Transdermal technology for skin penetration of dermo-cosmetic active substances

DESCRIPTION

The present invention relates to a transdermal technology for transcutaneous penetration of one or more dermocosmetic substances.

The present invention originates in the cosmetic field and in the field of transdermal systems for the controlled penetration of dermo-cosmetic active substances through the skin.

Specifically, the present invention relates to a penetration enhancer system for the skin penetration of dermocosmetic active ingredients without using hypodermic injections. The invention relates to the application in the cosmetic field of the transdermal technology, that is the penetration of dermocosmetic active substances through the skin.

Prior Art

Transcutaneous absorption of a dermocosmetic preparation includes the penetration of the preparation through the surface layers of the skin, which perform a barrier function, until reaching the first layers of the dermis.

The barrier-function of the skin is performed almost entirely by the horny layer. This consists of 15-25 layers of corneocytes trapped by a double-layered lipid matrix. All this forms a continuous layer, interrupted only by the sweat and sebaceous glands ducts and hair follicles. The horny layer forms the real barrier against penetration of foreign molecules of any type, thanks to its high density, low hydration and absence of vascularization. The very low permeability of the horny layer to water-soluble substances is due to the extracellular lipid matrix consisting of ceramides, cholesterol, long chain fatty acids.

Transdermal penetration, which identifies the transport of a substance through the horny layer and the epidermis, is a process that can take place through three routes: a) the intercellular, b) transcellular and c) through the skin appendages, such as the pilosebaceous apparatus and eccrine glands.

However, the access way through skin appendages represents a penetration route mainly for large molecules. The penetration surface is however relatively small; in fact, only 0.1-0.5% of the body surface is occupied by these structures. During this phase, a latency period is observed in which the substance comes to an equilibrium with the epidermis, with a first-order kinetics.

After reaching the substance-epidermis equilibrium, a steady penetration stream begins, whose scale is proportional to the concentration of the substance. At this point, the transcellular way becomes the main trans- epidermal penetration route, and penetration through the pilosebaceous apparatus is only responsible for a small fraction of the penetration, mostly for lipid or suspended in lipids substances.

Direct transcellular penetration envisages first the molecule passage through the cell wall of corneocytes and then the migration of the polar molecules to the aqueous areas of the corneocytes, and of the lipophilic molecules to areas with higher lipid content. It is clear that the whole process is based on the principle of passive diffusion.

Generally, lipophilic substances take the intercellular route, with a particularly tortuous path, while hydrophilic or polar compounds can penetrate in faster way through the transcellular route, taking advantage of the protein fraction of the keratinocytes.

Most of the biologically active substances permeates the horny layer through both pathways. Despite the winding via intercellular constitutes the greatest barrier for most of the substances, the essentially aqueous epidermis and dermis can represent a significant barrier for the penetration of highly lipophilic molecules.

In general, substances that pass through the skin are quite fat-soluble and not ionized. The transcellular or transepidermal route is followed by non-lipoid substances. As soon as the substance is brought by the carrier in contact with the glossy layer (which constitutes an obstacle to passages of chemical substances), a part thereof will cross it through the pilosebaceous route; then, when a certain concentration is reached on the other side, a trans-epidermal step will start.

Cutaneous absorption describes the transport of chemical substances from the outer surface of the skin up to the circulatory system. This process is often divided into:

- penetration: the entry of a substance in a particular layer or structure;

- permeation: the penetration of a compound in a second layer that is functionally and structurally different from the first layer previously crossed; - absorption: the absorption of the compound in the local vascular system and skin lymphatic system. This generally leads to an absorption into the systemic circulation (systemic absorption). As previously said, the transport across the horny layer is a passive diffusion process governed by Fick's diffusion law:

dq = R . Ds . A . CV

dt h

wherein: Cv = concentration of the active substance in the carrier; R = coefficient of distribution of the active ingredient between carrier and barrier; Ds = diffusion coefficient of the active substance through the epidermis; A = area affected by the absorption; h = thickness being crossed.

From this law it is clear that transport across the horny layer is directly proportional to the solubility and diffusibility of the substance applied and that the absorption rate is proportional to the difference of concentration of the substance of the high concentration region with respect to the region with lower concentration. The relative solubility of a solute is expressed by the partition coefficient horny layer/carrier and refers to the ratio between the solubility of the substance in the horny layer compared to that in the carrier. This parameter, therefore, indicates the affinity of the active ingredient to the horny layer and its ability to separate from the carrier. Diffusibility is the ability of a solute to cross the barrier and is influenced by various factors, including the convolutedness of the intercellular route.

