The present invention relates to a composition comprising tranexamic acid, at least one inhibitor or antagonist of the enzyme tyrosinase and at least one polyphenol source. The invention also relates to the cosmetic use of the composition for the prevention and/or treatment of alterations of skin pigmentation, in particular of skin hyperpigmentation.

BACKGROUND OF THE INVENTION

Melanocytes are highly specialized cells that have the function of protecting the epidermis from the damage of ultraviolet (UV) radiation by means of the production of melanin, through a process known as melanogenesis. Together with melanocytes, the epidermis is composed mainly of keratinocytes, which constitute up to 95% of the epidermis. During their differentiation, keratinocytes move progressively toward the more superficial layers of the skin and their nuclei are constantly exposed to high UV radiation levels. In order to protect the DNA of keratinocytes and therefore the integrity of the cells themselves, melanin is transferred from melanocytes to the surrounding keratinocytes by means of dendrites in response to exposure to UV radiation. By arranging themselves above the keratinocyte nuclei, melanin granules absorb UV radiation before it is able to reach the nucleus and damage the DNA.

Pigmentation disorders are conditions that have important aesthetic and psychological effects. When melanin production in human skin is altered, disorders such as vitiligo and melasma occur. Vitiligo is the most widespread pigmentation disorder (it affects 0.1-2% of world population) and is characterized by a loss of function of melanocytes, with consequent appearance of light patches on colored skin. Vice versa, the abnormal accumulation of melanin can lead to melasma, freckles and sun spots.

The appearance of dark areas and patches on the skin can also be a consequence of the normal skin aging process: the accumulation of reactive oxygen species (ROS) due both to ordinary metabolism and to extrinsic factors (exposure to sunlight, environmental pollution, lifestyle and habits such as smoking) can exacerbate the activity of various enzymes, such as tyrosinase, which is the main enzyme involved in melanin synthesis. These dark patches caused by an increase in UV exposure appear mostly in the regions of the body that are not covered and therefore more exposed, such as the face and hands, thus causing important psychological effects in people who have these skin disorders.

Skin pigmentation disorders are very frequent in all ethnic groups and cause medical problems (acceleration of the natural aging process) but most of all aesthetic and psychological problems, which affect the possibility to lead a normal life.

Skin pigmentation disorders are currently treated with nondefmitive methods based on invasive therapies which use laser, light or chemical peeling and on topical treatments that use creams based on active ingredients such as hydroquinone, azelaic acid, arbutin or kojic acid.

Hydroquinone is currently the most used and most effective active ingredient for the topical treatment of skin patches; however, it has some side effects, such as toxicity, allergic reactions, the development of skin contact dermatitis and also difficulty in preparing stable formulations.

Other active ingredients for topical use have been recently developed which have shown total or partial effectiveness in the treatment of hyperpigmentation in people of color; examples of these active ingredients are: soy extracts, licorice extracts, extracts from the leaves of Morus alba containing moracin M, 4-n-butylresorcinol, niacinamide (vitamin B3), ellagic acid, resveratrol, dioic acids and N-acetylglucosamine (Konda et al, New horizons in treating disorders of hyperpigmentation in skin of color, Sem Cutan Med and Sur, 31 : 133-139, 2012).

Active ingredients have also been developed recently which are effective for the treatment of hyperpigmentation of people of color and can be administered orally. Among these, mention can be made of procyanidins, tranexamic acid and extract of Polipodium Leucotomos (Konda et al., New horizons in treating disorders of hyperpigmentation in skin of color, Sem Cutan Med and Sur, 31 : 133-139, 2012).

Despite the recent discoveries that have just been described, in the field the need persists for new compositions for topical use for the treatment of skin hyperpigmentation (i.e., of skin patches) which can replace hydroquinone, demonstrating the same effectiveness (or a higher effectiveness), and at the same time have lower side effects.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a composition comprising tranexamic acid, at least one inhibitor or antagonist of the enzyme tyrosinase, and at least one polyphenol source.

In a preferred embodiment, the composition also comprises vitamin B3 or a derivative thereof.

The invention furthermore relates to the use of the composition in the cosmetic field for the prevention and/or treatment of skin pigmentation alterations, in particular for the treatment of skin hyperpigmentation.

