BR102022016835A2 - BIOMIMETIC PEPTIDES DERIVED FROM THYMOSIN-B4 FACTOR FUNCTIONALIZED WITH 5-HT AND 5-HTP*, DERMOCOSMETIC AND BIOPHARMACEUTICAL COMPOSITIONS CONTAINING THE PEPTIDES AND THEIR USES - Google Patents

Abstract

biomimetic peptides derived from thymosin-b4 factor functionalized with 5-ht and 5-htp*, dermocosmetic and bio-pharmaceutical compositions containing the peptides and their uses. The present invention involves biomimetic peptides derived from the multifunctional factor thymosin-b4 (tb4) functionalized with serotonin (5-ht- or 5-hydroxytryptamine) and/or its biological precursor 5-hydroxytryptophan (5-htp*). more particularly, the present invention refers to 5-ht-peptide and 5-htp*-peptide molecules that have in their binding sites a precise molecular configuration to give them high performance/efficacy in healing processes, tissue regeneration , cell renewal, and specifically in the process of angiogenesis (formation of new vessels from pre-existing vessels). Included as embodiments of the present invention are dermocosmetic and bio-pharmaceutical compositions containing these peptides and which exhibit the mentioned effects.

Description

[01] The present invention concerns biomimetic peptides derived from the multifunctional factor thymosin-B4 (TB4) functionalized with serotonin (5-HT- or 5-hydroxytryptamine) and/or its biological precursor 5-hydroxytryptophan (5- HTP*) . More particularly, the present invention refers to 5-HT-peptide and 5-HTP*-peptide molecules that have in their binding sites a precise molecular configuration to give them high performance/efficacy in healing processes, tissue regeneration , cell renewal, and specifically in the process of angiogenesis (formation of new vessels from pre-existing vessels). Included as embodiments of the present invention are dermocosmetic and bio-pharmaceutical compositions containing these peptides and which exhibit the mentioned effects.

[02] With the advancement of research in recent decades on the biology and function of platelets, several facets of these anucleated cells have been highlighted that indicate they have great biotechnological potential. As highly responsive, secretory cells, platelets can be explored to develop solutions that alter tissue injury microenvironments, through synthetic materials mirroring their content (platelet), and thus used as “clues” / extracellular signals instructive for morphogenesis and as consequence for the healing and tissue regeneration processes [1-3]. In this context, the biomimetic platform of platelet content emerges, the Biotech-educated platelets detailed and published in scientific articles, with platelet content being our “natural factory of growth factors” [2,4]. With this data, peptides with platelet signatures, bio-similar/biomimetic peptides from platelet content were designed through “high throughput screening” platforms, to produce synthetic biomimetic solutions for the dermocosmetics industry. It is known that platelet growth factors (emphatically in the plural) are a vast collection of compounds of high biological value, biomimetic models, and allow the convergence between biological systems (naturally occurring) and synthetic chemistry to create (in silico ) a new generation of biomimetic peptides, PlateSimilis, a library of peptides and other artificial signaling biomolecules capable of performing their known biomimetic functions or even new functions [(1)van der Meijden, P.E.J.; Heemskerk, J.W.M. Platelet biology and functions: new concepts and clinical perspectives. Nat. Rev. Cardiology. 2019, 16, 166179. (2) Andrade, S.S.; Faria, A.S.; Queluz, D.P.; Ferreira-Halder, C. Platelets as a ‘natural factory’ for growth factor production that sustains normal (and pathological) cell biology, Biological Chemistry 2020, 401, 471-476. (3) Faria, A.V.S.; Andrade, S.S.; Reijm, A.N.; Spaander, M. C. W.; de Maat, M.P.M.; Peppelenbosch, M.P.; Ferreira-Halder, C.V.; Fuhler, G. M. Targeting Tyrosine Phosphatases by 3-Bromopyruvate Overcomes Hyperactivation of Platelets from Gastrointestinal Cancer Patients. J. Clin. Med. 2019, 8, 936. (4) Andrade, S.A.; Faria, A.V.S.; Fuhler, G. M.; Peppelenbosch, M.P.; Ferreira-Halder, C.V. Biotech-educated Platelets: beyond tissue regeneration 2.0. in press. 2020].

[03] More specifically, in the present invention, with the use of the Biotech-educated platelets platform (platelet-dependent agonist) it was possible to identify platelet signatures for the healing and tissue regeneration processes such as i) biogenic amine serotonin (5-HT) , which is one of the strategic healing substances released from dense platelet granules, and ii) multifunctional polypeptide thymosin-B4 (TB4) which is released from alpha granules.