As expressed by Fick's law, the absorption rate - flow - is directly proportional to the concentration of the active substance dissolved in the carrier and to the surface of application, while it is inversely proportional to the thickness of the horny layer. Although all the parameters have been calculated, due to the complexity of the horny barrier Fick's law has only indicative value, as it does not take into account many factors both both biological, and biopharmaceutical and, as well as chemical and physical characteristics that influence the percutaneous absorption.

Transcutaneous penetration of a dermocosmetic active should consider some chemical and physical factors of the substance capable of penetrating the skin: -Molecular weight: the lower the MW, the greater the probability of penetration. The molecular size regulates the spread of the molecules used across biological membranes and can become a barrier to penetration.

-Physical structure, with the same molecular weight, a twisted and flexible molecule has higher probability of penetration than a stiff and bulky molecule. -Partition coefficient (i.e., hydrophilic or lipophilic properties of the substance). A lipophilic or amphiphilic substance is more likely to penetrate.

-Ionization; a charged substance is less likely to penetrate.

-Characteristics of the carrier. A carrier is defined by the type of preparation (cream, ointment, gel..) and by excipients (water, paraffin, propylene glycol ...). Carriers have an active and vital role in enhancing the percutaneous absorption as they can affect the characteristics of the horny layer and the partition/diffusion coefficient of the active ingredient,

as well as factors related to the application:

-Applied dose; the greater the amount or higher the concentration of substance applied to the skin, the greater the probability of penetration of that substance. -Exposed area; the greater the exposed skin surface the higher the probability of penetration. -Duration of the exposure; the longer the exposure time the highest the probability of penetration.

-Condition of the skin, thickness and integrity of the horny layer.

-Idratazione of the skin; the more hydrated the skin the more likely the penetration.

Notoriously, the emulsifier and surfactant substances accelerate penetration of a molecule carrying a complementary action to the carrier. Their mechanism of action consists in emulsifying sebum and surface liquids by incorporating them into the carrier, but later they may affect the barrier formed by the horny layer and weaken it.

Among different cosmetic forms, the hydrogels, the aqueous suspensions and the o/w emulsions behave on the skin as aqueous media, while ointments, anhydrous pastes and the w/o emulsions function as lipid systems.

If the aqueous component of the carrier evaporates, both the structure of the carrier and the concentration of the substance will vary. The phenomenon is typical of the o/w emulsions in which the water loss occurs. In the case of the w/o emulsion instead the water evaporates more slowly and the occlusive effect is more pronounced due to the greater affinity of the w/o emulsion in respect of the surface lipids.

To hydrate the skin, carriers with occlusive characteristics may be used, such that their application forms a film capable of preventing the evaporation of water. In this way, water that is present in the deeper layers of the skin reaches the surface layers thus increasing hydration. Water is the most natural carrier. Hydration seems to increase the transdermal transport of both hydrophilic and lipophilic permeants. Water within the horny layer alters the solubility of the permeants and therefore can change the carrier to membrane partition. In addition, hydration can open the structure of the horny layer leading to an increase in the penetration.

It is also known from the pharmaceutical industry the use of substances that increase penetration, in order to promote the penetration of active ingredients through the skin while reducing reversibly the barrier properties of the skin. In particular, these substances can to increase the transcutaneous penetration of active ingredients and change reversibly the skin barrier characteristics. The penetration enhancers act in several ways: by increasing the diffusibility of the substance inside the barrier through the interaction with the intracellular keratin, with desmosomes, with intercorneocyte lipids; increasing the solubility in the carrier and improving the partition coefficient.

For example, the use of vitamin E is known as a penetration enhancer for pharmaceutical active principles. It has been thought that vitamin E acts as a penetration enhancer by intercalating within the lipid bilayer region of the horny layer, so altering the membrane characteristics and influencing the permeability thereof, presumably disarranging the lipid phase.

In aesthetic medicine, branch of medicine close to the technological field of cosmetics that deals with treating the signs of aging, the need to deliver effective quantities of active principles in the skin areas affected by aging is overcome by resorting to subcutaneous injection of the active substances. Aesthetic medicine, however, has the drawback that the aesthetic treatment can not be carried out autonomously but only by qualified medical or paramedic personnel.

At present, therefore, a need is felt for having new technologies in the cosmetic field to perform beauty treatments of effectiveness comparable to those of aesthetic medicine without resorting to subcutaneous injection of active ingredients.