Another aspect of the present invention relates to the composition as described above for use as a remedy in the treatment and/or prevention of pathological conditions affecting the skin.

The Applicant has found surprising that by applying the composition according to the invention, particularly in the form of cream or serum, it is possible to improve/attenuate skin pigmentation alterations.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the survival of murine melanoma cells (B16) in a concentration-response curve with a positive control (SDS).

Figure 2 shows the production of melanin (pg/ml) by melanocytes following treatment with different compositions. Figure 3 shows photographs of tissues treated with various compositions, not treated or treated with a positive control.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a composition comprising tranexamic acid, at least one inhibitor or antagonist of the enzyme tyrosinase, and at least one polyphenol source.

In one embodiment, the composition comprises tranexamic acid, at least on inhibitor/antagonist of the enzyme tyrosinase, at least one source of polyphenols and vitamin B3 or a derivative thereof.

Preferably, the concentration of tranexamic acid varies between 0.2 and 10% by weight, preferably between 0.5 and 8% by weight.

The inhibitor or antagonist of the enzyme tyrosinase is chosen from trihydroxybenzoic acid glucoside, gallic acid (trihydroxybenzoic acid), catechin, epigallocatechin, epigallocatechin-3-0-gallate, methyl gentisate (methyl hydroxybenzoate), and 4-n-butylresorcinol.

In a preferred embodiment of the invention, the inhibitor or antagonist of the enzyme tyrosinase is chosen from gallic acid and trihydroxybenzoic acid glucoside.

Preferably, the concentration of said inhibitor or antagonist of the enzyme tyrosinase varies between 0.2 and 10% by weight, preferably between 0.5 and 8% by weight.

The at least one polyphenol source is chosen from soy extract and Polipodium extract (i.e., a fern extract).

Preferably, the at least one polyphenol source is an extract of Polipodium leucotomos, a particular species of fern.

The concentration of such at least one source of polyphenols varies between 0.05 and 5% by weight and preferably varies between 0.08 and 3% by weight.

The composition according to the present invention may furthermore comprise at least one excipient acceptable for pharmaceutical use or for cosmetic use that is useful in the preparation of the composition and is biologically safe and non-toxic.

Such excipient can be at least one conditioning agent, preferably at least one skin humectant, occlusive or emollient conditioning agent.

Preferably, such at least one skin humectant, occlusive or emollient agent is chosen among: glycerin, hyaluronic acid, capric/caprylic triglyceride, octyldodecanol, jojoba oil, macadamia oil, aspartic acid, decyl cocoate, soy oil, lactic acid, glyceryl monostearate, beeswax, glyceryl behenate, glyceryl dibehenate, tribehenin, betaine, stearic acid and combinations thereof. Preferably, such skin humectant, occlusive or emollient agent is chosen from: glycerin, jojoba oil, macadamia oil, capric/caprylic triglyceride, octyldodecanol, aspartic acid, glyceryl monostearate, lactic acid and combinations thereof.

The concentration of such conditioning agent varies preferably between 0.008 and 10% by weight, preferably between 0.05 and 6% by weight.

Preferably, the concentration of glycerin varies between 0.5 and 4% by weight and preferably varies between 1 and 3% by weight.

Preferably, the concentration of jojoba oil varies between 0.8 and 5% by weight and preferably varies between 1 and 4% by weight.

Preferably, the concentration of macadamia oil varies between 0.8 and 5% by weight and preferably varies between 1 and 4% by weight.

Preferably, the concentration of capric/caprylic triglyceride varies between 0.8 and 6% by weight and preferably varies between 1 and 4% by weight.

Preferably, the concentration of octyldodecanol varies between 0.8 and 5% by weight and preferably varies between 1 and 4% by weight.

Preferably, the concentration of glyceryl monostearate varies between 0.8 and 5% by weight and preferably varies between 1 and 4% by weight.

Preferably, the concentration of lactic acid varies between 0.008 and 2% by weight and preferably varies between 0.01 and 1% by weight.

Such excipient can be furthermore a stabilizing agent or a surfactant.

Such stabilizing agent or surfactant is preferably chosen from: aluminum and magnesium silicates, potassium cetyl phosphate, cetyl stearyl alcohol, cetyl alcohol, polyglyceryl-3 dicitrate/stearate, and combinations thereof.