[04] 5-HT (also known as 5-hydroxytryptamine) is produced from its biological precursor 5-hydroxytryptophan (5-HTP) by decarboxylation. The 5-HT produced is stored in platelet granules and is released by platelets in the event of intense tissue injury, especially in tissues with high regenerative power, such as the liver. It acts as a potent mitogen and modulates tissue remodeling [5-7]. Serotonin (5-HT) can also increase vascular permeability, induce dilation of capillaries, and cause contraction of nonvascular smooth muscle. Blocking its receptors with 5-HT2A and 2B receptor antagonists has been shown to inhibit liver regeneration [5]. These results suggest that platelet-derived serotonin is involved in the initiation of liver regeneration [(5)Lesurtel M, Graf R, Aleil B, Walther DJ, Tian Y, Jochum W, Gachet C, Bader M, Clavien PA. Platelet-derived serotonin mediates liver regeneration. Science. 2006 Apr 7;312(5770):104-7. doi: 10.1126/science.1123842. (6) T. Vitalis, J. G. Parnavelas, Dev. Neurosci. 25, 245 (2003). (7) M. Matsuda et al., Dev. Cell 6, 193 (2004)].

[05] The TB-4 factor, on the other hand, is a polypeptide that acts in the differentiation and acceleration of the migration of endothelial cells, with direct action in angiogenesis (the process of producing new vessels from pre-existing vessels, vascularization) [2- 7]. Thymosin-beta 4 (TB4) acts as a chemotactic, antimicrobial and regulatory agent of the hematopoietic system, increasing the rate of renewal, adhesion and dissemination of endothelial, epithelial and mesenchymal cells in the skin, which reinforces the healing and cell regeneration process, increasing the support of the skin (cutaneous tissue) by stimulating the synthesis of components of the extracellular matrix, with a homeostatic functional plus [8-10] [ (8) Faria, A.V.S.; Andrade, S.S.; Reijm, A.N.; Spaander, M. C. W.; de Maat, M.P.M.; Peppelenbosch, M.P.; Ferreira-Halder, C.V.; Fuhler, G. M. Platelets in aging and cancer double-edged sword. Cancer and Metastasis Reviews, 2020, v. 1, p.1205-1221. (9) Sorgdrager Freek J. H.; Naudé Petrus J. W.; Kema Ido P.; Nollen Ellen A.; Deyn Peter P. De.Tryptophan Metabolism in Inflammaging: From Biomarker to Therapeutic Target Frontiers in Immunology. 2019, 10, 2565. (10) Mannherz H. G.; Hannappel E. The ß-thymosins: intracellular and extracellular activities of a versatile actin binding protein family, Cell. Motil. Cytoskel. 2009, 66, 839-851].

[06] Keeping the context in organs with great regenerative potential, the skin has in its cutaneous structure (this includes its deepest layers) a very interesting model system for experimental studies of regenerative processes, this is due to its incredible capacity for regeneration and its ease and accessibility in obtaining fundamental data/results from processes related to regenerative biology. Discoveries from such skin research have contributed to the elucidation of new fundamental principles in regenerative biology. Highlighting its function as an external barrier, the skin is one of the most challenging organs in our body, due to a series of external stress factors that it is capable of withstanding, resulting in frequent damage to the cells and structures of the skin barrier. Thus, the skin has developed a set of complex natural mechanisms to protect itself and restore its integrity when injured, without resulting in septicemia. Under normal physiological conditions, restoration of a functional epidermal barrier is highly efficient, while repair of the deeper dermal layer is lesser and can result in scar formation with substantial loss of original tissue structure and function when the normal repair response does not work [11]. [ (11) Gould, J. Superpowered skin. Nature 563, S84-S85 (2018). doi: https://doi.org/10.1038/d41586-018- 07429-3 ] .

[07] There is, therefore, a technical need to identify new agents capable of triggering the skin regeneration process, not only for experimental purposes, but for dermocosmetic and therapeutic purposes in order to rescue the health and vitality of the damaged skin. for any loss, damage or compromise to the skin, such as scars, burns or wounds, but also to prevent and reverse signs of the aging process. In this sense, the present invention provides a solution to prevent, delay or reverse the signs of the skin aging process by promoting the recruitment of stem cells, with the use of peptide agents derived from the TB-4 factor functionalized with 5-HT. and/or its precursor 5-HTP*. More specifically, the invention acts to balance natural or induced senescence in favor of tissue or skin replacement, with access to the stem cell reservoir depending on the chronology of the skin analyzed.