One of the objects of the invention is to provide a penetration enhancer formulation suitable to make substances with dermocosmetic action penetrate in the superficial layers of the dermis without the use of transdermal or hypodermic injections.

Another object is to provide in the cosmetic sector an application of a transdermal technology typical of the pharmaceutical industry, in order to increase the penetration of dermocosmetic substances through the surface layers of the skin while limiting the risk of systemic absorption.

SUMMARY OF THE INVENTION

According to certain aspects, the invention concerns with the use of a transdermal technology to dermocosmetic treatments to permit the penetration of dermocosmetic active substances through the outer layers of the skin without the need of performing hypodermic or subcutaneous injections, obtaining cosmetic benefits similar to those of aesthetic medicine.

According to a general aspect, the present invention relates to a penetration enhancing system for topical use to increase the penetration of cosmetically active substances through the outer layers of the skin and to cosmetic compositions containing it.

In particular, by using the penetration enhancing system of the invention an increase of the penetration through the skin outer layers of dermocosmetic substances selected from the cosmetic ingredients INCI, CTFA, IECIC, JCLL, without performing the hypodermic or subcutaneous injections which are typical of the treatments of aesthetic medicine.

In a first aspect, the present invention relates to a penetration enhancing system or composition for the penetration of one or more cosmetic substances through the outer layers of the skin comprising a combination of decylene glycol, pentylene glycol, and caprylyl glycol.

Typically, the system of the invention increases skin absorption, specifically the absorption through the skin of cosmetic substances or preparations to which it is added.

According to some aspects of the present invention, the penetration enhancing system can be used in the formulation of cosmetic compositions for topical application to increase the penetration rate through the skin of one or more dermocosmetic substances.

According to another aspect the present invention, thus relates to a cosmetic composition for topical use provided with penetration enhanced features, including a penetration enhancing system comprising decylene glycol, pentylene glycol and caprylyl glycol, one or more dermocosmetic substances and a cosmetically acceptable carrier. Typically, the cosmetic composition with penetration enhancing properties of the invention has the following features: it is chemically inert, not toxic or irritating, not comedogenic or allergenic, cosmetically acceptable which means chemically and physically compatible with the cosmetic substances.

The cosmetic composition with penetration enhanced properties of the invention allows penetration of active substances through the outer layers of the skin without the need for hypodermic or subcutaneous injections, obtaining cosmetic benefits similar to those of aesthetic medicine.

The present invention relates to a penetration technology of substances for dermocosmetic use, therefore the topically applied cosmetic substances do not reach the systemic circulation. The absorption is thus limited to the outer layers of the dermis, thus limiting the systemic absorption of dermocosmetic substances which are applied by the topical route of administration.

The Applicant therefore provides a technology that allows checking and/or regulating the transcutaneous penetration of a dermocosmetic substance, to provide the aesthetic effect sought, while substantially avoiding systemic absorption.

According to a further aspect, the present invention relates to a transdermal technology or transdermic method for penetrating dermocosmetic substances through the skin, in particular through the epidermis and superficial layers of the dermis, comprising:

selecting cosmetically active substances with average molecular weight≤ 2000 Daltons, testing the cosmetically active substances with average MW≤ 2000 Daltons with Franz diffusion cell;

adding the cosmetically active substances selected by a penetration increasing system preferably comprising decylene glycol, pentylene glycol and glycol caprylyl,

adding a suitable carrier to the mixture of the penetration increasing system and cosmetically acceptable substance for topical application.

DETAILED DESCRIPTION OF THE INVENTION

According to certain aspects, the invention therefore relates to transdermal absorption or penetration through the skin of specific selected dermocosmetic actives.

The Applicant has found that selected glycols, when combined, result in an unexpected increase in the penetration rate of dermocosmetic substances through the surface layers of the skin, allowing the uses mentioned in claim 11. According to a first aspect, the present invention relates to a penetration enhancing system or a composition to increase the penetration of one or more cosmetic substances through the skin comprising a combination of decylene glycol, pentylene glycol, and caprylyl glycol.

Within the invention, the terms penetration enhancing system and composition increasing the penetration have the same meaning and are interchangeable. The terms dermocosmetic substances, as used herein, means substances provided with cosmetic action that are applied on the skin or its appendages. By using this transdermal technology/composition in the dermocosmetic field, penetration of the cosmetically active substance applied to the superficial layers of the skin is obtained with a cosmetic result similar to that obtained with some treatments typical of the aesthetic medicine. In fact, with respect to traditional cosmetic systems, the use of the penetration enhancing system of the invention induces penetration of high levels of dermocosmetic active substances through the outer layers of the skin, typically the epidermis.

