WO2012168519A1

WO2012168519A1 - Mixture of multifunctional peptides or polypeptides or combinations thereof that are capable of forming a 2d or 3d matrix for care of the skin, mucosae, scalp and/or hair, and the use thereof in cosmetic or dermopharmaceutical compositions

Info

Publication number: WO2012168519A1
Application number: PCT/ES2012/070356
Authority: WO
Inventors: Luis Javier Cruz Ricondo; Carmen Gutierrez Reyes; Gerardo Alexis Acosta Crespo; Fernando Albericio Palomera
Original assignee: Infinitec Activos, S.L.; Peptidepharma Nova, S.L.
Priority date: 2011-06-09
Filing date: 2012-05-18
Publication date: 2012-12-13

Abstract

Mixture of compounds of individual multifunctional peptides and/or polypeptides and/or combinations thereof, which contain biological peptide units capable of organizing structurally and forming a 2D or 3D matrix within the skin and/or the follicle, offering the properties of regeneration, anti-wrinkle capability and/or hair-follicle stimulation and repair. Furthermore, the invention relates to the topical application thereof for caring for the skin and the hair and to the use thereof in cosmetic or dermopharmaceutical compositions, in which said composition may comprise peptides or polypeptides of this type, alone or in combination.

Description

MIXING OF MULTIFUNCTIONAL PEPTIDES OR POLYPEPTIDES OR COMBINATIONS OF THESE CAPABILITIES OF FORMING A 2D OR 3D MATRIX FOR SKIN CARE, MUCOSES, HAIR LEATHER AND / OR HAIR AND ITS USE IN COSMETIC OR DERMOPHARMACEUTICAL COMPOSITIONS

DESCRIPTION

Mixture of individual multifunctional peptide and / or polypeptide compounds and / or combinations thereof and containing biological peptide motifs, capable of structurally organizing and forming a 2D or 3D matrix within the skin and / or follicle providing regenerative, anti-wrinkle and / or stimulants and repair of the hair follicle. As well as its application topically for the care of the skin and hair and its use in cosmetic or dermopharmaceutical compositions, where said composition can comprise peptides or polypeptides of this type of nature alone or in combination.

Object of the invention

The present invention consists of individual multifunctional peptides and / or polypeptides and / or combinations thereof and containing biological peptide motifs, capable of structurally organizing and forming a 2D or 3D matrix within the skin giving physiological support for the regeneration and reconstruction of a new skin by sending signals to cells to activate the necessary cellular functions such as proliferation, differentiation and cell multiplication. As well as regulating all processes related to old age and hair care and maintenance. Likewise, said multifunctional peptides and / or polypeptides are applied in the treatment and care of hair. As well as its use in cosmetic and dermopharmaceutical compositions.

The 3 critical components for skin regeneration are i) full scaffolding with the appropriate initial biomechanical properties and the ability to sustain the complete regenerative process, ii) the appropriate molecular ligament for growth factors, functional factors, regulators and stimulants within the network can sustain cell differentiation, proliferation, and migration; iii) and finally, cells capable of responding to growth factors and all biomechanical stimuli induced by the 2D or 3D matrix. As the newly regenerated skin achieves rapid cellular maturity and vascular status, resident cells they must respond to biological and physical stimuli by remodeling the initial scaffolding into a functional and metabolic tissue that performs the function of the original skin.

The 2D or 3D extracellular matrix of the present invention retains the basement of the membrane structurally intact, collagen fibers intact, and elastin-rich microfibers; also the biomechanical components, as well as the biochemical components, such as small leucine-rich proteoglycans (decorin and biglican) necessary to sustain angiogenesis, cell migration and the global intrinsic regeneration process.

Background of the invention

Cosmetic products are those products whose purpose is to provide care to the skin and hair by improving their properties, appearance and functions, as well as providing beauty and well-being to our body, mainly for the face and neck. Likewise, in cosmetics in general, those destined for decorative cosmetics, body products or sun care are also included. Aging and other external factors can affect the functions and appearance of the skin, even altering its physiological, biochemical or histological mechanism, so that a large number of cosmetics and pharmaceuticals are used to avoid such effects.

The world of cosmetics, which includes the development of a wide range of skin products, and therefore, helps to improve human life, is beginning a new era with technological and scientific help. The skin as an organ in all its integrity, and the scalp and in a correct interaction with everything, with daily life, with stress, with the normal aging process, and hair loss and with the lesions derived from it genetically or biologically. Your deep understanding is vital for the definitive solution of the problem of old age, and hair loss and care. The understanding of all these intrinsic processes of repair and natural regeneration open a revolutionary path in this field of cosmetics and dermopharmaceuticals.

