ITTO20080585A1 - COSMETIC COMPOSITION AND COSMETIC PROCEDURE FOR THE GROWTH OF TEGUMENTARY EQUIPMENT - Google Patents

Description

DESCRIPTION of the industrial invention entitled:

"Cosmetic composition and cosmetic procedure for the regrowth of integumentary systems"

TEXT OF THE DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a cosmetic composition and a cosmetic process suitable for promoting hair growth by recovering the original trophism of the integumentary systems, stimulating their growth and improving the vitality of the hair follicles.

TECHNICAL BACKGROUND OF THE INVENTION

It is known how with low intensity ELF (Extremely Low Frequency) fields it is possible to induce variations in the transport of various ionic species (e.g. calcium ions) across the cell membrane, obtaining variations in the rate of regeneration and repair of cells and tissues and changes in the redox potential of molecules SUMMARY OF THE INVENTION

The present invention has the purpose of identifying a cosmetic composition and a cosmetic process that can cause regrowth of hair and integumentary systems in general, resulting in a repair of atrophic hair follicles and / or regeneration of dormant hair follicles, accelerating their functional recovery. and competent cell turnover.

The present invention aims to find a valid and effective solution to combat the problems of hair loss and alteration of the integumentary systems in mammals, and in particular in humans, which until now have remained unsolved.

According to the present invention, this object is achieved thanks to the solution referred to specifically in the following claims. The claims form an integral part of the technical teaching provided herein in relation to the invention.

The invention relates to a cosmetic composition to be used to stimulate the growth of hair and integumentary systems in general which comprises:

at least one metal, its salts and / or its compounds, optionally in the form of a colloidal suspension; and

at least one proteolytic agent, preferably papain.

The invention also relates to a cosmetic process for stimulating the growth of the integumentary systems of a mammal which provides for the combined application i) of the cosmetic composition described above and ii) of low frequency ELF electromagnetic waves, magnetic fields and / or electrical static to a portion of tissue of interest of the mammal, in such a way that a specific and synergistic action is generated between the composition and the ELF application such as to promote the recovery of the original trophism of the integumentary systems in the portion of tissue of interest through:

1) the physiological activation of hair regrowth, or the stimulation of the hair bulbs to resume their normal activity;

2) direct action on hair follicles with recovery of atrophic follicles, even in scarred areas.

The present inventors have, in fact, demonstrated that static electric and / or magnetic fields associated with low-frequency ELF electromagnetic waves of relatively weak power determine, when used in combination with the cosmetic composition object of the present invention, the rapid physiological functional recovery of the follicles. quiescent or atrophic hair.

The present inventors have, in fact, applied simultaneously an electromagnetic radiation (in the frequency range 1 to 1000 Hz), and static magnetic fields (in the intensity range 1 to 1000 uT), and / or static electric fields (in the range intensity 1 to 1000 V / m) to different biopsies of animal hair skin appendages in culture media containing the cosmetic composition for the tissue application in question, and in vivo to different subjects affected by alopecia, effluvium or outflow. Surprisingly, a particular regeneration of atrophic or quiescent hair follicles was observed, accelerating their functional recovery and hair growth, as well as the recovery of the original pigmentation of the integumentary systems.

This phenomenon of hair growth also manifested itself in adverse experimental conditions, that is, in the tissue compartment recovering from post-surgical scarring.

A further object of the present invention is to provide a device suitable for implementing the methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, purely by way of non-limiting example, with reference to the attached figures, in which:

Figure 1 is a schematic representation of a device capable of generating static electric and / or magnetic fields associated with low frequency ELF electromagnetic waves to be used in the cosmetic process object of the present invention;

Figures 2 to 5 schematically illustrate different embodiments of coils to be used in the device of Figure 1.

In a currently preferred embodiment, the cosmetic process according to the present invention provides for the application of a specific-inducing cosmetic composition (preferably in the form of a lotion) and exposure for a period of time ranging from 5 minutes to 10 minutes. twice a day (morning and evening) to a device that delivers a set of static electric and / or magnetic fields and low intensity alternating electromagnetic fields (ELF).

The characteristics of the applied ELF fields (waveform, frequency, duty cycle, intensity) and of the static magnetic and electric fields eventually added to the ELF depend on the type of desired result (regeneration, repair, growth), and on the various conditions at the contour (type of tissue atrophy, type of substrate, time). The characteristics of the fields applied in the context of the process object of the present invention are given by:

the repetition frequency of the applied low-frequency electromagnetic wave and the waveform;

- the intensity of the alternating field (ELF) applied;

- the intensity of the applied static magnetic field; - the intensity of the applied static electric field; and - the time of application.