According to a further aspect, the invention relates to the use of a composition for facilitating and/or increasing the penetration of one or more cosmetic substances through the epidermis, comprising a combination of decylene glycol, pentylene glycol, and caprylyl glycol.

In accordance with one aspect, the invention relates to a dermocosmetic composition for topical application provided with penetration enhanced properties comprising a system for enhancing the penetration comprising decylene glycol, pentylene glycol, and caprylyl glycol, one or more dermocosmetic substances and a cosmetically acceptable carrier.

According to certain embodiments, the carrier of the dermocosmetic composition of the invention comprises a cosmetically acceptable alcohol with one to four carbon atoms, preferably ethanol.

The addition of a carrier in the system for enhancing the penetration of the invention increases the transcutaneous penetration by changing the organization of horny layer lipids and the structure of the barrier.

Furthermore, when using an alcohol as a carrier, the solvent properties of the alcoholic carrier positively affects the solubility of cosmetically active substances in the composition and the coefficient of partition. The penetration enhancing system of the invention may lead to the alteration of the critical molar ratio of ceramides, cholesterol and fatty acids present in the epidermis: if one of said 3 lipids is deleted or in excess, the lipid component in excess can not therefore preserve the lamellar organization, a separation of the phases is obtained with more permeable interstices and creation of further penetration route.

The penetration enhancing system of the invention provides a lipid extraction with formation of "pores" within the horny layer. In this way the skin hydration increases the solubility of the solute.

Typically, the penetration enhancing system increases the penetration rate of a cosmetically active substance through the skin, specifically the epidermis, by means of different mechanisms. A first penetration mechanism is the solvent action. The system for increasing penetration may solubilize the components of the skin tissues.

A second mechanism concerns the interaction of the system with the intercellular lipids leading to the destruction of the highly ordered lamellar structure, thereby increasing the diffusivity of cosmetic substances through the lipid domain. A further mechanism is the interaction of the system with the intracellular proteins to promote the permeation of drugs across the horny layer. A further mechanism is an increase in the distribution of the molecules, co-enhancers or co-solvents in the horny layer.

The analysis of the methods commonly used in aesthetic medicine have led the Applicant to explore the possibility to increase the penetration of cosmetically active substances through the skin without the use of needles, to obtain aesthetic results comparable to those obtainable with techniques of aesthetic medicine.

According to a further aspect, a dermocosmetic composition for topical application is provided, which increases the transcutaneous penetration of active cosmetic substances comprising a penetration enhancing system according to any one of the previously described embodiments and a cosmetically active substance.

The composition of the invention enables the penetration of dermocosmetic substances through the skin without the use of injections, as required in the field of cosmetic medicine to achieve an aesthetically appreciable result.

According to some embodiments, the penetration enhancing system and the composition of the invention are used to increase the penetration of cosmetically active substances with an average molecular weight MW≤ 2000 daltons, preferably ≤ 550, more preferably ≤ 380 daltons. Typically, the molecular weights of cosmetically active substances, as described in the present application, are expressed as the weighted average molecular weight (MW) (Da). The weighted average molecular weight of the cosmetic substances cited in the application may be determined using standard methods in the field, such as those disclosed in Ueno et al., 1988, Chem Pharm Bull. 36, 4971-4975; Wyatt 1993 Anal Chim Acta 272: 1-40; Watt Technologies 1999 "Dawn Light Scattering University Course Manual and" Dawn Eos Manual "Wyatt Technology Corp. Santa Barbara CA (USA).

In certain embodiments, the dermo-cosmetic or cosmetically active substance is present in the cosmetic formulation of the invention in an amount of 0.001 to 15% by weight, 0.05 to 10%, 0.1 to 5% by weight with respect the total weight of the composition.

In certain embodiments, the cosmetic composition contains an amount of the penetration enhancing system of 0.0001 to 10%, 0.001 to 3%, 0.05 to 1% by weight.

In accordance to further embodiments, the composition of the invention contains an additional glycol selected from the group including propylene glycol, hexylene glycol, ethoxydiglycol, ethylhexylglycerin, diethylene glycol and mixtures thereof.

In some embodiments, the composition of the invention further contains another component selected from the group including vitamin E, piperidine and mixtures thereof.

