WO2014106541A1

WO2014106541A1 - Dermal composition for use in the external genital area in females

Info

Publication number: WO2014106541A1
Application number: PCT/EP2013/050090
Authority: WO
Inventors: Katalin Daroczy
Original assignee: Ellen Ab
Priority date: 2013-01-04
Filing date: 2013-01-04
Publication date: 2014-07-10

Abstract

A dermal composition for use in the external genital area in females to treat or prevent dry vulva, the composition comprising a lipid base and lactic acid bacteria, wherein the water activity at 25°C of the dermal composition is less than 0.30.

Description

DERMAL COMPOSITION FOR USE IN THE EXTERNAL GENITAL AREA IN FEMALES

Field of the invention

The present invention relates to a dermal composition for use in the external genital area in females, being effective in maintaining and restoring a normal protective hydro lipidic barrier, as well as a flora of lactic acid bacteria, in the external genital area.

Background

The skin of human beings is protected by hydro lipidic film acting as a natural barrier. The function of the hydrolipidic film is to retain moisture and to protect against the entrance of unwanted microbes into the skin. An intact hydrolipidic film maintains the skin's natural suppleness. The hydrolipidic film consists of water, oils produced by the sebaceous glands, dead cells and beneficial, protective bacteria, also named as the body's natural flora. Partly due to the bacterial flora, the hydrolipidic film on the skin has an acidic pH about 5.0 to 5.5. Therefore the protective barrier sometimes is also denoted acidic mantel. The acidic environment is required to prevent non-resident bacteria from enter and grow on the skin. The hydrolipidic film varies in quantity and composition depending on the body region as well as exogenous and endogenous factors such as the time of day, the season and air humidity or nutrition, stress and illness.

The normal human skin microflora is composed of a limited number of microbial types including Proteobacteria, Proprionibacteria (e.g. P. acnes, P. avidum, and P. granulosum), Coagulase-negative Staphylococci (Staphylococcus epidermidis), Micrococci, Cory b acteria and Acinetobacter. Similar to lactic acid bacteria, also the growth of Proprionibacteria is favored by a slightly acid pH.

The genital tract of women is from nature colonized by lactobacilli, a kind of lactic acid producing bacteria. Moreover, it has been reported that more than 50 bacterial species colonize the healthy vagina. Studies have shown that urogenital cells are covered by dense bacterial bio films whose composition changes constantly but in which lactobacilli predominate at least until menopause. Benevolent lactic acid bacteria can also be found in the external genital area (vulva) there they contribute to creation of the protective hydrolipidic film sustaining a normal microbial flora, keeping the acidic pH normal and the skin and external mucous membranes soft and smooth.

If the protective system, i.e. the hydrolipidic film and the bacterial biofilm, is disturbed, dryness, irritation and even infections may develop, especially in the intimate areas, both in the internal and external genital tract, of the female body. Further, sustained or long-lasting disruption of the hydrolipidic film can imply that the skin/mucous membranes cannot regenerate themselves quickly enough, resulting in dryness and increased pH, which in turn can result in irritation and damage. Damaged skin and mucous membranes are more susceptible to pathogens, which can spread and also enter the vagina. Also a weakened lactobacillus flora in the external intimate area may contribute to an increased susceptibility to pathogens.

Urogenital infections (UI) include those that affect the bladder, kidneys, vagina, urethra, periurethra, and cervix, constitute a worldwide problem that every year affects more than 300 million women. The main clinical outcome is morbidity and discomfort amongst a large percentage of the female population, in addition to enormous costs to the health care system for treatment. Typical, urogenital infections include:

1) Bacterial vaginosis (BV), a condition wherein the Lactobacillus dominated vaginal microflora is replaced by a mix of other, mainly anaerobic bacteria species;

2) Yeast infections (Candida vaginitis (CV)/Vulvo vaginal candidiasis (VVC)), caused by overgrow of the yeast, most frequently Candida albicans: and

3) Urinary Tract Infection (UTI), a bacterial infection that affects part of the urinary tract. The origin of the uropathogens in uncomplicated UTI is the fecal flora, including, Escherichia coli (in about 80% of all UTI cases) as well as other bacteria (e.g. other enterobacteriaciae and Staphylococcus saprophyticus).

A dermal composition restoring or maintaining a flora of lactic acid bacteria in the external genital tract may be useful in counteracting urogenital infections. Similar, it would be of interest to maintain or restore a healthy microflora at the skin in general. Further, it would be of interest to preserve or restore the hydrolipidic film of the skin acting as a natural barrier to pathogens.