The concentration of such stabilizing agent or surfactant varies between 0.1 and 5% by weight and preferably varies between 0.3 and 3% by weight.

Preferably, the concentration of the aluminum and magnesium silicate varies between 0.08 and 2% by weight and more preferably varies between 0.1 and 1% by weight.

Preferably, the concentration of potassium cetyl phosphate varies between 0.1 and 2% by weight and more preferably varies between 0.2 and 1% by weight.

Furthermore, such excipient can be a preservative, preferably phenoxyethanol/ethylhexylglycerin or pentylene glycol.

Preferably, the concentration of the preservative varies between 0.5 and 5% by weight and more preferably varies between 0.75 and 3% by weight.

Furthermore, such excipient can be an antioxidant, preferably chosen from: allantoin, tocopherol, tocopherol acetate, vitamin E, vitamin C, sodium lactate, and combinations thereof.

The concentration of such antioxidant preferably varies between 0.08 and 5% by weight and more preferably varies between 0.1 and 3% by weight.

The composition of the present invention is prepared in a solvent, preferably in water.

Such solvent is present at a concentration comprised between 30% and 90% by weight, preferably between 40 and 80% by weight. In one embodiment of the invention, the composition comprises tranexamic acid at a concentration comprised between 1 and 5% by weight, the at least one inhibitor or antagonist of the enzyme tyrosinase at a concentration comprised between 1 and 5% by weight, and the at least one polyphenol source at a concentration comprised between 0.1 and 1% by weight.

In a preferred embodiment of the invention, the composition comprises tranexamic acid at a concentration comprised between 1 and 5% by weight, trihydroxybenzoic acid glucoside at a concentration comprised between 1 and 5% by weight, and Polipodium leucotomos extract at a concentration comprised between 0.1 and 1% by weight.

In one embodiment of the invention, the composition comprises tranexamic acid at a concentration comprised between 1 and 5% by weight, the at least one inhibitor or antagonist of the enzyme tyrosinase at a concentration comprised between 1 and 5% by weight, the at least one polyphenol source at a concentration comprised between 0.1 and 1% by weight, and vitamin D3 or a derivative thereof at a concentration comprised between 1 and 5% by weight.

In a preferred embodiment of the invention, the composition comprises tranexamic acid at a concentration comprised between 1 and 5% by weight, trihydroxybenzoic acid glucoside at a concentration comprised between 1 and 5% by weight, Polipodium leucotomos extract at a concentration comprised between 0.1 and 1% by weight, and niacinamide at a concentration comprised between 1 and 5% by weight.

A further aspect of the present invention relates to the composition according to the invention, formulated preferably for topical use as a cream, cream-gel, gel, serum, oil, emulsion, emulsion-gel (emulgel), ointment, spray or stick (such as lip balm). The formulation of a composition as an intensive serum for the face or as a cream, preferably as a hand cream or face cream, is particularly preferred. A further aspect of the present invention relates to the cosmetic use of the composition for preventing and/or attenuating skin pigmentation defects, more preferably skin hyperpigmentation defects, such as for example melasma (skin patches), freckles and sun spots.

A further aspect of the present invention relates to the composition as described above for use as a remedy.

A further aspect of the present invention relates to the composition as described above for use in the treatment and/or prevention of pathological conditions affecting the skin, preferably to prevent and/or treat pigmentation defects, more preferably skin hyperpigmentation defects, such as for example melasma, freckles and sun spots.

The composition according to the invention is capable of improving/attenuating alterations of skin pigmentation with a surprising effect. The composition according to the invention in fact comprises compounds which act on various pathways that lead to the forming of skin patches.

In particular, tranexamic acid, used in therapy as an anti-hemorrhagic, acts on plasmin activation and is capable of also inhibiting melanocyte activation. Tranexamic acid therefore inhibits the mechanisms that lead the melanocyte to synthesize melanin.

The tyrosinase inhibitor or antagonist is capable of interfering with the activity of the main enzyme involved in melanin biosynthesis.

Vitamin B3 or a derivative thereof acts by inhibiting the transport of melanin in the upper layers of the skin.