Typically, the cosmetically acceptable carrier of the composition of the invention is an excipient, carrier or diluent suitable for topical application.

In the present document, the term "carrier" refers to an excipient, carrier, diluent or adjuvant which may be present in the composition of the invention. Any carrier and/or excipient suitable for the form of preparation desired for administration is contemplated in the uses described herein.

The term "cosmetically acceptable" as used herein means that the dermo- cosmetic composition or its active components are suitable for cutaneous application in general, and when applied they do not cause an unwanted toxicity, allergic response, redness, incompatibility, instability, and the like reactions. The term dermocosmetic essentially means cosmetic as referred to the skin. In some embodiments, dermocosmetic substances contained in the composition of the present invention may be combined or mixed with a carrier or excipient according to the techniques of the cosmetic industry.

In some embodiments, the compositions or preparations of the invention may contain at least 0.0001% of each cosmetically active or cosmetically effective substance.

Typically, the composition of the invention uses active constituents substantially free of side effects, when applied by the topical route.

Typically, the cosmetic composition of the invention can be in any form suitable for topical application.

The composition for topical application can be in solid, semi-solid, fluid or semifluid form.

Suitable solid form formulations include creams, ointments, pastes, ointments, masks, transdermal paste or patch. A preferred embodiment provides a cream for topical application.

In other embodiments, the composition for local administration is in liquid form, for example, in the form of typically water based suspensions, solutions, lotions, gels, shampoos, emulsions.

In the dermocosmetic composition of the invention, one or more excipients typically used in the basic formulation of cosmetic products may be incorporated, such as oils, glycerin, emollients, emulsifiers, dispersing agent, in typical quantities for cosmetic compositions.

In the formulation of the dermocosmetic composition of the invention, vitamins, such as vitamin A, C or a B group vitamin may also be present, as wll as other cosmetically active substances such as lecithin, coenzyme Q, plant extracts, minerals.

In some embodiments, the composition of the invention may contain lipophilic substances or excipients, such as oils and butters, such as shea butter or coconut oil.

In the case of formulations in liquid form, water is present as a diluent or solvent, optionally mixed with other liquids used for the formulation of cosmetic compositions.

According to another embodiment, the cosmetic composition further comprises one or more amongst thickeners, solubilizers, preservatives, water, alcohols, glycerin, stabilizers, cosmetically acceptable antioxidants and antibacterials in quantities in line with those provided for common cosmetic formulations.

According to a further aspect of the invention, the cosmetic use of a composition is provided for topical application according to any one of the previously described embodiments for the treatment of wrinkles or corrugations and/or for maintaining mechanical properties and elasticity of the skin or making it brighter.

The invention methodology can be applied to different active molecules intended for dermocosmetic use, in order to make them similar in the penetration behavior in the epidermis and dermis, to those injected by the aesthetic doctor.

With the aim of testing the effectiveness of the system of the invention, the Applicant has carried out extensive researches aimed at verifying the epidermis penetration of cosmetically active substances or ingredients for dermocosmetic use, detecting separately the penetration percentage of such substances/active ingredients in the epidermis and/or dermis.

According to certain aspects, the Applicant has thus applied a method typically used in the pharmaceutical field, to assess and quantify the transcutaneous absorption, such as the Franz cells, to the dermocosmetic field, to check or quantify the epidermis penetration of the dermocosmetic substances contained in the composition of the invention.

In particular, by studying the conditions that promote transcellular penetration of cosmetic substances, the Applicant has also verified that the main factors that influence the transcutaneous absorption are the following:

1. Low/very low molecular weight, typically MW≤ 2000 dalton,

2. Concentration gradient, i.e. the difference of concentration of the active substances in the high concentration region with respect to the region with the lowest concentration. The increase of the concentration gradient increases the amount of active principle that can be transferred per unit time.

3. Characteristics of the carrier in which the cosmetic substance is dispersed, i.e. the characteristics of the cosmetic formulation necessary to achieve an adequate solubilisation in the lipid phase or in the aqueous phase. According to certain aspects, the present invention provides therefore a transdermal technology or method for the penetration of dermocosmetic substances through the skin, in particular through epidermis and superficial layers of the dermis, comprising:

-selecting cosmetically active substances with molecular weight≤ 2000 Daltons, -testing the cosmetically active substances with MW≤ 2000 Daltons with Franz diffusion cell and selecting the dermocosmetic active substances with an absorption gradient in the dermis lower than 20% in 24 hours;

-adding the selected cosmetically active substances with a penetration enhancing system preferably comprising decylene glycol, pentylene glycol and glycol caprylyl,

-adding a suitable carrier to the mixture of the penetration enhancing system and cosmetically acceptable substance for topical application.

