CN115768452A

CN115768452A - Coriolus versicolor extract for treating vaginal or cervical disorders caused by infectious pathogens

Info

Publication number: CN115768452A
Application number: CN202180044628.XA
Authority: CN
Inventors: F·洛萨多明格斯; S·帕拉科斯; Y·加斯莱恩
Original assignee: Iberia Nursing Health Co ltd
Current assignee: Iberia Nursing Health Co ltd
Priority date: 2020-04-28
Filing date: 2021-04-27
Publication date: 2023-03-07
Also published as: WO2021218964A1

Abstract

Use of a coriolus versicolor extract for the manufacture of a composition for the prevention and/or treatment of a vaginal or cervical condition caused by an infectious agent, wherein the prevention or treatment comprises vaginal or cervical administration of the composition to a female.

Description

Coriolus versicolor extract for treating vaginal or cervical disorders caused by infectious pathogens

This application claims benefit of international patent application PCT/CN2020/087445 filed on 28/4/2020.

Technical Field

The present invention relates to the field of vaginal or cervical disorders, in particular gynaecological disorders caused by infectious agents. The present invention provides compositions comprising extracts from Coriolus versicolor (Coriolus versicolor) and regimens for the prevention and/or treatment of such disorders.

Background

Gynecological infections are common in women of all ages. Typical gynecological infections include bacterial vulvovaginitis, vaginal yeast infection, trichomoniasis, nonspecific vulvovaginitis, and viral infections. Viral infections are among the most difficult to treat. In particular, human Papillomavirus (HPV) infections that may lead to cervical cancer are of increasing concern, particularly in sexually active women.

Cervical cancer caused by HPV infection has become the 2 nd most common female cancer in women 15-44 years of age in some developed countries. Over 30 to 40 types of HPV are commonly transmitted by sexual contact and infect the anovaginal or cervical region. However, most HPV types do not cause disease. Persistent infection by "high risk" HPV types may progress to premalignant and invasive cancers.

HPV types 16 and 18 together contribute to about 70% of all cervical cancers. However, it is noteworthy that the vast majority of high-risk HPV infections are cleared by the host immune system and regress subclinically within one year and regress by 90% within two years. However, a few subjects (5% to 10% of infected women) failed to control viral infection and developed HPV persistent infections. As mentioned above, this persistent infection is at risk of developing precancerous lesions of the vulva and cervix, which may develop into invasive cancer.

HPV lesions are thought to be caused by proliferation of infected basal keratinocytes. Infection usually occurs when the basal cells of the host are exposed to infectious virus that crosses the disturbed epithelial barrier, as would occur during sexual intercourse or after minor skin abrasion. After invasion of the cell by HPV virions, active infection occurs and the virus can be transmitted. Months to years may be required before Squamous Intraepithelial Lesions (SIL) develop and can be clinically detected. Persistent infection due to a disturbed epithelial barrier contributes to the development of cervical cancer. The overall pattern of cervical cancer evolution is shown in figure 1.

Progression from subclinical infection to clinical infection can take years, providing an opportunity for detection and treatment of precancerous lesions. Progression to invasive cancer can be prevented if subclinical HPV infection is detected early and examined regularly. To this end, cervical screening using the papanicolaou test (Pap) test or liquid-based cytology detects abnormal cells that may develop cancer. If abnormal cells are found, please ask women for colposcopy. During colposcopy, a biopsy can be taken and the abnormal area removed by a simple procedure, usually with a cautery ring or more commonly by freezing (cryotherapy) in developing countries. Treating abnormal cells in this manner can prevent them from developing cervical cancer. Pap smear tests have reduced the incidence and mortality of cervical cancer in developed countries, but even so, the incidence of cervical cancer in these countries has not decreased. In addition, in resource-poor areas, pap screening is generally not available for most women, and the mortality rate for cervical cancer is also high in these areas.

It should also be taken into account that the results of the Pap test are generally not conclusive and that in this case no surgical intervention is recommended, since this implies associated risks and increased costs. An inconclusive Pap test occurs when abnormal cells of type CIN1, indicative of mild cervical intraepithelial neoplasia, are detected. Typically, when CIN1 is detected by biopsy, the woman is infected with HPV, which may self-clear within 12-24 months, thus requiring subsequent testing rather than treatment. Women in this situation cannot know what will happen and may not follow preventive or therapeutic strategies that often lead to psychological stress and anxiety. Treatment or surgical strategy is initiated only when subsequent analysis shows malignant development (usually when cervical intraepithelial neoplasia type 2 CIN2 is detected first).

HPV vaccines (Cervarix and Gardasil) to prevent HPV type (16 and 18) infection can further reduce the incidence of cervical cancer. However, although these prophylactic vaccines encompass the two most common high-risk HPV types, other high-risk viruses that cause the remaining 30% of cervical cancer cases have not been encompassed. Furthermore, since HPV is the most common sexually transmitted disease, a large number of individuals are already infected and therefore cannot benefit from such prophylactic vaccination.

In summary, there is a need to provide alternative strategies to combat HPV infection and cervical cancer, particularly in the early stages of HPV infection.

Disclosure of Invention

The present inventors have developed strategies to combat HPV and other gynecological infections. The present strategy relates to enhancing local immunity of the vaginal or cervical system, in particular the vagina and cervix, by vaginal or cervical administration of a composition comprising a coriolus versicolor extract.

Coriolus versicolor extract contains two β -glucans, polysaccharide K (PSK) and polysaccharide peptides (PSP), which have been described as potent immunopotentiators (Kang SC et al, int J Biol macro.2013, vol.57, p.9-16 cui J, et al, biotechnol adv.2003, vol.21 (2), p.109-22.).

Oral formulations containing the extract are known in the art and have been used as an adjuvant to chemotherapy and radiotherapy of cancer and various infectious diseases, particularly in japan. However, coriolus versicolor extracts have never before been used for the topical treatment of vaginal or cervical disorders caused by infectious pathogens, including HPV infections and cervical cancer. The inventors have now found that the present composition comprising coriolus versicolor extract is particularly effective in combating vaginal or cervical infections such as vaginitis and HPV infections when applied directly to the vagina and/or cervix, and have further found a convenient administration regimen of the composition comprising coriolus versicolor extract to provide the best results.

Accordingly, one aspect of the present invention provides a composition comprising a coriolus versicolor extract for use in the prevention and/or treatment of a vaginal or cervical condition caused by an infectious pathogen by vaginal or cervical administration. This may be rewritten as the use of a coriolus versicolor extract for the preparation of a composition for the prevention and/or treatment of a vaginal or cervical condition caused by an infectious agent by vaginal or cervical administration. The invention also contemplates a method for preventing and/or treating a vaginal or cervical condition caused by an infectious agent in a female (including a human) in need thereof, the treatment comprising vaginal or transcervical administration of a composition comprising coriolus versicolor. The term "infectious agent" is understood to mean a microorganism in a broad sense, such as a virus, bacterium, prion, fungus or protozoan, which causes a disease in its host. In the present invention, the terms "infectious agent" and "pathogen" are used without distinction. Infectious pathogens in the sense of the present invention are typically selected from the group consisting of Human Papillomavirus (HPV), candida albicans (Candida albicans), chlamydia trachomatis (Chlamydia trachomatis), gardnerella vaginalis (Gardnerella vaginalis), campylobacter (Mobilucus), bacteroides (Bacteroides), mycoplasma (Mycoplasma), neisseria gonorrhoeae (Neisseria gonorrhoeae), trichosporoides vaginalis (Trichomonas vagianalis), herpes viruses (Herpes viruses), staphylococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis), escherichia coli (Escherichia coli), streptococcus alpha (Streptococcus alfa), bacillus (Bacillus), enterobacter (Enterobacter), proteobacter (Escherichia coli), escherichia coli (Klebsiella pneumoniae), staphylococcus epidermidis (Klebsiella pneumoniae), and Staphylococcus epidermidis).

Current strategies against HPV and other vaginal or cervical infections offer several advantages. First, local immune enhancement is achieved by applying the active ingredients of coriolus versicolor (particularly PSK and PSP) directly to the site of action. Since the vaginal or cervical system, mainly the vagina and cervix, is very sensitive to infectious diseases, it is very beneficial to enhance immunity in this particular environment. Locally enhancing vaginal or cervical immunity helps the body to fight a variety of vaginal or cervical infections, from non-specific vaginitis and candidiasis to HPV infections. Secondary side effects can be minimized because the active ingredients in the composition can be more easily tailored to achieve the desired effect, while at the same time an optimal dose response can be achieved. Further, administration by the vaginal/cervical route makes application easy and circumvents the need to protect the active compound from harsh environments such as the gastrointestinal tract.

The inventors have further found the most suitable administration regimen for the treatment of the above-mentioned vaginal or cervical disorders, such as cervical cancer. The prevention and/or treatment of vaginal or cervical conditions according to the invention is best when the composition is administered to a female human vaginally or transcervically according to the following protocol:

(a) One dose of the composition per day during 21-24 days, followed by

(b) No dose during 3 to 7 days, followed by

(c) One dose of the composition per day or every two days during 21-24 days, followed by

(d) No dose during 3 to 7 days, and

(e) Repeating (c) and (d) four times in sequence,

wherein each dose comprises 0.5 to 5mg of Coriolus versicolor extract.

As shown in the examples below, clinical trials have demonstrated the effectiveness of using the composition of the invention to treat low-grade cervical lesions caused by HPV and to clear HPV infections according to this administration regimen. In addition to being effective, the discontinuous treatment regimen of the present invention has a period of time during which no dose is administered, which also improves patient compliance with the treatment, resulting in increased patient compliance and therapeutic efficacy.

As will be described below, other ingredients, including other active ingredients, and excipients as carriers may preferably be added to the composition.

Drawings

Figure 1. Oncogenic protocol for cervical cancer. N represents normal, uninfected epithelial cells. Women are infected with HPV and develop low-grade squamous intraepithelial lesions (LSIL). No changes may be found in the epithelial cells during the first months of infection. Eventually, grade 1 cervical intraepithelial neoplasia (CIN 1) may be observed. After 12 to 24 months, the patient may develop high grade squamous intraepithelial lesions (HSIL). First, grade 2 cervical intraepithelial neoplasia (CIN 2) may be observed. Over the years, the lesion may evolve into grade 3 cervical intraepithelial neoplasia (CIN 3) and eventually invasive carcinoma (C). Percentages represent the estimated number of patients for whom HPV infection will reach these stages. The arrow labeled "t" represents an increase in time. The arrow labeled "1" indicates the stage at which the prevention and screening strategy is initiated. The arrow labeled "2" indicates the stage at which the affected patient begins treatment.

FIG. 2 cytokine expression in mouse vaginal epithelial cells determined by RT-qPCR after vaginal administration of control, GEL1 (0.05% Coriolus versicolor extract) or GEL 2 (0.25% Coriolus versicolor extract). A: TNF α, B: IL-1. Beta., C: IL-12, D: IL-6, E: IL-17.1: control, 2: GEL1, 10 day period, 3:

GEL

2,6 day period, 4:

GEL

2, 10 day period. The Y-axis represents the cytokine fold increase relative to baseline.

FIG. 3. Variation of epithelialization (epithelization) parameters before and after 12 days of treatment with PAPILOCARE GEL.

Figure 4.3 colposcopic photographs of patients before PAPILOCARE GEL administration (column a) and 12 days after PAPILOCARE GEL administration (column B).

Figure 5 efficacy of treatment with PAPILOCARE GEL in high risk HVP population as measured by Pap smear results and consistent colposcopic images.

FIG. 6 HPV clearance in high risk HVP populations following treatment with PAPILOCARE GEL.

Figure 7 images of patient cervical re-epithelialization during papiloreal GEL treatment.

Figure 8 degree of epithelialization according to the litker (Likert) scale after treatment with PAPILOCARE GEL.

Figure 9. Pressure levels reported by patients during treatment with PAPILOCARE GEL.

Detailed Description

In the present application, the term "extract" is used in the conventional sense to mean a concentrated preparation of a compound obtained by removing the active ingredient from a source (usually of plant origin) by suitable means. The extract contains one or more active ingredients and may be incorporated into pharmaceutical or cosmetic compositions in a variety of forms, including pure or semi-pure components, solid or liquid extracts, or solid plant matter.

Generally, plant extracts contain not only one component, but also multiple components, many of which are active. Sometimes, the beneficial effect derives from a combination of many of these active compounds. In other cases, one particular compound is primarily responsible for most activities.

Extracts as used herein also include "synthetic" extracts, i.e., various combinations of known components and/or ingredients that are combined to substantially mimic the composition and/or activity of a plant extract of natural origin. Synthetic extracts will have two or more, three or more, or four or more active ingredients that are the same as the active ingredients of natural origin. Natural or synthetic extracts enriched in one or more components are also considered part of the invention.

Coriolus versicolor (hereinafter, abbreviated as coriolus versicolor, also called Trametes versicolor) is a common polypore, belongs to Basidiomycotina (Basidiomycotina), and is found all over the world. The visible form of Coriolus is a mushroom sector with wavy edges and concentric colored zones. Coriolus versicolor is an obligate aerobic microorganism, and is commonly found on dead wood, stumps, trunks and branches throughout the year.

By "extract of coriolus versicolor" is understood a concentrated preparation of a compound obtained from coriolus versicolor, as defined above. The main active compounds in the extract of Coriolus versicolor are beta-glucan Coriolus versicolor glycopeptide (polysaccharide peptide, polysaccharopeptide) Krestin (PSK) and Coriolus versicolor glycopeptide (PSP). Both compounds are obtained from the extract of Coriolus versicolor mycelium.

As used herein, "corious versicolor extract" generally refers to a preparation containing biologically active chemical components and/or compounds isolated from corious versicolor, preferably corious versicolor glycopeptide Krestin (PSK) and/or corious versicolor glycopeptide (PSP). This includes the whole extract of Coriolus versicolor, but also includes pure or semi-pure preparations of the above bioactive compounds obtained from Coriolus versicolor.

Coriolus versicolor extracts containing biologically active compounds and preparations containing purified compounds are commercially available, for example, by SYMRISE corporation. Most commercial preparations of polysaccharopeptide use only intracellular polymers recovered from agaricus or submerged culture mycelia. Typical compositions and methods of extraction of Coriolus versicolor extract are described in Cui J et al, supra).

PSP and PSK are chemically similar β -glucan polysaccharides (KF Cheng, et al, cancer Therapy 2008, vol.6, p.117-130) linked to many proteins and they have similar physiological activity profiles. These compounds are known as potent immunopotentiators and cancer proliferation inhibitors (Kang SC et al, supra; cui J et al, supra).

The term "beta-glucan" in a broad sense refers to a polysaccharide of D-glucose monomers linked by beta-glycosidic bonds. Beta 0-glucan is a diverse group of molecules that may differ in molecular mass, solubility, viscosity, and three-dimensional configuration. Several β 1-glucans have been described as having various biological activities. In the present invention, the term "β 2-glucan" is understood to mean these biologically active β 3-glucans, in particular those derived from yeast and biological agaricus, including polystictus glycopeptides from polystictus versicolor. The present invention contemplates more biologically active beta 4-glucans, for example, beta-glucans from other agarics such as ganoderma lucidum, shiitake mushroom, chaga and grifola frondosa, beta (1, 3) D-glucans from baker's yeast, beta (1, 3) (1, 4) -glucans from oats and barley. Also contemplated are beta-glucan derivatives, such as carboxymethyl beta-glucan. These beta-glucans are also reported to have immunomodulatory properties. Carboxylated beta-glucans are also described as having wound healing properties. A convenient source of beta-glucan is the product of Naturalis Life Technologies, inc

Contains carboxymethyl beta-glucan, magnolol (magnolol) and honokiol (honokiol) carried by niosomal vesicles.

In a preferred embodiment, the prevention and/or treatment comprises enhancing the immunity of the vagina or cervix to infectious pathogens.

It is considered part of the present invention to provide a composition comprising a therapeutically effective amount of a coriolus versicolor extract for vaginal or cervical administration for enhancing the immunity of the vagina or cervix to infectious pathogens. This can be restated as the use of a composition comprising a therapeutically effective amount of coriolus versicolor extract in the manufacture of a medicament for enhancing the immunity of the vagina or cervix to infectious pathogens by vaginal or cervical administration. The present invention also provides a method of enhancing vaginal or cervical immunity to infectious pathogens in an animal, including a human, in need thereof, the method comprising vaginally or transcervically administering a composition comprising a therapeutically effective amount of a coriolus versicolor extract. The administration regimen is preferably as defined above.

The composition for vaginal or cervical administration of the present invention comprises coriolus versicolor extract. The compositions of the present invention may additionally incorporate other active ingredients that enhance the beneficial effects of coriolus versicolor extract by further enhancing immunity, reducing inflammation, facilitating beneficial vaginal flora, repairing disturbed epithelial barriers, or combating undesirable infectious pathogens. In this sense, the inventors have found that a synergistic interaction occurs between the coriolus versicolor extract and an active compound selected from the group consisting of moisturizers, probiotics, prebiotics, anti-inflammatory agents (inflammatory agents), tissue regeneration agents and antiviral agents. Thus, in one embodiment, the composition according to the invention, in addition to the coriolus versicolor extract, further comprises at least one active compound selected from a moisturizing agent, a probiotic, a prebiotic, a tissue regeneration agent, an anti-inflammatory agent and an antiviral agent.

Coriolus versicolor extract stimulates a protective immune response while suppressing unwanted immune responses that may lead to disease. For example, coriolus versicolor extract can restore or improve suppressed immune system function caused by, for example, administration of anticancer agents. Coriolus versicolor extract can stimulate a protective immune response against viral, bacterial and/or microbial infections. In addition, coriolus versicolor extract can suppress unwanted immune responses such as TNF-a production and induction of its metalloprotease production, which some tumor cells use to promote metastasis.

When applied directly to the vaginal or cervical region, in particular to the vagina or cervix, the composition according to the invention enhances the local immune response against typical pathogens which may be present in this region, such as Human Papilloma Virus (HPV), candida albicans, chlamydia trachomatis, gardnerella vaginalis, campylobacter, bacteroides, mycoplasma, neisseria gonorrhoeae, trichomonas vaginalis, herpes virus, staphylococcus aureus, staphylococcus epidermidis, escherichia coli, alpha streptococcus, bacillus, enterobacter, proteus, enterobacter cloacae, klebsiella, staphylococcus epidermidis, klebsiella pneumoniae and dermatophytes. These pathogens cause a range of conditions including, but not limited to, vaginitis, vulvovaginitis, dermatophytosis, candidiasis, vaginal or cervical herpes, gonorrhea, proctitis, infertility, cervicitis, pelvic inflammatory disease, ectopic pregnancy, acute or chronic pelvic pain, trichomoniasis and cervical cancer. Thus, in one embodiment, the composition for vaginal or cervical administration of the invention is for the prevention and/or treatment of any of the above conditions.

In another particular embodiment, the vaginal or cervical condition to be prevented or treated by the composition of the invention is vaginitis. "vaginitis" refers to inflammation of the vagina commonly caused by infection, which can lead to discharge, itching and pain, and is often associated with irritation or infection of the vulva. In the latter case, the condition is known as "vulvovaginitis".

Several types of vaginitis are known to affect women in general, including bacterial vaginitis, mycotic vaginitis, trichomoniasis, nonspecific vaginitis, and viral vaginitis.

Mycotic vaginitis is caused by fungal infection, usually by overgrowth of yeasts of the genus candida, but can also be caused by fungal infections of the skin. It is most commonly caused by a type of fungus known as candida albicans. The fungal Candida species (Candida species) occur naturally in the vagina and are generally harmless. However, if conditions change in the vagina, candida albicans may cause symptoms of candida vaginitis (thrash).

Trichomoniasis, sometimes referred to as "trichomoniasis (trich"), is another common cause of vaginitis. It is a sexually transmitted disease and is caused by the single-celled protozoan parasite trichomonas vaginalis, which exerts mechanical stress on host cells and then, after cell death, takes up cell debris. Symptoms include inflammation of the cervix (cervicitis), urethra (urethritis), and vagina (vaginitis), which produce itching or burning sensations.

Nonspecific vaginitis is a vaginal disease caused by an imbalance in the naturally occurring bacterial flora. The microorganisms associated with bacterial vaginosis are very diverse, but include gardnerella vaginalis, campylobacter, bacteroides and mycoplasma. Changes in the normal bacterial flora, which may be caused by antibiotic use, hormonal changes or pH imbalance, include a reduction in the genus lactobacillus (lactobacillus), allowing these harmful bacteria to stand and multiply. This type of vaginitis is extremely common, especially in women of childbearing age.

Viruses are also a common cause of vaginitis. One form caused by Herpes Simplex Virus (HSV) is often referred to simply as "herpes" infection. These infections are also transmitted by sexual contact. The main symptom of herpetic vaginitis is pain associated with lesions or "sores". These sores are usually visible on the vulva or vagina, but occasionally within the vagina, and can only be seen during gynecological examination.

Another source of viral vaginal infections is Human Papillomavirus (HPV). HPV, sometimes referred to as vaginal or cervical warts, can also be transmitted through intercourse. This virus causes painful warts to grow in the vagina, rectum, vulva or groin.

As mentioned above, persistent infection with HPV may also lead to cervical cancer. In a preferred embodiment, the composition of the invention is used to enhance vaginal or cervical immunity against HPV. In another preferred embodiment, the composition of the invention is used for the prevention and/or treatment of cervical cancer.

In particular, the compositions of the invention are useful in preventing persistent infection with HPV, thereby providing an effective strategy against cervical cancer at an early stage where no other therapeutic strategy is available.

As mentioned above, therapeutic interventions have hitherto occurred at the stage of detection of HSIL (high squamous intraepithelial lesions). Patients showing only LSIL (low-grade squamous intraepithelial lesions, consistent with CIN type 1 cells) were not treated for a time frame of 12 to 24 months until testing and confirmation of HSIL was repeated (see figure 1). The compositions of the invention are particularly suitable for patients who exhibit a positive test for LSIL/CIN1 but are not currently receiving treatment. The compositions of the invention may be administered at this stage and provide benefits in helping the body clear HPV infection by locally increasing immunity and avoiding persistent HPV infection, as well as in curing the above-mentioned LSIL/CIN1 lesions by enhancing epithelialization of the affected area. Thus, in a particular embodiment, the composition of the invention is for vaginal or cervical (vaginal or cervical) administration to women exhibiting low-grade squamous intraepithelial neoplasia (LSIL) or mild cervical intraepithelial neoplasia (CIN 1). This embodiment may also be expressed as a method of preventing or treating HPV infection or cervical cancer by vaginal or cervical administration of a composition of the invention in a female exhibiting LSIL or CIN 1.

Effective clearance of HPV infection at this stage is commonly referred to as "negativity". Thus, one embodiment of the invention relates to the use of the composition of the invention for the negativity of HPV-positive women showing LSIL or CIN1 by vaginal or cervical administration. This embodiment may also be expressed as a method of negating HPV-positive women exhibiting LSIL or CIN1 by vaginal or cervical administration of the composition of the invention.

In another specific embodiment, the composition of the invention is used to treat vaginal epithelial lesions caused by infectious agents, in particular LSIL or CIN1 lesions caused by HPV. In other words, the compositions of the invention are useful for re-epithelialization of vaginal tissue damaged by infectious agents, particularly for re-epithelialization of LSIL or CIN1 lesions caused by HPV.

Whatever the case above, the composition of the invention can be used in other pathological stages. For example, the compositions of the invention may be administered vaginally or transcervically to women at the low grade squamous intraepithelial lesion stage (HVP or CIN 1) for the prevention of persistent HPV infection and/or re-epithelialization of vaginal lesions. The compositions of the invention may also be used as an adjunct therapy to treat women suffering from HSIL or cancer.

For patients with HPV infection, a typical administration regimen comprises vaginal or cervical administration of the composition of the invention 21 to 75 times over a 6 month period. Thus, in a particular embodiment, the composition is for use in the treatment of HPV infections, wherein the treatment regimen comprises vaginal or cervical administration of the composition of the invention 21 to 75 times over a 6 month period. For patients with other vaginal or cervical infections, particularly recurrent vaginal or cervical infections, a typical administration regimen comprises vaginal or cervical administration of the composition of the invention 21 to 75 times over a 6 month period.

The inventors have found that the optimal administration regimen comprises: (ii) (a) one dose of the composition of the invention per day for a period of 21-24 days, followed by (b) no dose for a period of 3-7 days, followed by (c) one dose of the composition of the invention per day or every two days for a period of 21-24 days, followed by (d) no dose for a period of 3-7 days, and (e) repeating (c) and (d) four times in sequence.

In the present case, the term "dose" generally refers to the amount of a particular drug of a composition of the invention taken at one time. In most embodiments, 0.5 to 5mg of coriolus versicolor extract is included per dose.

In particular embodiments, the administration regimen comprises: a) one dose per day of the composition of the invention during 21-24 days, followed by (b) no dose during 3 to 7 days, followed by (c) one dose per two days of the composition of the invention during 21-24 days, followed by (d) no dose during 3 to 7 days, and (e) repeating (c) and (d) four times in sequence, wherein each dose comprises 0.5 to 5mg of coriolus versicolor extract.

In another particular embodiment, the administration regimen comprises: (ii) (a) one dose of the composition per day for a period of 21 to 24 days, followed by (b) no dose for a period of 3 to 7 days, followed by (c) repeating (a) and (b) twice in sequence, followed by (d) one dose of the composition every two days for a period of 21 to 24 days, followed by (e) no dose for a period of 3 to 7 days, and (f) repeating (d) and (e) twice in sequence, wherein each dose comprises 0.5 to 5mg of coriolus versicolor extract.

In another particular embodiment, the administration regimen comprises: (ii) (a) one dose of the composition per day over a period of 21 to 24 days, followed by (b) no dose over a period of 3 to 7 days, followed by (c) repeating (a) and (b) 5 times in sequence, wherein each dose comprises 0.5 to 5mg of coriolus versicolor extract.

The above-described administration regimen over a period of 6 months may be repeated one, two, three or four times, preferably once, in sequence, so that the entire treatment is as long as one year. If desired, the above treatment can be repeated once, twice, three or four times, preferably once, but with a rest of one to two months (treatment-free months) between each administration regimen.

In a specific embodiment, the doses in the above regimen are administered during 21 days and not administered during 7 days.

In a specific embodiment, each dose of the above administration regimen comprises 1 to 4mg ml of coriolus versicolor extract. Preferably, each dose contains 1.5 to 3.5mg of coriolus versicolor extract. More preferably, each dose contains 2 to 3mg of coriolus versicolor extract.

Compositions comprising coriolus versicolor extract for vaginal or cervical administration are part of the invention. In one embodiment, the composition of the present invention comprises a humectant. The term "moisturizer", also known as "emollient", is used herein in its general meaning in the pharmaceutical and cosmetic field as a compound that increases or maintains hydration of the skin or mucous membranes. In a preferred embodiment, the humectant is hyaluronic acid.

Hyaluronic acid (HA, also known as hyaluronic acid or hyaluronate, CAS number 9004-61-9) is an anionic, non-sulfated glycosaminoglycan, widely distributed in connective, epithelial and neural tissue. HA is a polymer of disaccharides, which themselves consist of D-glucuronic acid and D-N-acetylglucosamine, linked via alternating β -1,4 and β -1,3 glycosidic linkages. The presence of HA in epithelial tissue HAs been shown to promote keratinocyte proliferation and increase the presence of retinoic acid, resulting in skin hydration. The interaction of hyaluronic acid with CD44 promotes collagen synthesis and normal skin function. Hyaluronic acid is present in the extracellular matrix of basal keratinocytes and is critical to the structural integrity of the dermal collagen matrix. These benefits make hyaluronic acid a very effective humectant. Furthermore, HA is described as potentially beneficial for wound repair. When combined with the immunopotentiating activity of the extract of coriolus versicolor in the composition of the present invention, the moisturizing and wound repair activity of HA is very convenient to combat persistent infections of HPV as well as other infections causing e.g. vaginitis. HA is commercially available from a number of sources.

In one embodiment, the composition of the invention further comprises a prebiotic. The term "prebiotic" is used herein in its ordinary sense in the art as a non-digestible food ingredient that stimulates the growth and/or activity of colonizing bacteria of animals in a manner that claims to be beneficial to health. This definition does not emphasize that a particular bacterial population is the target of prebiotics. However, it is generally assumed that prebiotics should increase the number and/or activity of bifidobacteria and lactic acid bacteria, both of which have a variety of beneficial effects on the host, particularly in terms of maintaining a balanced microflora balance and the effectiveness and intrinsic strength of the immune system.

In a preferred embodiment, the prebiotic present in the composition of the invention is

Are alpha-glucan oligosaccharides obtained by enzymatic synthesis from natural sugars (sucrose and maltose). Due to the specificity of the glycoside chain thereof,

is a bioselective substrate for the beneficial microflora which is beneficial to the development of the skin saprophytic microflora and damages the undesirable opportunistic microflora whether pathogenic or not. Such prebiotics are commercially available, for example from Solavia company. Other non-limiting prebiotics contemplated for use in the compositions of the present invention are fructooligosaccharides, galactooligosaccharides and inulin.

In one embodiment, the composition of the invention comprises probiotic microorganisms. It is well known that "probiotics" are microorganisms that provide health benefits upon consumption. In the sense of the present invention, probiotics provide health benefits mainly by improving or restoring the vaginal flora. The probiotic bacteria may be provided as live microorganisms or non-live (e.g. dead) microorganisms. In a particular embodiment, the probiotic is inactive. Methods for rendering microorganisms inactive are known to those skilled in the art. For example, an effective method for rendering microorganisms inactive is tin plating (tinning).

In one embodiment, the probiotic bacteria in the composition of the invention are from the genus Lactobacillus. In a particular embodiment, the probiotic in the composition is selected from the group consisting of Lactobacillus plantarum (Lactobacillus plantarum), lactobacillus paracasei (Lactobacillus paracasei), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus crispatus (Lactobacillus crispatus) and Lactobacillus rhamnosus (Lactobacillus rahmnosus). In another embodiment, the Lactobacillus plantarum is Lactobacillus plantarum, the Lactobacillus paracasei is Lactobacillus paracasei Nu02 or Lactobacillus paracasei Nu-09, the Lactobacillus acidophilus is Lactobacillus acidophilus Nu-03 and the Lactobacillus rhamnosus is Lactobacillus rhamnosus Nu-04. In another embodiment, the composition comprises Lactobacillus plantarum Nu-06, lactobacillus paracasei Nu-02, lactobacillus paracasei Nu-09, lactobacillus acidophilus Nu-03, and Lactobacillus crispatus is Lactobacillus crispatus Nu-04.

The probiotic Lactobacillus strains described above are commercially available and sold by Nutris Ingredients SL. For example, nu-06, nu-02, nu-09, nu-03, nu-04 strains are included in the product NuProbi sold by this company

In (1). It has been found that the combination of coriolus versicolor extract with the above mentioned probiotics is particularly effective for the prevention/treatment of vaginal or cervical disorders such as those caused by HPV infections.

In one embodiment, the composition of the invention further comprises a tissue regeneration agent. By "tissue regeneration agent" is understood a compound that can promote the renewal, recovery and/or growth of body tissue, in particular damaged body tissue. The term includes, in a non-limiting manner, compounds that promote wound healing ("wound healing agents"), scarring ("scarring agents"), and compounds that enhance angiogenesis ("angiogenic agents").

In a preferred embodiment, the tissue regeneration agent is an extract of Centella asiatica (Centella asiatica). Centella asiatica (also known as centella asiatica (gotu kola) and Indian penywort) is a perennial creeping plant that grows around the Indian ocean. It is traditionally used locally to manage skin conditions, it is used to support faster healing of small wounds, scratches and superficial burns, and anti-inflammation for eczema, minor itching and insect bites. Tissue regeneration activity of centella asiatica is particularly relevant for repairing disturbed epithelial barriers (major wounds and/or minor abrasions) in the vagina and/or cervix to avoid and/or combat persistent viral infections (e.g. HPV). In this sense, the combination of a tissue regeneration agent (e.g. centella asiatica extract) with coriolus versicolor and optionally other active ingredients is particularly effective. Thus, in a particular embodiment, the present invention provides a composition comprising coriolus versicolor extract and centella asiatica extract for vaginal or cervical administration to repair a disturbed vaginal or cervical epithelial barrier. Another specific embodiment provides a composition comprising coriolus versicolor extract and centella asiatica extract for use in the prevention and/or elimination of vaginal and/or cervical persistent infections, in particular persistent HPV infections, caused by infectious pathogens as defined above, by vaginal or cervical administration.

"centella asiatica extract" refers to an extract obtained from centella asiatica that contains high concentrations of biologically active compounds, in particular pentacyclic triterpenoids. The triterpenes contained in these extracts are asiatic acid, madecassic acid and asiaticoside. As used herein, "centella asiatica extract" includes any of the available centella asiatica extracts, as well as the biologically active compounds from centella asiatica or purified or semi-purified preparations of specific biologically active compounds obtained from centella asiatica.

Published clinical studies describe the use of the following centella asiatica extracts: TTFCA, TECA and where the commercial extract names are mentioned,

(titrated extract of centella asiatica) or

(Total triterpenoid component of Centella asiatica) (EMA/HMPC/291177/200, 2010. "Association report on Centella asiatica (L.) -Urban, herba"). Studies have shown that the main component of centella increases collagenWhite synthesis, thereby facilitating tissue repair and angiogenesis.

In one embodiment, the composition of the invention further comprises an antiviral agent. An "antiviral agent" is understood in its general sense as a compound that can destroy or inhibit the development of a virus.

In a preferred embodiment, the antiviral agent is an extract of neem (Azadirachta indica). "Azadirachta indica," also known as Neem, nimtree (Nimtree) and Syringa oblata (Indian Lilac), is a tree of the family Meliaceae (family Meliaceae) mahogany (mahogany).

Various preparations obtained from different parts of neem have been found to exert antibacterial, antiviral, antimalarial, antioxidant, antifungal, antimutagenic, anticancer, contraceptive and antiulcer activities. Previous reports have documented that neem extract significantly inhibits poliovirus, HIV, coxsackie group B virus and dengue virus at an early stage of viral genome replication (Tiwari V, et al Phytother res.2010, vol.24 (8), p.1132-1140).

The main chemical components are azadirachtin, azadirachene (nimbinene), azadirachtin, azadirachtol (azadirachtol), azadirachnol (azadirachnol), deacetyl azadirachene (desacetonitrine), azadirachdiol (nimbiol), azadiracholide, quercetin, beta-sitosterol, n-hexacosanol, azadirachol, nimoracin (nimocin). As used herein, "neem extract" includes any of the available neem extracts, as well as a purified or semi-purified preparation of a biologically active compound from neem or one particular biologically active compound obtained from neem. These extracts are commercially available (e.g. available from Symrise).

In one embodiment, the composition of the invention further comprises an anti-inflammatory agent. An "anti-inflammatory agent" is understood in its general concept as a compound that reduces inflammation. Preferably, the compositions of the present invention comprise anti-inflammatory agents derived from natural sources. In a particular embodiment, the anti-inflammatory agent is derived from magnolia. In a preferred embodiment, the anti-inflammatory agent is Magnolol (Magnolol), honokiol (honoliol), or a mixture thereof. Magnolol (4-Allyl-2- (5-Allyl-2-hydroxy-phenyl) phenol) (magnol (4-Allyl-2- (5-Allyl-2-hydroxy-phenyl) phenol)) and honokiol (2- (4-hydroxy-3-propyl-2-alkenyl-phenyl) -4-propyl-2-alkenyl-phenol)) (honokiol (2- (4-hydroxy-3-prop-2-enyl-phenyl) -4-prop-2-enyl-phenol)) (Magnolol), which has been described as inhibiting nuclear factor kappa (kappa) -light chain enhancer (NF-kB) of activated B cells and also down-regulating the production of important inflammatory mediators such as interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha).

In one embodiment, the composition according to the invention comprises, in addition to the coriolus versicolor extract, probiotics, humectants, prebiotics, anti-inflammatory agents, tissue regeneration agents and optionally antiviral and/or anti-inflammatory agents. In a particular embodiment, the composition of the invention comprises hyaluronic acid, in addition to the coriolus versicolor extract,

NuProbi

And centella asiatica extract. In another specific embodiment, the composition of the invention comprises hyaluronic acid, in addition to the coriolus versicolor extract,

NuProbi

Centella asiatica extract and neem tree extract. In another specific embodiment, the composition of the invention comprises hyaluronic acid, in addition to the coriolus versicolor extract,

NuProbi

Centella asiatica extract, neem tree extract magnolol, and honokiol. In thatIn another embodiment, the composition of the invention additionally comprises carboxymethyl β -glucan.

In certain embodiments, the compositions of the present invention comprise coriolus versicolor extract and neem tree extract. The compositions of the present invention may also conveniently comprise a probiotic. In certain embodiments, the compositions of the present invention comprise a coriolus versicolor extract and at least one active compound selected from the group consisting of probiotics, neem tree extract, additional beta-glucans (other than PSP and PSK beta-glucans contained in the coriolus versicolor extract), and combinations thereof. In some embodiments, the composition comprises coriolus versicolor extract, neem tree extract, additional beta-glucans (such as carboxymethyl beta-glucan), probiotics, and at least one additional active ingredient selected from moisturizers, prebiotics, anti-inflammatory agents, tissue regeneration agents, and aloe vera. In some embodiments, the composition comprises coriolus versicolor extract, neem tree extract, additional beta-glucan (such as carboxymethyl beta-glucan), probiotic bacteria, and at least one additional active ingredient selected from hyaluronic acid, alpha-glucan oligosaccharides, magnolol, honokiol, centella asiatica extract, and aloe vera. In a particular embodiment, the composition of the invention comprises coriolus versicolor extract, neem leaf extract, carboxymethyl β -glucan, probiotics, magnolia bark, and honokiol. In a very specific embodiment, the composition of the invention comprises coriolus versicolor extract, neem leaf extract, carboxymethyl β -glucan, probiotics, hyaluronic acid, α -glucan oligosaccharides, magnolol, honokiol, centella asiatica extract, and aloe vera. The additional beta-glucan may be selected from carboxylated beta-glucans, beta (1, 3) D-glucans, and beta (1, 3) (1, 4) -glucans from several natural sources. In a particular embodiment, the additional β -glucan is carboxymethyl β -glucan. The probiotic bacteria may be selected from the group consisting of lactobacillus plantarum Nu06, lactobacillus paracasei Nu133, lactobacillus paracasei Nu653, lactobacillus acidophilus NuRF-2, lactobacillus rhamnosus NuRF-1, and combinations thereof.

The synergistic combination of the compounds in the above compositions determines that they are particularly effective for the treatment and/or prevention of vaginal or cervical conditions caused by infectious agents, in particular vaginitis and conditions caused by HPV infection such as cervical cancer. In a particular embodiment, the composition as defined above is for use in repairing a disturbed vaginal or cervical epithelial barrier by vaginal or cervical administration. Another particular embodiment provides a composition as defined above for the treatment of vaginal lesions caused by infectious agents, in particular LSIL or CIN1 lesions caused by HPV, by vaginal or cervical administration. Another particular embodiment provides a composition as defined above for use in the prevention and/or elimination of vaginal and/or cervical persistent infections, in particular persistent HPV infections, caused by infectious agents by vaginal or cervical administration.

In addition to the coriolus versicolor extract, other ingredients that may be incorporated into the compositions of the present invention are green tea leaf extract (or any of its well-known bioactive compounds), melaleuca alternifolia (or any of its well-known bioactive compounds) extract and aloe vera.

The present invention provides compositions comprising an effective amount of a coriolus versicolor extract and/or an effective amount of a biologically active chemical component and/or compound isolated from coriolus versicolor, preferably coriolus versicolor glycopeptide PSK and/or PSP. In a particular embodiment, the composition of the invention also contains an effective amount of at least one of the above-mentioned additional active ingredients (probiotics, moisturizers, prebiotics, anti-inflammatory agents, tissue regeneration agents and/or antiviral agents). The term "effective amount" as used herein refers to an amount of a compound that, when administered, is sufficient to prevent the development of, treat or alleviate to some extent the condition being addressed, which in the present invention is a vaginal or cervical condition caused by an infectious agent. In the sense of the present invention, an "effective amount" is also understood as an amount of the compound which is sufficient to enhance the vaginal or cervical immunity. For example, an effective amount of the coriolus versicolor extract in the composition may comprise 0.005 to 5%, or 0.01 to 1%, or 0.01 to 0.5%, such as 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, or 0.4%. An effective amount of a viral agent such as neem extract in a composition may comprise 0.001 to 5%, or 0.005 to 1%, or 0.005 to 0.1%, for example 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 or 0.09%. An effective amount of other β -glucans, such as carboxymethyl β -glucan, in the composition may comprise 0.01 to 10%, or 0.025 to 1%, or 0.05 to 0.5%, for example 0.08, 0.09, 0.1, 0.2, 0.3 or 0.4%. An effective amount of prebiotic, such as an alpha-glucan oligosaccharide, in the composition may comprise 0.01 to 10%, or 0.05 to 5%, or 0.1 to 1%, for example 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9%. The amount of humectant, such as hyaluronic acid, in the composition may comprise 0.01 to 10%, or 0.05 to 5%, or 0.1 to 1%, for example 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9%. An effective amount of an anti-inflammatory agent such as magnolol and/or honokiol in a composition can comprise 0.005 to 5%, or 0.01 to 2%, or 0.05 to 1%, e.g., 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9%. An effective amount of a tissue regenerating agent, such as a component derived from an extract of centella asiatica (ingredients), particularly asiaticoside, madecassic acid and/or asiatic acid, in the composition may comprise 0.001 to 5%, or 0.005 to 1%, or 0.005 to 0.1%, for example 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 or 0.09%. The composition may comprise aloe in an amount comprising 0.1 to 10%, or 0.5 to 5%, for example 0.8, 1, 1.5, 2, 3 or 4%. An effective amount of probiotic in the composition may be 0.05% to 10%, or 0.1% to 5%, or 0.2% to 3%, for example 3%, 2%, 1.5%, 1%, 0.5% or 0.25%.

The particular dose of the compound administered according to the present invention will, of course, be determined around the particular circumstances of the condition, including the compound administered, the encapsulation efficiency, the route of administration, and similar considerations.

As mentioned above, for the treatment of conditions caused by infectious pathogens such as HPV by the vaginal or cervical route, a convenient dose may comprise 0.5 to 5mg of coriolus versicolor extract, preferably 1 to 4mg or 1.5 to 3.5mg. More preferably, the dose comprises 2 to 3mg of coriolus versicolor extract. When other active ingredients are present in the composition, the amount of neem extract (if present) per dose may be 0.1-1mg, the amount of carboxymethyl β -glucan (if present) per dose may be 1.8-18mg, the amount of α -glucan oligosaccharides (if present)In) may be 5-50mg per dose, hyaluronic acid (if present) may be 0.24-2.4mg per dose, centella asiatica extract (if present) may be 0.3322-3.322mg per dose, and aloe vera (if present) may be 0.05-0.5mg per dose. The total amount of probiotic (if present) per dose may be 30 to 100mg (from 10 per gram of probiotic) ⁶ To 10 ⁸ One cell)

The compositions of the invention are for vaginal or cervical administration. Thus, the compositions of the present invention are preferably topical compositions.

Topical (including cervical or vaginal) formulations may be prepared according to methods well known in the art. Suitable carriers and amounts thereof can be readily determined by those skilled in the art depending on the type of formulation being prepared. Exemplary compositions for vaginal or cervical administration according to the invention may comprise: water, aloe barbadensis leaf juice, glycerin, propylene glycol, hydroxyethyl cellulose, hydrolyzed hyaluronic acid, sodium carboxymethyl β -glucan, α -glucan, oligosaccharides, coriolus versicolor extract, neem leaf extract, watermelon seed oil polyglycerin-6 ester, hazelnut oil polyglycerin-6 ester, almond oil polyglycerin-6 ester, kaempferol, polyglycerin-10 dilaurate, lecithin, magnolol, honokiol, polyglycerin-10 oleate, sorbitan, palmitate, dicetyl phosphate, sodium benzoate, potassium sorbate, lactic acid.

Preferably, the compositions of the present invention comprise suitable excipients and/or carriers for topical application, which may be topically acceptable pharmaceutical or cosmetic excipients and carriers generally known in the art. As used herein, "topically acceptable" refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the skin and/or mucous membranes of human and/or non-human animals without undue toxicity, incompatibility, instability, allergic response, and the like. Each "topically acceptable" carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Such excipients and carriers include, but are not limited to, agents that repair the barrier function of the skin, hydrating agents, emollients, emulsifiers, thickeners, humectants, pH adjusters, antioxidants, preservatives, vehicles (vehicles), or mixtures thereof. The excipients and/or carriers used have an affinity for the skin or mucus, are well tolerated, are stable, and are used in amounts sufficient to provide the desired consistency, and are easy to apply. In addition, the composition may comprise other ingredients such as perfumes, colorants and other ingredients known in the art for use in topical formulations.

The topical composition may be adapted for application to the skin and mucosa in the form of: non-ionic vesicle dispersions, emulsions, creams, emulsions, gels, aerosols, cream-gels, gel-creams, suspensions, dispersions, powders, solid sticks, wipes, poultices, foams, sprays, oils, ointments, liquids, soaps, sanitary napkins, ovules, pessaries, or any other form known in the cosmetic and pharmaceutical arts. In a particular embodiment, the composition of the invention is formulated as a gel and administered vaginally or transcervically via a cannula. In other embodiments, the compositions of the present invention are formulated as ovules. In other embodiments, the compositions of the present invention are formulated as pessaries.

The composition of the present invention may be suitably used for pharmaceutical products, cosmetics or hygiene products.

The effectiveness of the compositions of the present invention can be enhanced by encapsulating the active compound in liposomes.

The term "liposome" is to be understood as a self-assembled structure comprising one or more membranes consisting of bilayers, each of which comprises two monolayers comprising oppositely oriented amphiphilic molecules. Amphiphilic molecules can be polymers or lipids that comprise a polar (hydrophilic) head group region covalently linked to one or more non-polar (hydrophobic) chains. Energetic (energetically) unfavorable contact between hydrophobic acyl chains and the surrounding aqueous medium induces self-alignment of amphiphilic lipid molecules with their polar head groups oriented towards the bilayer surface, while the acyl chains are reoriented to the interior of the bilayer. Thus forming an energy stable structure in which the acyl chain is effectively protected from the aqueous environment.

The liposome encapsulates the aqueous region within a hydrophobic membrane; dissolved hydrophilic solutes cannot readily pass through lipids. Hydrophobic chemicals can be dissolved into the membrane and in this way liposomes can carry both hydrophobic and hydrophilic molecules. To deliver the molecule to the site of action, the lipid bilayer may be fused with other bilayers (e.g., cell membranes) to deliver the liposome contents. The use of liposomes has a wide range of advantages in cosmetics and pharmaceuticals, including increased biodegradability, biocompatibility, and prolonged release and reduced toxicity and side effects of encapsulated active agents. A further advantage is that compositions comprising liposome-encapsulated active agents generally exhibit an extended shelf life, since the liposomes protect the active agent from degradation.

Thus, in a particular embodiment, one or more of the coriolus versicolor extract and/or the additional active compound(s), if present, in the composition of the invention are encapsulated in liposomes.

Any type of liposome known in the art may encapsulate the active compounds in the compositions of the present invention. Particularly suitable types of liposomes are niosomes, phospholipid complexes and nanoparticles. Thus, in another particular embodiment, one or more of the extracts from coriolus versicolor and/or the additional active compounds comprised in the composition of the invention are encapsulated into niosomes, phospholipid complexes and ultrafine particles.

"Liposomes" are non-ionic surfactant-based liposomes. The lipoidal vesicles are formed primarily by cholesterol incorporation as an excipient, although other excipients may also be used. They are microscopically sized lamellar structures composed of nonionic surfactants of the alkyl or dialkyl polyglyceryl ether type and cholesterol, and are subsequently hydrated in an aqueous medium. The materials used for the preparation of the niosomes make them more stable, so that the niosomes have a number of advantages, for example, they have a very high penetration capacity. Liposomes containing several bioactive compounds are commercially available. For example, hyaluronic acid sold by Naturalis Life Technologies IncAcid lipid vesicle (NIO-Oligo)

). The same supplier sells Nio-Glucan lipoidal vesicles containing carboxymethyl β -Glucan (a water-soluble Glucan derivative), magnolol, and a mixture of honokiol.

A "phospholipid complex" is a complex of a natural active ingredient and a phospholipid, preferably phosphatidylcholine, usually also containing polyphenols, in appropriate molar ratios. Phospholipid complexes are solid dispersions of a natural active ingredient or a mixture of active natural ingredients (e.g., a plant extract) in a dietary phospholipid matrix (e.g., lecithin from soy). When encapsulated within a phospholipid complex, the active ingredient may correspond in some way to a component of the lipid membrane. Several natural active ingredients are commercially available in the form of phospholipid complexes, for example, centella, which contains triterpenes from Centella asiatica, is readily available from Indena

Also sold by the same supplier

It contains polyphenols from green tea leaves.

"ultrafine particles" are nanoscale single-bilayer liposomes containing a high percentage of Phosphatidylcholine (PC). The ultrafine particles effectively penetrate into the skin by topical application, facilitating the delivery of the active compounds encapsulated therein to skin cells.

According to the invention, the active compound can be contained directly in the composition or can be added in the form of liposomes. The invention also contemplates that some active compounds are included in the composition in the form of liposomes while others are not. Among the active compounds included in the composition in the form of liposomes, any type of convenient liposomes is contemplated, such as, for example, lipoidal vesicles, phospholipid complexes or ultramicroparticles, or mixtures thereof.

In a particular embodiment, the composition of the invention comprises coriolus versicolor extract, nio-Oligo

Centella

And optionally, neem tree extract and/or

Throughout the description and claims the word "comprise" and variations of the word are not intended to exclude other technical features, additives, components or steps. Furthermore, the term "comprising" encompasses the case of "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following drawings are provided by way of example and are not intended to limit the present invention. Moreover, the present invention encompasses all possible combinations of the specific and preferred embodiments described herein.

Examples

1. Composition for vaginal or cervical administration comprising coriolus versicolor extract

A gel for vaginal or cervical administration is formulated comprising 0.5% alpha-glucan oligosaccharide, 0.18% sodium carboxymethyl beta-glucan, 0.24% hydrolyzed hyaluronic acid, 0.05% coriolus versicolor extract, 0.01% neem extract, 1% aloe barbadensis leaf juice.

The composition is in the form of a gel and can be administered intravaginally or intracervically via a cannula.

2. In vitro study

The study was designed to investigate whether the compositions of the invention are capable of modulating the immune response of vaginal epithelial tissue by assessing the cytokine release from the tissue into the basal compartment after a defined treatment period. 3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (3- (4, 5-dimethylthizol-2-yl) -2,5-diphenyltetrazolium bromide) (MTT) measurements were also used to assess tissue viability.

Test system

Use of a reconstructed three-dimensional human vaginal epithelium model, epiVaginal ^TM VLC-100-FT (MatTek) was tested. The RhVE model EpiVaginal VLC-100-FT (MatTek) consists of normal human VEC epithelial cells cultured on an intrinsically laminar collagen matrix containing fibroblasts and Dendritic Cells (DCs). Thus, the test system represents an in vitro target organ of the species of interest and closely mimics the biochemical and physiological properties.

Material

The following gels were tested:

PAPILOCARE GEL composition of example 1 (P-7447)

The VEHICLE gel composition of example 1 contained no active ingredients: coriolus versicolor extract, azadirachta indica extract, carboxymethyl beta-glucan, magnolol and honokiol (P-7450).

VEHICLE +0.05% Coriolus versicolor extract (P-7451)

VEHICLE +0.01% Azadirachta indica extract (P-7452)

VEHICLE +0.18% carboxymethyl beta dextran sodium, 0.075% magnolol, 0.075% honokiol (P-7453)

LPS control (1. Mu.g/mL lipopolysaccharide, sigma catalog # L4391)

EpiVaginal ^TM Tissues were provided in a kit (VLC-100-FT, matTek) consisting of the following components relevant to this study:

1 Xsealed 24-well plate containing 24 inserts organized on agarose

1 bottle of assay Medium (DMEM-based Medium)

5. Other reagents

MTT solution:

-MTT stock solution: 3mg/mL MTT in Phosphate Buffered Saline (PBS)

-MTT medium: MTT stock solution is diluted 1+9 (final concentration 0.3 mg/mL) with DMEM medium

Isopropanol (I-propanol)

Method

a) Preliminary experiments

To examine the nonspecific MTT-reducing ability of the test article, 50mg of the test article was mixed per 2mL of MTT medium and incubated at 37. + -. 1 ℃ for 3 hours in the absence of light. If the mixture turns blue/purple, it is assumed that the MTT of the test article decreases. For quantitative correction of the results, the fraction of absorption due to nonspecific reduction of MTT was determined by using killed tissue. A test article is considered incompatible with the test method if the non-specific MTT is reduced by >30% relative to a negative control of viable epidermis.

To examine the coloring potential of the test article, 50mg of the test article was mixed in a transparent container for 15 minutes per 90. Mu.L of distilled water (aqua dest). The standard followed is that if staining is detected by visual assessment, the test article is examined for its tissue staining potential for quantitative correction of the results by using other biopsies without MTT staining. A test article is considered incompatible with the test method if the non-specific OD due to color development is >30% relative to the negative control.

b) Experimental procedures

Upon receipt, the tissues were transferred to 24-well plates containing 0.6mL of pre-warmed maintenance medium per well. The 24-well plate was subjected to 37. + -. 1 ℃ and 5.0% CO ₂ The humidified incubator below was incubated for at least 1 hour and up to 24 hours. The medium was then replaced with 0.6ml of fresh medium. Weigh 50. + -.2 mg (131.5 mg/cm) ² ) The amount of test article and applying it to the epithelial surface of each individual tissue. The test article was gently applied by repeated circumferential movements of the pipette to evenly cover the tissue surface but avoid any direct contact with the pipette tip. The final nylon mesh is ready for unrolling.

6h of experiment: starting from the negative control, tissues were treated with each dose group. The start time will be recorded with the administration of the first tissue. Then the tissue was subjected to 37. + -. 1 ℃ and 5.0% CO ₂ Incubate for 6 h. + -. 10min.

24h experiment: starting from the negative control, tissues were treated with each dose group. The start time was recorded with the first tissue dose. The tissue was then incubated at 37. + -. 1 ℃ and 5.0% CO2 for 24h. + -.1 h.

After the respective incubation periods, the media in contact with the tissue substrate surface was preserved and stored at-80 ℃ for further cytokine release analysis.

The tissue is then washed using, for example, a wash bottle that is gently rinsed about 15 times with 25mL PBS to remove any residual test article. Excess PBS was removed by gently shaking the insert and blotting the bottom with blotter paper. All inserts were treated in the same manner.

The treated tissue was then transferred to a prepared 24-well plate containing 2mL of preheated MTT medium and further processed at 37. + -. 1 ℃ 5.0% ₂ Incubate for 3h + -5 min.

After a 3 hour MTT incubation period, the tissue was placed on blotter paper to dry the tissue. Thereafter, a total biopsy of the epithelial cells is performed by a special biopsy using a punch. All tissue portions were transferred to appropriate tubes and 500 μ L isopropanol was added. The extraction is carried out at 2-8 ℃ in the dark, or at room temperature for at least 4 hours, throughout the weekend, with vortexing of each tube in the middle of the incubation period.

If any visible cell/tissue debris is in suspension, the tube is centrifuged at 300Xg to eliminate debris and avoid possible interference with the absorbance readings.

A 2x200 μ Ι aliquot of each tissue extract was transferred to a 96-well plate and OD would be measured in a flat-plate spectrophotometer without a reference wavelength at 570 nm.

Cytokine assays were performed according to the manufacturer of the Kit (V-PLEX Proinfilfamat Panel1 (hum) Kit, meso Scale Discovery, cat. No: K15049D-1) and the cytokines evaluated were IFN-. Gamma.IFN-. Beta.IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF-. Alpha..

As a result, the

a) Irritative potential of test article

After exposure times of 6h and 24h, no cytotoxic effect was observed when the tissues were treated with PAPILOCARE GEL (see table 1). Viability of treated tissue > 50% of negative control treated with PBS (105% and 122% at 6 and 24, respectively). Tissue treated with P-7450 (VEHICLE) showed lower viability of the treated tissue at 6h and 24h compared to papiloreal GEL, but it was considered non-irritating as viability was also > 50% of the negative control (93% and 70% at 6 and 24, respectively).

These results may indicate that neither papiloreal GEL nor VEHICLE exhibit stimulatory effects.

TABLE 1 viability of human vaginal epithelial cells

B) Immunomodulating effects

The cytokines evaluated are the pro-inflammatory group, which includes IFN- γ, IFN. β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF- α.

As shown in Table 2, an increase in cytokine levels was observed after treatment with positive control LPS at both concentrations tested (1 and 10. Mu.g/ml). In most cases, the levels of cytokines induced by papiloreal GEL were similar to the positive control LPS at 1 μ g/ml. In all cases, cytokine levels were lower in tissues treated with P-7450 (VEHICLE).

TABLE 2 cytokine levels in human vaginal epithelium

Conclusion

In tissues treated with PAPILOCARE GEL P-7447 and positive control LPS, a significant increase in cytokine levels was observed at 24 hours compared to 6 hours, indicating an increase in immunostimulatory effect with treatment time, up to 24 hours. No change in P-7450VEHICLE was observed between 6 hours and 24 hours. Synergistic interactions were observed between the assayed active ingredients of PAPILOCARE GEL.

3. Overview of Security

3.1 vaginal irritation test

Biological evaluation according to ISO 10993-10: irritation and skin sensitization test the vaginal irritation of the composition of example 1 was determined. The results show that the vaginal irritation index of the composition is 0.00. Thus, the composition of example 1 is not considered irritating to the vaginal mucosa.

3.2 cytotoxicity assays

Biological evaluation of medical devices according to ISO 10993-5: in vitro cytotoxicity assay, the composition of example 1 was tested for in vitro cytotoxicity. The results show that the mammalian fibroblasts, ATCC BalbC 3T3, treated with the composition of example 1 comprising the coriolus versicolor extract showed a 97.92% reduction in cell viability. Thus, the composition is considered to be cytotoxic.

3.3. Hypersensitivity test

Biological evaluation of medical devices according to ISO 10993-10: irritation and skin sensitization test the skin hypersensitivity of the guinea pigs with the composition of example 1 was determined. No skin reactions were observed in the animals after challenge with the composition of example 1 containing coriolus versicolor extract. These results indicate that the composition of example 1 can be considered non-allergenic.

4. In vivo studies

4.1. Immunomodulation in mice

The aim of this study was to evaluate the immunomodulatory properties of a composition comprising coriolus versicolor extract after topical administration in the vagina of mice. This was done in healthy mice. Local and systemic immune responses were evaluated. Two different compositions were used, GEL1 (PAPILOCARE GEL with complete final formulation as disclosed in example 1) and GEL 2 (formulation different from that disclosed in example 1, except that the concentration of coriolus versicolor extract was 0.25%).

The present study was conducted according to the National institutes of Health, guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Local ethical Committee of Grandda University (Local Ethic Committee of the University of Grandda).

The study was performed in healthy mice, obtained from Janvier (St Berthevin center, france), and raised in a makrolon cage (5 mice per cage), and kept in an air-conditioned atmosphere with 12-h light-dark cycle, and free access to tap water and food was available. Mice were randomly assigned to different experimental groups. Female CBA/J (J-2 α) healthy mice (8 to 10 weeks old) of different groups (n = 7) were vaginally inoculated with 50 μ l of GEL1 or GEL 2 daily. Control group (n = 6) was inoculated with PBS (control). Mice were sacrificed 6 and 10 days after treatment to assess the effect of treatment.

The vagina was excised for biochemical assays and the local immune response was assessed by RT-qPCR assay of different cytokines (TNF α, IL-1 β, IL-6, IL-12, IL-17) (Strum J et al, curr protocol pharmacol, 2002, chapter 6.9), which will detect mRNA copy amounts of each gene of interest. The technique involves the amplification of mRNA using a DNA template of the gene of interest to detect different transcription patterns of the gene being analyzed. mRNA is first reverse transcribed into cDNA and then amplified. This is detected when the reaction is run "in real time". To normalize the measurements, the target gene is studied in association with another gene, called the normalization gene, which is selected for its nearly constant expression level. Constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The data thus generated were analyzed by computer software to calculate relative gene expression (or mRNA copy number) in several samples.

As a result:

the results of qPCR showed that all cytokines tested (TNF α, IL-1 β, IL-6, IL-12, IL-17) were significantly increased (p < 0.05) in mice vaccinated with GEL1 and mice vaccinated with GEL 2 on

days

10 and 6, compared to the control group (FIG. 2). This indicates that the composition comprising coriolus versicolor extract at concentrations of 0.05% and 0.25% activates the immune response of vaginal epithelial cells in healthy mice.

The immune response elicited by coriolus versicolor extract (alone or in combination with other β -glucans) is non-specific. The non-specific immune system, also known as the innate immune system, provides immediate defense against infection and includes both humoral and cell-mediated immune components. The primary function of the innate immune system involves the recruitment of immune cells to the site of infection through the production of chemical factors, including cytokines. In this sense, although non-specific, the local non-specific immune response elicited by the compositions of the present invention in healthy individuals/tissues can be extrapolated to infected individuals/tissues.

4.2. Vaginal condition and cervical epithelialization in women.

The objective was to evaluate the effect of PAPILOCARE GEL on the following 12 days after GEL application: a) a change in vaginal microbial state, b) the degree of re-epithelialization of the cervical mucosa and c) vaginal health.

Method

Design of: an open-trial, non-comparative, prospective and pilot clinical study was conducted in two gynaecological clinics of madrid and barcelona (spain) under daily practical conditions. The purpose of this study was to evaluate the effect of PAPILOCARE GEL on the following parameters: a) epithelialization of cervical lesions (reversal of cervical ectropion), b) composition of vaginal microbiota, and c) vaginal health. These studies were conducted on the principle of protection of human subjects according to the Declaration of Helsinki and with oral informed consent of all participants. According to spanish regulations, studies with the following characteristics do not require ethical approval: real life, pilot studies, class I medical devices (non-drugs) have been marketed and used within the approved indications and are sponsored by major researchers rather than by private pharmaceutical companies.

Participants: women who participated in routine gynecological examinations, had no signs and symptoms of vaginal disease and were age between 18 and 45 years of age for which papanicolaou tablets were normal were included in the study. Epithelialization scores between 4 and 1 were also inclusion criteria. Exclusion criteria included vaginal infections, use of vaginal products other than the study compound, and pregnancy or lactation. Women with a history of disease or complications that the investigator considered disqualified were also excluded from the study.

Study procedure: women who agree and meet inclusion criteria are instructed to use PAPILOCARE vaginal gel correctly according to the manufacturer's information manual provided with the gel. The medical device is recommended to be used before sleep between periods, once a day and for 12 consecutive days. For sexual activity including use of condomWithout any limitation. No irrigation fluid or vaginal odor control agent is allowed. The vaginal gel was provided to all women for free throughout the treatment period. Participants were consulted at baseline (visit 1, inclusion study) and 12 days after treatment (visit 2). In two visits, the degree of epithelialization, vaginal microbiota composition, lactobacillus spp concentration, vaginal pH and vaginal health index were evaluated.

Evaluation of: the extent of cervical mucosal epithelialization was assessed by standard colposcopy and was rated by investigators using an ectopic epithelialization score, where 5 is no ectopy, 4: mild (<25% outer orifice), 3: moderate (25-50% external orifice), 2: severe (A) to (B)>50% outer orifice) and 1: severe ectopic and bleeding.

Vaginal microbiota was tested using VaginaStatus-Diagnostic (Institutet fur)

Herborn, germany) and further rated by investigators using a 5 point litterb scale, where 1 is a very severe deterioration of vaginal microbiota (all species evaluated changed); 2: severe exacerbations (changes in Candida spp.) or mycoplasma or more than three but not all species were detected); 3: moderate exacerbations (alteration of three species, but not the presence of candida, mycoplasma, atropisum vaginalis (Atopobium vagianae), escherichia coli, and gardnerella vaginalis); 4: mild exacerbations (1 or 2 species changes but no candida, mycoplasma, atopobacter vaginalis, escherichia coli, and gardnerella vaginalis may be present); 5: and (4) normal. Lactobacillus concentrations and pH values were also measured.

Vaginal health indices have also been evaluated (Bachman G.U. "physiological or biological administration: an old prophylactic newcastle", maturitas 1995, dec 22Suppl. The vaginal health index is a system for assessing vaginal elasticity, fluid volume, pH, epithelial integrity and moisture, and is rated on a scale of 1 to 5. For logistical and economic reasons, vaginal health index and vaginal microbiota were evaluated in only 11 patients at the barcelona checkpoint.

Statistical analysis: the sample volume calculation is not mandatory due to the exploratory nature of the study. Categorical variables are expressed as numbers and percentages, and quantitative variables are expressed as means and Standard Deviations (SD). The Wilcoxon signed rank test is used to compare pairs of samples of consecutive data. Data were analyzed using the 2011 version of the Power Analysis and Sample Size software program.

Results

Epithelialization of the cervix: a total of 21 women with an average age of 32.6 years (range 20-43 years) were involved in the assessment of exocervical epithelialization. Treatment with coriolus versicolor-based vaginal gel showed a positive effect in improving re-epithelialization of cervical mucosa with an average score at the last visit of 4.42 (P) compared to 3.09 at baseline<0.0001 And an overall improvement of 43% (fig. 3). Furthermore, at the end of the study, 95.3% of women showed an improvement in the degree of epithelialization, of which a score of 5 was observed in 11 women (52.4%) (table 3). Changes in colposcopy images before and after treatment are shown in figure 4.

TABLE 3A colposcopy epithelialization assessment results (CER score)

Vaginal health: a total of 11 women between the ages of 25 and 43 years (mean age 32.6 years) were included in the vaginal health assessment. After 12 days of treatment, the vaginal health index increased significantly from 19.0, the mean at baseline, to 22.3 (P = 0.007) at the last visit (fig. 3). The concentration of lactobacillus also increased in 6 patients (54.5%), and no change was observed in1 patient. In the remaining 3 women, a reduction in lactobacillus concentration was found. The mean vaginal pH showed a downward trend (4.32vs. 4.09, p = ns) (table 3B).

TABLE 3B results of vaginal microbiota

Vaginal health: a total of 11 women between the ages of 25 and 43 years (mean age 32.6 years) were included in the vaginal health assessment. After 12 days of treatment, the vaginal health index increased significantly from an average of 19.0 at baseline to 22.3 (P = 0.007) at the last visit (fig. 3). The concentration of lactobacillus also increased in 6 patients (54.5%), and no change was observed in1 patient. In the remaining 3 women, a reduction in lactobacillus concentration was found. The mean vaginal pH showed a downward trend (4.32vs.4.09, p = ns).

In summary, the results show that the PAPILOCARE vaginal gel based on coriolus versicolor has a clear beneficial effect on the epithelialization of the ectocervix, vaginal microbiota and vaginal health.

4.3. Cervical epithelialization in women.

This study was performed on 5 healthy women (i.e. without clinical vaginal symptoms). Each patient received PAPILOCARE GEL for a period of 12 days, once per day in the evening. The effect on cervical mucosal epithelialization was assessed by standard colposcopy. Colposcopic lesions were rated using the CER score (see above).

The results in Table 5 show the positive effect of PAPILOCARE GEL in improving cervical mucosal re-epithelialization with a 75% increase in CER score (baseline vs final: 2.4vs 4.2). A total of 2 out of 5 women showed complete recovery (CER score = 5).

TABLE 5 results of colposcopic evaluation (CER score)

Fig. 4 shows the changes observed in the cervical mucosa. The dark areas correspond to the epithelialization areas.

4.4. Clinical trial PAPILOCARE GEL

This is a multicenter, randomized, prospective, open-test, parallel-group and routine clinical practice control clinical trial to evaluate the efficacy of the composition according to the invention (PAPILOCARE GEL) defined in example 1 to repair the female cervical mucosa with HPV-related pap smear change and consistent colposcopic images.

Patients in the cohort were randomized into three groups (1:

papilocare, dosimetry a:

month 1:1 intubation +7 days rest during 21 days

Month 2 to 6: intubation for 1 time every other day (except menstrual day)

Papilocare, dosimetry B

Month 1 to 3: 1 intubation +7 days rest during 21 days

Month 4 to 6: intubation for 1 time every other day (except menstrual day)

C. Standard clinical practice (No Special treatment)

The number of patients scheduled to be included in the study was 96.32 per group. This is the amount of samples calculated for the primary endpoint.

Study duration was 12 months: treatment with Papilocare was followed up to 6 months and up to 1 year.

Throughout the study period, patients were monitored in 5 visits: v1 screening, V2 randomization, V3 month treatment, V4 month treatment, and V5 month follow-up.

In this preliminary final analysis, the results of efficacy are measured as follows:

primary endpoint

Regression of cervico-vaginal mucositis at 6 months, measured as the percentage of patients with normal pap smears and consistent colposcopic images.

Secondary endpoint

Regression of cervico-vaginal mucositis at 3 months, measured as percentage of patients with normal pap smears and consistent colposcopic images.

HPV clearance at 6 months. HPV clearance was considered when HPV detection was negative or positive, but some of the baseline genotype was cleared (partial clearance) and pap smear and colposcopy normalized.

Degree of re-epithelialization of the cervicovaginal mucosa at 3 and 6 months. Changes in cervical epithelialization were assessed by standard colposcopy (and rated by investigators using the litterbate scale from 0= severe ectopic + bleeding to 5= normal).

The level of stress experienced by the patient at 3 and 6 months. Validated PSS14 questionnaire was used.

Safety and tolerability of Papilocare at 3 and 6 months

Patient satisfaction at 3 and 6 months

By PCR (

HPV 4) and the evaluation of the pap smear samples (liquid cytology) studied were performed under "third party blind evaluation" conditions, focusing on the Institute of Cellular and Molecular Studies (ICM) of Lugo, spain.

For this analysis, groups a and B were combined and comparisons within (final vs baseline) and between (a + B vs C) groups were made. In addition, a subset of high risk HPV patients has been analyzed for lesion regression (primary target) and HPV clearance (secondary target).

Table 6 includes the number of patients and the duration of treatment for the population included in this pre-final analysis.

Table 6: analysis of included populations, including stratification by detected HPV risk

Number of patients (N)	First visit	3 months old	6 months old
				Total of	101	91	87
Papilocare (group A + B)	68	59	55
				Standard care (C group)	33	32	32
High risk HPV patients	79	70	68
				Papilocare (group A + B)	53	44	42
Standard care (C group)	26	26	26

Table 7 includes the number of patients evaluated for the assessment of efficacy endpoints in this pre-final analysis.

Table 7: including the population in the pre-final analysis of efficacy endpoints.

Therapeutic results

Repair of cervical mucosa

At 6 months, 84.9% of patients treated with Papilocare showed 64.5% of patients in the normal pap smear and consistent colposcopic image vs control group (primary endpoint) (p =0.031a + b vs C). After 3 months, 78.0% of the patients receiving treatment showed 54.8% of patients in the normal pap smear and consistent colposcopic image vs control group; the difference was also significant (table 8).

TABLE 8 therapeutic results for the general population

In the high risk population, the difference in a + B vs C at 6 months was 87.8% vs 56.0%, respectively (p = 0.0034). At 3 months, the results of A + B vs C were 79.5% vs 52%, respectively. The difference was also significant (table 9). These results are also graphically illustrated in fig. 5.

Table 9: therapeutic outcome in high risk HVP population

HPV clearance

As shown in table 10, after 6 months, 59.6% of total patients treated with Papilocare showed HVP clearance of 41.9% in the vs control group. Statistical trends of higher HPV clearance were observed in patients with the high risk HPV population receiving treatment: 62.5% vs. control in Papilocare group 40.0%, p =0.0767 (table 11). The results of table 11 are also graphically illustrated in fig. 6.

Table 10: HPV elimination in the general population

Table 11: HPV clearance in high risk HVP populations

Extent of epithelialization

A total of 91 patients at 3 months (V3) and 84 patients at 6 months (V4) have been analyzed. A significant difference was observed at baseline in the degree of epithelialization vs in patients treated with Papilocare at 6 months. In patients of 3 months, a trend of improvement compared to baseline was observed. Patients in the control group showed no significant improvement in baseline vs at 3 months and baseline vs at 6 months. The re-epithelialization results are shown in table 12. An image of the re-epithelialization of the patient's cervix is shown in fig. 7.

Table 12: re-epithelialization results at defined times

¹ Man-Whitney U (UMann-Whitney) test (independent samples)

² Wilcoxon test (Wilcoxon) (correlation samples: H0: difference =0vs H1: difference ≠ 0)

The percentage of treated patients with baseline improvement in the re-epithelialization scale vs at 3 and 6 months was 20.3% and 32.1%, respectively, while the control group was 25.0% and 25.8% at 3 and 6 months, respectively. No statistical significance was found in the treatment group at any time (table 13).

Table 13: percentage of patients showing changes in re-epithelialization

At 3 months, 88.1% of patients in Papilocare showed no ectopic or mild ectopic vs 65.6% in the control group, with statistical significance between groups (p =0.012, fisher test). At 6 months, a trend was shown: 90.6% in the patients receiving treatment vs 80.7% in the control group. The results are shown in table 14 and fig. 8.

Table 14: degree of epithelialization according to the Lister Scale

Patient reported stress levels

In the group treated with Papilocare, the measured level of PSS at 14 months showed from 21.13 (SD 8.77) to 19.71 (SD 9.04) at 3 months and 18.98 (SD 9.11) at 6 months (1.42 and 2.15 points at 3 months and 6 months, respectively). In the control group, the reported levels ranged from 17.72 (SD 7.18) to 17.42 (SD 6.33) at 3 months, and to 20.68 (SD 9.77) at 6 months (both at +0.3 and +2.96 points, respectively). Although not significant, a positive trend of pressure reduction was shown in the Papilocare group compared to the control group. The results are shown in table 15 and fig. 9.

Table 15: patient reported pressure

Satisfaction degree of product

After 3 or 6 months of treatment, 71.2% and 76.9% of patients reported complete or moderate satisfaction with Papilocare, respectively. At the same time, 83.1% and 86.5% of people, respectively, have mentioned a certain degree of satisfaction. At 3 months, only one patient reported a slight dissatisfaction with the product at the reported visit. The patient reported satisfaction results for the product as shown in table 16.

Table 16: patient reported satisfaction with product

Safety feature

91 patients can be evaluated for safety analysis. Details regarding treatment groups and follow-up periods for patients included in the safety analysis are contained in table 17.

Table 17: patient incorporating safety analysis

Crowd safety analysis	3 months old	6 months old
			In all	91	84
Papilocare (group A + group B)	59	53
			Standard of care (C group)	32	12

A total of 22 adverse events were reported by 11 patients, 21 of which were not severe and only 1 was graded as severe, but the event was not associated with current treatment.

Only 1 and 6 adverse events were very likely (probablity) and likely (posably) associated with treatment, respectively. 90% of adverse events were mild/moderate in intensity, and only 2 cases led to treatment withdrawal. See 18 for details.

Table 18: detailed adverse events reported in the study

The analysis result shows that:

significant differences in repair of HPV cervical lesions compared to controls at 3 and 6 months in populations of total HPV genotype and high risk HPV genotype

HPV clearance at 6 months shows a positive trend, which is almost significant in the high-risk HPV genotype population

Significant difference in cervical epithelialization at 6 months from the control group, and positive trend of pressure reduction

High degree of use satisfaction

Good therapeutic safety and tolerability. Pruritus, burning and candidiasis are treatment-related adverse events.

4.5. Clinical trial papiloreal gel + probiotic

This is a multicenter, randomized, prospective, open-test, parallel-group and control (PAPILOCARE gel) pilot clinical trial to evaluate the efficacy of the composition according to the invention (PAPILOCARE gel + probiotics) to increase the lactobacillus concentration at 3 months and to repair the cervical mucosa of women with HPV-related pap smear changes and consistent colposcopic images. PAPILOGEL gel compositions are as defined in example 1. The papilogera gel + probiotic composition contains, in addition to the components of PAPILOGEL, lactobacillus crispatus 100B (non-viable bacteria) in a concentration of 0.5%.

Patients in the cohort were randomized (1:

A.Papilocaregel：

Month 1:1 intubation +7 days rest during 21 days

Month 2 to 6: intubation for 1 time every other day (except menstrual day)

B.PapilocareGel+ Probiotics

Month 1:1 intubation +7 days rest during 21 days

Month 2 to 6: intubation for 1 time every other day (except menstrual day)

The number of patients scheduled for inclusion into the study was 40 per group. This is the amount of samples calculated for the primary endpoint.

Study duration was 6 months: follow-up with Papilocare treatment for 6 months and up to 1 year. Throughout the study period, patients were monitored in 4 visits: v1 screening, V2 randomization, V3 month treatment, V4 month treatment.

primary endpoint

Baseline vs, increase in Lactobacillus concentration by percentage at 3 and 6 months

Secondary endpoint

Cervico-vaginal mucositis regressed at 3 and 6 months, measured as percentage of patients with normal pap smears and consistent colposcopic images.

HPV clearance at 3 and 6 months. HPV clearance was considered when HPV detection was negative or positive, but some of the baseline genotype was cleared (partial clearance) and pap smear and colposcopy normalized.

By PCR (

HPV 4), assessment of pap smear samples (liquid cytology) and real-time PCR assays of lactobacillus vaginalis populations (lactobacillus crispatus, lactobacillus jensenii (l.jensenii), lactobacillus inerticus (l.iners), lactobacillus gasseri (l.gasseri) were determined) all performed under "blind third party assessment".

Table 19 includes the number of patients and the duration of treatment for the population included in this pre-final analysis.

Table 19: population for inclusion in analysis

Therapeutic results

Increased concentration of lactobacilli in vaginal mucosa

At 3 months, the mean percentage increase in lactobacillus concentration compared to baseline was 53% in patients treated with Papilocare gel + probiotics (group B), while this increase was 27% in patients treated with Papilocare gel (group a). This corresponds to a significant improvement of 96% between the two groups at 3 months. At 6 months, the mean percentage increase in lactobacillus concentration was 76% in patients treated with Papilocare gel + probiotics (group B) and 55% in patients treated with Papilocare gel (group a), which corresponded to a significant improvement of 38% (table 20).

TABLE 20 efficacy results of the main criteria for improvement of Lactobacillus concentration

Primary standard at three months
			Improvement of Lactobacillus concentration (changing vs baseline)	3 months old	6 months old
Group A	27％	55％
			Group B	53％	76％
Improvement of A vs B	96％	38％

Repair of cervical mucosa

At 3 months, 78% of the patients treated with Papilocare gel + probiotics (group B) showed normal pap smears and consistent colposcopic images, while 58% of the patients with Papilocare gel (group a) corresponded to a significant improvement of 74% between the two groups at 3 months. After 6 months, 95% of the patients treated with Papilocare gel + probiotics (group B) showed normal pap smears and consistent colposcopic images, while 78% of the patients treated with Papilocare gel (group a) corresponded to a significant improvement of 65% between the two groups at 6 months (table 21).

Table 21: therapeutic results

HPV clearance

As shown in table 22, after 3 months 73% of the patients treated with Papilocare gel + probiotics (group B) showed HVP clearance compared to 48% of the patients with Papilocare gel (group a). Statistically significant differences in higher HPV clearance were observed in treated patients. At 6 months, 83% of patients treated with Papilocare gel + probiotics (group B) showed HVP clearance compared to 63% of patients with Papilocare gel (group a). Statistically significant differences in higher HPV clearance were observed in treated patients.

Table 22: HPV clearance

In summary, the incorporation of probiotics into PAPILOCARE gel provides a surprising increase in the efficacy of the composition according to the invention as a measure, in particular by repairing the cervical mucosa and HPV clearance. Interestingly, mucosal repair and clearance had improved significantly after 3 months of treatment. This means that the addition of probiotics not only improves the treatment, but also significantly speeds up the treatment.

5. Compositions of extracts and other products for use in the compositions of the invention

Suitable sources of active ingredients for use in the compositions of the invention are listed below.

Coriolus versicolor extract (Yunzhi) from (Symrise)

Coriolis) has the following composition: water (a)>50 percent of glycerin (25-50 percent), coriolus versicolor extract (1-5 percent), sodium benzoate (0.5 percent), lactic acid (0.215 percent) and potassium sorbate (0.2 percent).

Azadirachta indica extract from Jasminum deltoides (Symrise) ((R))

Neem) has the following composition: water (a)>50%), glycerol (25-50%), neem extract (0.1-1%), sodium benzoate (0.5%), lactic acid (0.36%) and potassium sorbate (0.2%).

Prebiotics from Naturalis Life Technologies

Has the following composition: carboxymethyl β -glucan sodium: 2.0-6.0%, magnolol/honokiol: 1.0-3.0%, polyglycerol-10 dilaurate: 10.0-15.0%, watermelon seed oil polyglycerol-6 ester: 10.0-15.0%, hazelnut oil polyglycerol-6 ester: 10.0-15.0%, sorbitan oleate: 4.0-6.0%, lecithin: 3.0-5.0%, water: 45.0 to 50.0 percent.

The probiotic bacteria Lactobacillus plantarum Nu06, lactobacillus paracasei Nu-09, lactobacillus paracasei Nu-02, lactobacillus acidophilus Nu-03 and Lactobacillus rhamnosus Nu-04, and Lactobacillus crispatus 100B are obtainable from Nutris Ingredients SL.

References cited in the application

"Effects of β -glucans from Coriolus versicolor on macropathological cytology area related to the Akt and CK2/Ikaros", int J Biol Macromol.2013, vol.57, p.9-16.

Cui J, et al, "polysaccharides of Coriolus versicolor: physiological activity, uses, and production". Biotechnol adv.2003, vol.21 (2), p.109-22.

KF Cheng, et al, "General review of polysaccharides from. C. Versicolor: pharmaceutical and clinical students", cancer Therapy 2008, vol.6, p.117-130.

EMA/HMPC/291177/200,2010.“Assessment report on Centella asiatica(L.)Urban,herba”.

Tiwari V, et al, "In vitro anti viral activity of neem (Azardirachta index L.)" Bar extract aggregate virus type-1infection ". Phytother Res.2010, vol.24 (8), p.1132-1140-virus type-1infection

ISO 10993-10:2010-Biological evaluation of medical devices part 10:tests for irritation and skin sensitization

ISO 10993-5:2009Biological evaluation of medical devices Part 5:Tests for in vitro cytotoxicity

"Tissue expression profiling real-time PCR", curr protocol Pharmacol.2002, chapter 6.9, doi.

Bachmann G.“Urovaginal or cervical ageing:an old problem newly recognized”,Maturitas1995,Dec；22Suppl:S1-S5

Bachman G.“A new option for managing urovaginal or cervical atrophy in post menopausal women”,Contemp Obstet Gynecol 1997,vol.42,p.13-28

VaginaStatus-Diagnostic test,Instiüt für

Herborn,Germany.Available at.http://www.mikrooek.de.Accessed date 24Feb 2016.

Claims

1. Use of coriolus versicolor extract for the preparation of a composition for the prevention and/or treatment of a vaginal or cervical condition caused by an infectious agent selected from the group consisting of: human Papillomavirus (HPV), candida albicans, chlamydia trachomatis, gardnerella vaginalis, flexibacter, bacteroides, mycoplasma, neisseria gonorrhoeae, trichomonas vaginalis, herpesvirus, staphylococcus aureus, staphylococcus epidermidis, escherichia coli, streptococcus alphabetus, bacillus, enterobacter, proteus, enterobacter cloacae, klebsiella, staphylococcus epidermidis, klebsiella pneumoniae, and dermatophytes,

wherein the preventing or treating comprises vaginal or cervical administration of the composition to the female according to the following protocol:

(a) One dose of said composition per day during 21-24 days, followed by

(b) No dose for a period of 3 to 7 days, followed by

(d) No dose during 3 to 7 days, and

(e) Repeating (c) and (d) four times in sequence,

wherein each dose comprises 0.5 to 5mg of Coriolus versicolor extract.

2. The use of claim 1, wherein (c) is one dose every two days during 21-24 days.

3. The use of claim 1, wherein the treatment comprises vaginal or cervical administration of the composition to a female according to the following protocol:

(a) One dose of the composition per day during 21 to 24 days, followed by

(b) No dose during 3 to 7 days, followed by

(c) Repeating (a) and (b) twice in sequence, followed by

(d) One dose of the composition every two days during 21-24 days, followed by

(e) No dose during 3 to 7 days, and

(f) Repeating (d) and (e) twice in sequence,

wherein each dose comprises 0.5 to 5mg of Coriolus versicolor extract.

4. The use of any one of the preceding claims, wherein the dose is administered during 21 days and not administered during 7 days.

5. Use according to any one of the preceding claims, wherein each dose comprises 1 to 4mg of coriolus versicolor extract.

6. The use according to claim 5, wherein each dose comprises 1.5 to 3.5mg of Coriolus versicolor extract.

7. The use according to claim 6, wherein each dose comprises 2 to 3mg of coriolus versicolor extract.

8. Use according to any one of the preceding claims, wherein the composition further comprises at least one ingredient selected from: probiotic microorganisms, neem tree extract, carboxymethyl beta-glucan, alpha-glucan oligosaccharides, hyaluronic acid, centella asiatica extract, and aloe vera.

9. The use of claim 8, wherein the probiotic microorganism (if present) is in an amount of 30 to 100mg per dose, the neem tree extract (if present) may be in an amount of 0.1-1mg per dose, the carboxymethyl β -glucan (if present) is in an amount of 1.8-18mg per dose, the α -glucan oligosaccharide (if present) is in an amount of 5-50mg per dose, the hyaluronic acid (if present) is in an amount of 0.24-2.4mg per dose, the centella asiatica extract (if present) is in an amount of 0.3322-3.322mg per dose, and the aloe vera (if present) is in an amount of 0.05-0.5mg per dose.

10. Use according to any one of claims 8-9, wherein the probiotic bacteria are selected from the group consisting of lactobacillus plantarum, lactobacillus paracasei, lactobacillus acidophilus, lactobacillus crispatus and lactobacillus rhamnosus.

11. The use of claim 10, wherein the lactobacillus plantarum is lactobacillus plantarum Nu-06, being lactobacillus paracasei Nu-02 or lactobacillus paracasei Nu-09, being lactobacillus acidophilus Nu-03, lactobacillus crispatus 100B and being lactobacillus rhamnosus Nu-04.

12. The use of any one of claims 8-11, wherein the composition comprises lactobacillus crispatus, such as lactobacillus crispatus 100B.

13. The use according to any one of claims 8 to 9, wherein the composition comprises the product NuProbi

14. The use of any one of claims 8-13, wherein the composition comprises neem tree extract.

15. The use of any one of claims 8-14, wherein the composition comprises carboxymethyl β -glucan.

16. The use of any one of claims 8-15, wherein the composition comprises an alpha-glucan oligosaccharide.

17. The use of any one of claims 8-16, wherein the composition comprises hyaluronic acid.

18. The use of any one of claims 8-17, wherein the composition comprises an extract of centella asiatica.

19. The use of any one of claims 8-18, wherein the composition comprises aloe.

20. Use according to any one of the preceding claims, wherein one or more of the coriolus versicolor extract and/or additional active compounds (if present) in the composition are encapsulated into liposomes.

21. The use according to any one of the preceding claims, wherein the infectious pathogen is selected from the group consisting of human papilloma virus, candida albicans, chlamydia trachomatis, gardnerella vaginalis, trichomonas vaginalis and herpes virus.

22. The use of claim 21, wherein said infectious agent is human papilloma virus.

23. Use according to any one of the preceding claims, wherein the vaginal or cervical condition is selected from cervical cancer, vulvovaginitis, dermatomycosis, candidiasis, genital herpes, gonorrhea, proctitis, infertility, cervicitis, pelvic inflammatory disease, ectopic pregnancy, acute or chronic pelvic pain and trichomoniasis.

24. The use according to claim 23, wherein the vaginal or cervical disorder is selected from cervical cancer, vulvovaginitis, trichomoniasis, candidiasis and genital herpes.

25. The use of claim 24, wherein the vaginal or cervical disorder is cervical cancer.

26. The use of claim 25, wherein the vaginal or cervical cancer is low grade squamous intraepithelial lesion (LSIL) or mild cervical intraepithelial neoplasia (CIN 1).

27. Use according to any one of the preceding claims, wherein the composition is a gel.

28. The use of any one of the preceding claims, wherein the dose is administered by intubation.