Women today have an active lifestyle playing sports and working out regularly. Further, they travel a lot, both to work and during holidays. An active lifestyle requires careful body hygiene. Frequent water contact, extensive use of hygienic products affects the natural microbial flora and the fragile and sensitive skin, especially of the external genital tract including its mucus membranes. Further, also the increasing trend, especially among younger women, of shaving parts or all of their intimate area affects the protective hydrolipidic film including a natural microbial flora in the intimate are of the body negatively. Especially, as shaving typically includes the use of shaving creams and foams. As a consequence many women experience dryness and also a sense of not being fresh in their intimate area.

Further, also age, phase of menstrual cycle, sexual activity, contraceptive choice, pregnancy, antibiotic treatments, and excessive use of hygienic products may affect the microbial ecosystem both in the internal and external genital tract negatively.

A dermal composition soothing and moisturizing the mucus membranes and replenishing the natural microbial flora and hydrolipidic film in the external intimate area would thus be of interest.

Various moisturizing dermal compositions are known in the art. Typically, they are creams, i.e. semi-solid emulsions comprising a mixture of oil and water (equal proportions), ointments, i.e. homogeneous, viscous, semi-solid preparation typically comprising oil and water (80:20), or lotions, i.e. a low- to medium-viscosity (i.e. less viscous than creams) emulsions comprising a mixture of oil and water. Commonly, moisturizing dermal compositions of the art comprises water as well as to provide composition with good spreadability and to be effective in providing moisturizing effect. Further, moisturizing dermal compositions of the art commonly comprises propylene glycol.

Probiotics, such as lactic acid bacteria, have found a wide spread use, for example in functional food. As they are living organism and as their use as probiotics is linked to the viability of the bacteria, the carrier of the bacteria has to be carefully selected. Typically, a unique carrier system has to be developed for each new application. The carrier should typically provide a sufficient shelf life. Commonly, lactic acid bacteria are either supplied as an aqueous solution, typically being a chilled product with a fairly limited shelf life, or as a freeze dried powder, to be reconstituted before use. Further, examples of cosmetic yoghurts, i.e. aqueous dairy products, are known in the art. Similar to functional foods, the shelf life of such yoghurts are limited.

Thus, there is a need in the art for a dermal composition to preserve or restore the hydrolipidic film, including the flora of lactic acid bacteria, of the skin acting as a natural barrier to pathogens.

Summary

Consequently, the present invention seeks to mitigate, alleviate, eliminate or circumvent one or more of the above-identified potential deficiencies in the art and disadvantages singly or in any combination by providing a dermal composition for use in the external genital area in females, said composition comprising a lipid base comprising a long-chain triglyceride, or a mixture of long-chain triglycerides, and medium-chain triglyceride, or a mixture of medium-chain triglycerides, and lactic acid bacteria. In order to provide the dermal composition with proper spreadability it should be semi-solid at 20°C. Further, the water activity at 25 °C of the dermal composition should be less than 0.30 in order to provide sufficient shelf life.

According to a preferred aspect of the invention, the dermal composition is packed in a package with a metallic barrier layer to increase the shelf life of the dermal composition.

According to a further aspect of the invention, such a dermal composition is used to treat or prevent bacterial and/or yeast infections, such as dermal or vaginal infections in a mammal, e.g. a human being, or infections in the external genital area of a mammal, e.g. a human being. Further, the dermal composition may be used therapeutic treatment or prevention of dry skin and/or dry mucus membranes, such as dry vulva.

According to a further aspect of the invention, such a dermal composition is non-therapeutically used to restore or maintain a flora of lactic acid bacteria of the skin and/or of the mucous membranes of a mammal, such as human being. In such use the dermal composition may be topically, e.g. cutaneously, applied.

According to a further aspect of the invention, such a dermal composition is non-therapeutically used to counteract dryness of the skin or of the mucus membranes of a mammal, such as to counteract dryness of the external genital organs of a female mammal, e.g. human being. Also in such use the dermal composition may be topically, e.g. cutaneously, applied.

Further advantageous features of the invention are defined in the dependent claims. In addition, advantageous features of the invention are elaborated in

embodiments disclosed herein.

Detailed description of preferred embodiments

The present inventor has found that a dermal composition comprising a lipid base, such as a vegetable fat and/or vegetable oil, and lactic acid bacteria is efficient in maintaining and restoring a normal protective hydrolipidic barrier in the external genital area. Without being bond to any theory, it is believed that the lipid base keeps the moisture in the skin and mucous membranes in the external intimate area, softens and soothes this sensitive and fragile part of the body, while the lactic acid bacteria (e.g. lactobacilli) complements and restores the natural beneficial bacterial flora. However, in order to provide sufficient shelf life to the dermal composition, it was found that the lipid base has to be carefully selected. Especially, it was found that the water content of the formulation has to be low. Further, in order to find use as a dermal composition, the viscosity of the composition at room temperature, as well at the temperature of the skin in the external genital area of females, has to be carefully selected. The viscosity should be sufficient low to provide spreadability, while it should be sufficient high to not rinse of the skin once applied. Formulating a lipid base comprising no or very low amounts of water and also having suitable rheological properties is by no means straight forward. It was found that a combination of a vegetable fat, e.g. a long-chain triglyceride, a vegetable oil, e.g. a medium-chain triglyceride, and optionally a CI 2-22 aliphatic monohydric alcohol provided a suitable lipid base.