Suitable carriers are those previously described in reference to any one of the embodiments of the composition of the invention.

According to certain embodiments, the transdermal technology, in accordance with an aspect of the invention, requires the following 5 features to increase the penetration of dermocosmetic active ingredients through the skin:

- Low molecular weight of the active principle, typically≤ 2000 Daltons - Concentration gradient

- Suitable carrier for fat-soluble or water-soluble substances

- Use of dermocosmetic active ingredients tested by Franz cell

- Use of a penetration enhancing system preferably comprising decylene glycol, pentylene glycol and caprylyl glycol.

The transdermal technology can be applied to any cosmetic forms, for example, emulsion, lotion, water-alcohol solution and to any area of the human body, such as the face, scalp, body.

The Applicant, in order to highlight the penetration capability of different substances for dermocosmetic use, has carried out experiments of transdermal penetration by the Franz Diffusion cell method, the results of which are reported in the following.

By this technology or method, the penetration of cosmetically active ingredients in measured amounts can be checked by designing dermocosmetic treatments specific as to their function and with the benefit brought to the skin very similar to the cosmetic medicine.

The present invention is described in detail in the following examples.

Example 1

The purpose of this study is to investigate the ability of different molecules with different biological and chemical nature, for example, amino acids, trace elements, nucleic acids and vitamins, to penetrate and be absorbed through the skin, by quantifying over 24 hours the quantities available in the epidermis and in the dermis.

The experimental system is represented by a Franz Diffusion cell, on which a suitably treated pig skin has been mounted; the anatomical portions of skin used in this test were: epidermis and epidermis + dermis.

in particular, penetration tests were carried through by Franz diffusion cells which, using cutaneous membranes composed of epidermis and dermis, allow the assessment of the amount of actives penetrated in the 30 minute, 8 hours, and 24 hours experimental times. The Franz diffusion cell is the ex vivo system suitable for evaluation of the degree of absorption of a substance through the skin.

The cells used for the experiment, thermostated thanks to a continuous flow at 37°C of propylene glycol, are characterized by two compartments, a donor, the upper one, and a receiver, the lower one, between which a portion of the skin is placed, and the whole is fixed with hooks in such a way that there can be no leakage of the solution during the test sequence.

The dermocosmetic substance to be tested is placed in the upper compartment, donor, in contact with the skin, while the receiving compartment is filled with a saline solution (32 g for each cell), maintaining stirring by means of a magnetic stir bar.

The receiving compartment is equipped with a sampling tube through which withdrawals of the receiving phase can be carried out at established experimental times, and with an external jacket connected to a thermostatic system. The whole assembly is housed in a special glass set on a magnetic plate allowing rotation of the magnetic stirring, keeping the cell properties.

0.2 ml of each solution prepared were applied on the tissues; the application surface was 4 cm ² ; at the end of each monitored experimental time (30 minutes, 8 hours, and 24 hours after application) the tissue was removed from the cell, homogenated and used for the analysis. Untreated tissues have been used as blanks for the evaluation of basal levels of each of the molecules in the study and the subsequent subtraction with respect to the processed experimental data.

The epidermis is obtained from whole skin by subjecting the tissue to a heat treatment with the only purpose to soften the tissue for better handling (60°C for 45 minutes) followed by chemical peeling with 80% lactic acid until it "frosting" occurs (appearance of white patches on the skin, a sign of the epidermis detaching from the dermis in the region of the dermo-epidermal membrane).

Epidermis + dermis are obtained by removal of the hypodermis by surgical instruments.

The methods used for the determination of substances being subject of the study are herein described.

Homogenates so prepared were appropriately treated with the swabs contained in the kit (QIAGEN) being used, so that DNA and RNA were obtained from biological matrices with a high level of purification from all proteins; nucleic acids were extracted from homogenated tissues by means of disposable chromatographic columns, in which the solid phase binds the DNA/RNA by affinity while the mobile phase washes it and eliminates any contaminant; then the DNA/RNA was eluted with the appropriate buffer and subsequently assayed.

Analysis for the trace elements have been carried out in accordance with the ICP-OES technique. The reading was carried out on the homogenates as such, after calibration of the instrument with a series of standard solutions containing the molecules to be determined.

