* * *

TEXT OF THE DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a cosmetic composition, activatable by means of the application of a low frequency alternating electromagnetic field (ELF) , and the relative cosmetic process apt to promote the growth of hair through recovery of the original trophism of the integumentary system, to stimulate its growth and to improve the vitality of hair follicles.

TECHNICAL BACKGROUND OF THE INVENTION

It is known that, with low intensity ELF {Extremely Low Frequency) fields variations can be induced in the transport of various ionic species (for example, calcium ions) through cell membrane, obtaining variations in the rates of regeneration and repair of cells and tissues and variations in the redox potential of molecules [1-4].

SUMMARY OF THE INVENTION

The present invention has the object of identifying a cosmetic composition that can determine a regrowth of hair and of the integumentary system in general when activated by means of the application of a low frequency alternating electromagnetic field (ELF) and the relative cosmetic process, determining a repairing of the atrophic hair follicles and/or the regeneration of quiescent hair follicles, accelerating their functional recovery and competent cell turnover, when activated.

The present invention has the object of finding a valid and efficacious solution to the problems of hair loss and alteration of the integumentary system in mammals, and in particular in humans, which to date are unsolved.

According to the present invention, such object is achieved by means of the solution specifically recalled in the claims that follow. The claims form an integral part of the technical teaching provided herein relative to the invention.

The invention relates to a cosmetic composition, in combination with the application of a low frequency alternating electromagnetic field (ELF) and the relative process described below, to be used for stimulating the growth- of hair and of the integumentary system in general,- comprising: at least one metal and its salts and/or compounds, possibly in the form of a colloidal suspension; and

-at least one proteolytic agent (preferably papain) .

Furthermore, the invention relates to a cosmetic process to stimulate the growth of the integumentary system of a mammal, envisioning the combined application of i) the above-described cosmetic composition and ii) low frequency electromagnetic waves (ELF) , static magnetic and/or electric fields to a portion of mammalian tissue of interest, so that a specific and synergistic action is generated between the composition and the ELF application such to promote the recovery of the original trophism of the integumentary system in the portion of tissue of interest through: 1) physiological activation of the regrowth of hairs, that is, stimulation of the hair bulbs to return to their normal activity;

2) a direct action on the hair follicles with recovery of the atrophic follicles even in scarred zones. In fact, the present inventors have demonstrated that static electric and/or magnetic fields associated with low frequency electromagnetic waves ELF of relatively weak power determine, when used in combination with the cosmetic composition object of the present invention, the rapid physiological functional recovery of quiescent or atrophic hair follicles.

In fact, the present inventors have simultaneously applied electromagnetic radiation (in the frequency interval from 1 to 1000 Hz) and static magnetic fields (in the intensity interval from 1 to 1000 uT) , and/or static electric fields (in the intensity interval from 1 to 1000 Vm) to various biopsies of animal cutaneous hairy adnexae in culture media containing the cosmetic composition in object for application to tissue, and in vivo to various subjects affected by alopecia, effluvium or defluvium. Surprisingly, a particular regeneration of the atrophic or quiescent hair follicles was observed, accelerating their functional recovery and hair growth, as well as recovery of the original pigmentation of the integumentary system.

Such hair growth phenomenon was evident even under adverse experimental conditions, such as in tissue areas with post surgical scars.

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

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, by way of non-limiting example only, with reference to the annexed figures, wherein: figure 1 is a schematic representation of a device capable of generating static electric and/or magnetic fields associated with low frequency electromagnetic waves ELF to be use 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 in figure 1.

In a currently preferred embodiment, the cosmetic process according to the present invention envisions the application of a specific-inducing cosmetic composition

(preferably in the form of a lotion) and the exposure for a period of time varying between 5 minutes and 10 minutes twice a day (morning and evening) to a device delivering a combination of static electric and/or magnetic fields and low frequency alternating electromagnetic fields

(ELF) of low intensity.

The features of the applied ELF fields (waveform, frequency, duty cycle, intensity) and of the static magnetic and electric fields possibly added to the ELF depend on the type of result desired (regeneration, repair, growth) , and on the various background conditions

(type of tissue atrophy, type of substrate, time) . The features of the fields applied in the process object of the present invention are given by:

- the frequency of repetition of the low frequency electromagnetic wave applied and the waveform; the intensity of the alternating (ELF) field applied; - the intensity of the static magnetic field applied; the intensity of the static electric field applied;

- the application time. The in vitro and in vivo tissue study materials are :

1. in vitro biopsies of animal skin with alopecia or extensive fur effluvium - defluvium, refractory to common treatments; 2. in vivo alopecic human scalp or hair effluvium - defluvium, refractory to common treatments. Instruments and physical specifications The device 1 to be used in the cosmetic process object of the present invention is schematically illustrated in figure 1.

The device 1 comprises a chamber 2 which may possibly be shielded from the static or low frequency alternating magnetic field by means of a mu-metal shield, and from the static electric and high frequency alternating electromagnetic fields by means of an additional brass shield contained in the mu-metal one.

The device 1 is connected to the outside through narrow openings 5 to allow the passage of the electrical connections 15 (coils, video camera) . In a preferred embodiment, the device 1 comprises inside:

- two coaxial coils 4 generating the static magnetic field; two coaxial coils 7 generating the alternating electromagnetic field; two aluminium plates 3 generating the static electric field with axis perpendicular to that of the cited coils.

The static magnetic field is generated by an external electric circuit 10, in the "current pump" configuration, providing the desired - adjustable - current to the dedicated coils.

The static electric field is generated by applying an appropriate continuous voltage difference to the metallic plates by means of a source 9.

The alternating electromagnetic field is generated by a current generator 11 piloted by an arbitrary waveform generator applied to the dedicated coils . The coils 4 and 7 contained in the device 1 may be realised in one of the following configurations: a) a particularly preferred embodiment of the device 1 comprises a double wound coil as illustrated in figure 2, wherein one winding is used to produce the alternating electromagnetic field and the other to produce the static magnetic field. b) a different particularly preferred embodiment of the device 1 comprises at least two double wound coils arranged in an array as above as illustrated in figure 3 so to obtain a zone as wide as desired in which it is possible to irradiate the field. c) another particularly preferred embodiment of the device 1 comprises a double wound coil wound around a magnetic nucleus in ferrite, as illustrated in figure 4. d) a further particularly preferred embodiment of the device 1 comprises at least two double wound coils each wound around a magnetic nucleus of ferrite, wherein all said components (coils + nuclei) are arranged in an array as illustrated in figure 5 so to obtain a zone as wide as desired in which it is possible to irradiated the field.

A further preferred embodiment is that in which all of the embodiments a) , b) , c) , d) described above are realised with simple (single wound) coils instead of double wound coils, in which the possible superimposition of a static magnetic field and an alternating electromagnetic field are obtained electronically, with an algebraic summation operation of the desired signals. Furthermore, figure 5 illustrates the presence of an electromagnetic shield in mu-metal or similar material around the double wound or single wound coils with nucleus or without nucleus so to annul the irradiation on all sides, except the side destined to irradiate the portion of tissue of interest (numeric reference 12 in figure 1) , allowing a possible operator to hold the device 1, however constituted, in hand without being needlessly irradiated.

Irradiation was interrupted every hour and a part of the sample was removed and analysed.

Total exposure of the cellular sample to the alternating electromagnetic field/static magnetic and/or electric field is comprised between 15 minutes and 96 hours . Note that the irradiation conditions reported herein identify a specific operational window of the device used for irradiation, such window being susceptible of generating the effects described below on cell cultures. It is also clear that such irradiation conditions are not intended as limiting and it will be possible for one skilled in the art 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 made by the present inventors of a particular pro-regenerative eutrophicating 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, for example, papain, on human scalp, human or, in general, animal cutaneous tissue with hairy cutaneous adnexae.

In fact, the present inventors surprisingly verified that the metals, their colloidal suspensions, their salts and their compounds participate, synergistically with the delivery of a combination of static electric and/or magnetic fields and low frequency alternating electromagnetic fields (ELF) of low intensity, in various enzymatic processes and have a regulating action on metabolism and catabolism of the hair follicle microenvironment , inducing the control and regeneration of the atrophic or quiescent hair bulbs.

In parallel to the above described action, the present inventor could verify in performing the experiments that the proteolytic enzyme (preferably papain) promotes two main functions:

(i) activation of the growth factors physiologically present in the microenvironment of the damaged or senescent tissues, and

(ii) chemotactic induction of the resident stem cell compartment to a rapid repairing differentiation.

Furthermore, it was discovered that (i) the metals, their colloidal suspensions, their salts and compounds, (ii) together with the delivery of a combination of static electric and/or magnetic fields and low frequency alternating electromagnetic fields (ELF) of low intensity and together with (iii) the administration of papain have different synergistic functions: uptake of the nutrients present in the preparation is accelerated, tissue repair is induced, a physiologically correct microenvironment is regenerated, and, finally, the disemia that appears in the state of follicular atrophy is corrected. Furthermore, the present inventors discovered that with papain the Langerhans cells (stratum spinosum) are stimulated to take up foreign substances (excess collagen) and digest them. Such mechanism was shown to be particularly useful in the treatment of quiescent follicles . These totally surprising and unexpected observations led the present inventors to create a process provided with a corresponding device and composition suitable to promote the recovery of the original trophism of the integumentary system, to stimulate its growth and to improve the vitality of hair follicles through the use of metals, their colloidal suspensions, their salts and compounds, together with the delivery of a combination of static electric and/or magnetic fields and low frequency alternating electromagnetic fields (ELF) of low intensity and together -with the administration of papain.

Treatment of biopsies of animal cutaneous tissue with hairy adnexae in vitro with the process, the device and the composition of the invention results in a surprising repair of the damage and in the complete recovery of the original trophism of the integumentary system. All tissues represented in the biopsy samples treated are subjected to a complete functional and morphological regeneration (see Example 1). The in vivo histological results obtained starting from 30-60 days of treatment with the composition object of the present invention and its tissue-specific formulations, at histological examination confirm the formation of regeneration and re-trophisation of human scalp and in general of human cutaneous tissue with hair adnexae with excellent histo-functional features, morphologically comparable to intact tissue in vivo, (see section relative to Clinical Studies) .

In detail, the composition object of the present invention comprises at least the following components: at least one metal, possibly in the form of a colloidal suspension, its salts and its compounds; and at least one proteolytic agent, preferably papain. The proteolytic enzyme is present in the composition at a concentration comprised in the interval from 0.1 μg/L to 100 mg/L, more preferably from 10 mg/L to 80 mg/L.

The metal is present in the composition at a concentration comprised in the interval from 0.01 mg/L to 100 mg/L, more preferably from 0.1 mg/L to 20 mg/L.

In a further preferred embodiment the composition comprises additional active ingredients, selected for their enhancing and synergising activity for ELF and for the active principles defined above, specifically for each tissue type and relative human or animal integumentary system to be treated. Such additional active ingredients may be selected from an amino acid, a sugar, a vitamin, selenium.

The composition according to the present invention may be in the form of a lotion, gel, cream, liquid infusion, spray, suspension, emulsion.

Examples of in vitro and in vivo tissue-specific compositions Table 1.

Human scalp, human and animal cutaneous tissue with hairy adnexae .

CrPl-MD-GEL composition (medical device)

Table 2. Human scalp, human and animal cutaneous tissue with hairy adnexae.

Composition CrPl-MD-Lq (Lotion)

Table 3. Human scalp, human and animal cutaneous tissue with hairy adnexae.

Composition CrP2-MD-Lq (Lotion)

Table 4. Human scalp, human and animal cutaneous tissue with hairy adnexae.

Composition CrPl-MD-INFUS (Liquid infusion)

The composition object of the present invention may further comprise additional active components, such as:

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

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

The metals are preferably introduced into the compositions of the invention in the form of salts or colloidal suspensions. In addition to Zinc oxide, to colloidal Silver and to colloidal Gold mentioned in the previous tables, it is possible to use Iron salts or Ferritin, Sodium, compositions containing metals, their combinations and derivatives, among others, with the same results.

As proteolytic enzymes, in addition to the previously cited papain, for example, collagenases may be used (preferably of type Ia, type II, type IV), serratiopeptidases, elastases, bromelain, bradykinase, Clostridium peptidases, enzymes expressed by

Lactobacillus acidophilus , enzymes expressed by the genus Aspergillus , proteases, alliinases, fibrinolysin .

Mixtures of amino acids, preferably containing L-

Methionine, L-Cystine, L-Cysteine, L-Glycine, L- Leucine, L-Lysine, L-Proline, L-Glutamine, constitute pro-eutrophic elements that function as important growth factors for the scalp, skin and adnexae, regulating cellular and tissue turnover.

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

In addition to the amino acids, the compositions of the invention may also comprise other elements, such as Selenium, carbonates, bicarbonates, glycyrrhetic acid, glycyrrhizin, glucose, glutathione, glycocolic acid, soy lecithin, collagen, elastin, wheat extract, and the like.

Among the vitamins that may be present in the composition we cite, by way of example only, 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 was discovered by the present inventors that vitamin A, used at doses that are free of any pharmacological effect, enhances the reestablishment of the correct pH and antioxidant potential in tissues such as skin, hypoderm, and mucosa.

Always based on the experiments of the present inventors, it was verified that in the composition object of the present invention the B vitamins, preferably Bl, B2, B5, B6, B9 and B12 contribute to the normalisation of the local-regional microenvironment .

Always based on the experiments of the present inventors, it was verified that in the composition object of the present invention vitamin C promotes the reestablishment of a peri-follicular physiological microenvironment, while vitamin D at physiological values plays an auxiliary role in the consolidation of an ideal sodium-calcium ion exchange favouring adjustment to physiological values of the altered electrolytic concentration around the quiescent or atrophic hair follicles.

The present inventors have demonstrated that vitamin E, used at doses free of any pharmacologic effect, leads to the gradual physiological restoration of the microenvironment, mainly hypodermal, actively countering as a co-adjuvant the physiological biosynthesis of hair.

Concerning the activity of vitamin H (Biotin) in the composition object of the present invention it was discovered that it favours the activation of two repair mechanisms in the organism: attraction of stem cells from the circulatory stream and an accelerated specialisation of the resident stem cells in the skin towards a rapid differentiation into mature cells destined to restore the damaged hair follicles. Furthermore, the present inventors have discovered that vitamin PP (Nictinamide) , in association with the various components of the composition object of the present invention, local-regionally induces several mechanisms of action: 1) it functions as a constituent of enzymes involved in redox reactions;

2) it participates in energy metabolism of carbohydrates and proteins;

3) it takes part in stimulating capillary turnover. The cosmetic and the medical device type compositions object of the present invention may also comprise further accessory elements such as excipients and vehicles whose choice and employment fall within the capacity of one skilled in the art with no need to exert inventive activity. Biopsies

Twenty biopsies of animal skin affected by alopecia, effluvium or defluvium refractory to common treatments were analysed. All samples were washed three times with physiological solution containing antibiotics

(100 Units/ml of penicillin + 100 μg/ml streptomycin + 40 mg/L gentamicin) for ten minutes at room temperature.

Each biopsy was sectioned in six parts (two controls -1, two controls -2 and two samples) . The samples (3) were suspended in a solution of 20 ml of CrPl-MD-INFUS at a final concentration of Ix in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) .

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

1. Control biopsies were suspended in 20 ml of physiological solution in 10 cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) .

2. Control biopsies 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-Plough, Italy) 2 mM L-glutamine (Life Technologies) .

3. All samples were placed in a Heraeus incubator thermostatically controlled at a temperature of 37 ⁰ C with an atmosphere containing a constant supply of 5% CO ₂ (v/v in air) for 180 days.

Every 21 days of culture the supernatants of the cultures under study were collected and such supernatants were stored in a freezer at -80°C for subsequent Laboratory analysis: total NO concentration, nitrites and nitrates. For each biopsy taken, six tissue portions were set up, of which: two samples and four controls (two controls 1 and two controls 2) .

A) Three experimental replicates, each formed by two controls (1, and 2) and one sample, were incubated at 37 ⁰ C in an atmosphere containing a constant supply of 5% CO ₂ (v/v in air) , for a time interval of 180 days .

B) Three additional experimental replicates, each formed by two controls (1, and 2) and one sample, were subjected to an ELF magnetic field at a frequency comprised in the interval from 1 and 1000 Hz, and static magnetic fields (in the intensity interval from 1 to 1000 uT) , and/or static electric fields (in the intensity interval from 1 to 1000 V/m) for a time interval of 10 minutes twice per 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; r ^> waveform: sinusoidal;

^- time of application 10 minutes twice per day every 12 hours.

Such conditions were determined experimentally by optimising the impedenziometric feedback by scanning the intensity of the alternating magnetic field from 0 to 30 uT, and correlating the frequency of the total field intensity thus obtained.

The biopsies were incubated, performing sampling and corresponding tests every 21 days for a maximum of 180 days. Immunofluorescence protocol After washing three times for 10 minutes at room temperature in PBS (pH 7.4), the samples were resuspended in a fixing solution of 4% paraformaldehyde in RPMI1640 at pH 7.4 for one hour at room temperature. After embedding in paraffin, the samples were sectioned and positioned on slides. The sections were stained with haematoxylin/eosin, anti-cytokeratin 10 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA) , anti-cytokeratin 11 monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA) . For each monoclonal antibody, specific controls were set up with the corresponding isotypes (Santa Cruz Biotechnology, Santa Cruz, California, USA) . All samples positioned on slides were observed with light microscopy after being sealed with moviol and coverslips.

Measurement of NITRITES (NO ₂ ^' ) and NITRATES (NO ₃ ^' )

Since the greater part of the nitrogen monoxide (NO) , produced by NO synthase enzymes is oxidised to nitrites and nitrates, the concentration of these anions was used as a quantitative measure of NO production. This method is based on the enzymatic conversion of nitrates to nitrites through use of the enzyme nitrate reductase.

Next the nitrites are identified spectrophotometrically by means of a colorimetric reaction that, in the presence of the Griess reagent (N-naphthylethyl-enediamine in 2N HCl and sulfanilamide in 2N HCl), provides an azo-derivative that absorbs light at 540 nm as a final product. The NO concentration is calculated indirectly evaluating both the nitrite and nitrate levels separately. This method allows evaluation of the quantity of endogenous nitrites present in the samples and subtraction of this from the total value obtained. Measurement of endogenous nitrites in the samples 100 μl samples of supernatant were added to 100 μl of Griess reagent, and after 10 minutes of incubation the 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 made with fresh medium and the titration curve with known concentrations of sodium nitrite. The nitrite concentration was expressed in μmoles of nitrite per mL . Measurement of total nitrites in the samples following conversion of the nitrates to nitrites

50 μl samples of supernatant were added to 25 μl of nitrate reductase and 25 μl of NADH in 96-well plates and left to incubate for 30 minutes at 37°C. Next, 100 μl of Griess reagent were added and after 10 minutes of incubation the 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 made with fresh medium and the titration curve with known concentrations of sodium nitrate. The nitrite concentration was expressed in μmoles of nitrite per mL. Determination of nitrate concentration in the samples

In order to obtain the nitrate concentration it is necessary to subtract the value obtained in the method for the measurement of endogenous nitrites from that resulting from the method for the measurement of total nitrites (after complete conversion of the nitrates to nitrites) . The measurement of the nitrites and nitrates was performed with a kit: Nitric Oxide (NO2-/NO3-) Assay, cat. No. DE1500, (R&D Systems Inc., Minneapolis MN 55413, USA) . Measurement of total nitrogen monoxide (NO) This method is based on the enzymatic conversion of nitrates into nitrites by the use of nitrate reductase enzyme. The nitrites obtained react with the Griess reagent (N-naphthylethyl-enediamine in 2N HCl and sulfanilamide in 2N HCl) yielding as a final product an azo-derivative optically visible at 540 ran. The guantity of NO is calculated indirectly based on the concentration of nitrites obtained from the total conversion of nitrates into nitrites. For the measurement, 50 μl samples of supernatants were taken and were added to 25 μl of nitrate reductase and 25 μl of NADH in 96-well plates and left to incubate for 30 minutes at 37°C. Next 100 μl of Griess reagent were added, and after 10 minutes of incubation the 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 made with fresh medium and the titration curve with known concentrations of sodium nitrate. The nitrite concentration was expressed in μmoles of nitrite per mL. The measurement of total NO was performed with the kit : Total Nitric Oxide Assay, cat. No. DE1600, (R&D Systems Inc., Minneapolis MN 55413, USA) . Western blot for cytokeratins

The biopsies were suspended in lysis buffer (1% SDS, 30 mM TRIS pH 6.8, 5% glycerol) to which protease inhibitors were added (Protease Inhibitor Cocktail, Calbiochem, San Diego, CA), they were subjected to homogenisation, followed by incubation of the samples for 30 minutes at 4°C. The lysates obtained were centrifuged at 12,000 RPM for 20 minutes 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) . Before electrophoresis, the samples were boiled for 5 minutes in the presence of beta-mercaptoethanol and bromophenol blue. The samples were subjected to electrophoresis in a 12% gel (SDS-PAGE) and transferred onto PVDF membranes (Perkin Elmer Inc.). The membranes were saturated with methanol at room temperature and then incubated with the following primary antibodies diluted in PBS containing 5% powdered skim milk: anti- cytokeratin 14 with dilution titre of 1:500 (Santa Cruz Biotechnologies Inc., , Santa Cruz, California, USA), anti-cytokeratin 18 with dilution titre of 1:500 (Santa Cruz Biotechnologies Inc., Santa Cruz, California, USA), anti-cytokeratin 19 with dilution titre of 1:500 (Santa Cruz Biotechnologies Inc., Santa Cruz, California, USA), overnight at 4°C. After five washes, the membranes were incubated with the corresponding secondary antibodies (1:1000) conjugated with horseradish peroxidase (HRP, Santa Cruz Biotechnologies Inc., Santa Cruz, California, USA) for 1 h at room temperature. The corresponding bands were detected with chemiluminescence liquids (Super Signal Western Pico solution, Pierce Biotechnology Inc., Rockford, Illinois, USA) and fixed on photographic films. EXAMPLES Six month incubation

All type 1 controls, both for the treatment A groups and for the treatment B groups, suspended in physiologic solution only, as described in materials and methods, underwent a rapidly progressing necrotic process. They were then eliminated at day 15 of the study (Table 5) . All type 2 controls, both for the treatment A groups and for the treatment B groups, suspended in culturing media only, as described in materials and methods, underwent a process of progressive exfoliation, without presenting any repair of the areas affected by alopecia, effluvium or defluvium, until a global autolysis resulted in about 30-60 days. They were then eliminated at the onset of the irreversible necrotic processes on average at day 45 (Table 5)

EXAMPLE 1. Light microscopy These results indicate a phenomenon of reactivation or regeneration of the hair follicles in the biopsy samples, both for the treatment groups A and for the treatment groups B.

The results relative to expression of cytokeratins 10 and 11 and histological staining of the biopsies with haematoxylin/eosin (used for the visualisation of follicular vitality) are reported in table 5 (CONTROLS) and in table 6 (SAMPLES) and were expressed on a qualitative and semi-quantitative scale as a ratio of the results found in treatment group A (CrPl-MD-INFUS) with respect to the results shown by the treatment group B (CrPl-MD-INFUS + ELF) .

Analysis of the light microscopy results in Table

6 reveals a net increase in the number of follicles in the samples from group B (CrPl-MD-INFUS + ELF) treated for six months with respect to the samples from group A

(CrPl-MD-INFUS) treated for six months.

In addition, all of the follicles appeared trophic, vital and active in the samples from group B (CrPl-MD-INFUS + ELF) with respect to the samples from group A (CrPl-MD-INFUS) , where hypotrophism of the follicle itself is noted.

Finally, a net predominance of cytokeratins 10 and

11, typical of normal integumentary tissues with vital and active follicles, is pointed out (Table 6) in the samples from group B (CrPl-MD-INFUS + ELF) treated for six months with respect to the samples from group A

(CrPl-MD-INFUS) .

Table 5. Type 1 controls and type 2 controls both for the treatment groups A and the treatment groups B. Mean day of irreversible necrosis is equal to day 15 for the type 1 controls. Mean day of irreversible necrosis is equal to day 45 for type 2 controls .

Legend = absence of fluorescence

+ = slight fluorescence per optical field ++ = light fluorescence per optical field +++ = medium fluorescence per optical field ++++ = high fluorescence per optical field +++++ = very high fluorescence per optical field

Table 6. Samples from treatment groups A and treatment groups B. Mean semi-quantitative values at the incubation days 90 and 180.

Legend = absence of fluorescence

+ = slxght fluorescence per optical field ++ = light fluorescence per optical field +++ = medium fluorescence per optical field ++++ = high fluorescence per optical field +++++ = very high fluorescence per optical field

EXAMPLE 2. Western blot

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

The results were strongly positive for the production of cytokeratin 1, cytokeratin 2, cytokeratin 10, cytokeratin 11 and cytokeratin 14, in the treated samples, and in particular in treated samples from group B, for 180 days in vitro with the composition object of the present invention, ChPl-MD-INFUS, with respect to a weakly positive result in samples from treatment group B, for 180 days in vitro, as is 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 cellular differentiation, of hair follicle growth and control of hair formation [3-8].

Table 7. Samples treated in vitro for 180 days from group A and group B, expression of cytokeratins

1,2,10,11,14.

Legend = absence of the band

-/+ = light band present + = thin band present ++ = medium band present +++ = extensive band present ++++ = strong band present

EXAMPLE 3. Measurement of nitrites, nitrates and total NO.

Nitrogen monoxide (NO) , improperly called nitric oxide, is a reactive chemical species of radical nature centred on nitrogen. NO is transformed into a series of derivatives, such as nitrites and nitrates, which accumulate in the blood and in other extracellular fluids as a function of the quantity of the primary mediator produced, to then be definitively discarded from the organism in 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] . Table 8.

Therefore, overall, the experimental data analysed herein, demonstrate that the composition, the process and the device object of the invention, used in vitro can improve some physio-pathological conditions through an increase in endogenous NO synthesis, demonstrated by the corresponding increase in its catabolites nitrites and nitrates. The NO, the derived nitrites and the nitrates appear to assume the role of biological mediators participating indirectly in the regulation of the proliferation and differentiation of the integumentary structures and specifically in the cyclic modifications of the hair follicle [3-8]. From the analysis of the results in table 8 a significant increase in the concentrations of nitrites and nitrates and therefore of the production of NO was observed in the samples from group B treated in vitro for 90 days (three months) and 180 days (six months) with the process, the device and the composition objection of the invention ChPl-MD-INFUS with respect to the controls 1-2 and to the sample from group A. Such results are typical of normal integumentary tissues with vital and active follicles.

Naturally, the details of realisation and the embodiments may vary appreciably with reference to what has been described and illustrated, without departing from the scope of the present invention as defined by the annexed claims.

BIBLIOGRAPHY

1. Blackman CF et al. A role for the magnetic field in the radiation induced efflux of calcium ions from brain tissue in vitro. Bioelectromagnetics . 1985; 6: 327-337.

2. Nikolova T, Czyz J, Rolletschek A, Blyszczuk P, Fuchs J, Jovtchev G, Schuderer J, Kuster N, Wobus AM. Electromagnetic fields affect transcript levels of apoptosis-related genes in embryonic stem cell- derived neural progenitor cells. FASEB J. 2005

0ct;19 (12) :1686-8.

_; 3. Nordberg J and Arner ESJ. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radical Biol Med. 200; 31:1287-312. 4. Fujiwara N, Osanai T, Kamada T, Katoh T, Takahashi K, Okumura K. Study on the relationship between plasma nitrite and nitrate level and salt sensitivity in human hypertension modulation of nitric oxide synthesis by salt intake. Circulation. 2000; 101:856-861.

5. Comaish S. "Metabolic disorders and hair growth" Br J Dermatol 1971; 84:83.

6. Kossard S. "Postmenopausal frontal fibrosing alopecia: Scarring alopecia in a pattern distribution" Arch Dermatol 1994 ; 130 : 770-774.

7. Schmidt J. B. "Hormonal basis of male and female androgenetic alopecia: Clinical relevance". Skin Pharmacol 1994 ; 7 : 61-66.

8. Robinson M, Reynolds AJ, Gharzi A, Jahoda CA. In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture. J Invest Dermatol. 2001 Sep; 117(3) :596- 604.