In vitro and in vivo tissues under study are: 1. in vitro biopsies of alopecic animal skin or with large effluvium-outflow of hair, refractory to common treatments;

2. in vivo alopecic human scalp or with effluvium-outflow of hair, refractory to common treatments.

Instrumentation and physical specifications

The device 1 to be used in the context of the cosmetic process object of the present invention is schematically illustrated in Figure 1.

The device 1 comprises a chamber 2 which can possibly be shielded from the static or alternating low frequency magnetic field thanks to a mumetal screen, and to the static and electromagnetic alternating high frequency electric field thanks to an additional brass shield contained in the one in mu-metal.

The device 1 is connected to the outside through narrow openings 5 to allow the passage of the electrical connections 15 (coils, camera).

In a preferred embodiment, the device 1 comprises internally:

two coils 4 generating the static magnetic field placed on the same axis;

two coils 7 generating the alternating electromagnetic field placed on the same axis;

two aluminum plates 3 generating the static electric field with axis perpendicular to that of the aforementioned coils.

The static magnetic field is generated by means of an external electric circuit 10, in a "current pump" configuration, which supplies the desired adjustable current to the appropriate coils.

The static electric field is generated by applying a suitable continuous potential difference to the two metal plates by means of a source 9.

The alternating electromagnetic field is generated by means of a current generator 11 piloted by a generator of arbitrary waveforms, applied on the appropriate coils.

The coils 4 and 7 contained in the device 1 can be made in one of the following configurations: a) a particularly preferred embodiment of the device 1 comprises a bifilar coil as illustrated in figure 2, in which a winding is used to produce the electromagnetic field alternating and the other to produce the static magnetic field.

b) a different particularly preferred embodiment of the device 1 comprises at least two bifilar coils as above arranged in arrays as shown in Figure 3 so as to obtain an area as wide as desired in which it is possible to irradiate the field.

c) another particularly preferred embodiment of the device 1 comprises a bifilar coil wound around a ferrite magnetic core, as illustrated in figure 4.

d) a further particularly preferred embodiment of the device 1 comprises at least two bifilar coils each wound around a magnetic ferrite core, where all said assemblies (core coils) are arranged in arrays as illustrated in figure 5 so as to obtain a area as wide as desired in which the field can be irradiated.

A further preferred embodiment is that in which all the embodiments a), b), c), d) described above are made with simple (single-wire) coils instead of two-wire coils, in which the possible overlap of static magnetic field and alternating electromagnetic field are obtained electronically, with an operation of algebraic sum of the desired signals.

Figure 5 also illustrates the presence around the two-wire or single-wire coils, with core or without core, of an electromagnetic screen in mu-metal or similar material such as to make the irradiation null on all sides, except the side intended to irradiate. the portion of fabric of interest (reference number 12 in Figure 1), allowing a possible operator to hold the device 1, however constituted, in his hand without being unnecessarily irradiated.

Irradiation was suspended every hour and part of the sample was removed and analyzed.

The total exposure to the alternating electromagnetic / static magnetic and / or electric field of the cell sample is between 15 minutes and 96 hours.

It should be noted that the irradiation conditions reported here identify a specific window of operation of the device used for irradiation, this window being capable of generating the effects described below on cell cultures. It is clear, however, that these irradiation conditions are not to be construed as limiting and it will be possible for the skilled technician in the field to experimentally identify other irradiation conditions and other working windows.

ELF-specific composition for the regeneration of hair follicles.

The invention is based on the surprising observation conducted by the present inventors of a particular eutrophicating pro-regenerative stimulus exerted by the combination of at least one metal, preferably a zinc salt, colloidal silver or colloidal gold, and at least one proteolytic agent, such as papain , on human scalp, skin tissue with human or animal hair appendages in general.

The present inventors have, in fact, surprisingly verified that metals, their colloidal suspensions, their salts and their compounds, synergistically with the delivery of a set of static electric and / or magnetic fields and alternating low frequency electromagnetic fields (ELF) of low intensity, they intervene in various enzymatic processes and regulate the metabolism and catabolism of the hair follicular microenvironment, inducing the control and regeneration of atrophic or quiescent hair bulbs.