The atomic emission spectroscopy (AES or OES: Optical emission spectroscopy) is an emission spectroscopic technique used in chemical analysis. It exploits the administration of relatively high energy, so as to cause dissociation into atoms and the excitation of the latter. Based on the emitted wavelength, it is possible to trace the unknown species, since the spectra are characteristic of each substance, while the quantitative analysis can also be performed by measuring the intensity of the emission.

The use of an inductively coupled plasma source (ICP) is characterized by high peculiar temperatures (6500-10000 K) of the plasma that help atomizing and ionizing or exciting almost all elements. Detection limits are very low, ranging from a few units of μg/L up to centesimal fractions of μg/L. The signal results to be stable and highly reproducible, with the ability to easily perform analysis of multicomponents having low chemical interferences.

For amino acids and vitamins homogenates were derivatized with fluorenylmethyloxycarbonyl chloride (FMOC-CI); the derivative so obtained was injected into HPLC, according to chromatographic modes specific for each amino acid.

High-performance liquid chromatography or high pressure liquid chromatography, more simply known by the English acronym HPLC, is a type of liquid chromatography that represents the instrumental evolution of liquid phase chromatography on classical column.

This is a chromatographic technique that allows separation of two or more compounds present in a solvent exploiting the affinity balance between a "stationary phase", placed inside the chromatographic column, and a "mobile phase" flowing through it. A substance more similar to the stationary phase compared to the mobile phase employs a longer time to traverse the chromatographic column (retention time), compared to a substance with low affinity for the stationary phase and high for the mobile phase. At the end of the column there is applied a detector (IR, UV-VIS, spectrofluorometric, mass spectrometer) and a computer that allow a continuous analysis of the output from the column, and then quantification and/or identification of the substances injected is possible through a proper chromatogram.

Specifically, the analytical determinations were carried out using:

• Chemical (HPLC) and spectrophotometric (Mass Spectrophotometer) analysis for amino acids, vitamins and trace elements,

• GPC - TDA analysis for nucleic acids.

More series of Franz cells were set up with five cells for each substance to be tested.

A series of five cells was set up for each experimental time (30 minutes, 8 hours, and 24 hours) because the determinations on portion of skin are destructive determinations of the sample, and therefore each determination prevented the continuation of the test and the evaluation of the molecule penetration in the next experimental time.

Therefore for each product being tested three series of Franz cells were performed, five cells each, relative to the 30 minutes, 8 hours, and 24 hours withdrawals.

The substances tested were as follows:

Calculation of results

Using the concentrations of each molecule obtained analytically in any experimental system, the amount of the individual amino acids, trace elements, vitamins and nucleic acids absorbed by tissue in monitored experimental times has been calculated expressed as a percentage of penetration compared to the applied quantity.

In order to subtract the amount of endogenous molecules of the tissues with respect to what made by the application of the products, each determination was performed against the respective blank: all the determinations on the content of molecules penetrated in the epidermis were performed using the homogenate of epidermis from non-treated skin as the blank. The obtained results are shown below. For each tested molecules the percentage of penetration in the epidermis, dermis and in the two structures altogether are reported.

The penetration kinetics recorded do not appear to follow common trends, therefore it can be assumed that each of the tested molecules penetrates and is distributed in the skin between epidermis and dermis following tissue-specific interactions, as well as according to the chemical-physical characteristics typical to each molecule.

Penetration data can be summarized by dividing the results into categories of substances:

- Nucleic acids have shown an overall penetration of 29%.

- Trace elements have shown an overall penetration in the range of 29.3% to 90.9%.

- Vitamins have shown an overall penetration in the range of 30.6% to 83.3%.

- Amino acids have shown an overall penetration in the range of 22.4% to 75.9%.

- Peptides have shown a maximum overall penetration of 80%. - The remaining substances have shown an increasing penetration of over 30%. The absence of traces of the molecules being studied in the receptor fluid is indicative of the fact that there is not a systemic absorption and that the substances applied to the epidermis do not go beyond the dermis, where they are metabolised.

EXAMPLE 2

Comparative tests of in vitro penetration or of cutaneous absorbance of cosmetically active substances added with a system according to the invention. The purpose of this study was to investigate the ability of the three selected glycols: Glycol Caprylyl, Decylene Glycol and Pentylene Glycol to modulate- increase penetration through the skin of a polypeptide molecule: Sh- Polypeptide 3, by quantifying over 24 hours the amounts available in the epidermis and/or in the dermis.

