EP3380103A1

EP3380103A1 - Cicatrizing pharmaceutical composition for topical use

Info

Publication number: EP3380103A1
Application number: EP16815558.8A
Authority: EP
Inventors: Marianna ALTIERI
Original assignee: Nathura SpA
Current assignee: Nathura SpA
Priority date: 2015-11-25
Filing date: 2016-11-24
Publication date: 2018-10-03
Also published as: WO2017089475A1; ITUB20155902A1

Abstract

The present invention relates in general terms to a pharmaceutical composition for topical application, comprising an effective combination of hyaluronic acid, beta-glucan, hydrolyzed sericin, and glycerophosphoinositol or a pharmaceutically acceptable salt thereof, useful in particular as a cicatrizing agent for the treatment of micro- cracks and vulvar and/or vaginal epithelium wounds.

Description

Title: Cicatrizing pharmaceutical composition for topical use

DESCRIPTION

The present invention relates in general terms to a cicatrizing composition for topical use comprising an effective combination of active ingredients, useful in particular for the treatment of micro-cracks and vulvar and/ or vaginal epithelium wounds.

Background art

In the pharmaceutical field, the association between hyaluronic acid and beta-glucan has been known for quite some time for the vessel regenerating properties at skin level, for the vaginal treatment, for example for the treatment of atrophy or similar conditions.

Hyaluronic acid (HA) is a glycosaminoglycan present at high concentrations in the connective tissue and in the skin. The high hygroscopicity of HA is essential to recognize his action of tissue hydration. The chemical and physical characteristics of hyaluronic acid are the basis of the important lubricating properties of this substance, and the derived clinical applications were primarily viscous supplement, tissue regenerator, antioxidant and chondroprotector.

Further, in the skin, HA plays an important role as a free radicals scavenger and antioxidant. High molecular weight HA makes a viscous border around the cells in a dose dependent manner, and this restricts the movement of the oxygen reactive species. During the cicatrization process, hyaluronic acid is involved in various cellular responses. In the first inflammatory phase HA is accumulated in the injured tissue and, because of interactions with specific binding molecules, it induces the release of inflammatory cytokines improving the infiltration of cells that migrate in the injury site. During the granulation phase, the HA high levels promote cell proliferation and migration, while in the remodelling step HA contributes to the cicatrization process. As for beta-glucan [(1 ,3)-(1 ,6)-P-D-Glucan generally derived from fungus Plerotus Ostreatus or from yeast (100% Saccharomyces cerevisiae)], this is a high molecular weight polysaccharide, known as hydrophilic colloid capable of binding water and transfer it to the stratum corneum. It is a moisturizing and protective functional substance which is also used as an adjuvant in the wound cicatrization, in particular for the application on sensitive skin. Beta-glucan has a key role in the modulation of the local immune system, which is compromised in case of chronic inflammation.

Beside to the application for cutaneous treatments in the vagina, hyaluronic acid and beta-glucan are used for other topical applications, especially if in combination with other active ingredients, such as, sericin and/ or bisabolol. In this regard, the hydrolyzed sericin is known for its film-forming, moisturizing and protective properties. In fact, sericin helps to improve the skin elasticity, has anti-stretch mark, anti-aging, and antioxidants properties.

Alpha-bisabolol, instead, is a monocyclic sesquiterpene alcohol which, together with the chamazulene, represents an important component of the camomile essential oil. It shows decongestant and soothing properties. The bisabolol (purified as alpha form) is an active ingredient, known to exert an antiflogistic action found in dermatological, stomatological and otorhinolaryngological literature .

By way of example, WO2014/ 170239 describes a composition comprising sclareolide, a tyrosinase inhibitor, a sun protection factor, an antiinflammatory agent, and a desquamating agent which may contain, in addition to hyaluronic acid and beta-glucan also alpha-bisabolol and hydrolyzed sericin.

WO2014/202851 describes a composition comprising honey and other components, including beta-glucan, hyaluronic acid and bisabolol. The composition is formulated in the form of vaginal suppositories.

US2014/315995 discloses a composition for topical application comprising vitamin C, vitamin E and a polyphenolic antioxidant, suitable also for a vaginal application. Among the possible further components, such composition may comprise hyaluronic acid, beta-glucan and alpha bisabolol.

EP2762139 relates to a composition with antimycotic activity, containing luliconazole and/ or lanoconazole that may also comprise hyaluronic acid and sericin, acting as moisturizing agents, and which can be applied to the vaginal mucosa.