The objective of the present invention is the development of peptide assets that are capable of forming within the skin or the hair follicle a 2D or 3D matrix, combined with active fragments such as growth factors, adhesion molecules that simultaneously provide different signals to the cells for activation and stimulation. Mainly it acts on those processes that normally deteriorate and diminish over the years. Reactivate and Reversing these processes indefinitely is the goal of this innovative and revolutionary product.

The discovery and application in cosmetics and / or dermopharmaceuticals by topical administration of multifunctional peptides or polypeptides capable of forming within the skin or hair follicle an organized, planned and exact structure of peptides that form a 2D or 3D matrix and in conjunction with others active fragments Contributing to the sending of signals to the cells to activate the main cellular functions necessary in each case (skin and hair) such as proliferation, differentiation and cell multiplication. These processes contribute to the regeneration and reconstruction of a new skin and the growth of new hair. This active peptide or polypeptide at the molecular level penetrates the skin -dermis and epidermis-root of the hair follicle- and forms a 2D or 3D network, which as a regenerating matrix, completes the process of regeneration, replacement and reconstruction of the skin and the scalp It allows to support the different cells, mainly involved in the specific functions of each application. In this way the simultaneous production of all the important proteins in the skin is reactivated, such as the different types of collagen, elastin, proteoglycans, etc. And keratin and melanin in the case of the hair follicle.

Another innovation of the present invention is the stabilization of these active peptides and polypeptides in their monomeric form within cosmetic or dermopharmaceutical formulations and their penetration into the skin and / or hair follicle root with subsequent spontaneous formation of the polymer network (matrix 2D or 3D) for the anchoring of cells and generation of the different activation signals thereof.

The 2D or 3D matrix product of the invention not only maintains a physiological support for internal regeneration in the skin and / or root of the hair follicle but also for the creation of a biomimetic matrix with biological functions that induce superficial regeneration active

The 2D or 3D matrix product of the invention can form autonomous mono, di and three-dimensional networks and / or in combination with those existing in the skin under natural physiological conditions, in concentrations of the order of 0.0000001% up to 100% of the weight of the structure.

The 2D or 3D matrix product of the invention preserves the extracellular matrix and mainly induces fibroblasts to the production of elastin, proteoglycans, laminin, tenacin, and collagen type I, III, IV, VIII. It also preserves the vascular and communication channels of the dermis and epidermis. And in the case of the hair follicle it induces proliferation, regulation of dermal porridge and stem cells which are key in the generation of new hair.

The 2D or 3D matrix product of the invention allows rapid revascularization and cell repopulation and maintains tension and vigor.

The 2D or 3D matrix product of the invention helps the recovery of discontinuities in the skin such as wrinkles, fine lines, cracks, irregularities or roughness, increased pore size, loss of elasticity, loss of firmness, loss of firmness. smoothness, loss of the ability to recover from deformation, sagging of the skin such as sagging of the cheeks, the appearance of bags under the eyes or the appearance of double chin, among others, changes in skin color such as spots, redness, dark circles , bags under the eyes or appearance of hyperpigmented areas such as age spots or freckles among others, hair loss, loss of collagen structuring and other histological changes of the stratum corneum, dermis, epidermis, vascular system.

During the last decade, many examples of peptides with the ability to self-assemble to supramolecular molecules based on their amphiphilic properties as monomers have been published. ^{1, 2}

There are four main categories of peptides based on molecular bricks for the formation of nanostructures. ³ Among them we have i) amphiphilic peptides, ii) alkylated or acetylated peptides by large aliphatic chains, iii) phospholipopeptide conjugates and finally iv) peptide block based copolymers.

Amphiphilic peptides

Amphiphilic peptides are made up of amino acids only and are organized in successions of hydrophobic and hydrophilic domains. These peptide bricks comprising two different surfaces, one hydrophilic side and the other hydrophobic allowing a certain supramolecular organization. There are different types of designs according to the distribution of positive and negative charges in the peptide sequence. ⁴ These types of amphiphilic peptides have been used primarily in the preparation of hydrogels. ⁵

Aggeli and colleagues investigated the same spontaneous assembly in beta-leaf peptides in the fibers. ^{6, 7} They demonstrated the formation of polyelectrolyte beta sheet complexes that would occur when mixing aqueous solutions of cationic and anionic peptides, producing the same spontaneous self-assembly of fibrillar networks and the formation of hydrogels at an appropriate pH. ⁸ Well-defined assemblies of beta sheet structures have also been achieved at the air-water interface. Rapaport et al ⁹ investigated a family of amphiphilic peptides comprised of alternating hydrophobic and hydrophilic halves with the generic sequences of XY- (ZY) nX where the N and C-terminal (X) residues correspond to ammonium and carboxylate respectively, while Y and Z consist of alternating hydrophilic and hydrophobic amino acids.