In parallel to the action described above, the present inventors were able to verify in the conduct of the experiment that the proteolytic enzyme (preferably papain) promotes two fundamental functions, namely: (i) the activation of growth factors physiologically present in the microenvironment of the tissues damaged or senescent, and (ii) the chemotactic induction of the residential stem cell to a rapid reparative differentiation.

Furthermore, it was discovered that (i) metals, their colloidal suspensions, their salts and their compounds, (ii) together with the delivery of a set of static electric and / or magnetic fields and alternating low frequency electromagnetic fields ( ELF) of low intensity and concomitantly with (iii) the administration of papain perform various synergistic functions: it accelerates the absorption of nutrients present in the preparation, it induces tissue repair, the regeneration of a physiologically correct microenvironment, and finally, peripheral dysemia that appears in follicular atrophy states is corrected. Furthermore, the present inventors have discovered that with papain the Langerhans cells (thorny layer) are stimulated to incorporate foreign substances (excess collagen) and digest them. This mechanism has proved particularly useful in the treatment of dormant follicles.

These absolutely surprising and unexpected observations have led the present inventors to create a procedure accompanied by the relative device and a composition designed to promote the recovery of the original trophism of the integumentary systems, to stimulate their growth and to improve the vitality of the hair follicles through the use of metals, their colloidal suspensions, their salts and their compounds, together with the supply of a set of static electric and / or magnetic fields and low intensity alternating electromagnetic fields (ELF) and in conjunction with the administration of papain .

The treatment of skin tissue biopsies with animal hair appendages in vitro with the method, the device and the composition of the invention results in a surprising repair of the damages and in a total recovery of the original trophism of the integumentary systems. All tissues represented in the treated biopsy specimens are subjected to a complete, functional and morphological regeneration (see Example 1).

The histological results obtained in vivo starting from 30-60 days of treatment with the composition object of the present invention and its tissue-specific formulations, confirm on histological examination the formation of a regeneration and retrophication of human scalp and in general of skin tissue with human hair appendages with optimal histofunctional characteristics, morphologically comparable and intact in vivo, (see the section on Clinical Studies).

In detail, the composition object of the present invention comprises at least the following components:

at least one metal, optionally in the form of a colloidal suspension, its salts and / or its compounds; and at least one proteolytic agent, preferably papain.

In a further preferred form of the invention, the composition comprises further active ingredients, selected for their enhancing and synergistic activity of ELF and of the active principles defined above, specifically for each type of tissue and related human or animal integumentary systems to be treated.

Examples of specific tissue compositions in vitro and in vivo

Table 1. Human scalp, skin tissue with human and animal hair appendages.

Table 2. Human scalp, skin tissue with human and animal hair appendages.

propylene glycol 5% w / v q.s. for L of solution

Alcoholic solution at 30% w / v q.b. for L of solution

Table 4. Human scalp, skin tissue with human and animal hair appendages.

Composition CrPl-MD-INFUS (Infusion liquid)

Substance Concentration

Active principles sqm / L

Colloidal gold solution at 46% w / v 10 mg / L

Papain FU 20.00 mg / L

Excipients mg / L

Blend of L-amino acids (preferably

L-Methionine, L-Cystine, L-Cysteine,

2,820 mg / L

L-Glycine, L-Leucine, L-Lysine, L-Proline,

L-Glutamine)

Vitamin A 4 mg / L

Vitamins B (Bl, B2, B5, B6, B9, B12) 48mg / L

Vitamin c 4500 mg / L

Vitamin D 0.0055 mg / L

Vitamin E 10 mg / L

Vitamin H 0.069 mg / L

Vitamin PP 46 mg / L

Selenium 0.1 mg / L

propylene glycol 5% w / v q.s. for L of solution

Alcoholic solution at 30% w / v q.b. for L of solution

The composition object of the present invention can also comprise further active components, such as:

- mixtures of amino acids, preferably L-Methionine, L-Cystine, L-Cysteine, L-Glycine, L-Leucine, L-Lysine, L-Proline, L-Glutamine L-Glycine, L-Leucine, L-Lysine, L -Proline;

- vitamins A, B, C, D, E, H and PP;

- Selenium.