WO2012/076409 relates to a gel for vaginal application containing a fraction of colostrum enriched with immunoglobulins and chemotactic factors and antimicrobial factors and/ or antiviral agents partially encapsulated in mucus adhesive microbeads. The gel may also contain sericin, with moisturizing, film-forming and buffering activity.

CN 10380159 describes a gel for treating acne comprising, among other ingredients, hyaluronic acid, beta-glucan and alpha-bisabolol. Although the compositions in the art represent effective remedies for the treatment of inflammations and are capable of exerting a cicatrizing action at topical level in different action sites, it remains, however, the need to find a cicatrizing and/ or vessel regenerating composition which can be formulated as a cream, which can be used through topical administration, and having a specific efficacy for the treatment of micro-cracks and wounds, in particular of the vulvar (external use) and vaginal (internal use) epithelium. The Applicants have now found that an appropriate combination of hyaluronic acid, beta-glucan, hydrolyzed sericin, and glycerophosphoinositol allows to obtain an improved vessel regenerating action, which makes the thus obtained composition particularly suitable for skin administration for the treatment of cicatrization problems, for example postpartum, post physical treatments and/ or sores of different origin.

Summary of the invention In a first aspect, the invention relates to a dermo cosmetic or pharmaceutical composition for topical application, comprising: hyaluronic acid, beta-glucan, hydrolyzed sericin, and glycerophosphoinositol or a pharmaceutically acceptable salt thereof, and, optionally, bisabolol. In a further aspect, the invention relates to the use of said composition as a medicament, preferably as a cicatrizing and/ or vessel regenerating agent, even more preferably as a cicatrizing agent, in particular for the treatment of micro-cracks and wounds of vulvar (external use) and vaginal (internal use) epithelium of different origin.

In a preferred embodiment, the composition is intended for topical use, and it is in a pharmaceutical form suitable for skin applications, for example in the form of a cream or gel. In a further aspect, the invention relates to a process for the preparation of the above composition, which comprises the steps of: a) preparation of an aqueous phase comprising liquid and powder not- thermolabile ingredients; b) preparation of a fat phase by fat melting and oil mixing; c) emulsion of the two phases; d) addition of the other ingredients dissolved in aqueous solution.

It is a further aspect of the invention a pharmaceutical composition comprising hyaluronic acid, beta-glucan, and glycerophosphoinositol, preferably for use as a medicament for topical administration, even more preferably as a cicatrizing and/ or vessel regenerating agent.

Detailed description

Unless otherwise specified, the term "weight percentage" (% w/w) indicates the weight of the substance relative to the total weight of the composition.

"Pharmaceutically acceptable salt" includes in its meaning those derivatives of the concerned compound able to maintain or improve the tolerability and/ or the biocompatibility of the compound from which they are derived.

The term "topical application" means an administration by direct contact of the present composition with the action site to which it is targeted, typically skin and/ or mucous membranes, exerting in this way an action at the local level.

As indicated above, the composition of the invention, comprising hyaluronic acid, beta-glucan, and hydrolyzed sericin, is characterized in that it contains at least glycerophosphoinositol or a pharmaceutically acceptable salt thereof, optionally in the presence of bisabolol. This association of components allows the use of the composition as cicatrizing agent, as a healing agent for atrophic and dystrophic conditions, and as balancing agent of the vaginal environment in general. Moreover, the present composition is suitable for the topical application, since it can be formulated so to allow the cutaneous application, for example in the form of mucus adhesive cream, allowing the local application in a simple and functional way.

The composition of the invention, moreover, allows to obtain a combined and synergistic action of the ingredients which compose it, exerting, in particular, an increased and effective cicatrizing function.

The hyaluronic acid ingredient is preferably at high molecular weight, defined as having a molecular weight (MW) of at least 1.5 x 10⁶ Dalton, preferably comprised between 1.5 x 10⁶ and 2.2 x 10⁶ Dalton. In any case, the hyaluronic acid is present in the pharmaceutical composition of the invention in amounts between about 0.1% and 0.4% w/w being the values between about 0.1% and 0.3% w/w especially preferred.

Similarly to hyaluronic acid, also beta-glucan and hydrolyzed sericin are present in the composition of the invention in amounts comprised between about 0.1% and 0.4% w/w, being the values comprised between about 0.1% and 0.3% w/w particularly preferred.

Therefore, in one embodiment, the composition of the invention comprises hyaluronic acid, beta-glucan and hydrolyzed sericin in the same quantities % w/w, preferably comprised between 0.1% and 0.3% w/w.