[08] In searches carried out in patent banks, documents were found that describe the use of Thymosin-B4 in compositions for inhibiting or reversing skin aging and tissue regeneration, such as document EP2311485A1 presents compositions of Thymosin B4 for wound repair comprising thymosin beta 4 (TB4) having the sequence SDKP DMAEI EKFDK SKLKK TETQE KNPLP SKETI EQEKQ AGES (SEQ ID NO:2), an isoform of said TB4, an isoform of TB4 comprising LKKTET (SEQ ID NO:1) ; Document US2013149397A1 presents methods to prevent, improve or reduce dermatological signs of aging that employ active agents, in addition to a retinoid, that stimulate the expression of thymosin beta - 4 in the skin. Also provided are methods for screening substances that stimulate thymosin beta-4 expression levels and methods for using active agents identified by the screening protocol in skin treatment; Document CA2426200A1 describes the inhibition or reversal of skin aging by actin-sequestering peptides. Aging-associated skin degradation is inhibited or reversed by administration of an actin-sequestering peptide such as thymosin beta-4, an isoform of thymosin beta-4, or oxidized thymosin beta-4 alone or administered in conjunction with at least one agent which assists or stimulates the reduction of skin aging, wherein said agent is selected from the group consisting of vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-B), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 ( IL-1), prothymosin alpha and thymosin alpha 1; Document KR102079067 describes a cosmetic skin care composition comprising T4 fusion protein.

[09] However, no documents were found in the prior art that report the use of Thymosin-B4 with 5-HT and/or its precursor 5-HTP* in skin tissue regeneration, renewal and healing. As mentioned above, the effect of seretonin on liver regeneration is known, but this monoamine is little explored in relation to the skin, in addition, its release in this organ is not the same as that in the liver.

[010] Therefore, considering that Serotonin (5-HT) (and/or its precursor 5-HTP*) and Thymosin-B4 are molecules released from different platelet compartments upon stimulation of different agonists, in situations different times and at different times, added to the fact that the use of serotonin and/or its precursor in skin tissue regeneration and healing is little explored, the great advantage of the present invention is the molecular junction of this monoamine and/or its precursor (5- HTP*) with the Thymosin-B4 protein or its peptide derivatives, through a non-covalent cross-link reaction, originating new peptides with high potential for dermocosmetic and biopharmaceutical use.

[011] Therefore, this invention demonstrated for the first time peptide sequences derived from TB-4 functionalized with a non-covalent cross-link with 5-HT and/or 5-HTP (its biological precursor), presenting direct or indirect renewal effects cellular, promoting the healing process and tissue regeneration, proving to be a potent active ingredient for dermocosmetic formulations, as they are biomimetic (synthetic) to platelet factors and molecules that act in tissue regeneration and wound healing.

Brief Description of Figures

[012] Figure 1. Map of interactions between 50 intra-platelet proteins (black dots), to start the Biotech-educated platelets platform. Complex interactions of central multifunctional proteins in multiple physiological processes: in blue. Border color proportional to MiST score; edge thickness proportional to spectral counts. Physical interactions between platelet proteins (thin black lines) were selected from CORUM, IntAct and Reactome.

[013] Figure 2. HFF-1 fibroblast growth/proliferation assay by cell counting in the presence of the sequences in Table 1. Fibroblasts (HFF-1) were treated with the sequences highlighted in Table 1. Growth/proliferation was monitored by cell count for up to 50 hours of cultivation, (triplicate assay) *p<0.001.

[014] Figure 3. Analysis of cell viability by MTT reduction, cells exposed to PlateSimilis 1 and 2. Human skin keratinocytes (HaCat) highlighted in the figure were treated with PlateSimilis 1 and 2 1% w/v (A and B respectively ). Treatment of cells with PlateSimilis 1 and 2 did not alter cellular metabolism, the cells remained viable and with active metabolism, (triplicate assay) *p<0.001.

[015] Figures 4. Analysis of Healing by the Cell Migration Process, HaCat lineage analyzed in 24h and 48h under PlateSimilis 1 treatment. The “wound healing” assays by “scratch” in HaCat culture (human skin keratinocytes, with greater than 90% confluency) were performed in the presence (A-C) or absence of (D-F) PlateSimilis 1 (1%). We observed a wound reduction/increase in healing of 50% in 24 h of treatment with PlateSimilis 1. Thus, as we observed in (C) a healing rate of 80 ± 1.2. (D-F) Control with SFB (fetal bovine serum). Representative images are shown at 10X magnification. The migration area was quantified using ImageJ software. (G) Representative graph of wound quantification versus time (24 and 48 h), and SFB control. ANOVA GraphPad statistics. Representative images from three independent experiments.