Ghadiri's group studied the principles of design and preparation to achieve synthetic organic nanotubes, with special emphasis on the non-covalent process such as self assembly and self-organization. ^{10, 11} As an example, we have the beta sheet of tubular structure that was formed by stacking rings of the cyclic peptide. The cyclic peptide contains an alternate number of amino acids D and L, in which the amide bond is perpendicular to the plane of the ring.

Papapostolou and collaborators ¹² designed a second generation of self-assembling fibers, the system was based on two complementary leucine zippers. Peptides rented with long aliphatic chains

Stupp's laboratory has made an important contribution to the understanding of amphiphilic beta-leaf peptides in cylindrical or spherical micelles. ^{13, 14, 15, 16} This same group has demonstrated the use of amphiphilic peptide fibers in a wide range of applications.

The ^"wik et al ¹⁷ showed that certain sequences of peptides conjugated to C18 aliphatic chains of both the N-terminal or C resulted in control of their secondary structure incorporation into a liposome membrane.

Giralt et al ¹⁸ incorporated aliphatic chains in a proline-rich peptide sequence, from a family of peptides capable of penetrating at the cellular level. It was demonstrated by confocal microscopy and flow cytometry that when a tail of the myristyl group was coupled to the peptide, it significantly improved internalization in HeLa cells. On the contrary, this improvement was not observed in the case of a caproil (C6) -peptide conjugate Mono capable group studied with dioleoylphosphatidylcholine (DOPC) as a membrane model and electron transmission microscopy (TEM) demonstrates the importance of length aliphatic chain in the penetration of the cell membrane.

The alkylated aliphatic chain amphiphilic peptide has also been used for non-covalent functionalization of carbon nanotubes. Arnold et al. ¹⁹ showed that carbon nanotubes are insoluble in water, and when they were functionalized with amphitic peptides they were soluble in solutions. aqueous. In addition, the solubility of functionalized carbon nanotubes could be controlled by pH adjustment.

Phospholipid Conjugates to Peptides

Musiol et al. ²⁰ have demonstrated the preparation of semi-synthetic lipoproteins by lipidation of the C-terminal using Huisgen copper I catalysis in the 1,3-dipolar cycloaddition. ²¹ A 214-231 fragment of the human prion protein with a C-terminal proparglyglycine was reacted with a 1, 2-dimeristoyl-sn-glycerol-3-phosphaethanolamine (DMPE) azido-modified, mimicking the GPI anchor, to form a stable product of 1,4-disubstituted 1,3,3-triazole. The "click" reaction was used for the binding of the N-cysteinyl-lipopeptide and the fluorescent N-terminal dizzying. ^{22, 23} Incubation of HeLa cells with the micellar solution of the lipopeptide confirmed its rapid internalization of semi-synthetic lipoprotein, as visualized by confocal fluorescence microscopy.

Peptides based on copolymer blocks

The fourth class of amphiphilic peptides comprises block-based peptides of copolymers. ^{24, 25} Klok et al. ²⁶ synthesized a series of hybrids and triblocks of copolymers comprising sequences of amphiphilic peptides and polyethylene glycol. The copolymer blocks were synthesized by solid phase, obtaining segments of monodisperse peptides with a defined amino acid composition. The self-assembly properties of the new peptide sequence were retained despite binding with PEG molecules. An alternately superstructured order of PEG domains and peptide sequences with a high structural structure in beta sheet was observed. This work suggests that the combination of biological structural motifs with synthetic polymers can be a versatile strategy for the development of new self-assembling materials with complex interior structures and their potential to apply it to biology.

Smeenk et al. ²⁷ described the preparation and assembly of an ABA-type triblock copolymer consisting of a block of peptide of beta core plate composed of a repetitive sequence of [(AG) 3EG] n conjugated at the end with a block of PEG.

In the state of the art, documents are shown showing polypeptides alone or accompanied that allow their application with regenerative properties as can be seen in European Patent Publication No. EP 1419764 which refers to a composition adapted to a topical application on the skin, which comprises, in a physiologically acceptable medium (i) at least one peptide or a mixture of peptides (ii) at least one inhibitor of calcium channels, where it has been shown that said association allows to neutralize the formation of wrinkles of facial expression: this association neutralizes the effects of micro-tensions on the skin, Relaxing the contractile fibroblasts of the dermis that are supposed to be involved in the genesis of expression wrinkles and thus reduce expression wrinkles and prevent them from deepening, respecting the expression of the face. As the preferred peptide within the scope of the invention, the sequence acetyl hexapeptide-3) hexapeptide can be used .SEC ID No. 2;

Description of the figures

A group of figures that allow clarifying the concepts described are described below.

FIGURE 1. Study of the structures formed by the peptides of this invention. The scale of the bar in all the photos was 100 um. A, (amplification, 40 x); B, (amplification, 60 x); C, (amplification, 200 x); D, (amplification, 40 x); E, (amplification, 180 x); F, (amplification, 1900 x).

FIGURE 2. Study of cell amplification and multiplication using matrix forming peptides. The results were evaluated after 2 and 4 days of treatments with the matrix forming peptides.

FIGURE 3. Study by flow cytometry of the affinity of fibroblasts for matrix forming peptides. The results were evaluated after 2 and 4 days of treatments with the matrix forming peptides at different concentrations.

FIGURE 4. A) Collagen I expression in an untreated control of human dermal fibroblasts. B) Expression of collagen I in human dermal fibroblasts treated with a mixture of polypeptides of the invention C) Expression of collagen VI in an untreated control of human dermal fibroblasts. D) Expression of collagen VI in human dermal fibroblasts treated with the polypeptide mixture of the invention. FIGURE 5. Left: Untreated human dermal fibroblasts. Right: human dermal fibroblasts treated with a mixture of polypeptides of the invention

FIGURE 6. Induction of the production of type I collagen by the peptide matrix at different concentrations. Description of the invention

The present invention fully solves the problem of skin aging and problems related to hair loss and maintenance. Applicants of the present invention have discovered that multifunctional peptides or polypeptides (hybrid peptides or signal fragments) or combinations thereof capable of spontaneously forming and / or inducing a 2D or 3D matrix within the skin and / or hair follicle contributes to the improvement, renewal and care of the skin and / or hair follicle. It involves the formation of complex matrices and superstructures that support, anchor and activate the different types of cells involved in the production of different types of collagen, elastin, glycoproteins, etc. As well as this matrix is able to form in the root of the hair serving as a support, anchor and vascular system for the dermal papilla cells and the stem cells present there. Therefore all the multifunctional peptides and polypeptides that are capable of forming a 2D or 3D matrix and / or scaffold and in combination with peptides, activation signals, regulation of specific functions and that serve as support and stimulation of the cells within the skin and / or hair follicle, providing a simple, effective and risk-free solution for the treatment and / or care of the skin, mucous membranes and / or, different types of alopecia, which includes application on the skin, mucous membranes, and / or leather scalp by different types of administration.

In a first aspect, the invention relates to a multifunctional peptide or polypeptides or combinations thereof and with biological peptide motifs capable of forming a matrix, 2D or 3D, according to the following general formulas (1-9).

(1) R- (AAiAA ₂ AA ₃ ) nY

(2) R- (AA ₁ AA ₂ AA ₃ ) n- (BB ₁ BB ₂ BB ₃ ) nY

( ³ ) R- (CC CC ₂ CC3) n- (AAiAA ₂ AA ₃ ) nY

(4) R- (C Ci CC ₂ CC ₃ ) n- (AAi AA ₂ AA ₃ ) n- (BBi BB ₂ BB ₃ ) nY

R- (AA AA ₂ AA ₃ ) n- (BB ₁ BB ₂ BB ₃ ) nY

(9)

R- (CCi CC ₂ CC ₃ ) n- (AA- | AA ₂ AA ₃ ) nY

AA ^ AaAAs- refers to peptides or polypeptides capable of forming a 2D and 3D matrix within the skin and hair follicle

BBí BBaBBs- refers to peptides or polypeptides with active biological motifs capable of producing or inducing any signal or activation of interest to the skin and hair follicle, which also includes antibiotics, antimicrobials, anti-inflammatories, growth factor fragments, stimulators of Healing, cytokine fragments. CC1CC2CC ₃ - refers to another peptide that simultaneously with BB ^ BaBBs is capable of inducing or inhibiting any important activity within the skin and hair follicle, also includes fibronectin, vitronectin, polylysine, laminins, RGD fragment, polypeptides derived from the extracellular matrix.

And it can be: -H, -OH, -NH ₂ , -OR1, -NR -SR1 _, methyl, ethyl, hexyl, dodecyl, hexadecyl, PEG.

Where R1 is independently selected from the group consisting of H, substituted or unsubstituted aliphatic group, substituted alicyclyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted araiquyl.

R is selected from the group consisting of H, acetyl, tert-butanoyl, hexanoyl, hexanoyl, 2-methylhexanoyl, cyclohexanecarboxyl, can be: aliphatic compounds or not, such as octanoic acid butyric acid, decanoic acid, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, saturated or unsaturated C2-C4 alkenyls, C2-C24 substituted alkynyl, aryl or araiquyl groups

n is a natural number in 1, 2, 3, ... 100

Another aspect of the present invention is that these peptides or polypeptides may have a structure, either branched, dendrimeric, branched by the side chains, in combination with other polymers or proteins. Another aspect of the present invention is directed to a cosmetic or dermopharmaceutical composition comprising an effective cosmetic or dermopharmaceutical amount of at least one peptide of general formula (1), its stereoisomers, mixtures thereof or its acceptable cosmetic or dermopharmaceutical salts and the less a cosmetic or dermopharmaceutical excipient or adjuvant for the treatment and / or care of the skin, mucous membranes, scalp and treatments for alopecia.

After much research, the results obtained allow us to quantitatively determine a range of the ideal proportions that the composition must have.

- The composition of the invention is composed of a percentage of the mixture of: -peptides between 0 and 100%, preferably between 0.0000001% and 20% and more preferably at a concentration between 0.0001% and 5% in weight, with respect to the total weight of the composition.

- Preservatives between 0 and 5%

- Various assets 0 to 20% including vitamins, plant extracts and sunscreens - Alcohols, glycols, rheology modifiers, emollients, emulsifiers, silicones, surfactants, film-forming agents and various polymers between 0 and 20%

- Perfumes and essential oils 0 to 2%

- Additives including antioxidants, chelants and salts between 0 and 5%

Detailed description of the invention

The peptides of the present invention are multifunctional peptides or polypeptides (peptide hybrids), which have an important ability to spontaneously and / or induce a 2D or 3D matrix and their combination with peptides or signal fragments within the skin and / or hair follicle and therefore they are useful for the treatment of skin, mucous membranes and scalp.

Polypeptides and peptides are terms used interchangeably herein to designate a linear series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The polypeptide and the peptide may be a synthetic peptide or polypeptide, a recombinant peptide or recombinant polypeptide, or a peptide or polypeptide derived from enzymatic segmentation of the naturally occurring full-length protein of natural origin.

Synthetic peptide refers to a chain of chemically produced amino acid residues and linked together by peptide bonds, free of naturally occurring proteins and fragments thereof.

Multifunctional peptides or polypeptides (peptide hybrids) refers to the combination of different peptides with the same and / or different activity and / or function. These peptides can be arranged in different forms: linear, branched, crosslinked

The following specific examples provided herein serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention.

EXAMPLES OF REALIZATION

Example 1.

Methodology. Synthesis and characterization of the peptides of this invention

The peptides were synthesized using the solid phase methodology and the Fmoc / t-Bu strategy. As coupling agents for the synthesis, different combinations such as DIC / HOBt, TBTU / HOAt or HATU / HOAt were used depending on the chemical complexity of the corresponding amino acid. The elimination of the Fmoc group was performed with 20% piperidine in DMF. After synthesized, the Peptides were separated from the resin and purified by RP-HPLC. Finally, the peptides were characterized by mass spectrometry and amino acid analysis.

The peptides were characterized by RP-HPLC in a Water 996 photodiode detector series instrument. This instrument is equipped with a Water 2695 modular separator and a Millenium program. The reverse phase column that was used was C18 (C18 symmetry inverse phase HPLC columns, 4.6 x 150 mm, 5 μηι) (Waters, Ireland). The peptides were detected at 220 nm, and a linear gradient of 5 to 100% CH3CN (+ 0.036% TFA) and H20 (+ 0.045% TFA) of 8 min was used at a flow rate of 1.0 mL / min. The peptides were analyzed by MALDI-TOF and ES (+) - MS in an Applied Biosystems Voyager DE RP, using a matrix of 2.5 dihydroxybenzoic acid (DHB), and a Micromass VG-quattro spectrometer.

Example 2

The morphology of the matrix forming peptides was studied by scanning electron microscopy. The matrix forming peptides were adhered to a thin glass plate. This plate was then placed on a metal plate with a double conductive tape and the peptides were coated with gold nanoparticles for 120 seconds in a vacuum system. The NIH ImageJ software was used to process the photos obtained.

Results

The images above show the self-assembly and structuring of the peptides of this invention, which allow to support and biological signals to the cells. And create an extracellular network and interactive communication.

Example 3

Cell amplification and multiplication using matrix forming peptides

HeLa cells were cultured in DMEM with low glucose content, containing 10% fetal bovine serum, 5% ultraglutamine, 2.5% penicillin, and 2.5% streptomycin at 37 ° C in a C0 ₂ controlled atmosphere incubator. 96-well plates (Nalge Nunc) were previously coated with matrix-forming peptides and wells without peptides were used as experimental control. HeLa cells were seeded at 1 x 10 ³ cells / cm ² in 96-well plates and cultured for 2 and 4 days. The final percentage of cell proliferation was determined with respect to the initial value of the same treated and not treated with the peptides. Results

This experiment demonstrates the population increase in the number of cells that is favored with the presence of the matrix forming peptides of the present invention on the plates tested. Also, the cell population was significantly increased as the days go by.

Example 4

Affinity test for fibroblasts using matrix forming peptides

Human dermal fibroblasts were used to study affinity for matrix forming peptides. Thus the binding of the peptides to the fibroblast cells was studied at different concentrations (5 ug / mL, 10 ug / mL, 20 ug / mL and 40 ug / mL respectively) of the fluorescein-labeled peptide matrix for 1 h at room temperature in culture medium. Then, the human dermal fibroblasts treated with peptides and untreated were washed several times in type V plates at 1500 rpm for 2 minutes to remove peptides that did not bind to the cells. Next, the treated and untreated cells were analyzed by flow cytometry.

Results

This study demonstrates the affinity of matrix forming peptides for fibroblasts. It also demonstrates a dose-dependent behavior, as the amount of peptides increases the cellular affinity for the peptide matrix increases.

Example 5

Study of the formation of collagen I and VI by fluorescence microscopy. Human dermal fibroblasts were seeded at low confluence on glassware. 24 hours after seeding, they were treated with a concentration of 1.0% matrix forming peptides of the 100ppm solution.

The samples were incubated with the corresponding antibodies against collagen I and VI

Subsequently, the untreated cell samples were analyzed and compared with the treated cells.

The equipment used was a Confocal Spectral Microscope FV1000 Olympus. Results

As can be seen in the previous images, after treatment with the matrix-forming peptides of the present invention, there is a greater intensity of fluorescence because the signal of collagen I and VI is higher.

Example 6

Fibroblast binding studies by scanning electron microscopy

Human dermal fibroblasts were seeded at low confluence on glassware. 24 hours after sowing, they were treated with a product concentration of 0.3% of the 100ppm solution.

Samples were fixed 24 hours after treatment with glutaraldehyde and successive additions of ethanol at different proportions in water.

The equipment used was Scanning Electron Microscope. It is of the Field Emission Filament type. NOVA NANOSEM2 model.

Outcome

In the SEM image on the right we can see an increase in the filaments that the cell uses to anchor, as well as an increase in membrane proteins.

Example 7

Determination of collagen biosynthesis by matrix forming peptides.

Human dermal fibroblasts (FDH) were seeded in 96-well plates (3000 cells per well), and grown in DMEM containing 5% fetal bovine serum (FBS) for 24 hours. Then, the medium was replaced by fresh medium containing 0.1% FBS and the corresponding treatment to determine collagen production. After incubation with the matrix-forming peptides for 72 hours, equal amounts of supernatant were added to a plate coated with human type I collagen antibody. The plate was incubated at room temperature for 2 hours to allow the antibody reaction and the collagen produced. Then, the plate was washed three times with PBS containing 0.5% Tween 20 to remove the remaining sample that was not bound. Next, a human antibody that recognizes biotin-labeled type I collagen was added for 1 hour. After washing the plate with PBS containing 0.5% Tween 20 to eliminate nonspecific interactions, streptavidin peroxidase was added "horseradish" (HRP) (St Louis, Sigma). The amount of collagen in each well was determined using tetramethylbenzidine (St Louis, Sigma) as a substrate for HRP. The reaction between HRP and TMB was stopped by the addition of 1 N HCI, and the OD was measured at 450 nm. Untreated cells represented the negative control of the experiment. The results have been analyzed using a single dose of treatment of matrix forming peptides.

Results

As seen in Figure 4, matrix forming peptides were able to induce the activation of type I collagen.

Example 8

Study of the cytotoxic effect of matrix forming peptides on different cell lines

Cell lines tested: 1064SK (CRL-2076), human cell line; skin; morphology: fibroblasts; normal Hs68 (CRL-1635), human cell line; skin; Morphology: fibroblasts, HeLa (CCL-2), human cell line; Morphology: epithelial.

Results

Table 1.

As shown in Table 1, matrix forming peptides are not toxic. They begin to be toxic around 850 μΜ. Therefore, these results mean that matrix forming peptides offer a wide window for cosmetic or dermopharmaceutical application.

The compositions of the invention are capable of being incorporated into a vehicle or an acceptable cosmetic or pharmaceutical sustained release system selected from the group consisting of liposomes, millicapsules, microcapsules, nanocapsules, sponges, vesicles, micelles, microspheres, microspheres, nanospheres, lipospheres, microemulsions, nanoemulsions, miliparticles, my ero particles, nanoparticles and solid lipid nanoparticles, are also incorporated into any protein hydrolyzate whether they are of vegetable, synthetic, natural or animal origin.

The compositions can also be found adsorbed on a solid organic polymer or cosmetically or pharmaceutically acceptable solid support. selected from the group consisting of talc, bentonite, silica, starch and maltodextrin, and dextran, and their derivatives

The form of presentation is selected from the group consisting of creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balms, foams, lotions, gels, hydroalcolic solutions, liniments, serums, soaps, shampoos, ointments, mousses, ointments , powders, bars, pencils, vaporizers, aerosols, capsules, gelatin capsules, tablets, sugar-coated tablets, powders, granulated forms, chewing gums, solutions, suspensions, emulsions, syrups, jellies and gelatin.

Other ways in which the product can be incorporated is in a woven material, a non-woven fabric or a sanitary product which is selected from the group consisting of bandages, gauze, shirts, socks, socks, underwear, girdles, gloves, diapers, compresses, dressings, bedspreads, wipes, hydrogels, adhesive patches, non-adhesive patches and facial masks.

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Claims

1- Mixture of multifunctional peptides or polypeptides or combinations of these capable of forming a 2D or 3D matrix, for skin care providing regenerative properties such as stimulating and repairing anti-wrinkles of the hair follicle that is characterized by the fact that the peptides or polypeptides are selected within from the group that contains the general formulas:

(1) R- (AA ₁ AA ₂ AA ₃ ) nY

(2) R- (AA ₁ AA ₂ AA ₃ ) n- (BB ₁ BB ₂ BB ₃ ) nY

( ³ ) R- (CC ₁ CC ₂ CC3) n- (AA ₁ AA ₂ AA ₃ ) nY

(4) R- (CC! CC ₂ CC ₃ ) n- (AA! AA ₂ AA ₃ ) n- (BB, BB ₂ BB ₃

R- (AAiAA ₂ AA3) n- (BBiBB ₂ BB ₃ ) nY

(9)

R-ÍCCiCCzCCsJn-ÍAAiAAzAAsJn-Y

Where: AAiAAaAAs- refers to peptides or polypeptides capable of forming a 2D and 3D matrix within the skin and hair follicle,

BBí BBaBBs- refers to peptides or polypeptides with active biological motifs capable of producing or inducing any signal or activation of interest to the skin and the hair follicle, they can also be antibiotics, antimicrobial, anti-inflammatory, growth factor fragments, Healing stimulators, cytokine fragments.

CC1CC2CC ₃ - refers to another peptide that simultaneously with BB ^ BaBBs is capable of inducing or inhibiting any important activity within the skin and hair follicle, 10 may also be fibronectin fragments, vitronectin, polylysine, laminins, RGD fragment, polypeptides derived from the extracellular matrix.

Where R 1 is independently selected from the group consisting of H, substituted or unsubstituted aliphatic group, substituted alicyclyl, substituted or unsubstituted heterocycle, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted araiquyl.

R is selected from the group consisting of H, acetyl, tert-butanoyl, hexanoyl, hexanoyl, ^ 2-methylhexanoyl, cyclohexanecarboxyl, can be: aliphatic compounds or not, such as octanoic acid butyric acid, decanoic acid, oleic acid, lauric acid , capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, C2-C4 saturated or unsaturated alkenyls, C2-C24 substituted alkynyl, aryl or araiquyl groups.

n = a number between 1, 2, 3, ... up to 100

25 2- Mixture of multifunctional peptides or polypeptides or combinations thereof capable of forming a 2D or 3D matrix, for skin care by providing regenerative properties as anti-wrinkles stimulating and repairing the hair follicle according to claim 1, characterized in that the mixture It may contain more than one peptide or polypeptide.

30 3- A cosmetic or dermopharmaceutical composition for skin and / or hair care, where said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination, characterized by being constituted by;

-Peptides or polypeptides between 0 and 100%

35 - Preservatives between 0 and 5% - Various assets 0 to 20% including vitamins, plant extracts and sunscreens

- Alcohols, glycols, rheology modifiers, emollients, emulsifiers, silicones, surfactants, film-forming agents and various polymers between 0 and 20%

- Perfumes and essential oils 0 to 2

- Additives including antioxidants, chelants and salts between 0 and 5%

4- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim 3, characterized in that the peptides or polypeptides They are selected from the group formed by the general formulas:

(1) R- (AAiAA ₂ AA ₃ ) nY

(2) R- (AA ₁ AA ₂ AA ₃ ) n- (BB ₁ BB ₂ BB ₃ ) nY

(3) R- (CC CC ₂ CC3) n- (AAiAA ₂ AA3) nY

(4) R- (C Ci CC ₂ CC ₃ ) n- (AAi AA ₂ AA ₃ ) n- (BBi BB ₂ BB ₃ ) nY

R- (CCi CC ₂ CC ₃ ) n- (AA- | AA ₂ AA ₃ ) nY

Where:

AA ^ AaAAs- refers to peptides or polypeptides capable of forming a 2D and 3D 3D matrix within the skin and hair follicle,

BBí BBaBBs- refers to peptides or polypeptides with active biological motifs capable of producing or inducing any signal or activation of interest to the skin and hair follicle, they can also be antibiotics, antimicrobials, anti-inflammatories, growth factor fragments, stimulators of healing, cytokine fragments 10.

CC1CC2CC ₃ - refers to another peptide that simultaneously with BB ^ BaBBs is able to induce or inhibit any important activity within the skin and hair follicle, they can also be fibronectin fragments, vitronectin, polylysine, laminins, RGD fragment, polypeptides extracellular matrix derivatives.

R is selected from the group consisting of H, acetyl, tert-butanoyl, hexanoyl, hexanoyl, 2-methylhexanoyl, cyclohexanecarboxyl, can be: aliphatic compounds or not, such as octanoic acid butyric acid, decanoic acid, oleic acid, lauric acid, ^ capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, C2-C4 saturated or unsaturated alkenyls, C2-C24 substituted alkynyl, aryl or araiquyl groups. n = a number between 1, 2, 3, ... up to 100.

5- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim 3, characterized in that the general formula peptide or polypeptides It is preferably at a concentration between 0.0000001% and 20% by weight, based on the total weight of the composition.

6- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim 3, characterized in that the peptide or polypeptides of formula In general, it is preferably at a concentration between 0.0001% and 5% by weight, based on the total weight of the composition.

7- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim 3, characterized in that they are incorporated into a vehicle or an acceptable cosmetic or pharmaceutical sustained release system selected from the group consisting of liposomes, millicapsules, microcapsules, nanocapsules, sponges, vesicles, micelles, microspheres, microspheres, nanospheres, lipospheres, microemulsions, nanoemulsions, miliparticles, microparticles, nanoparticles and solid nanoparticles lipid

8- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim 3, characterized in that they are adsorbed on a solid organic polymer or cosmetically or pharmaceutically acceptable solid support selected from the group consisting of talc, bentonite, silica, starch and maltodextrin, and dextran, and their derivatives.

9- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim 3, characterized in that they are incorporated in any hydrolyzate of proteins whether of plant, synthetic, natural or animal origin. 10- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim characterized in that its presentation form is select from the group consisting of creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, lotions, gels, hydroalcolic solutions, liniments, serums, soaps, shampoos, ointments, mousses, ointments, powders, bars, pencils , vaporizers, aerosols, capsules, gelatin capsules, tablets, sugar-coated tablets, powders, granulated forms, chewing gums, solutions, suspensions, emulsions, syrups, jellies and gelatin.

1 1- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim 3 characterized in that they are incorporated in a tissue, a non-woven fabric or a medical device.

12- A cosmetic or dermopharmaceutical composition for skin and / or hair care, wherein said composition comprises peptides or polypeptides capable of forming a 2D or 3D matrix alone or in combination according to claim according to previous claim characterized in that the tissue, tissue-not - Fabric or sanitary product is selected from the group consisting of bandages, gauze, t-shirts, socks, socks, underwear, girdles, gloves, diapers, pads, apositos, bedspreads, wipes, hydrogels, adhesive patches, non-adhesive patches and facial masks.