Beta-glucan can be used with a purity comprised between 85% and 93%, being preferably obtained by a bio-fermentation route from the yeast Saccharomyces cerevisiae, or by extraction from the fungus Plerotus Ostreatus. It gives to the composition of the invention improved moisturizing and protective properties, especially helpful in the cicatrization of the treated vaginal areas. Hydrolyzed sericin is preferably obtained by silk purification and it makes it possible to increase the film-forming and moisturizing properties of the present composition, ensuring, moreover, an excellent biocompatibility. It is used in the composition of the invention in amounts comprised between about 0.1% and 0.4% w/w, being the values comprised between about 0.1% and 0.3% w/w particularly preferred.

The present composition is also characterized in that it contains as active ingredient also the glycerophosphoinositol (GPI), or, preferably, a pharmaceutically acceptable salt thereof. In one embodiment, said salt is selected from: lysine and choline salt, being the latter particularly preferred. The Applicant has in fact noticed that the use of choline salt is particularly useful in case the present composition is intended as a medical device.

The glycerophosphoinositol salt of choline is an active ingredient of vegetal origin, usually purified from sunflower lecithin and commercially available.

Preferably, the glycerophosphoinositol or a pharmaceutically acceptable salt thereof, is present in the composition of the invention as an aqueous solution in amounts comprised between about 0.1% and 0.3% w/w, being the values comprised between about 0.2% and 0.3% w/w particularly preferred.

In one embodiment, the composition of the invention may also contain bisabolol, in racemic form or, preferably, as alpha isomer. Bisabolol possibly used in the present composition is preferably extracted by steam distillation from bark of the plant Vanillosmopis erythropappa (also known as Candeia wood).

The composition of the invention comprises bisabolol in an amount comprised between 0% and 2% w/w, preferably between about 0.1% and 1.5% w/w, even more preferably between about 0. 1% and 1% w/w. In this regard, the use of bisabolol is particularly convenient as it is able of exerting a decongestant and soothing action even at small percentages, without causing substantially any side effects.

In one embodiment, the composition of the invention, preferably in the form of a hydrophilic cream, comprises at least: hyaluronic acid 0.1-0.3% w/w

beta-glucan 85% 0.1-0.3% w/w

hydrolyzed sericin 0.1-0.3% w/w

alpha bisabolol 0.1-0.2% w/w

glycerophosphoinositol 0.2-0.3% w/w

(salt of choline in Aq. solut. 10%) 0.25% w/w

Purified water q.s. to 100%

Alternatively to beta-glucan 85%, the above preferred composition may contain beta-glucan 93% in the same indicated amounts % w/w.

In addition to the described components, the composition of the invention may further contain other functional excipients known in the art and used by the skilled in the art for the preparation of a cream, such as typically: sweet almond oil (prunus dulcis), glycerine, decyl oleate, hydroxyethyl acrylate/ sodium acryloyldimethyl taurate copolymer, caprylyc glycol, hexane- l ,2-diol, crosslinked polyacrylic acid, ethylhexylglycerin , sorbitan isostearate, polysorbate 60, lecithin, lactic acid, phytic acid, sodium hyaluronate, sodium hydroxide, tocopherol, ascorbyl palmitate, citric acid, hydroxyethyl acrylate/ sodium acryloyldimethyl taurate copolymer, alone or, preferably, in mixture thereof. Excipients are present in an overall amount comprised between about 10% and 18% w/w, being the values comprised between about 13% and 15% w/w particularly preferred. Preferred excipients are sweet almond oil and/ or an acrylic acid polymer crosslinked with divinyl alcohol. In particular, the sweet almond oil contains a high percentage of oleic and linoleic acid, and minor amounts of palmitic, stearic, lauric and myristic acid. Even more preferably, said sweet almond oil is used in amounts between about 3 and 5% w/w, while the acrylic acid polymer crosslinked with divinyl alcohol is used in amounts between about 0.4 and 0.6% w/w.

Preferably, the present composition for topical use is in the form of mucus adhesive hydrophilic cream, even more preferably having a pH comprised between about 3.5 and 5. The cream formulation not only allows to have an easily applicable on the skin product, as it is rapidly absorbed, but also it allows an application that is not greasy, since it is a water-based formulation. The density of the hydrophilic cream is preferably comprised between about 0.8 and 1.2 g/ml with a viscosity at 25°C (R6/20, 49%) comprised between 20,000-30,000 mPas.

According to a further aspect, the invention relates to the present composition, designed for example as a medical device, for use as a medicament, preferably as a cicatrizing and/ or vessel regenerating agent, even more preferably for the treatment of micro-cracks and vulvar epithelium (external use) and vaginal (internal use) wounds of different origin. In other words, the present composition is useful for the preparation of a medicament with cicatrizing and/ or vessel regenerating action, for topical application.

The composition of the invention is also effective for the treatment of postpartum cicatrizing problems, post physical treatments and/or sores of different origin, and atrophy, for example post menopause or due to hormonal imbalances in general. In one embodiment, the invention relates to the above composition, as a medicament for topical application (or cutaneous), preferably in the form of a hydrophilic cream. Specifically, the Applicant has noticed that the composition of the invention may assist in the treatment of symptoms associated with atrophic vaginitis, such as dryness, itching, irritation and pain/ discomfort, improving elasticity and tone of tissues and encouraging the restoring of a moist and more lubricated environment. It should be noted that, in addition to exert the regenerative actions of the skin, the composition of the invention also allows to exert an improved soothing and anti-inflammatory action, associated with a convenient cytotoxicity, due to the combination of the active ingredients, as described above in detail, and as reported in the experimental part herein (see Examples 2 and 3).

Furthermore, the data collected in the Example 1 clearly show that the present composition may support the migration of keratinocytes and thus promote the regeneration of the skin. The Applicant has, in fact, studied the ability to repair artificial wounds induced in vitro in cell monolayers, since this ability can be considered as an evidence of the skin reparation activity in vivo. In more detail, when the cells are exposed to substances that stimulate their ability to migrate (indispensable element in the process of vessel regeneration and wound healing) they change shape and modify the cytoskeleton organization, changing stationary phenotype in migratory phenotype . Microfilament bundles containing adhesion molecules as viniculine and actin and stress fibers that keep the cells anchored to the substrate disappear or migrate to the cell periphery, allowing for cell migration. Furthermore, the present composition is also convenient due to the ease of preparation.

In this regard, it constitutes a further aspect of the invention a process for the preparation of the composition for topical use as described above in detail, said process which comprises the preparation of an aqueous phase comprising non-thermolabile components such as hyaluronic acid, and glycerophosphoinositol, and of a fatty or oily phase, followed by the emulsion of the two phases and the addition of the remaining components in an aqueous solution.

In a further aspect, the invention also relates to a pharmaceutical composition comprising hyaluronic acid, beta-glucan, and glycerophosphoinositol, preferably for topical use as a cicatrizing and/ or vessel regenerating agent. Said composition is useful in cutaneous cicatrizing processes and in the treatment of micro-cracks and vulvar and vaginal epithelium wounds in general. The invention will now be described with the following experimental part, intended to better understand the content, without however limiting the scope.

EXPERIMENTAL

EXAMPLE 1: IN VITRO EVALUATION OF WOUND HEALING ON CELL CULTURES

Example 1.1: test execution

Adopted cell model The cell model used for the in vitro test is represented by stabilized human keratinocytes (HaCaT cells). The cell line derives from adult skin that maintains full epidermal differentiation capacity.

Sample preparation The sample was dissolved in growth medium at a concentration of 1 mg/ml and subsequently diluted in the culture medium and tested at the following concentrations:

- 1000 Mg/ml

- 100 Mg/ml - 10 Mg/ml

Treatment and exposure

The cells were treated for 24 hours with the product to the final concentrations indicated in the culture medium (DMEM + 10% FBS).

Cells treated with medium alone were used as negative control. Cells were kept in incubator at 37 °C with 5% CO2. The test was performed in triplicate.

Preparation of cell cultures

Cells were plated at 30,000 cells/ well into sterile 96-well plates and allowed to grow to confluence in the above described culture medium, before performing the wounds and apply the samples.

Execution of the wound and exposure of cells to the products

Once confluency was obtained, an artificial wound was carried out for the entire length of the monolayer with a sterile plastic tip, on each sample. The cell culture medium was removed and replaced with the medium containing the substances to be tested, except in the negative controls.

Evaluation of wound healing

Immediately after the execution of the wound (TO) and at different times within 24 h fresh observations are carried out in phase contrast microscopy of the treated samples and controls with a Leica DM microscope IL-IMC with magnification between 10X and 4X, with registration of the wound size in μπι. The images are documented with digital camera Primocam 5000 with the ISCapture image software. At different times, normally after 2, 6, 24 h of treatment wound measurements in each sample were made again. By calculating the average values recorded at different times, the percentage of wound healing was calculated and this was compared between the treated samples versus the negative control to rate effectiveness. Example 1.2: results and conclusions

Table 1: average wound reduction % compared to T0(ds)

* The used composition of the invention comprises the required ingredients for its formulation as a cream for topical use, i.e.: hyaluronic acid 0.2% w/w

beta-glucan 85% 0.2% w/w

hydrolyzed sericin 0.2% w/w

alpha bisabolol 0.1% w/w

glycero phosphoinositol 0.25% w/w

(10% choline salt)

excipients 13- 15% w/w

purified water q.s. to 100%

It can be noted that the present composition shows an increase in the wound healing ability in vitro after 6 hours of treatment already.

EXAMPLE 2: IN VITRO EVALUATION OF THE CYTOTOXICITY AND THE ANTI-INFLAMMATORY POTENTIAL ON 3D EPIDERMIS

Example 2.1: Cytotoxicity The cytotoxicity was assessed by MTT assay after incubation; cell survival test with 3D reconstructed human epidermis in vitro for the evaluation of the cytotoxic potential caused by the sample.

Cytotoxicity test purpose

The cytotoxicity assay may be performed to evaluate the irritation potential of a product or a substance using keratinocytes cultures taken from human skin. The in vitro test conducted on cells derived from skin tissue appears to be a simplified experimental method, but able to give a lot of information on the reactions that may occur in vivo. Moreover, the use of a three-dimensional model of human skin, where a well- differentiated stratum corneum is present, effectively mimics the percutaneous absorption that can occur in vivo, and allows to evaluate the biological effects that occur in the deeper layers of the epidermis.

Under the impulse of toxic and stressful stimuli, such as exposure to UV or quite aggressive biological and chemical substances, the keratinocytes increase the production and the release of cytokines and prostaglandins, such as interleukin- 1 a (IL- la), interleukin 1 β (IL- Ι β), tumor necrosis factor-a (TNF-a) or interferon-a (IFN-a), Interleukin-6 (IL-6), prostaglandin E2 (PGE2) and prostaglandin 2a (PGE2a). These molecules are soluble mediators involved in the genesis of the cutaneous inflammation process.

The appearance or the increase of these markers in the cell culture medium after exposure to certain substances, suggests the triggering of a cascade of pro-inflammatory events and can therefore be considered a good marker of pro-inflammatory/ pro-irritating potential.

The set of functionality tests carried out by the Applicant has been studied to assess the protective and inhibitory potential of the tested product against skin irritation induced by moderate irritant agents such as sodium lauryl sulfate (SLS), using a pattern of cells of the human skin grown multi-layered in vitro. To evaluate the anti-inflammatory effect, using specific ELISA, the inhibition of the release of a cytokine (IL- la) caused by SLS was assessed, in presence of the tested substance compared with untreated skin samples. Under normal physiological conditions, non- stimulated skin keratinocytes constitutively produce small doses of cytokines and prostaglandins.

Treatment and exposure

30μ1 of the sample as it is (δθμΐ/cm²) were applied on each unit of epithelium, in duplicate, and the exposure was continued for 24 hours at 37 °C, 5% CO2. After the exposure, the product was eliminated by washing in phosphate buffer (PBS) and cell viability was assessed by MTT assay. The skin irritation characterized by the release of IL- la was caused by pre-treatment for 40 minutes with 0.25% SLS. Not SLS irritated and treated with 30μ1 of PBS units were used as negative control. For IL- 1 alpha analysis, medium samplings were performed at 6 and 24 hours. As negative control the PBS treated unit maintenance medium and the medium of two sample treated not irritated skin units were taken, while as a positive control the culture medium of two SLS 0.25% solution treated skin units was used. Cytotoxicity test (description)

The MTT test is simple, accurate and provides reproducible results. This method was originally developed by Mossman. The key reagent is 3- [4, 5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide or MTT, a substance which gives a yellow colour in aqueous solution. The mitochondrial dehydrogenase of the viable cells cuts the tetrazolium ring, leading to the formation of purple formazan crystals insoluble in water. The crystals are dissolved with a MTT solubilizing solution.

The resulting purple solution is measured spectrophotometrically. An increase or decrease of the viable cells leads to a concomitant change in the amount of the formed formazan and which can be considered as an indicator of the degree of cytotoxicity caused by exposure to irritants.

At the end of treatment, the cell viability is assessed by incubating the tissues with MTT for 3 hours in a 24-well plate (1 mg/ml; 0.3 ml per well). The precipitated formazan crystals were extracted using isopropanol (2 ml) and quantified spectrophotometrically in a 96-well plate (200 μΐ/well).

The plate was shaken on a shaker plate assuring that all crystals have dissolved and have formed a homogeneous solution. The absorbance at 550 nm was read with a colorimeter (Tecan Sunrise remote model) equipped with a plate reader subtracting the background reading at 690 nm.

The result is expressed as cell viability in percent according to the formula:

% cell viability = [OD(550 nm - 690 nm) tested product/ OD (550 nm - 690 nm) negative control] x 100.

Example 2.2: results

The 3D epidermis treatment with 0.25% SLS for 40 minutes caused a cell mortality of 15% after 24 hours. The tested sample of the present composition showed a cell mortality of 61% after 24 hours of treatment.

Table 2: cell survival on 3D epidermis after 24 hours of exposure vs. untreated control (ds)

* The used composition of the invention includes:

hyaluronic acid 0.2% w/w

beta-glucan 85% 0.2% w/w

hydrolyzed sericin 0.2% w/w

alpha bisabolol 0.1% w/w

glycero phosphoinositol 0.25% w/w

(10% choline salt) excipients 13- 15% w/w

purified water q.s. to 100%

EXAMPLE 3: IN VITRO EVALUATION OF THE ANTI-INFLAMMATORY POTENTIAL ON 3D EPIDERMIS The anti-inflammatory potential of the composition of the invention was evaluated by inhibiting the release of inflammatory cellular mediators (IL- la) after SLS induced stress.

Example 3.1: cytokine production IL- la was assayed in the culture medium of the treated and not treated epidermis units, using direct ELISA (Enzyme-linked immunosorbent assay), that is, the colour development was directly proportional to the cytokine amount present in the culture medium.

50 μΐ of sample were incubated for 1.5 hours at room temperature in a plate charged with the specific primary antibody to the tested cytokine. After incubation, the samples were washed 5 times with a washing solution. Then 100 μΐ of biotinylated secondary antibody were added and incubated for one hour at room temperature. The samples were washed again 5 times with the washing solution and incubated for 30 minutes with the solution of peroxidase conjugated streptavidin (HP). After 5 washes the colorimetric substrate was added for the detection of the cytokine bound to the plate. The reading was made at 450 nm and the cytokine concentration was determined using a standard curve as reference. The sensitivity limit of this test was less than 3 pg/ml. Example 3.2: results The samples were tested on the epidermis to assess a possible decrease in the production of IL- la, induced by 0.25% SLS pre-treatment. (Table 3.1.2.1), index of the anti-inflammatory effect.

Table 3: inhibition of the IL-1 alpha release after treatment with the sample.

* The used composition of the invention includes:

hyaluronic acid 0.2% w/w

beta-glucan 85% 0.2% w/w

hydrolyzed sericin 0.2% w/w

alpha bisabolol 0.1% w/w

glycero phosphoinositol 0.25% w/w

(10% salt of choline)

excipients 13- 15% w/w

purified water q.s. to 100%

The release of IL- 1 alpha in the 3D epidermis culture medium after 6 h and 24 h of exposure to the tested product and after 0.25% SLS solution stimulation is highlighted in pg/ml.

As it can be noted, in the presence of 0.25% SLS there is a release of IL- 1 that increases progressively from 6 to 24 hours of exposure. The present composition was found to reduce the production of 0.25% SLS induced IL- 1 alpha. The sample causes an interleukin release superior to the negative control, probably due to the cell mortality observed at 24h. EXAMPLE 4: IN VITRO EVALUATION OF SYNERGISTIC EFFECT ON TISSUE MATRIX RESTORATION ON A VAGINAL CELL LINE.

The synergistic effect of the composition of the invention on vaginal tissue matrix restoration was evaluated comparing gene expression of collagen VI in cells treated with each individual component of the composition respect to that in cells treated with the complete composition of the invention.

The expression of collagen VI is a mark of cellular regenerating ability and tissue matrix restoration.

In order to assess the gene expression of collagen VI in cells, the extracted total mRNA was subjected to reverse transcription and cDNA of collagen VI thus obtained is quantified by highly sensitive real-time RT-PCR.

Adopted cell model

The cell model used for the in vitro test is represented by stabilized human VK2/E6E7 vaginal epithelial cell line (commercially available from GIBCO) derived from adult skin that maintains full epidermal differentiation capacity.

Treatment and exposure

VK2/E6E7 vaginal epithelial cell line was cultured in plastic plates at 37°C, 5% CO₂ in a DMEM:F12 culture medium containing: 2.5 mM L- glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and 1200 mg/L sodium bicarbonate supplemented with 10% of fetal bovine serum).

The cell line was maintained at 37°C, 5% CO2 in a sterile incubator for a period of 24 hours.

Experiments were carried out adding each singular component of the composition or the complete composition to the cell line maintained at 37°C, 5% CO2. To assess the ability of extracellular matrix synthesis and molecular process of repairing, cells were analyzed after 24 hours from the addition of above-mentioned components to be tested

Cells treated with medium alone were used as negative control. Concentrations of each component tested individually were: hyaluronic acid 0.2% w/w glycero phosphoinositol 0.25% w/w

(10% choline salt) beta-glucan 93% 0.2% w/w hydrolyzed sericin 0.2% w/w

The same concentrations were maintained in the formulation of complete composition.

Extraction total RNA and RT-Real-time PCR

Quantitative RT-PCR— Total RNAs were extracted from cells with TRI reagent (Invitrogen), retro-transcribed using the High Capacity cDNA synthesis kit (Invitrogen) (containing the relevant specific primers), and amplified on an Abi Prism 7000 instrument (Applied Biosystems).

Pre-developed Taqman gene expression assays (Invitrogen) were used to quantify transcripts gene of interest. Ac tin was used as internal reference.

The relative gene expression was determined by comparing 2-AACt (Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-AAC(t) method. Methods 25, 402-408). Results

Gene expression of collagen VI in treated and untreated cells was evaluated by comparing the values of Ct (Cycle threshold) to determine a change of target gene expression in a sample compared with another sample selected as calibrator (control, untreated). For each experiment in relative quantification the following were necessary:

• TARGET ("sample"): DNA sequence to be analyzed. [The target corresponds to individual experiments carried out with each ingredient] • CONTROL: The sample to be used as reference for the comparative analysis (control, untreated)

• ENDOGENOUS CONTROL - A gene constitutively expressed (beta-actin in this case) in all the analyzed samples, which was needed to normalize data with respect to the loaded DNA amount and to changes of reaction efficiency.

Calculations:

Normalize the value of each sample (control, HA, BETOX, GPI, sericin) with a constitutively expressed endogenous control (beta-actin):

Ct average value (sample) - Ct beta actin = dCt sample

Compare each dCt thus obtained, with the dCt control (untreated): dCt sample - dCt control= ddCt sample R= final result= 2 - ddctsampie

Table 4: amount of collagen VI after treatment with each component of the composition or the complete composition.

Evaluation of synergistic e ffect

To evaluate if a synergistic effect occurred, the following calculation (comparative with the control) was adopted.

Sum of collagen VI gene expression (COL6A1) of the individual ingredients compared to the control: (HA COL6A1 - CNT COL6A1) + (GPI COL6A1 - CNT COL6A1) + (BGL COL6A1 - CNT COL6A1) + (SERIC COL6A1 - CNT COL6Al)= 0.91

Difference between gene expression of the complete formula and control:

FINAL COMPOSITION COL6A1 - CNT COL6Al= 1.3

This value (1.3) is greater than that of the sum of the individual compounds (0.91); this confirms the hypothesized synergistic effect.

The synergistic effect, thus, was evaluated in terms of final results of the total composition compared to those of individual compounds on gene expression of a target gene (in this case, collagen VI), as reported in the following publications: Chiasson J., Naditch L., Diabets Care, 2001 Jun;24(6):989-94 and Burashnikov A., et al. J Am Coll Cardiol. 2010 Oct 5; 56(15): 1216-1224. The synergistic activity of all the components causes an evident increase of the amount of collagen VI in the cells treated with such composition.

EXAMPLE 5: IN VITRO EVALUATION OF ADDITIVE EFFECT ON CELLULAR REPAIR ON A VAGINAL CELL LINE.

The additive effect of the composition of the invention on cellular repair was evaluated comparing gene expression of interleukin 6 (IL-6) in cells treated with each individual component of the composition with the gene expression of interleukin 6 (IL-6) in cells treated with the complete composition of the invention.

The expression of IL-6 is commonly associated with events promoting cellular repair.

In order to assess the gene expression of IL-6 in cells, the extracted total mPvNA was subjected to reverse transcription and cDNA of IL-6 thus obtained was quantified by highly sensitive real-time RT-PCR.

The method used to carry out this experiment is the same as that used in Example 4.

Results:

Gene expression of IL-6 in treated and untreated cells was calculated as follows:

Calculations:

Normalize the value of each sample (control, HA, BETOX, GPI, sericin) with an endogenous control (beta-actin) constitutively expressed:

Ct average value (sample) - Ct beta actin = dCt sample Compare each dCt thus obtained, with dCt control (untreated): dCt sample - dCt control = ddCt sample R= final result= 2 ^{~ ddctsam}P^le

Table 5: Amount of IL-6 after treatment with each component of the composition or the complete composition.

Evaluation of addivite e ffect:

To evaluate if an additive effect occurred, the following calculation (comparative with the control) was adopted.

Sum of IL-6 gene expression of the individual ingredients compared to the control:

(HA IL6 - CNT IL6) + (GPI IL6 - CNT IL6) + (BGL IL6 - CNT IL6) + (SERIC IL6 - CNT IL6)= 2.03

Difference between gene expression of the complete formula and control:

FINAL COMPOSITION IL6 - CNT IL6= 1.9 This value (1.9) is similar to sum of individual compounds (2.03) and the hypothesized additive effect is thus confirmed; in fact, the result of the action of the compounds is a pharmacological response equal to the algebraic sum of the individual responses, without any antagonistic effect among themselves. The additive activity of all the components causes an evident increase of the amount of IL-6 in cell treated with such composition and thus of the capability to promote cellular repair through the activation of the gene expression of interleukin 6.

Claims

1. A pharmaceutical composition for topical application, comprising: hyaluronic acid, beta-glucan, hydrolyzed sericin, and glycerophosphoinositol or a pharmaceutically acceptable salt thereof.

2. The composition according to claim 1 , further comprising bisabolol, preferably in amounts comprised between 0 and 2% by weight with respect to the total weight of the composition.

3. The composition according to claim 2, wherein said bisabolol is in purified form as alpha isomer.

4. The composition according to the preceding claims, wherein said pharmaceutically acceptable salt of glycerophosphoinositol is a choline salt.

5. The composition according to the preceding claims, wherein said hyaluronic acid is present in amounts comprised between 0.1% and 0.4%, preferably between 0.1% and 0.3% by weight with respect to the total weight of the composition.

6. The composition according to the preceding claims, wherein said beta- glucan is present in amounts comprised between 0.1% and 0.4%, preferably between 0.1% and 0.3% by weight with respect to the total weight of the composition.

7. The composition according to the preceding claims, wherein said hydrolyzed sericin is present in amounts comprised between 0.1% and 0.4%, preferably between 0.1% and 0.3% by weight with respect to the total weight of the composition.

8. The composition according to the preceding claims, wherein said glycerophosphoinositol or a pharmaceutically acceptable salt thereof is present in amounts comprised between 0.1% and 0.3%, preferably between 0.2% and 0.3% by weight with respect to the weight the total of the composition.

9. The composition according to the preceding claims, comprising at least: hyaluronic acid 0.1-0.3% w/w beta-glucan 0.1-0.3% w/w hydrolyzed sericin 0.1-0.3% w/w alpha bisabolol 0.09 to 0.12% w/w glycero phosphoinositol (10% choline salt) 0.2-0.3% w/w purified water q.s. to 100%

10. The composition according to claim 9, wherein beta-glucan has a purity of at least 85%, preferably a purity of 85% -93%.

1 1. The composition according to the preceding claims, further comprising one or more excipients selected from: sweet almond oil (prunus dulcis), glycerine, decyl oleate, hydroxyethyl acrylate/ sodium acryloyldimethyl taurate copolymer, caprylic glycol, hexane- l ,2-diol, crosslinked polyacrylic acid, ethylhexyl glycerine, sorbitan isostearate, polysorbate 60, lecithin, lactic acid, phytic acid, sodium hyaluronate, sodium hydroxide, tocopherol, ascorbyl palmitate, citric acid, hydroxyethyl acrylate/ sodium acrylyldimethyl taurate copolymer, alone or, preferably, in mixture thereof.

12. The composition according to claim 1 1 , wherein said excipients are present in amounts comprised between 10% and 18% by weight with respect to the total weight of the composition.

13. The composition according to the preceding claims, in the form of gel or cream, preferably hydrophilic cream.

14. The composition according to the preceding claims, for use as a medicament.

15. The composition for use according to claim 14, as a cicatrizing and/ or vessel regenerating agent.

16. The composition for use according to claim 15, as a cicatrizing agent.

17. The composition for use according to claim 16, as a cicatrizing agent for postpartum conditions, post physical treatments and/ or sores of different origin, atrophy, post menopause or due to hormonal imbalances, or for the treatment of micro-cracks and vulvar and vaginal epithelium wounds.

18. A composition comprising: hyaluronic acid, beta-glucan, and glycerophosphoinositol or a pharmaceutically acceptable salt.

19. The composition according to claim 18, for use as a medicament, preferably by topical administration.

20. The composition for use according to claim 18, as a cicatrizing and/or vessel regenerating agent.