The at least one polyphenol source has an antioxidant and photoprotective effect, thus reducing the stimulus to produce melanin.

The result achieved as a consequence of the application of the composition according to the invention for 7 days on volunteer test subjects (study performed in 14 days with alternating applications of the product) and with a single application per day (normally the dose is double, i.e., one application in the morning and one in the evening) is surprising; melanin reduction is in fact 26.21%, the dark tone of the skin patches decreases significantly over 7 days, despite irradiation with a continuous UV source, which does not occur normally, since during the treatment of patches with known compositions the patches are usually protected with a sunscreen in cream form.

Therapies of approximately 120/180 days are normally needed to achieve similar results.

EXAMPLES

Assessment of cytotoxicity of the composition by means of an in vitro assay on melanocyte cell cultures

The MTT assay was performed in order to assess the potential cytotoxic effect of the composition on a cell line of B16 melanocytes.

The test was conducted on murine melanoma cells (B16), a well- known line commonly used in melanogenesis studies. The cells were cultured in DMEM (Dulbecco's modified Eagle medium), containing 2 mM glutamine, 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) and incubated in standard culture conditions (37°C, 5% CO2). Good cell culture practice was applied.

The composition being considered was dissolved and then diluted in culture medium to the desired final concentrations, comprised between 0.0391 and 5.0 mg/ml. The negative reference (internal standard) was tested at concentrations comprised between 0.0391 and 5.0 mg/ml, while the positive control (SDS) was tested at concentrations comprised between 0.00313 and 0.4 mg/ml.

An adequate number of cells was seeded in plates with 96 wells. Once a semiconfluent monolayer was achieved, the cells were treated with the various concentrations of the composition and of the standards and incubated for 24 hours in standard conditions. Untreated cells kept in culture medium represent the negative controls. After 24 hours of contact, the plates were examined under a phase contrast microscope, the medium was delicately removed from each well, the cells were then treated with MTT (1 mg/ml) and incubated for 3 hours in standard conditions. At the end of this period, the MTT solution was eliminated and 100 mΐ of isopropanol were added to each well in order to dissolve the formazan crystals that had formed. Absorbance (optical density, OD) was determined by means of a spectrophotometer reading at the wavelength of 540 nm.

Cell viability inhibition at each tested concentration was expressed as a percentage with respect to the negative control (untreated cells) according to the following formula:

inhibition % = 100 - [(ODX / ODNC) x 100]

ODX = Average optical density of the cells treated with the sample being considered at concentration X;

ODNC = Average optical density of the negative controls.

The values thus calculated were charted against the concentrations themselves. The resulting concentration-response curves allow to extrapolate both for the samples and for the standards the theoretical IC50 value. The IC50 value indicates the concentration of the tested compound that is necessary to inhibit cell viability by 50%. IC50 is a parameter that allows to assess the cytotoxicity of a compound according to Table 1 :

Table 1 Table 2: Concentration-response curve of SDS (positive control)

*Decrease in cell viability <10% = not significant

The tested concentrations comprised between 0.1 and 0.4 mg/ml decreased cell viability significantly. A value of IC50 < 0,5 mg/ml indicates a cytotoxic effect. The same results of Table 2 are shown in the chart in Figure 1.

Table 3: Concentration-response curve of the negative standard

*Decrease in cell viability <10% = not significant

None of the tested concentrations significantly decreased cell viability. A value of IC50 > 0.5 mg/ml indicates an absence of cytotoxic effect. Table 4: Concentration-response curve of the composition according to the present invention

*Decrease in cell viability <10% = not significant

The tested concentrations did not decrease cell viability. A value of IC50 > 0.5 mg/ml indicates absence of cytotoxic effect.

Assessment of depigmenting activity in vitro on reconstructed human tissues

Tested samples:

A: Base cream + 4% niacinamide

B: Base cream + 2% diglucosyl gallic acid

C: Base cream + 0.5% Polipodium

D: Base cream + 4.5% tranexamic acid

E: Base cream + 4% niacinamide, 2% diglucosyl gallic acid, 0.5% Polipodium and 4.5% tranexamic acid.

