal immunoadjuvants.

[5] A vaginal or vulval cleansing solution comprising a composition according to any of claims 1 to 3.

[6] A vaginal product or a sanitary supply for women comprising an immuno- adjuvant capable of inducing mucosal immunity.

[7] The vaginal product or sanitary supply for women according to claim 6, wherein the vaginal product or sanitary supply for women is in the form selected from the group consisting of vaginal tablet, lotion, cream, ointment and solution.