The metals are preferably introduced into the compositions of the invention under f ^ o rma d- <1> i - <'> s-a-l <1> i <'> or colloidal suspensions. In addition to the zinc oxide, colloidal silver and colloidal gold mentioned in the previous tables, it is possible to use with the same results, among others, iron or ferritin salts, sodium, compositions containing metals, their combinations and derivatives.

As proteolytic enzymes, in addition to the aforementioned papain, collagenase (preferably type I, type II, type IV), serratiopeptidase, heparanase, DNAse, elastase, bromelain, bradykinase, Clostridium peptidase, enzymes expressed by Lactobacillus acidophilus can be used, enzymes expressed by the genus Aspergillus, protease, aliinase, fibrinolysin.

The mixtures of amino acids, preferably containing L-Methionine, L-Cystine, L-Cysteine, L-Glycine, L-Leucine, L-Lysine, L-Proline, L-Glutamine L-Glycine, L-Leucine, L-Lysine, L-Proline, constitute eutrophicating elements that act as important growth factors for the scalp, skin and appendages, regulating cell and tissue turnover.

Among the amino acids that can be present in the compositions of the invention we mention by way of example methionine, cystine, N-acetylcysteine, cysteine, glieine, leucine, isoleucine, proline, glutamine, arginine, glutamic acid, histidine, histidine-HCl, lysine, lysine -HCl, phenylalanine, serine, threonine, tryptophan, tyrosine, tyrosine-disodium salt, goine, proline, hydroxyproline. Such amino acids are often used in blends comprising a large number of different amino acids.

In addition to the amino acids, the compositions of the invention can also comprise other elements such as selenium, carbonates, bicarbonates, glycyrrhetic acid, glycerizin, glucose, glutathione, glycolic acid, soy lecithin, collagen, elastin, wheat extract, and the like.

Among the vitamins that may be present in the compositions of the invention we mention, by way of example, retinoic acid, retinaldehyde, retinol, alpha-tocopherol, beta-carotene, cholecalciferol, ascorbic acid, pantothenic acid, dexpanthenol, D-calcium pantothenate, cocarboxylase tetrahydrate , pyridoxine, pyridoxine-HCl, folic acid, niacinamide, nicotinamide, riboflavin, riboflavin dihydrate sodium phosphate, cyanocobalamin, para-aminobenzoic acid, biotin.

It has been discovered by the present inventors that vitamin A, used in doses without any pharmacological effect, enhances the restoration of the correct pH and antioxidant power in tissues such as skin, subcutis and mucous membranes.

Still on the basis of the experiments of the present inventors it has been verified that in the context of the composition object of the present invention the vitamins of group B, preferentially B1, B2, B5, B6, B9 and B12, contribute to the normalization of the locoregional microenvironment.

Still on the basis of the experiments of the present inventors it has been verified that in the context of the composition object of the present invention, vitamin C promotes the restoration of a perifollicular physiological microenvironment, while vitamin D at physiological values plays an auxiliary role in the consolidation of an ideal exchange. sodium-calcium ionic favoring the correction of the altered electrolyte concentration around the quiescent or atrophic hair follicles to physiological values.

The present inventors have shown that vitamin E, used in doses without any pharmacological effect, leads to the gradual physiological restoration of the microenvironment, especially subcutaneously, actively contrasting by assisting the physiological biosynthesis of the hair.

In relation to the activity of vitamin H (Biotin) within the composition object of the present invention, it has been discovered that this favors the activation in the organism of two reparative mechanisms: the recall of stem cells from the bloodstream and an accelerated specialization of residential stem cells in the skin to rapidly differentiate into mature cells intended to restore damaged hair follicles.

Furthermore, the present inventors have discovered that vitamin PP (Nicotinnamide), in association with the various components of the composition object of the present invention, induces loc-regionally different mechanisms of action:

1) it performs functions as a constituent of enzymes involved in redox reactions;

2) it intervenes in the energy metabolism of glycides and proteins;

3) is part of the ferments of the capillary turnover.

The cosmetic and medical device compositions object of the present invention can also comprise further accessory elements such as excipients and vehicles, the choice of which and the use of which fall within the skills of the skilled in the art without this requiring the exercise of any inventive step.

Biopsies

20 biopsies of animal skin affected by alopecia, effluvium or outflow refractory to common treatments were analyzed. All specimens were washed three times with saline and antibiotics (100 units / ml penicillin 100 ug / ml streptomycin 40 mg / L gentamicin) for 10 min at room temperature.

Each biopsy was sectioned into six parts (two controls-1, two controls-2 and two specimens).

The samples (3) were suspended in a solution of 20 ml of CrPl-MD-INFUS with final concentration IX in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark).

Two types of controls were prepared, negative controls-1 (1) were treated exclusively with physiological solution and antibiotics (as described above); negative controls-2 (2) were treated with cell culture media:

1. Control biopsy findings were suspended in 20 ml of saline in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark).

2. Control biopsy findings were placed in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in 20 ml of D-MEM medium supplemented with:

10% FCS (Celbio, Milan, Italy)

40 mg / L gentamicin (Schering-Plow, Italy)

2 mM L-glutamine (Life Technologies.

3. All the samples were placed for 180 days in a thermostatically controlled Heraeus incubator at a temperature of 37 ° C with an atmosphere containing a constant 5% CO2 supply (v / v in air).

Every 21 days of culture, the supernatants of the cultures under study were collected and these supernatants were stored in a freezer at minus 80 ° C for the following laboratory analyzes: total NO concentration, nitrites and nitrates.

Six tissue portions were prepared for each biopsy sample, of which: two samples and four controls (two controls 1 and two controls 2).

A) Three experimental copies, each consisting of two controls (1, and 2) and a sample, were incubated at 37 ° C with an atmosphere containing 5% of constant CO2 supply (v / v in air}, for a time interval of 180 days.

B) Further three experimental copies, each consisting of two controls (1, and 2) and a sample, were subjected to an ELF magnetic field at a frequency ranging from 1 to 1000 Hz, and static magnetic fields (in the range of intensity 1 to 1000 uT), and / or static electric fields (in the intensity range 1 to 1000 V / m), for a time interval of 10 minutes twice a day for 180 days.

In a preferred embodiment of the process object of the present invention, the conditions applied are the following:

alternating magnetic field 30 uT;

> static magnetic field 15 uT;

> waveform repetition frequency: 35 Hz; waveform: sine wave;

> application time 10 minutes twice a day every 12 hours.

These conditions have been obtained experimentally by optimizing the impedance feedback by scanning the intensity of the alternating magnetic field from 0 to 30 uT, and correlating the frequency to the total intensity of the field thus obtained.

The biopsies were incubated, taking samples and related tests every 21 days, for a maximum of 180 days.

Immunofluorescence protocol

After three washes for 10 min at room temperature in PBS (pH .7.4), the samples were resuspended in a 4% paraformaldehyde fixative solution in RPMI 1640 with pH 7.4, for one hour at room temperature. After embedding in paraffin, the samples were sectioned and placed on microscope slides. Sections were stained with hematoxylin / eosin, anti-cytokeratin 10 monoclonal antibodies (Santa Cruz Biotechnology, America, California), anti-cytokeratin 11 monoclonal antibodies (Santa Cruz Biotechnology, America, California). Specific controls were set up for each monoclonal antibody with the relative isotypes (Santa Cruz Biotechnology, America, California). All samples placed on microscope slides were observed under the optical microscope after being sealed with moviol and cover slips.

Measurement of NITRITES (NO ^) and NITRATES (NO <~>)

Since most nitrogen monoxide (NO) produced by NO synthetase enzymes is oxidized to nitrites and nitrates, the concentration of these anions has been used as a quantitative measure of NO production. This method is based on the enzymatic conversion of nitrates into nitrites using the nitrate reductase enzyme.

Subsequently the nitrites are identified spectrophotometrically by means of a colorimetric reaction which, in the presence of the Griess reagent (naphthylethylenediamine dihydrochloride in 2N HC1 and sulfonylamide in HC12N), provides as final product an azo-derivative which absorbs light at 540 rati. The NO concentration is indirectly calculated by evaluating both nitrite and nitrate levels separately. This method allows to evaluate the quantity of endogenous nitrites present in the samples and therefore to subtract it from the total values obtained.

Measurement of endogenous nitrites in samples

Samples of 100 μΐ of supernatant were added to 100 μΐ of Griess's reagent, and after 10 minutes of incubation the absorbency of the solution was read at 540 nm with a model EL340 microplate reader from Packard. The blank, to be subtracted from each sample, was carried out with fresh medium, and the calibration curve with known concentrations of sodium nitrite. The nitrite concentration was expressed as μιηοΐϊ of nitrite per mL. Measurement of total nitrite in samples following conversion of nitrates to nitrites

Samples of 50 μΐ of supernatant were spiked with 25 μΐ nitrate reductase and 25 μΐ of NADH in 96-well plates and allowed to incubate for 30 min at 37 ° C. Then 100 μΐ of Griess's reagent was added, and after 10 minutes of incubation 1 <1> absorbance of the solution was read at 540 nm with a Packard model EL340 microplate reader. The blank, to be subtracted from each sample, was carried out with fresh medium, and the calibration curve with known concentrations of sodium nitrate. The nitrite concentration was expressed as μπιοΐί of nitrite per mL.

Determination of the concentration of nitrates in the samples To obtain the concentration of nitrates it is necessary to subtract the value obtained in the method for measuring endogenous nitrites from that obtained in the method for measuring total nitrites (after complete conversion of nitrates to nitrites).

The measurement of nitrites and nitrates was performed with a kit: Nitric Oxide (NC> 2 <~> / N03-) Assay, cat. n <D> DE1500, (R&D Systems Ine., Minneapolis MN 55413, USA). Measurement of total nitrogen monoxide (NO)

This method is based on the enzymatic conversion of nitrates into nitrites using the nitrate reductase enzyme. The nitrites obtained react with Griess's reagent (naphthylethylenediamine dihydrochloride in HC1 2N and sulfanilamide in HC12N giving as final product an optically visible azoderivative at 540nm. The quantity of NO is indirectly calculated on the basis of the concentration of nitrites obtained from the total conversion of nitrates into For measurement, samples of 50 μΐ of supernatant were taken and added to 25 μΐ nitrate reductase and 25 μΐ of NADH in 96-well plates and allowed to incubate for 30 min at 37 ° C. Then 100 were added μΐ of Griess's reagent, and after 10 minutes of incubation 1 <1> absorbance of the solution was read at 540 nm with a plate reader model EL340 from Packard. The blank, to be subtracted from each sample, was carried out with fresh medium , and the calibration curve with known concentrations of sodium nitrate. The concentration of nitrite was expressed as μιηοΐϊ of nitrite per mL. Total NO was performed with the kit: Total Nitric Oxide Assay, cat. n ° DE1600, (R&D Systems Ine., Minneapolis MN 55413, USA).

Western Blot for Cytokeratins

The biopsies were suspended in lysing buffer (1% SDS, 30 mM Tris pH 6.8, 5% glycerol) to which protease inhibitors (Protease Inhibitor Cocktail, Calbiochem, San Diego, CA) were added, underwent a homogenization, followed by an incubation of the samples for 30 minutes at 4 ° C. The obtained lysates were centrifuged at 12,000 rpm for 20 min at 4 ° C and the supernatant was collected; the protein concentration of the samples was evaluated with the Bio-Rad method (Benchmark Plus assay, Bio-Rad). Prior to the electrophoretic run, the samples were boiled for 5 minutes in the presence of beta-mercaptoethanol and bromophenol blue. The samples were subjected to electrophoretic run in a 12% gel (SDS-PAGE) and transferred to a PVDF membrane (Perkin Elmer Ine.). The membranes were saturated with methanol at room temperature and subsequently incubated with the following primary antibodies diluted in PBS with 5% skimmed milk powder: anti-cytokeratin 14 with dilution titre 1: 500 (Santa Cruz Biotechnologies Ine., Santa Cruz , California, USA), anti-cytokeratin 18 with 1: 500 dilution (Santa Cruz Biotechnologies Ine., Santa Cruz, California, USA) and anti-cytokeratin 19 with 1: 500 dilution (Santa Cruz Biotechnologies Ine., Santa Cruz, California, USA) overnight at 4 ° C. After five washes, the membranes were incubated with related secondary antibodies (1: 1000) conjugated with horseradish peroxidase (HRP, Santa Cruz Biotechnologies Ine., Santa Cruz, California, USA) for 1 h at room temperature. The corresponding bands were highlighted with chemiluminescence liquids (Super Signal Western Pico solution, Pierce Biotechnology Ine., Rockford, Illinois, USA) and fixed on photographic plates.

EXAMPLES

Six month incubation

All controls belonging to type 1, both for treatment groups A and for treatment groups B, suspended only in physiological solution, as described in materials and methods, underwent a rapidly progressive necrotic process. They were then eliminated on day 15 of the study (Table 5).