[016] Figures 5. Analysis of Healing by the Cell Migration Process, HaCat lineage analyzed in 24h and 48h under PlateSimilis 2 treatment. The “wound healing” assays by “scratch” in HaCat culture (human skin keratinocytes, with greater than 90% confluence) were performed in the presence (A-C) or absence of (D-F) PlateSimilis 2 (1%). We observed a wound reduction/increase in healing of 40% in 24 h of treatment with PlateSimilis 1. Thus, as we observed in (C) a healing of 70 ± 1.2. (D-F) Control with SFB (fetal bovine serum). Representative images are shown at 10X magnification. The migration area was quantified using ImageJ software. (G) Representative graph of wound quantification versus time (24 h and 48 h), and SFB control. ANOVA GraphPad statistics. Representative images from three independent experiments.

[017] Figure 6. Average Hydrodynamic Diameter (DHM - nanopolymer size) versus zeta potential. PlateSimilis 1 in its two-phase nanopolymeric system (PoL-2), analyzed at different storage times.

[018] Figure 7. Average Hydrodynamic Diameter (DHM - nanopolymer size) versus zeta potential. PlateSimilis 1 in its two-phase nanopolymeric system (PoL-2), under different analysis conditions/variables.

[019] Figure 8. Analysis of cell viability by MTT reduction, cells exposed to PlateSimilis 1 (1x conc. and 4x conc.) in its biphasic nanopolymeric system. Human skin keratinocytes (HaCat) highlighted in the figure were treated with PlateSimilis 1 (1x and 4x conc.) (A-B). The treatment of cells with PlateSimilis 1 did not (1x and 4x conc.) alter cellular metabolism, the cells remained viable and with active metabolism, (triplicate assay) *p<0.001.

[020] Figure 9. Spheroid formation of HaCat cells previously treated with PlateSimilis 1 in a two-phase nanopolymeric system. HaCat cells were plated according to methods for spheroid formation in the presence and absence of PlateSimilis 1 in a two-phase nanopolymeric system. (A) Light contrast microscopy images at 10X magnification (A1- PlateSimilis 1 and A2- Control). (B) Graph showing average spheroid size and spheroidal mass growth rate. Representative images of experiments performed in triplicate. Number of experiments performed (spheroid formation, n=3). Scale 100 µm, graphs represent the increase in spheroid size in the presence of PlateSimilis 1 and in its absence. Bars represent ± means and standard deviation, test used ANOVA GraphPad.

[021] Figure 10. Formation of HaCat cell spheroid for subsequent treatment with PlateSimilis 1 in its two-phase nanopolymeric system. HaCat cells were plated according to methods for spheroid formation in the presence and absence of PlateSimilis 1 in a two-phase nanopolymeric system. (A) Light contrast microscopy images at 10X magnification. (B) Graph showing average spheroid size and spheroidal mass growth rate. Representative images of experiments performed in triplicate. Number of experiments performed (spheroid formation, n=3). Scale 100 µm, graphs represent the increase in spheroid size in the presence of PlateSimilis 1 and in its absence. Bars represent ± means and standard deviation, test used ANOVA GraphPad.

[022] Figure 11. Human skin keratinocyte spheroids treated with PlateSimilis 1 in a biphasic nanopolymeric system - quantification of laminin and type I collagen production post treatment. HaCat cells were plated according to methods for spheroid formation in the presence and absence of PlateSimilis 1 in a two-phase nanopolymeric system. (A) Light contrast microscopy images at 10X magnification, spheroids treated with PlateSimilis 1 plus laminin. (B) Light contrast microscopy images at 10X magnification, spheroids treated with PlateSimilis 1 plus type I collagen. (C) Graph with quantification of total laminin and collagen in spheroids treated with PlateSimilis 1. Representative images of the experiments carried out in triplicate. Number of experiments performed (spheroid formation, n=3). 100 µm scale of images, controls represent the treatment of spheroids with laminin and type I collagen without PlateSimilis 1. Bars represent ± means and standard deviation, test used ANOVA GraphPad.

[023] Figure 12. Human skin keratinocyte spheroids treated with PlateSimilis 1 and PlateSimili in a biphasic nanopolymeric system - coordination of cell renewal in the presence of PlateSimilis functionalized or not with 5-HT and/or 5-HTP*. HaCat cells were plated according to methods for spheroid formation in the presence of PlateSimilis functionalized or not with 5-HT in a biphasic nanopolymeric system. (A) Light contrast microscopy images at 10X magnification, spheroids treated with PlateSimilis 1. (B) Light contrast microscopy images at 10X magnification, spheroids treated with PlateSimilis 2. Representative images of experiments performed in triplicate. Number of experiments performed (spheroid formation, n=3). Scale 100 µm of the images, controls represent the treatment of spheroids with laminin and type I collagen without PlateSimilis 1. Bars represent ± means and standard deviation, test used ANOVA GraphPad.

[024] Figure 13. Representative gels of total protein extraction from skin keratinocyte spheroids treated with PlateSimilis 1 in a biphasic nanopolymeric system - detection of p53 and ki67. The cell samples were subjected to total protein extraction for subsequent measurement of total proteins using the BCA kit for Western Blotting analysis. (A) Result for detection of p53 (dilution 1:1000) cell signaling and human secondary antibody at a dilution of 1:10000, from the spheroid samples in Figures 9 and 10. (B) Result for detection of ki67 (dilution 1:500 ) ThermoFisher and human secondary antibody at a dilution of 1:10000, from the spheroid samples in Figures 9 and 10.

[025] Figure 14. Co-culture with Feeder of human skin fibroblasts (HFF-1) and endothelial cells (HUVEC) in the presence of PlateSimilis 1 for the formation of angiogenic tubular structures. Endothelial cells (4 x 103 cells/well) were plated on a layer of skin fibroblasts (feeder/feeder) and stimulated with PlateSimilis 1 (1%). Images (duplicates) were taken by Luma Scope microscope (Etaluma Inc, Carlsbad, CA, USA), yellow circles indicate tubular structures; and the red arrows indicate the connection point between endothelial cells. Images at 10X magnification (scale bar 50 µm) after 24 hours of culture. Representative images from three independent experiments.

[026] Figure 15. PlateSimilis 1 of the Biotech-educated platelets platform - consolidating the phenomenon of formation of “crowded cellular environment” with endothelial cells (HUVECs). Interactions activated by PlateSimilis 1 (1%), images illustrate the perspective of the Fibrin Merck matrix and organization of F-actin bundles in the cytoskeleton (A), the nuclear perspective and the overlap of the images. Immunofluorescence markers for nucleus (DAPI, blue) (B) and F-actin filaments (phalloidin, green) (C) represented in overlay/localization. Images were obtained by Luma Scope microscope (Etaluma Inc, Carlsbad, CA, USA) at 10X magnification (scale bar 100 µm) after 72 hours of culture.

[027] Figure 16. Adhesion and proliferation of HaCat under treatment with PlateSimilis 1, ascorbic acid and niacinamide, on a “time-lapse” cytation platform. Representative images from videos captured for 24 h (1 image captured every 10 min for 24 h). (A) HaCat PlateSimilis 1 (1%); (B) HaCat PlateSimilis 1 (1%) + 5% ascorbic acid (Nano Vit C); (C) HaCat PlateSimilis 1 (1%) + Niacinamide PC 4%; (D) HaCat SFB 10% control; (E) 5% ascorbic acid control; (F) 4% niacinamide control. *Videos can be requested.

[028] Figure 17- Incorporation of PlateSimilis 1 into cosmetic formulations - spray, cream and gel

[029] Figure 18. Images taken from the reports/reports of in vitro safety and toxicity tests by the company InvitroCell. First image: results of the test with Epskin’s reconstituted human skin. Second image: summary with the characteristics of the irritability/opacity and permeability test, and result of the PlateSimilis 1 evaluation using bovine cornea. Third and final image: summary of the in vitro phototoxicity test and results of the analysis of PlateSimilis 1. All tests showed the safety and biocompatibility of PlateSimilis 1 = PlateSimilis Éris.

[030] Figure 19. Images taken from the final toxicity and phototoxicity test reports/reports. (A) evaluation of the potential for primary, accumulated irritability and skin sensitization of a product under controlled and maximized conditions (RIPT) and (B) testing the potential for photoallergy and skin phototoxicity of PlateSimilis 1. Both tests on volunteers supported the claims /attributes of dermatologically tested and hypoallergenic.

[031] Figure 20. Result of anti-aging tests, carried out by a specialized company. The image taken from the final report/report supports the anti-aging, anti-aging and anti-wrinkle claims.

Detailed Description of the Invention

[032] By identifying the targets 5-HT (and/or precursor 5-HTP*) and thymosin-B4, through studies carried out with platelets stimulated by different types of agonists, biomimetic peptides derived from the multifunctional protein thymosin were designed and synthesized -B4 functionalized, through a non-covalent cross-link reaction, with serotonin (5-HT) and/or with the precursor of the bioactive amine serotonin (5-HTP*). This non-covalent cross-link allows the design of a variety of functional peptide fiber networks thanks to dynamic supramolecular binding modes and reversibility. From then on, feasibility studies were carried out on the use of the peptide molecule derived from Thymosin-B4-5HTP and/or peptide derived from Thymosin-B4-5HT, linked by non-covalent cross-link, in skin tissue repair.

[033] Therefore, the present invention is the unprecedented functionalization of the thymosin-B4 protein with serotonin (5HT) and or its precursor 5-HTP and its use in the preparation of dermocosmetic compositions and for use in the healing process, cell renewal, tissue repair and tissue regeneration.

[034] The dermocosmetic and bio-pharmaceutical compositions of the present invention are characterized by comprising at least one of the sequences represented by SEQ ID No. 1 to SEQ ID No. 17 and acceptable excipients for pharmaceutical and/or cosmetic use. They can come in the form of a spray, gel, cream.

[035] The spray compositions are characterized by consisting of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 from 0.2 to 3%, panthenol 0.2 to 3%, vegetable glycerin from 0.1 to 1.0%, phenoxyethanol from 0.1 to 1.0%, deionized water (q.s.p.).

[036] The cream compositions are characterized by consisting of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 from 0.2 to 3%, polawax wax from 5 to 20%, TCM from 1 to 10%, Vegetable Glycerin 1 to 10%, Vitamin C 1 to 10% or Niacinamide PC 1 to 10%, Liquid Vitamin E 0.1 to 3%, EDTA 0.01 to 2%, Chloromethylisothiazolinone /Methylisothiazolinone from 0.1 to 3%, Phenoxyethanol from 0.1 to 3% and deionized water (q.s.p.).

[037] Gel compositions are characterized by consisting of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 from 0.2 to 3%, natrosol from 0.5 to 5%, panthenol from 0.5 to 5%, vegetable glycerin from 0.5 to 6%, Chloromethylisothiazolinone/Methylisothiazolinone from 0.1 to 3%, Phenoxyethanol from 0.1 to 3% and deionized water (q.s.p.).

[038] The invention can be better understood from the non-limiting examples that follow:

[039] Example 1- Identification of the platelet signature and synthesis of molecules

[040] Omics analyzes - analysis of high biological complexity systems - of agonist-dependent platelet content, focus on Proteomics, Shedomics and Peptidomics

[041] From each agonist-dependent activation condition, the recruited platelet content was analyzed, both intra and extra platelet (secretome). Once organized, the samples were subjected to analysis by high-performance liquid chromatography and mass spectrometry, proteomics of multifunctional intraplatelet peptides and modulators of platelet functions. Analysis of peptides generated by the action of membrane proteases (shedomics) was also carried out. In a simplified manner, we describe the method used: LC-MS analysis was performed on an Easy-nLC II nanoHPLC system coupled to an LTQ-Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) via a nanoelectrospray ion source. Each sample was applied to a C18 chromatography column with 10 µm beads (Phenomenex, Inc., Torrance, CA). The separation was carried out on a C18 analytical column. The elution of the peptides was carried out through a gradient from 5% to 45% acetonitrile, in 0.1% formic acid, in 90 min at 200 nL / min. The spectrometer was operated in data-dependent mode, and the 10 most intense peaks were selected for collision-induced dissociation (CID) fragmentation after acquiring each full scan with specific predetermined parameters of injection time, resolution, and fragmentation scan.

[042] Our data analysis followed the DIKW (Data-Information-Knowledge-Wisdom) model, widely used in biotechnological analyses, such as "omics". Raw data files were subjected to searches against NCBI Databases nr_20130601, UniProt, and Moonlightpro filtered for D. rerio taxonomy using PEAKS Studio (version 8; Bioinformatics Solution, Waterloo, Canada).

[043] Our analyzes using omics tools to feed the Biotech-educated platelets platform allowed us to correlate agonist-dependent platelet content with platelet signaling in the functional context of networks integrated in strategic processes for skin maintenance. We explore information on protein-protein interactions and platelet growth factor relationships and multifunctionality in physiological processes related to tissue regeneration (including the wound healing process). The use of graphical visualization for specific subnetworks allowed and still allows the investigation of the main signaling modules involved in platelet activation. We highlight in figure 1 a fraction of our analyzes in a platelet protein interactome model, such as cytokines, chemokines, coagulation factors and growth factors.

Biotech-educated platelets platform - In silico models and peptide synthesis

[044] With the results of the omics analyzes and the MagPix multiplex platform, we proceeded to the in silico models. For the construction and validation of the Biotech-educated platelets Platform, molecular docking and molecular dynamics tools were used to analyze in detail the proteins and peptides (platelet factors) targets in our shedomics and peptidomics analysis, and thus select the most promising ones. active candidates for dermocosmetic formulations, through the biological characterization of this new class of bioactive molecules, biomimetic peptides (of intraplatelet origin) and biomimetic peptides released by the action of metalloproteases, all called PlateSimilis. In the present invention, biomimetic peptides were functionalized with a platelet signature, with serotonin (5-HT), the biogenic bioamine identified in tissue remodeling processes, being the most promising for non-covalent cross-link functionalization, as well as its biological precursor 5 -HTP. Next, in vitro cellular tests for safety and efficacy were carried out. In this way, we describe in a simplified way the synthesis method used.

[045] The synthesis followed the solid phase methodology by Fmoc (9-fluorenylmethoxycarbonyl), as described by Hirata et al [12]. The ends of the peptides were appropriately blocked according to our cellular targets. The synthetic peptides were characterized in high-performance analytical liquid chromatography, on an Econosil C18 reversed phase column (4.6 mm X 150 mm), coupled to a pump system and UV-Vis detector (215 and 280 nm) both Shimadzu. (12) Hirata, I.Y., Cezari, M.H.S., Nakaie, C.R., Boschcov, P., Ito, A.S. & Juliano, M.A. Internally quenched fluorogenic protease substrates: solid phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor -acceptor pairs. Lett. Pept. Sci. 1, 299-308 (1994). The synthesized peptides were analyzed by mass spectrometry, using the method of measuring the time of flight of the ion in a mass spectrometer model Microflex LT, equipped with MALDI-TOF technology (Matrix Laser Disorprtion Ionization Time-of-Flight). In all analyses, the matrix used was a-cyano-4-hydroxycyanaminic acid. The hydrolysis fragments were determined using high-performance liquid chromatography associated with an “electron spray” mass spectrometer, using LC/MS-2010 equipment, ShimadzuSi. The samples were analyzed by chromatography using UV detectors (215 to 365 nm), Shimadzu UV-vis SPD-20AV, the column used was a C18 Ultrasphere (4.6 mm validated: (A) TFA/H2O (1:1000) and (B) TFA/ACN/H2O (1:900:99).

[046] From multiple analyzes we were able to highlight through in silico analyzes the peptides derived from platelet factors, described in table 1, and highlighted here: Palmitoyl-octapeptide-5-HT or -5-HTP* - [Palmitoyl or Acetyl]- TXXEHXXD-5-HT or -5-HTP* or [Palmitoyl or Acetyl]- TXXEHXXD- COOH- (which can also be M) or Palmitoyl-TETQEKND-5-HT or -5-HTP* - [Palmitoyl or Acetyl]- TETQEKND-5-HT or 5-HTP* or [Palmitoyl or Acetyl]- TETQEKND-COOH- Example 2- Formation of the Cross-link between the molecules Thymosin- B4, Serotonin (5-HT) and/or its precursor 5-HTP * Structure of TB4-based peptides containing metal-binding sites and coordination of binding to the serotonin precursor 5-HTP* and 5-HT

[047] For peptide sequences derived from factor TB-4 (see table I) Palmitoyl or Myristoyl or Acetyl- TXXEHXXD-5-HT (Myristoyl-Thr-X-X-Glu-His- X-X-Asp-5-HT, X= Ala-Glu Glu-His-Asp-5-HT) or Palmitoyl or Myristoyl or Acetyl- TXXEHXXM-5-HT or 5-HTP (Myristoyl-Thr-X-X-Glu-His-X-X-Met-5-HT or 5- HTP, of sodium phosphate and 400 µM to 1 mM Zn2+ Cu2+ or Ni2+ to obtain a peptide concentration of 230 µM. The peptides (0.00001% stock solution) were dissolved in pH 7.4 and 20 mM sodium phosphate buffer and mixed with various metal ions (160 µM) and incubated with serotonin (5-hydroxytryptamine, 5-HT) or its precursor 5- hydroxytryptophan (5-HTP*). All peptide solutions were incubated under controlled temperature conditions at 4°C for at least 24 h before incorporation into the delivery system and subsequent cellular analysis.