The purpose of the test is to assess whether the tested product has a depigmenting activity in vitro on reconstructed human tissues by assessing melanin synthesis. It is deemed that this capacity renders the product a potential "depigmenting" candidate in vivo, potentially capable of reducing patches caused by skin hyperpigmentation. The reconstructed tissues are formed by normal keratinocytes (NHEK) and human melanocytes (NHM) cultured to form a multilayer and highly differentiated model of human epidermis. The melanocytes inside the co-cultures undergo melanogenesis, which leads to tissue pigmentation. The present system provides a useful in vitro instrument for assessing cosmetic and pharmaceutical agents designed to modulate skin pigmentation.

25 mΐ of the product being considered or of the positive control were applied topically (2 mg/cm ² ) every other day to the tissues. Each experiment was performed on at least two tissues.

At the end of the treatment period (10-14 days), all the tissues

(treated, treated with positive control and untreated) were photographed using a digital camera for visual inspection of depigmentation (Figure 3).

The tissues were then removed from the insert by cutting the filter with a scalpel. Each filter was immersed in 360 mΐ of Solvable and heated at 100°C for 45 minutes. Optical density was measured on 80 mΐ of extract at

490 nm by using synthetic melanin as a reference. The results are expressed in pg/ml of melanin.

In order to check that the tissues are still viable at the end of the treatment, an MTT test was conducted. For each condition, two tissues were placed in 300 mΐ of MTT and incubated for 3 hours at 37°C, 5% CO2. Extraction was performed in 1.5 ml of isopropanol at room temperature, for a minimum of 2 hours. Optical density is measured at 570 nm.

Table 5: Melanin dosage

Absorbance measured at 490 nm is directly proportional to the quantity of melanin extracted from the tissues. The values were interpolated with a synthetic melanin standard curve in order to obtain the melanin content (pg/ml) of each tissue. In Figure 2, the chart expresses the percentage content of melanin with respect to the untreated negative control.

NC = negative control (untreated tissues); A = Base cream +

4% niacinamide; B = Base cream + 2% diglucosyl gallic acid; C =

Base cream + 0.5% Polipodium; D = Base cream + 4.5% tranexamic acid; E = Complete depigmenting cream; CQ = internal positive control.

The "Complete depigmenting cream" product reduces by 26.21% the production of melanin in reconstructed human tissues, while the other tested products had an effect comprised between 14.73% and 20.90%. Assessment of possible irritation power of the composition according to the present invention according to the amended Draize classification

This test allows to assess the tolerability of the composition according to the present invention by identifying and classifying the irritation power.

The assessment of any irritation or sensitizing power of the tested products subjected is a valid aid in checking for the existence of a potential unwanted effect during use, as required by (EC) Regulation no. 1223/2009 of the European Parliament and Council of 30 November 2009 on cosmetic products.

The test is conducted on ambulatory volunteers according to the following inclusion criteria:

a) good general state of health;

b) absence of skin disorders;

c) absence of pharmacological treatments in progress;

d) commitment not to vary from ordinary daily routine; e) negative anamnesis for atopy.

The samples were applied, as a function of their characteristics of use: as is or at a standard concentration of 10%. For liquid products, 20 mΐ are applied to the skin; 20 pg are applied for solid/semisolid products.

The device used to perform an occlusion patch test is the Finn

Chamber (a patch containing an aluminum disc with a diameter of 7 mm and absorbent paper discs).

In the case of liquid products, the Finn Chamber contains absorbent paper discs impregnated with a known quantity (20 mΐ) of sample being considered, in the case of solid products the product to be tested is in direct contact with the skin.

Execution of the test

- the skin area intended for the assay (skin of the back) was cleaned with a 70% alcohol solution;

20 pg or mΐ/cm ² of cosmetic product are applied to the skin;

- the skin region intended for the assay is covered with the Finn Chamber;

- the product is kept in contact with the skin for 24 hours;

- the Finn Chamber is removed;

- skin reactions are assessed 15 minutes, 1 hour and 24 hours after removal of the Finn Chamber.

Assessment and calculation of results

The reactions are assessed on the basis of the arbitrary scale listed in table 6. Table 6: Assessment scale

For each volunteer it is possible to assign scores for erythema and edema at various times after the removal of the Finn Chamber.

The total skin irritation index (IIM tot) is calculated by averaging the irritation indices for erythemas (IIM Er) and edemas (IIM Ed) after 24 hours.

The product being studied is classified by applying table 7. Table 7: Classification of the average irritation index (according to amended Draize)

Table 8: Results

From the results obtained, it is found that the composition according to the present invention is not irritant.

Table 9: Example of formulation of the composition according to the present invention

Table 10: Example of formulation of a depigmenting cream

Method for preparing a depigmenting cream

The various "Phases" listed in table 10 are weighed and mixed in various steps.

In particular, the method for preparing the cream comprises the steps of:

- weighing the ingredients of "phase O" and introducing them in a mixer, preferably in a turbo emulsifier;

- bringing to a temperature comprised between 50 and 85°C; - weighing the ingredients of "phase A";

- bringing to a temperature comprised between 55 and 80°C;

- incorporating phase A with phase O under agitation;

- cooling to a temperature comprised between 30 and 50°C;

- incorporating the ingredients of phase B under agitation;

- keeping under agitation up to complete dissolution of phase B.

Clinical evaluation of the effect of a composition according to the present invention in reducing the visibility of dark spots

A depigmentation cream, according to the invention, prepared as described above, was tested on 20 female subjects, aged between 18 and 60 years old, selected with the following inclusion criteria:

- presence of age spots and skin discolorations on face;

- good state of health/absence of psychological and/or cognitive disorders;

- no dermatopathies and allergic pathologies (to cosmetics or other specific excipient), or other pathologies (as unknown irritant responses);

- no ongoing pharmacological treatments that could affect the result of the test;

- no participations in other clinical trial during the previous 30 days;

- signature of the informed consent form.

Samples of the depigmentation cream were applied on skin area affected by discoloration twice per day for 84 consecutive days by massaging gently until absorbed, using the amount to cover the discoloration.

Experimental procedure

INSTRUMENTAL PARAMETERS

- Melanin (responsible for skin color) of dark spot was measured with MEXAMETER®MX 18 based on absorption. The special probe of the instruments emits light of defined wavelengths. A receiver measures the light reflected by the skin. As the quantity of emitted lights is defined, the quantity of light absorbed by the skin can be calculated.

The melanin is measured by two wavelengths. These wavelengths have been chosen in order to achieve different absorption rates by the melanin pigments. The achieved results are shown on digital displays on a scale from 0-999.

CLINICAL PARAMETERS

The following parameters were measured at tO (basal value) and after 14, 28, 56 and 84 days of use of the depigmentation cream (tl4, t28, t56 and t84) :

- Skin complexion evenness

- Visibility of dark spots

- Reduction of shape of dark spots

- Reduction of size of dark spots

In order to carry out a statistical survey and to be able to evaluate the skin variations in a specific period, the following skin parameters have been analysed:

INSTRUMENTAL and CLINICAL PARAMETERS

Data show that face dark spots/melanin decreases by:

- 9% after 14 days of treatment;

- 15% after 28 days of treatment;

- 17% after 56 days of treatment;

- 21% after 84 days of treatment.

Data show that skin complexion evenness improves:

- in 40% of the volunteers after 14 days of treatment;

- in 55% of the volunteers after 28 days of treatment; - in 60% of the volunteers after 56 days of treatment;

- in 65% of the volunteers after 84 days of treatment;

Data show that dark spot visibility decreases:

- in 45% of the volunteers after 14 days of treatment; in 55% of the volunteers after 28 days of treatment; - in 60% of the volunteers after 56 days of treatment;

- in 65% of the volunteers after 84 days of treatment;

Data show that the reduction of size of the dark spot is visible:

- in 10% of the volunteers after 14 days of treatment;

- in 35% of the volunteers after 28 days of treatment;

- in 40% of the volunteers after 56 days of treatment;

- in 45% of the volunteers after 84 days of treatment;

Data show that the variation of the shape of the dark spot is visible:

- in 10% of the volunteers after 14 days of treatment;

- in 35% of the volunteers after 28 days of treatment;

- in 40% of the volunteers after 56 days of treatment;

- in 40% of the volunteers after 84 days of treatment.

The disclosures in Italian Patent Application No. 102018000003428 from which this application claims priority are incorporated herein by refer - ence.