The test was carried out in two steps: in the first step glycols behaviors were analyzed individually; in the second step 3 different mixtures of glycols were analyzed in order to verify a possible synergism of action.

The experimental sample used was pig skin (obtained once the animal has been slaughtered for food purposes); the absorption system employed consists of the Franz Diffusion Cell.

Mixtures of Caprylyl Glycol + Sh-Polypeptide 3, Decylene Glycol + Sh-

Polypeptide 3 and Pentylene Glycol + Sh-Polypeptide 3 were initially prepared and applied on the surface of the tissue in the ratio 1:1.

In the second phase of the work, the following mixtures have been created and tested: - Pentylene Glycol 60%, Caprylyl Glycol 30%, Decilene Glycol 10%

- Pentylene Glycol 70%, Caprylyl Glycol 25%, Decilene Glycol 5%

- Pentylene Glycol 80%, Caprylyl Glycol 15%, Decilene Glycol 5%

applied to the surface of the tissue in a ratio 1:1 with SH-Polypeptide 3.

The experimental system is formed by a Franz Diffusion cell, on which the skin suitably treated is mounted; anatomical portions of skin being used in this test were: epidermis and epidermis + dermis (the hypodermis has been deleted as per OECD Test No. 428: Skin Absorption: In Vitro Method).

The Franz cell is an apparatus used in the skin permeation test. It consists of two chambers separated by a membrane. The substance, for which the permeation is to be measured, is applied to the membrane by means of the donor chamber; the receiving room instead contains a fluid in which the substance permeated through the membrane is dispersed and is then sampled for subsequent analysis. The receiving chamber is kept at a controlled

temperature (temperatures other than the ambient one are obtained by a flow of liquid in the jacket for temperature control).

0.4 ml of each mixture were applied on the tissues; the application surface was 4 cm ² ; at the end of the monitored experimental time (24 hours after

application) the tissue was removed from the cell, mechanically homogenated in a phosphate buffer and used for the analysis.

Untreated tissues have been used as blanks for the evaluation of basal levels of each of the molecules in the study and the subsequent subtraction with respect to the processed experimental data. After 24 hours of treatment, the solution inside the receiving chamber was analyzed for verification of the presence/absence of the molecules of interest. The treatment took place at room temperature and in duplicate.

The homogenates of the obtained tissues and the solution contained in the receiving chamber at the end of the test were analyzed, as to the dose of SH- Polypeptide 3, by a colorimetric technique.

COLORIMETRIC ASSAY

The analysis principles are based on the fact that each substance has a maximum characteristic absorption of electromagnetic radiation in certain wavelengths, and consists in the measure of the absorption of a radiation in the visible range. Starting from the Lambert-Beer law, which correlates absorption to the concentration of the substance through a constant, which is derived from the product of a characteristic quantity of the absorbing species for the length of the optical path, determination of the concentration of the substance in the test solution is therefore possible.

By using the concentrations of Sh-Polypetide 3 analytically obtained in each experimental system, the amount of penetrated molecule through the tissue has been calculated (epidermis and epidermis + dermis) expressed as a percentage of penetration compared to the amount applied. The percentage of penetration through the skin was calculated by subtraction with respect to what has been found in the epidermis + dermis and epidermis.

The solution inside the receiving chamber was analyzed for verification of the presence/absence of the molecules of interest. In order to subtract the amount of endogenous molecules in the tissues with respect to what the application of the products has produced, each

determination was performed against the respective blank: the determinations on the content of molecules penetrated into the epidermis were performed using the homogenate of untreated skin from epidermis as a blank, while the determinations on the content of molecules penetrated into the epidermis + dermis were carried out using the epidermis + dermis homogenate from non- treated skin as a blank.

The result was calculated as the average of the measurements.

Shown below are the results obtained. For each molecule being tested the percentage of penetration in the tissues (epidermis, dermis and the two structures altogether) are reported.

As can be seen, there is no substantial difference in the skin penetration between the active substance alone and the active substance associated individually with the three boosters.

Conversely, the association of the cosmetically active ingredient (cosmetic agent) with the mixture of 3 glycols with different composition results in a significant increase in the penetration rate, as shown in the following table:

The experimental data proves that a mixture of the three glycols acts synergistically, thereby significantly increasing the degree of penetration through the epidermis of the Sh-3 polypeptide molecule.

EXAMPLE 3 Penetration enhancing system for crossing one or more dermocosmetic substances through the cutaneous outer layers having the following formulation:

EXAMPLE 4

Composition in the form of a cream containing the system for increasing penetration: