HK1206975B - Inhibition of the adhesion of pathogenic microorganisms by a sucrose stearate and/or a sorbitan ester in the cosmetic treatment of cutaneous atopy - Google Patents

Description

Inhibition of the adhesion of pathogenic microorganisms by sucrose stearate and/or sorbitol esters in the cosmetic treatment of atopic skin

Scope of the Invention

The present invention relates to a cosmetic treatment method for human atopic dermatitis, which method is based on applying to the skin a cosmetic composition capable of forming a film thereon. According to the invention, this film—by virtue of its components—inhibits the adhesion of pathogenic microorganisms to the skin and strengthens the skin barrier.

More specifically, the cosmetic composition contains esters of sucrose and/or sorbitan, which can block the adhesion of Staphylococcus aureus to the skin and nasal mucosa, thereby inhibiting the proliferation of pathogenic bacteria. This inhibitory effect protects the skin barrier by limiting the breakdown of lipids, particularly ceramides, indirectly caused by Staphylococcus aureus.

Existing technology

Atopic dermatitis, or atopic skin, is an inflammatory, pruritic skin condition associated with a genetic immune predisposition and skin barrier abnormalities. Clinically, this genetic predisposition can also manifest as allergic rhinitis and asthma.

Atopic dermatitis results in hypersensitivity to environmental allergens that are tolerated by healthy subjects.

In atopic dermatitis, eczema represents a delayed-type hypersensitivity reaction mediated by Th2 lymphocytes (which produce IL-4 and IL-5) and antigen-presenting cells.

Similar to any immune response mediated by antigen-specific T lymphocytes, the inflammatory response in atopic dermatitis eczema progresses through three phases.

The first stage is asymptomatic sensitization. This phase is not clinically apparent and results in the expansion of antigen-specific T lymphocytes. Typically, sensitization occurs in adolescence through exposure to environmental allergens captured by dendritic cells.

In the second phase, eczema develops. Following re-exposure to the allergen in question, Langerhans cells migrate and activate antigen-specific cytokine-producing Th2 lymphocytes, which in turn activate various cell types in the skin. This mechanism helps recruit white blood cells into the dermis and epidermis, where they produce inflammatory mediators. This phase, which lasts for a variable period, is characterized by eczematous lesions. In most patients, it is accompanied by high levels of antigen-specific IgE antibodies, some of which are bound to the surface of Langerhans cells.

Finally, the lesions resolve in the third stage. Atopic dermatitis eczema develops in bursts, separated by spontaneous remission periods. The mechanisms involved in regulating inflammation remain poorly understood.

Atopic dermatitis affects 10–30% of the population, but its incidence is increasing in developed countries, having doubled or tripled in the past two decades.

Atopic dermatitis is characterized by sudden, itchy eruptions of acute eczema, interspersed with periods of resolution. It is most common in children. It develops approximately a few months after birth, appearing as lesions on the cheeks and skin areas exposed to friction. It then develops into flare-ups between the ages of one and two. During this period, the skin becomes dry, red, and oozing, especially on the inner surfaces of skin folds. After age five, flare-ups tend to subside, but the skin remains dry and sensitive. In some people, atopic dermatitis can persist into adulthood.

Two phenomena occur behind atopic dermatitis: first, the skin barrier is weakened, and second, the skin becomes colonized by pathogenic microbiota.

The main tissue of the skin barrier is the stratum corneum, the outermost layer of the epidermis, which includes corneocytes and specific lipids, including cholesterol, free fatty acids, cerebrosides, and ceramides. These lipids act as "cerebrosides" between cells and make the stratum corneum waterproof.

Analysis of the stratum corneum of patients with atopic dermatitis reveals significant deficiencies in certain proteins and skin lipids. Both filaggrin, a protein involved in assembling keratin filaments in corneocytes, and involucrin, a protein essential for building the protein skeleton of the stratum corneum, are deficient. The most notable lipid deficiencies are ceramides 1 and 3.

Ceramides are particularly important in regulating the skin barrier function by limiting water evaporation.

The deficiency of these proteins and lipids means that corneocytes are less able to adhere to each other, affecting the cohesion and integrity of the stratum corneum. This is manifested as a significant reduction in the thickness of the stratum corneum in these patients.

Atopic dermatitis is characterized by a significant loss of skin proteins and lipids, which, as mentioned above, leads to evaporation of water and dryness of the skin. This, of course, makes it easier for allergens to penetrate the skin.

Thus, atopic dermatitis triggers a vicious cycle: evaporation of water from cells leads to dehydration and dryness of the skin, which in turn increases skin permeability. Increased skin permeability makes it easier for allergens to penetrate the tissues that maintain skin irritability.

The skin's barrier function can also be strengthened by a local ecosystem composed of saprophytic flora.

Saprophytic flora are a natural, permanent feature of the skin surface. The density of bacteria on the skin surface is estimated to be between 10 ² and 10 ⁵ / cm2. The most common species are Staphylococci, particularly the coagulase-negative Staphylococcus epidermidis and related species (Micrococcus), and coryneform bacteria, both aerobic (Corynebacterium, Brevibacterium) and anaerobic (Propionibacterium acnes). These bacteria establish themselves in the stratum corneum, where they adhere to corneocytes to form a protective biofilm.

This saprophytic flora thus strengthens the barrier function, as it occupies potential adhesion sites for other (possibly pathogenic) microorganisms - and inhibits their proliferation.

However, if the skin is damaged as in atopic dermatitis, the impaired skin barrier function promotes water evaporation and creates a surface environment that is conducive to the colonization of bacteria, viruses, or fungi. This favorable environment is exacerbated by the increase in skin temperature that occurs during an eczema flare.

Thus, pathogenic bacterial flora can temporarily grow on the surface of the skin. Most of these species are found in the normal environment, including species belonging to the genera Pseudomonas and Acinetobacter; other species are obtained from the digestive or oral flora, such as enterobacteria, Streptococcus or other species of Clostridium.

These bacteria are not normally able to proliferate on the surface of human skin, but they can cause infection if the skin barrier function is compromised as in atopic dermatitis, where they can form a pathogenic bacterial biofilm.

Among the bacteria of the transient pathogenic flora, Staphylococcus aureus (S. aureus) is the most potentially pathogenic species, being the most common member of the genus found in the nose and throat of 15%-30% of healthy people. This Gram-positive bacterium colonizes the skin by adhering to skin cells.

Recent studies have shown that 90% of patients with atopic dermatitis have their skin colonized by Staphylococcus aureus, compared to only 5% of healthy subjects.

Of note, atopic dermatitis has also been shown to be a risk factor for colonization of the nasal mucosa with S. aureus membranes, which provide a reservoir in the environment of atopic patients (Pascolini et al., 2011).

Some studies have also shown that ceramides (enzymes) produced by skin flora are activated in the presence of Staphylococcus aureus. Ceramidase hydrolyzes ceramides in the stratum corneum, suggesting that colonization of the skin by Staphylococcus aureus may contribute to a deficiency of this lipid in the atopic epidermis (Kita et al., 2002).

Staphylococcus aureus is also a source of superantigens (protein toxins) that interact with immune cells in subjects with atopic dermatitis, amplifying the inflammatory response and thereby triggering flare-ups.

Thus, there is a tangible link between the proliferation of Staphylococcus aureus on the epidermal surface and the impaired skin barrier function caused by insufficient ceramides, especially in subjects with atopic dermatitis (remember, impaired barrier function promotes sensitization to environmental allergens and the maintenance of skin inflammation). The two main viruses responsible for infection are herpes simplex virus (HSV) and vaccinia virus. Colonization of the Malassezia yeast has also been observed in patients with atopic dermatitis.

Current treatments for atopic dermatitis are based on managing inflammation and controlling bacterial colonization.

Medications aimed at managing inflammation include glucocorticoids and calcineurin inhibitors, such as pimecrolimus and tacrolimus. These products are often combined with moisturizers.

Bacterial control, particularly involving the administration of broad-spectrum antibiotics and the application of disinfectants, has been widely reported. First-line treatment has been made difficult by the development of bacterial resistance. Furthermore, both types of treatment kill not only pathogens residing on the skin, but also beneficial saprophytes.

The main disadvantage of both approaches is that they destroy components required for effective barrier function, namely the saprophytic flora.

Recent studies have shown that topical application of compositions containing certain sugars can inhibit the adhesion of pathogenic bacteria to the skin.

Document WO 2006/106220 reports that certain sugars, mono- and oligosaccharides such as rhamnose, galactose, mannose and lauryl glucoside, are able to inhibit the adhesion of bacteria, in particular of the genus Staphylococcus (Staphylococcus intermedius), to the keratinocytes of atopic (dermatitis) dogs.

WO 96/23479 suggests that certain sugars can be used to inhibit the adhesion of certain microorganisms. Examples of these sugars include monosaccharides such as raffinose, mannose, and rhamnose, disaccharides, oligosaccharides, aminated sugars, and esters of various sugars, including glucose esters such as cetearyl glycoside, octyl glycoside, and decyl glycoside. These various sugars can be incorporated into cosmetic or dermatological compositions to inhibit bacterial adhesion, such as that of Staphylococcus aureus, and/or for therapeutic purposes, particularly the treatment of atopic dermatitis.

Document EP0875239A2 lists various sugar esters, including sucrose esters, which exhibit resistance against certain Gram-positive bacteria, such as Staphylococcus epidermidis, Staphylococcus aureus, Corynebacterium spp., and Propionibacterium acnes, which are involved in a range of pathologies that manifest as atopic dermatitis. The esters listed are palmitic/stearic acid diesters, stearic acid diesters, lauric acid monoesters, myristic acid monoesters, and stearic acid triesters and tetraesters.

Document EP1340486A1 lists various sugar esters, including esters of sucrose, fructose, glucose, and trehalose. Only the esters of fructose, glucose, and trehalose were tested for their ability to inhibit the growth of Staphylococcus aureus. Sucrose esters were not tested for this activity. Only one representative of this family, sucrose stearate, was tested, and this was only for bleaching activity (see Table 7). Finally, this document does not mention atopic dermatitis.

EP0815841A1 reports on a composition for controlling skin redness caused by diaper friction. This is not a treatment for atopic dermatitis. The document describes the inhibitory effect of a mixture of sucrose monoesters with palmitic acid and stearic acid, or with palmitic acid and lauric acid, on the growth of Staphylococcus aureus and Staphylococcus epidermidis.

Document EP 2 210 588 A1 describes foaming compositions containing polysorbate 80, an ester of sorbitol. Polysorbate 80 is a very common emulsifier.

Mintel document (XP002693208) describes moisturizing compositions containing sucrose stearate.

Document WO 4/037225 A2 describes compositions containing no ethanol or propylene glycol for use as foaming aerosol carriers. Atopic dermatitis is mentioned among other conditions that can be treated with aerosols supplemented with specific active substances. Sucrose stearate and polysorbate 80, mentioned in the examples, are commonly used surfactants.

Document FR 2 798 591 A1 describes the use of specific plant oils to increase the synthesis of skin lipids, in particular for the treatment of atopic dermatitis. Sucrose distearate and sorbitan tristearate are commonly used as surfactants.

MERCK document (XP-002693209) describes anti-wrinkle compositions containing sucrose stearate.

Document EP 1 639 989 A1 describes the production of microemulsions containing sugar or sorbitan esters used as surfactants.

In Examples 5 and 8 of US 2005/158348 A1, the compositions contain polysorbate 80 and sucrose esters, respectively. These compositions are used to treat pain and inflammation. Sorbitan and sucrose esters are used as excipients.

Document US 2010/080768 A1 describes various ceramides and sphingolipids present in compositions for the treatment of various diseases, including atopic dermatitis.

Document US 2011/101135 describes the use of sorbitan monocaprylate as a preservative for cosmetic compositions. Various bacterial strains were tested, including Staphylococcus aureus.

Despite this, there remains a need for more effective alternatives to the various modalities currently used to manage atopic dermatitis.

It is in this context that the applicant has surprisingly discovered that certain sugar esters, such as esters of sucrose and sorbitan, strongly inhibit the adhesion of pathogenic microorganisms to the skin and mucous membranes.

Inhibiting the adhesion of S. aureus to skin surfaces prevents the proliferation of pathogenic microorganisms, even in the absence of antibiotics or topical antiseptics.

One of the advantages of inhibiting the adhesion of pathogenic microorganisms, especially Staphylococcus aureus, to the skin is to maintain the saprophytic flora and enhance the barrier function of the skin by reducing the decomposition of its component lipids.

Glycoesters can advantageously combine with naturally occurring lipids in the epidermis to form a membrane that can more effectively combat the breakdown of the epidermal barrier and the colonization of the skin by pathogens.

Other objects and aspects of the present invention will become apparent from the following detailed description, which is intended for purposes of illustration and not limitation in any way.

Detailed Description of the Invention

A first aspect of the present invention is directed to a cosmetic composition for topical application containing at least one sucrose ester and/or sorbitan ester as an agent for inhibiting the adhesion of Staphylococcus aureus to human skin and nasal mucosa.

More particularly, the present invention relates to a composition for topical application comprising at least one sucrose ester and/or sorbitan ester as an agent for inhibiting the adhesion of Staphylococcus aureus to human skin and nasal mucosa for the treatment of atopic dermatitis.

A sugar ester is a sugar in which at least one free alcohol group has been esterified with a fatty acid chain.

A number of different sugar ester families have been described. Notably, there are esters of glucose, sucrose, and sorbitan (Piccicuto et al., 2001). These have the advantage of being non-toxic and non-irritating and are widely used in the food processing, pharmaceutical, and cosmetic industries, often due to their surface-active properties.

Of these three different families, this application relates only to esters of sucrose and sorbitan.

These nonionic surfactants have a hydrophilic group consisting of sucrose or sorbitan bound by an ester bond to a hydrophobic group consisting of fatty acids. The surface active properties of these esters also make them easy to combine with lipids in cosmetic compositions, especially emulsions.

α-D-glucopyranose-β-D-fructofuranoside or sucrose carries eight free alcohol groups and can therefore be esterified with up to eight fatty acids.

Sorbitan is produced by the dehydration of sorbitol (itself a polyol obtained by reducing the aldehyde group of glucose to produce an alcohol group). Sorbitan carries four free alcohol groups, each of which can be esterified with a fatty acid.

In the context of the present invention, sorbitan esters are esters of the "Span" type, such as those shown in the following formula:

Where R is a fatty acid, in this case, monoesterified Span.

The present invention also relates to polyethoxylated sorbitan esters of the "Tween" type, for example, the following formula:

Where R is a fatty acid, in this case, monoesterified Tween.

The carbon chains of fatty acids can be short or long, for example, lauric, myristic, palmitic and stearic acids have corresponding chains of _C12 , _C14 , _C16 and _C18 . Fatty acids can also be selected from saturated and unsaturated fatty acids, for example, oleic acid (at the _C18 carbon chain).

Therefore, according to another feature of the invention, the cosmetic composition comprises sucrose and/or sorbitan in which at least one alcohol group is esterified with a fatty acid chosen from fatty acids with a carbon chain between C ₁₂ and C ₂₂ , advantageously between C ₁₂ and C ₁₈ .

Depending on the ratio of fatty acids to sucrose, esterification can give monoesters, diesters, triesters or polyesters (up to octyl esters), or mixtures of all these forms. The esterification of sorbitan can give monoesters, diesters, triesters, tetraesters or mixtures of these different forms.

According to another embodiment, the cosmetic composition of the present invention comprises sucrose and/or sorbitan, wherein a part or all of the alcohol groups have been esterified with the same fatty acid.

In this case and advantageously, the sucrose esters are chosen from: sucrose laurate, sucrose myristate, sucrose palmitate and sucrose stearate, and the sorbitan esters are chosen from: sorbitan laurate (Span 20), sorbitan monostearate (Span 60), sorbitan oleate (Span 80) and sorbitan sesquioleate (Montane 83).

In an advantageous embodiment, the sucrose ester is sucrose stearate containing at least 70% by weight, advantageously at least 75% by weight, of sucrose and stearic acid monoesters, the remainder being di-, tri- and polyesters of sucrose and stearic acid.

Advantageously, the cosmetic composition of the present invention comprises a polyethoxylated sorbitan ester. Mention may be made of ethoxylated sorbitan monolaurate (polysorbate 20), polyoxyethylene sorbitan monostearate 20 (polysorbate 60), and polyoxyethylene sorbitan monooleate (polysorbate 80). In a preferred embodiment, the sorbitan ester is polysorbate 20 or polysorbate 80.

According to an alternative embodiment, the cosmetic composition may consist of sucrose and/or sorbitan, wherein a part or all of the alcohol groups have been esterified with at least two different fatty acids.

In this case and advantageously, the sucrose esters are chosen from: sucrose palmitate stearate and sucrose tetrastearate triacetate, and the sorbitan esters are chosen from: oleate/stearate esters of sorbitan (Montan 481) and sorbitol esterified with fatty acids from olive oil (sorbitan olivate).

According to a preferred embodiment, the sucrose ester in the cosmetic composition of the present invention is sucrose stearate, ie, sucrose esterified with at least one molecule of stearic acid (C ₁₈ fatty acid).

It is well known to those skilled in the art that for a given fatty acid carbon chain length, the proportion of monoesters in the mixture will affect the hydrophobicity/hydrophilicity of the sugar ester.

These properties are characterized by the hydrophilic/lipophilic balance (HLB) index. Values between 1 and 10 indicate lipophilic sugar esters, corresponding to relatively low amounts of monoesters and, therefore, a high proportion of polyesters. However, values between 11 and 20 indicate hydrophilic sugar esters, corresponding to a high proportion of monoesters and, therefore, a low proportion of polyesters.

In a particular embodiment of the present invention, sucrose stearate has an HLB value of 16, corresponding to a relative proportion of sucrose monoester and stearic acid between 75% and 80% by weight. Therefore, the product is referred to as sucrose monostearate.

Preferably, the sucrose ester and/or sorbitan ester represents 0.1-5%, advantageously between 1% and 3% by weight of the composition.

Advantageously, the cosmetic composition also comprises lipids capable of restoring the skin barrier, namely:

- at least one lipid that does not naturally occur in the skin, advantageously an oil; and/or

- A blend of ingredients naturally found in the skin, including ceramides 1, 3, 6, cholesterol, free fatty acids, and phytosphingosine.

In fact, supplementation with certain specific lipids can help restore the lipid layer between skin cells, i.e., the extracellular binder, thereby helping to restore the skin barrier. As a result, the evaporation of skin moisture and the risk of S. aureus adhering to the skin surface and multiplying are reduced. This effect is combined with the effectiveness of the esters of the present invention in inhibiting S. aureus adhesion and preventing the hydrolysis of ceramides in the stratum corneum.

Lipids, such as oils, which are not naturally present in the skin, mediate short-term moisturization of the tissue. They are advantageously selected from sunflower oil and rapeseed oil (Brassica rapa) because of the high concentration of essential omega-6 and omega-3 fatty acids in these oils.

In a particular embodiment of the present invention, the cosmetic composition comprises lipids not naturally occurring in the skin, sunflower oil in an amount of 3-15% by weight of the composition and rapeseed oil (Brassica) in an amount of 0.05-10% by weight of the composition.

Replenishing naturally occurring lipids in the skin could correct the deficiency of certain stratum corneum lipids in patients with atopic dermatitis. This could help restore the skin's long-term barrier function, especially in combination with the effects of the esters described above.

In a specific embodiment, the mixture of naturally occurring components corresponds to a lipid composition designated by INCI, namely, water, ceramide 3, ceramide 6II, ceramide 1, phytosphingosine, cholesterol, sodium lauroyl lactylate, carbomer, and xanthan gum. This lipid composition is advantageously provided as the commercial product SK Influx V ^™ (Evonik Industries).

Preferably, the lipid composition comprises 0.01-5% by weight of the total composition.

Advantageously, the cosmetic composition further comprises at least one additional polyol selected from rhamnose, xylitol and mannitol.

These additional polyols, such as rhamnose and xylitol, help inhibit the adhesion of pathogenic bacteria such as Staphylococcus aureus to human skin and nasal mucosa. Mannitol also has free radical quenching activity.

In a particular embodiment of the present invention, the cosmetic composition comprises a mixture of three of the above-mentioned additional polyols.

Advantageously, rhamnose comprises 0.01-1% by weight of the composition, xylitol comprises 0.05-2% by weight of the composition and mannitol comprises 0.005-1% by weight of the composition.