An embodiment of the invention thus relates to a dermal composition for use in the external genital area in females comprising a lipid base, the lipid base comprising a long-chain triglyceride, or a mixture of long-chain triglycerides, a medium-chain triglyceride, or a mixture of medium-chain triglycerides, and optionally a CI 2-22 aliphatic monohydric alcohol, and lactic acid bacteria. In order to provide the composition with good spreadability, the lipid base should be semi-solid at 20°C.

Further, the water activity at 25 °C of the dermal composition should be less than 0.30, such as less than 0.25 or less than 0.20. According to an alternative embodiment, the water content of the dermal composition is less the 0.5 wt%, such as less than 0.1 wt%.

Such a dermal formulation was found to be effective in maintaining and restoring a normal protective hydrolipidic barrier, including a normal bacterial flora, acting as a natural barrier to pathogens. Due its typically natural ingredients including fats and oils having vegetable origin, the dermal composition is very gentle against the fragile and sensitive tissues in the intimate part of the body. Further, the shelf life, i.e. the viability of the lactic acid bacteria after storage, was also found to be sufficient.

However, while the lipid base did provide a carrier system protecting the labile lactic acid bacteria, it was anyhow found that the selection of the package for the dermal composition was important to its shelf life. It was surprisingly found that use of a standard aluminum tube sealed only by folding its end, provided improved shelf life, compared to a welded extra tight plastic tube, having a barrier layer of ethylene vinyl alcohol, acting as an effective oxygen barrier.

According to an embodiment, the dermal composition is packed in a package with a metallic barrier layer. Preferably, the dermal composition is packed in an aluminum tube. However, the dermal composition may also be packed in package of laminate type, the laminate including a metallic barrier layer. Tubes made of laminates including a metallic barrier layer are known in the art and are publically available. Similar to plastic tubes, tubes made of laminates are typically sealed by welding.

Further, also the oxidation stability of the lipid base may affect the shelf life.

According to an embodiment the long-chain triglyceride thus has an iodine number according to IUPAC 2.205 lower than 3.0. Further, the medium-chain triglyceride may have an iodine number according to IUPAC 2.205 lower than 0.5.

As used herein, the term medium-chain triglyceride refers to a triglyceride comprising residues of carboxylic acids comprising 6 to 12 carbon atoms. The term is intended to include separate triglycerides as well as mixtures of triglycerides. According to an embodiment, medium-chain triglycerides are C6-12 fatty acid esters of glycerol. The fatty acid may be the same or different and they may be saturated, mono- unsaturated or polyunsaturated. A preferred example of a medium-chain triglyceride is an oil having vegetable origin.

As used herein, the term long-chain triglyceride refers to a triglyceride comprising residues of carboxylic acids comprising more than 12 carbon atoms. The term is intended to include separate triglycerides as well as mixtures of triglycerides. According to an embodiment, long-chain triglycerides are CI 3-30, such as CI 6-22, fatty acid esters of glycerol. The fatty acid may be the same or different and they may be saturated, mono-unsaturated or polyunsaturated. A preferred example of a long-chain triglyceride is a fat having vegetable origin.

The spreadability of the dermal composition is affected by the ratio of the various lipid components of the lipid base. Further, also the inherent viscosity of the long-chain triglyceride as well of the medium-chain triglyceride will affect the spreadability of the dermal composition. It was found that the dermal composition preferably should have solid content of at least 5%, but less than 40% at 20°C, according to IUPAC 2.150(a). Further, the solid content of the dermal composition at 30°C, according to IUPAC 2.150(a), should preferably be at least 5%.

According to an embodiment, the dermal composition comprises 20 to 70 wt.%), such as 40 to 60 wt.%, of the long-chain triglyceride, and 10 to 60 wt.%, such as 20 to 40 wt.% of the medium-chain triglyceride. Preferably, the long-chain triglyceride has a solid content of less than 5% at 40°C, but of more than 30% at 20°C, according to IUPAC 2.150(a). Further, the slip melting point, according to AOCS Cc 3-25, of the long-chain triglyceride is preferably between 25 and 40°C, such as between 30 and 35°C. An example, of a suitable vegetable fat is mixture of hydrogenated coco- glycerides, e.g. Akosoft 36 sold by AarhusKarlshamn AB

According to an embodiment, the medium-chain triglyceride is a

caprylic/capric acid triglyceride. In such a triglyceride, the acid ratio of caprylic/capric acid triglyceride may be 50:50 to 80:20. Further, the content of other carboxylic acid residues than caprylic acid and capric acid residues may be less than 5 molar%.

By adding a CI 2-22 aliphatic monohydric alcohol to the dermal composition, its moisturizing properties may be improved. Further, a CI 2-22 aliphatic monohydric alcohol will affect the spreadability of the dermal composition. According to an embodiment, the dermal composition thus comprises a CI 2-22 aliphatic monohydric alcohol. The dermal composition may comprise 5 to 40 wt.%, such as 10 to 30 wt.%, of a CI 2-22 aliphatic monohydric alcohol.

According to an embodiment, the CI 2-22 aliphatic monohydric alcohol is selected from the group consisting of dodecanol (CI 2), 1 -tetradecanol (CI 4), cetyl alcohol (CI 6), stearyl alcohol (CI 8), arachidyl alcohol (C20), and docosanol (C22). Preferably the CI 2-22 aliphatic monohydric alcohol is cetyl alcohol (CI 6).

Moisturizing dermal compositions of the art commonly comprises

preservatives, such as parabens. According to an embodiment, the dermal composition comprises essentially no parabens. Probably due to the low water activity of the composition, it was found that the need for a preservative, such as a paraben, may be dispensed with. Possibly, also the presence of lactic acid bacteria affects the shelf life of the composition.

It was also found the presence of glycols, such as ethylene glycol, propylene glycol, typically used in moisturizing dermal compositions of the art, negatively affected the viability of the lactic acid bacteria. According to an embodiment, the dermal composition thus comprises essentially no ethylene glycol or propylene glycol.

Lactic acid bacteria (LAB) embrace a variety of genera of bacteria producing lactic acid as a major metabolic end-product of carbohydrate fermentation. An important example of a genus of lactic acid bacteria is Lactobacillus. The normal, healthy bacterial flora in the inside intimate area (i.e. in the vagina) of a fertile women is characterized by a complex ecology of microorganisms. The predominant bacteria species of this complex ecology of microorganisms are from the genus Lactobacillus. These benevolent bacteria also colonize the outside/external part of the intimate area and contribute to formation of a protective hydrolipidic film which covers the external intimate area such a barrier, keeping the skin and mucosa soft and smooth. In addition to providing an acidic pH, i.e. typically 3.8 - 4.2 in the vaginal fluid, Lactobacilli secretes antimicrobial substances (e.g. hydrogen peroxide, bacteriocins and bacteriocin- like substances) also protecting the genital tract from colonization and/or overgrowth of unwanted, potentially pathogenic microorganisms. Commonly occurring Lactobacillus species in the vagina are L. crispatus, L. gasseri, L. iners and L. jensenii followed by L. rhamnosus, L. fermentum, L. plantarum, and L. vaginalis. The bacteria in the protective barrier of external intimate area are mainly lactobacilli of the same species as in the vagina. According to an embodiment, the lactic acid bacteria in the dermal composition are selected from the genus Lactobacillus.

According to an embodiment, the lactic acid bacteria are selected from the group consisting of the strains:

- Lactobacillus gasseri, LN 40, deposited under number LMG P-20560;

- Lactobacillus casei subsp rhamnosus (also known as Lactobacillus rhamnosus), LN 113, deposited under number LMG P-20562; and

- Lactobacillus fermentum, LN 99, deposited under number LMG P-20561 ; or

- mixtures thereof.

Those strains were deposited at the Belgian Coordinated Collections of Microorganisms (BCCM), Laboratorium voor Microbiologie - Bacterien verzameling (LMG) Universiteit Gent (June 14, 2001). Further, properties of these strains are disclosed in WO 2003/038068.

Freeze dried powder of the LN strains have been tested for in vitro cytotoxicity in cultured mammalian cells (L 929 mouse fibroblasts). The test was performed in accordance with the US Pharmacopeia 30th edition, Method <87> and the ISO 10993-5 Elution Test guideline. Based on these results, it is concluded that the freeze dried powder of LN strains passes the requirements of the USP<87> (safe to use regarding cytotoxicity).

In dermal compositions for use on the skin in other parts of human body than the external genital area of females, the lactic acid bacteria lactic bacteria may be replaced or complemented by bacteria of bacterial genera present in the normal human skin microflora, such as by P. acnes, P. avidum, P. granulosum and/or Staphylococcus epidermidis. The lipid base may be the same as the one used in compositions to be applied in the genital area of females. According to an embodiment, the lactic acid bacteria of the dermal compositions disclosed herein are replaced or complemented by Proprionibacteria. The growth of Proprionibacteria is favored by a slightly acidic pH. The amount of lactic acid bacteria in the composition may vary. According to an embodiment, the dermal composition comprises at least 10 2 , such as at least 103, 104,

5 6 7 7 8 9

10^J, 10 , 10', 10', 10° or 10 CFU lactic acid bacteria per gram of the composition. For cosmetic use, the dermal composition may comprise at least 10 CFU lactic acid bacteria per gram of the dermal composition. According to an embodiment, wherein the dermal composition is intended for therapeutic use, it comprises at least 10⁴, such as at least 10⁶, CFU lactic acid bacteria per gram of the composition.

In preparing the dermal composition, the lactic acid bacteria are typically lyophilized before being added to the lipid base. In order to maintain a high viability and protect the lactic acid bacteria during the lypohilization, a lyoprotectant may be added to the lactic acid bacteria before being lyophilized. Commonly employed lyoprotectants comprise polyhydroxy compounds, e.g. a mono-, di-, or polysaccharide, polyalcohols, e.g. sorbitol, and inorganic agents, such as calcium chloride.

By applying the dermal composition to the external genitals of a female, typically a human being, it can protect the intimate area from unwanted

microorganisms, e.g. bacteria and yeast, which may enter from the rectum. Further, the dermal composition may also assist in maintaining and restoring the body's own, occasionally weakened, Lactobacillus flora. Further, the dermal composition may also assist in maintaining and restoring a healthy dermal flora of bacteria at other parts of the body than the external genitals. The lactic acid bacteria of the dermal composition may also themselves proliferate and colonize in the external genitals, becoming part of the healthy bacterial flora. Especially, colonization may be triggered if the composition enters the vagina, having an environment suitable for growth of probiotic bacteria.

Thus, the dermal composition may be used for treating or preventing bacterial and/or yeast infections, such as dermal or urogenital infections in a mammal, e.g. a human being, or infections in the external genital area of a mammal, such as a human being. Further, the dermal composition may find non-therapeutic or cosmetic use in restoring or maintaining a flora of lactic acid bacteria of the skin and/or of the mucous membranes of a mammal; especially, in the external genital area. In such use, the dermal composition is typically topically applied to the external genital organs of a female mammal.

As already described, the dermal composition is also effective in moisturizing the skin, as well as mucous membranes; especially the mucous membranes of the external genital organ. Thus, the dermal composition may be used for therapeutic treatment or prevention of dry skin and/or dry mucus membranes, such as dry vulva. Further, the dermal composition may find use in non-therapeutically or cosmetically counteracting dryness of the skin or of the mucus membranes of a mammal; especially in counteracting dryness of the external genital organs of a female mammal, e.g. a human being. Also in such use, the dermal composition is typically topically applied to the external genital organs of a female mammal.

According to an embodiment, preventing is intended to mean reducing the incidence of the condition of concern.

In topically applying the dermal composition to the external genital organ, a pea-sized amount the composition may be applied. The composition may be applied one to two times daily. It may be applied as long as needed to relieve discomfort. Due to the properties of the dermal composition, it can be safely applied around/close to the vaginal opening, especially by women who also feel dryness inside the intimate or/and want to strengthen her vaginal flora.

As described, the dermal composition may be used in methods of:

- treating or preventing bacterial and/or yeast infections;

- restoring or maintaining a flora of lactic acid bacteria of the skin and/or of the mucous membranes of a mammal;

- treating or preventing dry skin and/or dry mucus membranes; and

- counteracting dryness of the skin or of the mucus membranes.

In such methods, the dermal composition is topically administrated to a mammal, such as human.

Further, the dermal composition may be used in the manufacture of a medicament for:

- treating or preventing bacterial and/or yeast infections;

- treating or preventing dry skin and/or dry mucus membranes; and

- counteracting dryness of the skin or of the mucus membranes.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preferred specific embodiments described herein are, therefore, to be construed as merely illustrative and not limitative of the remainder of the description in any way whatsoever. Further, although the present invention has been described above with reference to specific embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and, other embodiments than the specific above are equally possible within the scope of these appended claims, e.g. different than those described above.

In the claims, the term "comprises/comprising" does not exclude the presence of other elements or steps. Additionally, although individual features may be included in different claims or different embodiments, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous.

In addition, singular references do not exclude a plurality. The terms "a", "an", "first", "second" etc do not preclude a plurality.

Experimental

The following examples are mere examples and should by no mean be interpreted to limit the scope of the invention. Rather, the invention is limited only by the accompanying claims.

Dermal composition

Preparation of freeze dried powder of lactic acid bacteria

After fermentation of each of the lactic acid bacteria strain {Lactobacillus gasseri (LN 40) deposited under number LMG P-20560, Lactobacillus casei subsp rhamnosus (LN 113) deposited under number LMG P-20562, and Lactobacillus fermentum (LN 99) deposited under number LMG P-20561), the bacterial cells from each bacteria culture were separated from the medium, and mixed with a protective medium consisting of sucrose, trehalose and calcium chloride. Thereafter they were frozen (-40°C to -60°C) under vacuum, whereby water and any other volatile components were removed by sublimation. The bacteria were freeze dried to obtain powders with a water activity of less than 0.1, each comprising approximately 60 wt.% lactic acid bacteria {Lactobacillus gasseri (LN 40) deposited under number LMG P- 20560, Lactobacillus casei subsp rhamnosus (LN 113) deposited under number LMG P-20562, and Lactobacillus fermentum (LN 99) deposited under number LMG P-20561, respectively) and approximately 23 wt% sucrose, 13wt.% trehalose, and 3 wt.% calcium chloride.

The freeze dried powders were then milled to a particle size < 0.315 mm, and then mixed to obtain bacteria pool comprising of 10 CFU%> Lactobacillus gasseri (LN 40) deposited under number LMG P-20560, 45 CFU%> Lactobacillus casei subsp rhamnosus (LN 113) deposited under number LMG P-20562, and 45 CFU%

Lactobacillus fermentum (LN 99) deposited under number LMG P-20561.

Preparation of a dermal composition for use in the external genital area in females

To obtain 100 g of a dermal composition 49.5 g Akosoft 36, a mixture of hydrogenated coco-glycerides sold by AarhusKarlshamn AB (AAK), 19.8 g cetyl alcohol, and 29.70 g Bergabest MCT oil 60/40, a mixture of Caprylic/capric

Triglycerides sold by Sternchemie GmbH & Co KG, were heated to 50-60°C in a mantled glass reactor under stirring. Subsequently, the resulting mixture was allowed to cool down under vacuum. Upon reaching 38°C, freeze dried bacteria (1 g) from the previous example was added and the stirring under vacuum was maintained until the mixture had cooled down to about 20°C.

The resulting dermal composition was filled into a plastic bag and allowed to further solidify at room temperature.

In a similar manner, a dermal composition comprising 3 wt% of the freeze dried bacterial powder was prepared. The relative weight ratio of the lipophilic components was the same, i.e. 48.5 g Akosoft 36, 19.8 g cetyl alcohol, and 29.10 g Bergabest MCT oil 60/40 were mixed, thereafter freeze dried bacteria (3 g) from the previous example was added.

Package into tubes

The solidified compositions (1 wt. % and 3 wt. %, respectively) in the plastic bags were kneaded. Subsequently, they were filled (15 g) into Coex-tubes (25*75 mm, 50% PE-003- 50% HDPE-B4520, EVOH Barrier; from Tuboplast/CTL packaging) and aluminum tubes (19* 119 mm from Tectubes), respectively. The Coex-tubes were welded while the aluminum tubes were folded to seal the tubes.

Shelf-life

Example 1

The shelf life of the dermal composition after storage at various conditions (1 5°C; 2: 25°C / 60% RH; and 3: 30°C / 65% RH) were evaluated after 2, 4, 6, and 8 weeks by determining the number of remaining viable bacteria cells. Table 1 - shelf life

Conditions Time (weeks) 1%-Al 1%-COEX 3%-Al 3%-COEX

0 3.50E+08 3.40E+08 1.22E+09 1.07E+09

5°C

8 4.00E+08 ND 9.70E+08 1.02E+09

0 3.50E+08 3.40E+08 1.22E+09 1.07E+09

25°C / 60% H 4 2.05E+08 9.25E+07 6.74E+08 2.84E+08

8 1.00E+08 ND 6.25E+08 3.05E+08

0 3.50E+08 3.40E+08 1.22E+09 1.07E+09

2 2.50E+07 5.00E+07 3.75E+08 6.50E+08

30°C / 65% RH 4 5.70E+07 2.63E+06 2.51E+08 2.26E+08

6 5.05E+07 1 .70E+06 5.80E+08 4.45E+07

8 5.20E+07 1 .OOE+05 3.10E+08 5.20E+06

As can be seen from Table 1 (cf. numbers in bold), the aluminum tube provided a better shelf life than the plastic tube, although the plastic tube being extra tight and provided with a barrier layer of ethylene vinyl alcohol. Further, it is to be noted that while the plastic tube was welded the aluminum tube was only folded to seal it. It is thus surprising to note such a marked difference in shelf life. No difference in visual appearance or texture of the formulations was noted between the two types of tubings.

Example 2

Further, a long term study of the shelf life of the composition comprising 3 wt% of the freeze dried bacterial powder has been initiated. After storage at 30°C / 65% RH for 12 months in an aluminum tube, the number of viable bacteria had only been reduced from 1.22E+09 to 1.90E+07 CFU per gram of the composition. Applying the same conditions for storage in a Coex tube, reduced the number of viable bacteria from 1.07E+09 to less than 1.00E+02 CFU per gram, after 9 months, whereafter the test was aborted. Example 3

Similarly, a long term study of the shelf life of the composition comprising 3 wt% of the freeze dried bacterial powder at 25°C / 60% RH has been initiated. After storage at 25°C / 60% RH for 18 months in an aluminum tube, the number of viable bacteria had only been reduced from 1.22E+09 to 5.22E+07 CFU per gram of the composition. Applying the same conditions for storage in a Coex tube, reduced the number of viable bacteria from 1.07E+09 to less than 2.50E+01 CFU per gram after 9 months, whereafter the test was aborted. Thus, a striking difference in terms of shelf life, determined as the number of remaining viable bacteria cells after storage at 30 C / 65% RH and 25°C / 60% RH, respectively, was observed (cf. example 2 and 3). Packing in aluminum tubes was found to provide superior shelf life compared to packing in Coex tubes.

In summary, it is clearly seen that the dermal composition disclosed herein is preferably to be packed in a package with a metallic barrier layer.

Consumer study

In order to evaluate the dermal composition, a consumer study was performed. The aim of the consumer study was to generate information on the use of product for the intimate hygiene with particular emphasis of the suitability of the new dermal composition to soothe the outer intimate area of females and induce a feeling of freshness.

The study was conducted in compliance with ethical principles of the latest revision of the Declaration of Helsinki and the data protection law of individuals (SFS 1998:204). For the consumer study, the 3 wt. % composition according to the previous examples, packed in 15 ml aluminum tubes, was used.

Study design and outline

The dermal composition was studied in a single-blind consumer study of 29 women, median age 52 years, mean 47 years, SD 14, range 18-66 years (two missing data).

The composition (a pea size amount) was used twice daily for one week on the outer mucous membrane in the genital area, around the opening of the vagina, but not in the vagina. The women were asked to rate their experiences (using a 4- or 5 -point ordinal scale) and commented on the properties of the product in a questionnaire which then was sent anonymously to the institute responsible for the study.

Result

At inclusion to the study, the majority of the women had some discomfort in their intimate area, such as dryness, odor, itching or discharge. After use of the dermal composition, the majority of women expressed a positive attitude to the properties of the product, such as odor, viscosity and the after- feel of the outer mucous membranes. The use of the cream also improved the feeling of dryness, freshness and odor. Further, a vast majority of the women (approx. 80%) expressed purchase intentions, whereas only approximately 20% showed no interest.

A sub-group analysis of the 41% of the women who had felt dryness in their intimate area at inclusion showed that:

- the majority (75%) of these women have been improved a lot or slightly by the cream regarding dryness.

A sub-group analysis of the 31% of the women who at the inclusion did not feel fresh in their intimate area and also felt to have a bad odor showed that:

- 78% reported an improvement of the freshness after use of the dermal composition; and

- 66% reported a reduction in the odor after use of the dermal composition.

It was thus confirmed that the dermal composition is effective in counteracting dryness of the external genital area in females. Further, it is also effective in reducing odor and increasing freshness of the external genital area in females. It thus is clear that the dermal composition provides synergistic effects previously not reported in the art.

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(yes or no)

-4-1 Authorized officer

Pasche, Constantinus

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Claims

1. A dermal composition for use in the external genital area in females, said composition comprising:

- a lipid base comprising a long-chain triglyceride, or a mixture of long-chain triglycerides, and medium-chain triglyceride, or a mixture of medium-chain

triglycerides, the lipid base being semi-solid at 20°C; and

- lactic acid bacteria;

wherein the water activity at 25°C of the dermal composition is less than 0.30, such as less than 0.25 or less than 0.20.

2. The dermal composition according to claim 1, wherein said lipid base has a solid content of at least 5%, but less than 40% at 20°C, according to IUPAC 2.150(a).

3. The dermal composition according to claim 1 or 2, wherein said lipid base has a solid content of at least 5% at 30°C, according to IUPAC 2.150(a).

4. The dermal composition according to any one of the preceding claims, wherein said lipid base further comprises a CI 2-22 aliphatic monohydric alcohol.

5. The dermal composition according to any one of the preceding claims, wherein the dermal composition is packed in a package with a metallic barrier layer.

6. The dermal composition according to the preceding claim, wherein said package is an aluminum tube.

7. The dermal composition according to any one of the preceding claims, wherein said long-chain triglyceride has an iodine number according to IUPAC 2.205 lower than 3.0, and/or said medium-chain triglyceride has an iodine number according to IUPAC 2.205 lower than 0.5.

8. The dermal composition according to any one of the preceding claims, wherein said long-chain triglyceride is a mixture of long-chain triglycerides, said mixture having a solid content of less than 5% at 40°C, but of more than 30% at 20°C, according to IUPAC 2.150(a).

9. The dermal composition according to any one of the preceding claims, wherein the dermal composition comprises 20 to 70 wt.%, such as 40 to 60 wt.%, of said long-chain triglyceride.

10. The dermal composition according to any one of the preceding claims, wherein said medium-chain triglyceride is a caprylic/capric acid triglyceride.

11. The dermal composition according to claim 10, wherein the caprylic xapric acid ratio in said caprylic/capric acid triglyceride is 50:50 to 80:20, and wherein the content of other carboxylic acid residues than caprylic acid and capric acid residues are less than 5 molar%.

12. The dermal composition according to any one of the preceding claims, wherein the dermal composition comprises 10 to 60 wt.%, such as 20 to 40 wt.%, of said medium-chain triglyceride.

13. The dermal composition according to any one of the preceding claims, wherein the dermal composition comprises 5 to 40 wt.%, such as 10 to 30 wt.%, of a CI 2-22 aliphatic monohydric alcohol.

14. The dermal composition according to claim 13, wherein said C12-22 aliphatic monohydric alcohol is selected from the group consisting of: dodecanol (CI 2), 1-tetradecanol (CI 4), cetyl alcohol (CI 6), stearyl alcohol (CI 8), arachidyl alcohol (C20), and docosanol (C22); preferably said CI 2-22 aliphatic monohydric alcohol is cetyl alcohol (CI 6).

15. The dermal composition according to any one of the preceding claims, wherein the dermal composition comprises essentially no parabens.

16. The dermal composition according to any one of the preceding claims, wherein the dermal composition comprises essentially no ethylene glycol and essentially no propylene glycol.

17. The dermal composition according to any one of the preceding claims, wherein the lactic acid bacteria are selected from the genus Lactobacillus.

18. The dermal composition according to claim 17, wherein the lactic acid bacteria are selected from the group consisting of the strains Lactobacillus gasseri, LN 40, deposited under number LMG P-20560, Lactobacillus casei subsp rhamnosus, LN 113, deposited under number LMG P-20562, and Lactobacillus fermentum, LN 99, deposited under number LMG P-20561, or mixtures thereof.

19. The dermal composition according to any one of the preceding claims, wherein the dermal composition comprises at least 10², such as at least 10³,10⁵, 10⁶, 10⁷,

10 7', 108° or 109", CFU lactic acid bacteria per gram of the composition.

20. The dermal composition according to any one of the preceding claims, wherein the lactic acid bacteria have been lyophilized before being added to the lipid base.

21. The dermal composition according to the claim 20, wherein the dermal composition further comprises a lyoprotectant, such as a polyhydroxy compound, e.g. a mono-, di-, or polysaccharide, a polyalcohol, e.g. sorbitol, and/or an inorganic agent, e.g. calcium chloride, for protecting the lactic acid bacteria during the lypohilization.

22. The dermal composition according to any one of the preceding claims, for treating or preventing bacterial and/or yeast infections, such as dermal or urogenital infections in a mammal, e.g. a human being, or infections in the external genital area of a mammal, e.g. a human being.

23. The dermal composition according to any one of claims 1 to 21, for therapeutic treatment or prevention of dry skin and/or dry mucus membranes, such as dry vulva

24. The dermal composition according to any one of the claims 1 to 21, for use according to claim 22 or 23, wherein the dermal composition is topically applied to the external genital organs of a female mammal, e.g. a human being.

25. Non-therapeutic use of the dermal composition according to any one of the claims 1 to 21 to restore or maintain a flora of lactic acid bacteria of the skin and/or of the mucous membranes of a mammal, such as human being, comprising topical, e.g. cutaneous, application of the dermal composition.

26. The use according to the claim 25, wherein the dermal composition is topically applied to the external genital organs of said female mammal.

27. Non-therapeutic use of the dermal composition according to any one of the claims 1 to 21 to counteract dryness of the skin or of the mucus membranes of a mammal, such as to counteract dryness of the external genital organs of a female mammal, e.g. human being, comprising topical, e.g. cutaneous, application of the dermal composition.

28. The use according to the claim 27, wherein the dermal composition is topically applied to the external genital organs of said female mammal.