All the controls belonging to type 2, both for treatment groups A and for treatment groups B, suspended only in culture medium, as per the description in materials and methods, underwent a progressive flaking process, without presenting any repair of the areas affected by alopecia, effluvium or outflow, up to a global auto-lytic outcome in about 30-60 days. They were then eliminated upon incurring irreversible necrotic processes on average on day 45 (Table 5).

EXAMPLE 1. Optical microscopy

These results indicate in the biopsy samples, both for treatment groups A and for treatment groups B, a phenomenon of reactivation or regeneration of the hair follicles.

The results relating to the expression of cytokeratin 10 and 11 and the histological staining of biopsy preparations with hematoxylin / eosin (used for the visualization of follicular viability) are reported in table 5 (CONTROLS) and in table 6 (SAMPLES) and have been expressed with a qualitative and semi-quantitative scale, comparing the results manifested by treatment group A (CrPl-MD-INFUS) versus the results manifested by treatment group B (CrPl-MD-INFUS ELF).

From the analysis of the results in Table 6, optical microscopy (eosin and hematoxylin staining) shows a net increase in the number of follicles in the samples treated for six months belonging to group B (CrPl-MD-INFUS ELF) compared to samples treated for six months. belonging to group A (CrPl-MD-INFUS).

Furthermore, all follicles appeared trophic, viable and active in the samples belonging to group B (CrPl-MD-INFUS ELF) compared to the samples belonging to group A (CrPl-MD-INFUS) where hypotrophism of the follicle itself is noted.

Finally, it is reported (Table 6) a clear predominance of cytokeratins 10 and 11, typical of normal integumentary tissues with viable and active follicles in the samples belonging to group B (CrPl-MD-INFUS ELF) treated for six months compared to the samples belonging to the group A (CrPl-MD-INFUS).

Table 5. Controls belonging to type 1 and controls belonging to type 2, for both treatment groups A and treatment groups B. Median day of irreversible necrosis is equal to day 15 for controls belonging to type 1. Median day of irreversible necrosis is equal to day 45 for controls belonging to type 2.

Control 1 Control 1 Control 2 Control 2 Markers

GROUP A GROUP B GROUP A GROUP B

day 15 day 15 day 45 day 45

Nuclei / cytoplasm necrosis

necrosis necrosis preserved colliquative necrosis

diffuse massive vacuolar

Eosin / diffuse

iZmatoxylin

Legend

- - absence of fluorescence

+ = slight fluorescence for optical field

++ = subtle fluorescence for optical field

+++ = medium fluorescence for optical field fluorescence for optical field

++++ = high fluorescence for optical field

+++++ = diffuse fluorescence for optical field

Table 6. Samples belonging to the typology of treatment groups A and treatment groups B. Median semi-quantitative values at incubation days 90 and 180.

EXAMPLE 2. Western blot

The samples were subjected to phenotypic analysis with Western Blot for the markers cytokeratin 1, cytokeratin 2, cytokeratin 10, cytokeratin 11 and cytokeratin 14, as discussed below.

The results show a strong positivity for the production of cytokeratin 1, cytokeratin 2, cytokeratin 10, cytokeratin 11 and cytokeratin 14, in the treated samples, and in particular in the treated samples belonging to group B, for 180 days in vitro with the composition object of the present invention, ChPl-MD-INFUS, compared to only a slight positivity in the treated samples belonging to group B, for 180 days in vitro, as reported in table 7.

Cytokeratins 1, 2, 10, 11 and 14 are expressed in normal integumentary tissues with vital and active follicles during the states of cell differentiation, hair follicle growth and control of hair formation [3-8].

Table 7. Samples treated for 180 days in vitro belonging to group A and group B, expression of cytokeratins 1,2,10,11,14.

EXAMPLE 3. Measurement of nitrites, nitrates and total NO

Nitric oxide (NO), improperly called nitric oxide, is a reactive chemical species of a nitrogen-centered radical nature. NO is transformed into a series of derivatives, such as nitrites and nitrates, which accumulate, depending on the amount of the primary mediator produced, in the blood and other extracellular fluids and then be definitively removed from the body through the urine; the plasma and urinary levels of nitrites / nitrates correlate quite well in vivo with the "endogenous" production of NO, even after particular therapies [4].