Advantageously, the composition also contains at least one antipruritic agent selected in particular from the group consisting of:

-Palmitoylethanolamine, CAS No. 544-31-0;

-Hydroxy-α-sanshool, CAS No. 83883-10-7, part of the composition of zanthalène ^™ ;

- tyrosylarginine-based lipodipeptide, part of the calmosensine ^™ composition;

- Ichtyol or ammonium ichthosulphonate, CAS number 8029-68-3.

Advantageously, the cosmetic composition of the invention may also comprise at least one anti-inflammatory agent, preferably chosen from:

-β-Sitosterol, CAS number 83-46-5;

- Enoxolone or glycyrrhetinic acid, CAS No. 471-53-4.

Advantageously, the cosmetic composition also includes vitamin PP, which is known to promote the synthesis of lipids in the stratum corneum, including ceramides, free fatty acids and cholesterol. Vitamin PP works by stimulating the activity of serine palmitoyltransferase, a key enzyme in the synthesis of sphingosine, a precursor for the production of ceramides.

Another aspect of the present invention relates to a cosmetic composition for the cosmetic treatment of atopic dermatitis.

The invention also relates to a cosmetic treatment for atopic dermatitis based on the protection of the epidermis by forming a film on the skin with the composition described below.

In an advantageous embodiment, the treatment regimen comprises applying to the skin a film-forming composition, in particular containing esters of sucrose and/or sorbitan, to inhibit the adhesion of Staphylococcus aureus to the skin surface, together with lipids such as fatty acids, ceramides and cholesterol, to strengthen the skin's defense against microbial damage, and in particular to target enzymes secreted by such microorganisms that hydrolyze components of the stratum corneum.

This mask can be applied to the skin, depending on the stage of the disease, in order to:

- Arrest of progress;

- Acts during the flare-up phase of the disease;

-Prevent relapse.

Advantageously, the composition for stopping a process comprises (in percentages by weight of the composition):

- between 1% and 5% sucrose monostearate and/or sorbitan esters;

- mixtures of between 3% and 25% oil;

-SK Influx V ^™ between 0.01% and 5%;

- A mixture of between 0.06% and 4% rhamnose, xylitol and mannitol.

Advantageously, the composition for treating an attack comprises (in percentages by weight of the composition):

- between 1% and 5% sucrose monostearate and/or sorbitan esters;

- mixtures of between 3% and 25% oil;

-SK Influx V ^™ between 0.01% and 5%;

- a mixture of rhamnose, xylitol and mannitol between 0.06% and 4%;

- Antipruritic agents between 0.01% and 1%;

- Anti-inflammatory agents between 0.5% and 1%.

Advantageously, the composition, in particular for preventing relapse, contains (in percentages by weight of the composition):

- between 1% and 5% sucrose monostearate and/or sorbitan esters;

- mixtures of between 3% and 25% oil;

-SK Influx V ^™ between 0.01% and 5%;

- a mixture of rhamnose, xylitol and mannitol between 0.06% and 3%;

- Antipruritic agents between 0.01% and 1%;

- Between 0.01% and 2% vitamin PP.

These various compositions are film-forming, that is to say they are capable of producing a thin film on the surface of the skin protecting it from the invasion of pathogenic microorganisms.

Many advantages can be seen from reading this invention:

- Reduces the adhesion of Staphylococcus aureus to atopic skin to prevent the growth of pathogenic bacterial flora while maintaining the saprophytic flora;

It is known that bacterial biofilms grow on the skin through a series of distinct phases, from bacterial adhesion to film growth, maturation, and expansion. Therefore, inhibiting the adhesion of Staphylococcus aureus to atopic skin results in the inhibition of pathogenic biofilm formation;

- Use of non-toxic, non-irritating sucrose and/or sorbitan esters to reduce the need for antimicrobial agents such as antibiotics and topical antiseptic products;

- The presence of lipids that are not naturally present in the skin restores the barrier function and helps reduce the evaporation of water from the epidermis;

- The complementary effects of sucrose and/or sorbitan esters and lipids reduce the burden of S. aureus by inhibiting adhesion, and then restore the barrier function of the stratum corneum;

- Thus, sucrose and/or sorbitan esters and lipids form a film on the epidermis that protects it from pathogenic microorganisms and prevents disruption of the barrier function.

Example

1/Effect of sucrose stearate on the adhesion of Staphylococcus aureus to solid substrates in vitro.

1) Method

Use the following scheme:

Staphylococcus aureus was grown in “Nutrient Broth N°2’ (Oxoid) at −37°C for 12 h at the Clinical Bacteriology Laboratory of La Timone Hospital (CIP65-8);

- The culture was centrifuged at 9000 g for 10 min and the bacterial pellet was resuspended in phosphate-buffered saline (PBS);

- dilute the suspension 50-fold in aqueous solution or in an aqueous solution containing the tested polyol. This is xylitol or sucrose stearate HLB 16 (SURFHOPE ^™ C1816);

The bacteria were preincubated with the test polyols for 45 min at −37°C;

The mixture (100 μl) was then spotted into the wells of a glass cytology slide with a fixed surface area (0.8 cm²) and walls, i.e., special slides for cell culture (Chamber Slide System, Labtek);

- the bacterial cultures were then incubated at 37° C. for 60 minutes, after which they were rinsed with UltraPurewater to wash off any bacteria that had not adhered to the surface of the wells;

- Adherent bacteria were stained with crystal violet for 10 seconds, rinsed, and dried at 25°C for 12 hours;

-Finally, the bacteria were examined under a microscope (oil immersion) at ×1000 magnification with a completely uniform field of view;

-Finally, the number of bacteria in each well is automatically counted using appropriate software.

2) Results

In this method, the effect of the polyol was analyzed in comparison to the inhibition of Staphylococcus aureus adhesion to a solid substrate. The test was performed with xylitol at a final concentration of 0.5% and HLB16 sucrose stearate (sucrose monostearate, SURFHOPE C18-C16) at a final concentration of 1%.

Table 1: Effects of xylitol (0.5%) and HLB16 sucrose stearate (1%) on the adhesion of Staphylococcus aureus to glass slide surfaces. The results are expressed as the mean counts of three fields together with the corresponding standard deviations.

3) Conclusion

A strong inhibition of S. aureus adhesion to the slide surface was observed in the presence of 1% HLB 16 sucrose stearate.

2/ Effect of the esters of the present invention on the adhesion of Staphylococcus aureus to human keratinocytes in vitro.

To confirm the in vitro findings, an ex vivo adhesion assay was performed on human keratinocytes adapted from the dog method (McEwan et al., 2005).

1) Bacterial strains

The clinical strain of S. aureus (CIP65-8) was isolated from a patient in the clinical bacteriology laboratory of La Timone Hospital in Marseille.

2) Method

Staphylococcus aureus was inoculated into 10 ml of 'Nutrient Broth No. 2' (Oxoid) and incubated with stirring at 37°C for 12 hours. The culture was centrifuged and the bacterial pellet was suspended in PBS. The bacterial density was adjusted to approximately 10 ⁶ CFU/ml with osmotic purified water.

Target areas were marked on the arm of a human volunteer. At each area, skin fragments were removed using five consecutive "pre-stripping" procedures with Sellotape ^™ Original discs (22 mm in diameter). Keratinocytes were then recovered from each target area using a D-Squame ^™ adhesive disc (22 mm in diameter) with a D-Squame disc applicator at a pressure setting of 150 g/cm².

The discs were placed in a Petri dish (35 mm in diameter) and overlaid with 0.5 ml of water or 0.5 ml of a suspension of Staphylococcus aureus containing the tested polyol at a density of 10 ⁶ CFU/ml (triplicate fields each).

The polyols tested in this manner were:

- Xylitol, at a final concentration of 0.5%;

-HLB16 sucrose stearate (sucrose nitrogen stearate, SURFHOPE C1816) at a final concentration of 1%,

- Sucrose laurate monoester (SURFHOPE C1216, 80% monoester), at a final concentration of 0.1%,

- Polysorbate 60, at a final concentration of 0.1%;

- Polysorbate 60, at a final concentration of 1%;

- Polysorbate 20, at a final concentration of 0.1%.

All solutions were prepared in water and exposed to S. aureus at room temperature 45 minutes before the adhesion assay began. The discs were incubated at 37°C for 60 minutes. The discs were then rinsed in osmotic purified water to remove any bacteria not adhered to the keratinocytes. Adherent bacteria were stained with oxalated crystal violet for 10 seconds. Finally, the discs were rinsed in osmotic purified water, placed on microscope slides, and air-dried for 24 hours.

3) Image acquisition and bacterial counting

Adherent bacteria were photographed using an Olympus BX53 microscope equipped with a digital camera. These images were then analyzed using UTHSCSA Image Tool software (Reindeer Graphics Inc.). The average number of bacteria per disc was calculated from 10-12 images, corresponding to the surface area of approximately 100 corneocytes. Mean values were compared using STATGRAPHICS Plus software (Manugistics).

4) Results and Conclusions

These test results showed that xylitol inhibited the adhesion of Staphylococcus aureus to keratinocytes by approximately 20% (Table 2). Very strong inhibitory activity—50% or more—was observed with HLB16 sucrose stearate and polysorbate 60 at a final concentration of 1% (Table 2). Polysorbates 20 and 60 showed very promising inhibitory activity at 0.1%. In contrast, sucrose monolaurate induced a significant increase in adhesion.

Table 2: Effect of xylitol and sugars of the present invention on the adhesion of Staphylococcus aureus to human keratinocytes.

For xylitol and sucrose stearate (1%), each sugar was tested in two independent experiments.

Thus, these ex vivo results confirm the in vitro results showing that the presence of HLB16 sucrose stearate in the surrounding environment inhibits the adhesion of S. aureus to solid substrates such as cytology slides and human keratinocytes.

3/Examples of topical compositions

ATODERM ^™ STOP EVOLUTIVE

product % composition by weight HLB 16 Sucrose Stearate (SURFHOPE C1816) 2.0. lipid phase 11.0. Other polyols 0.61. water Sufficient quantity (Q.s.) 100

ATODERM ^™ STOP CRISE

product % composition by weight HLB 16 Sucrose Stearate (SURFHOPE C1816) 2.0. lipid phase 11.0. Other polyols 0.61. Antipruritic agents 0.3. anti-inflammatory agents 0.5. water 100

ATODERM ^™ STOP RECIDIVE

product % composition by weight HLB 16 Sucrose Stearate (SURFHOPE C1816) 2.0. lipid phase 11.0. Other polyols 1.1. Antipruritic agents 0.3. Vitamin PP 0.3. water 100

References

Kita K., Sueyoshi N., Okino N., Inagaki M., Ishida H., Kiso M., Imayama S., Nakamura T., Ito M. 2002. “Activation of bacterial ceramidase by anionic glycerophospholipids: possible involvement in ceramide hydrolysis onatopic skin by Pseudomonas ceramidase.” Biochem J., 362, pp. 619-626.

McEwan N.A., Kalna G., and Mellor D. 2005. "A comparison of adherence by four strains of Staphylococcus intermedius and Staphylococcus hominis to canine corneocytes collected from normal dogs and dogs suffering from atopic dermatitis." Research in Veterinary Science, vol. 78, pp. 193-198.

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Claims (6)

1. Use of polysorbate 20, characterized in that it is used as an active ingredient in the preparation of compositions that inhibit the adhesion of Staphylococcus aureus to human skin and nasal mucosa. 2. The use as described in claim 1, wherein the polysorbate 20 comprises 0.1-5% by weight of the composition. 3. The use as described in claim 1, wherein the polysorbate 20 comprises between 1% and 3% by weight of the composition. 4. The use as described in claim 1, characterized in that it further comprises lipids capable of restoring skin barrier function, namely: - At least one lipid that is not naturally present in the skin; and/or - A mixture of naturally occurring ingredients in the skin, including ceramides 1, 3, and 6, cholesterol, free fatty acids, and phytosphingosine. 5. The use as described in claim 4, wherein the lipid is an oil. 6. The use as claimed in claim 4, characterized in that the mixture of the naturally occurring components in the skin corresponds to a lipid composition having an INCI name, namely: water, ceramide 3, ceramide 6II, ceramide 1, phytosphingosine, cholesterol, sodium lauroyl lactylate, carbomer, xanthan gum, accounting for 0.01%-5% by weight of the composition.