Overall, therefore, the experimental data analyzed here show that the composition, the procedure and the device, object of the invention, used in vitro can improve some pathophysiological conditions through an increase in the endogenous synthesis of NO, demonstrated by the relative increase of the its catabolites nitrites and nitrates. NO, nitrites and nitrate derivatives appear to assume the role of biological mediators that indirectly participate in the regulation of proliferation and differentiation of the integumentary structures and specifically in the cyclical changes of the hair follicle [3-8].

The analysis of the results in Table 8 shows a significant increase in the samples of group B treated in vitro for 90 days (three months) and 180 days (six months) with the procedure, the device and the composition object of the invention ChPl-MD -INFUS, compared to controls 1-2 and to the samples of group A, of the concentrations of nitrites and nitrates and, therefore, of the production of NO. Such results are typical of normal integumentary tissues with viable and active follicles.

Naturally, the details of construction and the embodiments may be widely varied with respect to what has been described and illustrated without thereby departing from the scope of protection of the present invention, as defined by the attached claims.

BIBLIOGRAPHY

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Claims (19)

CLAIMS 1. Cosmetic composition comprising: at least one metal, its salts and / or its compounds; and at least one proteolytic enzyme. 2. Composition according to claim 1, wherein said at least one metal is selected from zinc, silver, gold, iron, sodium. 3. Composition according to claim 1 or claim 2, wherein said at least one proteolytic enzyme is selected from papain, collagenase, serratiopeptidase, heparanase, DNAse, elastase, bromelain, bradykinase, Clostridium peptidase, enzymes expressed by Lactobacillus acidophilus, enzymes expressed by the genus Aspergillus, protease, aliinase, fibrinolysin, preferably papain. 4. Composition according to claim 2, wherein zinc is present as zinc oxide. 5. Composition according to claim 2, wherein the iron is present as ferritin. Composition according to any one of the preceding claims, wherein said at least one proteolytic enzyme is present in a concentration ranging from 0.1 μg / L to 100 mg / L, more preferably from 10 mg / L to 80 mg / L. Composition according to any one of the preceding claims, wherein said at least one metal is present in a concentration in the range from 0.01mg / L to 100mg / L, more preferably from 0.1mg / L to 20mg / L. Composition according to any one of the preceding claims, wherein said composition further comprises at least one of a physiologically acceptable vehicle, an amino acid, a sugar, a vitamin, selenium. 9. Composition according to any one of the preceding claims, wherein said composition is in the form of lotion, gel, cream, infusion liquid, spray, suspension, emulsion. 10. Cosmetic process to stimulate the regeneration and / or recovery of the original trophism of the integumentary system and / or improve the vitality of the hair follicles in a portion of mammalian tissue comprising the following steps: i) applying on said portion of fabric a composition according to any one of claims 1 to 9; ii) applying on said portion of tissue an alternating electromagnetic field at low frequency (ELF) and at least one of a static magnetic field and a static electric field; so as to stimulate the regeneration and / or recovery of the original trophism of the integumentary apparatus and / or improve the vitality of the hair follicles in said portion of tissue. 11. Process according to claim 10, wherein said alternating electromagnetic field is applied with a frequency comprised between 1 and 1000 Hz, preferably between 1 and 100 Hz. Method according to any one of claims 10 or 11, wherein said alternating electromagnetic field is applied with an intensity comprised between 1 and 200 µT. 13. Process according to any one of claims 10 to 12, wherein said alternating electromagnetic field is applied with a wave form such as to generate a harmonic content up to 50 KHz. 14. Process according to any one of claims 10 to 13, wherein said static magnetic field is applied with an intensity comprised between 10 and 2000 µT, preferably comprised between 10 and 500 µT. 15. Process according to any one of claims 10 to 14, wherein said static electric field is applied with an intensity between 1 and 1000 V / m, preferably between 10 and 100 V / m. 16. Process according to any one of claims 10 to 15, wherein said step ii) takes place for a period of time between 2 minutes / Bis in die and 2 hours / Bis in die, preferably between 5 minutes / Bis in die and 20 minutes / Bis in die. 17. Process according to any one of claims 10 to 16, wherein said composition is applied topically on integuments and covering or supporting tissues of mammals in the human or veterinary field. 18. Process according to any one of claims 10 to 17, wherein said mammal is a human being. 19. Use of the composition according to any one of claims 1 to 9, in the form of a cosmetic, medical device or drug, to stimulate the regeneration and / or recovery of the original trophism of the integumentary system and / or improve the vitality of the follicles hair in a mammal, preferably in man.