[048] According to a first aspect of the present invention, peptides derived from the TB-4 factor are provided with binding sites for serotonin (5-HT) and/or its precursor 5-HTP with a dissociation constant of less than 10- 7M. Specifically, these binding sites comprise the amino acid residues methionine or aspartate or glutamate in position I (in relation to the C-terminal region), and their functional equivalents. All peptides are synthesized with a purity of > 98%. Table 1- Thymosin-B4 and its derivatives, synthesized and linked to serotonin (5HT) and/or its precursor 5-HTP

[049] Growth/proliferation assays were carried out with skin fibroblasts (HFF-1) with the sequences mentioned in Table 1.

[050] In the following text the derivatives will be called:

[051] PlateSimilis 1 or PlateSimilis Éris [Palmitoyl or Acetyl]-TXXEHXXD-5- HT and/or 5-HTP (which can be D or M in the C-terminal region)

[052] PlateSimilis 2 [Palmitoyl or Acetyl]- TXXEHXXD-COOH- (D or M) without functionalization.

Example 3- Cellular assays with TB-4-derived sequences functionalized (PlateSimilis 1) or not (PlateSimilis 2) with 5-HT and/or 5-HTP Cell Lines

[053] The HaCat cell line (Ha = human adult, Cat = Calcium temperature), normal human skin keratinocytes, was maintained/cultured in DMEM high glucose medium (Invitrogen, USA), supplementation with 10% FBS and/or InGrowth- S and antibiotics (penicillin (100 U/mL) and streptomycin (100 µg/mL)). As it presents a proliferation index of 3.8 in 48h, which corresponds to a high level of cell renewal, the cell medium was replaced with each subculture, and with the cells adhered every 2 days. The normal human skin fibroblast line, HFF-1, maintained/cultured in DMEM high glucose medium (Invitrogen, USA), supplementation with 10% FBS and/or InGrowth-S and antibiotics (penicillin (100 U/mL) and streptomycin (100 µg/mL)). The HUVEC (Human Umbilical Vein Endothelial Cells) line was cultivated in Roswell Park Memorial Institute (RPMI) culture medium, supplemented with 10% antibiotics (penicillin (100 U/mL) and streptomycin (100 µg/mL)). All cells were cultivated/maintained at 37 °C under 2.5% CO2 tension and we continued with subcultures and monitoring of morphology at each cell passage.

HFF-1 fibroblast growth/proliferation assay by cell counting in the presence of the sequences in Table 1

[054] Human skin fibroblasts (HFF-1) were cultured in a 96-well plate, until they reached ~80% confluency, in DMEM high glucose medium (Invitrogen, USA), supplementation with 10% FBS and antibiotics (penicillin ( 100 U/mL) and streptomycin (100 µg/mL)), for subsequent treatment with the sequences in the Table. As a result, we observed in Figure 2 that some sequences stood out effectively in relation to the growth/proliferation of skin fibroblasts, such as SEQ 5 and SEQ 14, PlateSimilis 1 and 2, respectively.

Cell viability by analysis of MTT reduction under treatment with peptides derived from TB-4 functionalized (or not with 5-HT - and/or 5-HTP)

[055] HaCat cells (normal skin keratinocytes) were plated at a density of 5 x 103 cells/mL in 96-well plates (plating volume per well: 250 uL), supplemented with 10% FBS, and incubated at 37o C , 5% CO2 for 48h. Subsequently, the MTT reduction capacity of the cells (5 x 103cells/mL) was analyzed using the Promega kit. The effects of PlateSimilis 1 and 2 peptides, at a concentration of 1% (w/v), on the viability of the HaCat line were evaluated. For this, the medium was removed and medium without supplementation containing MTT (0.5 mg/mL) was added to the wells, following the manufacturer's instructions. After incubation for 4h at 37o C, the medium was carefully removed and then added to the formazan inactivation and solubilization solution (100 µL). The plates were shaken for 10 min and the absorbance corresponding to each well was read on a spectrophotometer (Elx 800 BIO-TEK) at a wavelength of 570 nm. The values in the graphs were expressed as percentage of cell viability. The SFB was used for comparison and the reduction in MTT in this condition was considered as 100%.

[056] In Figure 3 we can see that treatments with PlateSimilis 1 and 2 (both at 1% w/v) did not alter the metabolic capacity of human skin keratinocytes, using SFB as a control. Therefore, PlateSimilis 1 and 2 adequately preserved the morphology/phenotype and cellular metabolism of the HaCat lineage. Indicating an active and regulated cellular metabolism with healthy cells, without loss of viability, after treatment with PlateSimilis 1 and 2 in vitro in a monolayer system.

Cellular Migration using the “wound healing” or “wound migration” method

[057] HaCat cells (normal skin keratinocytes) (5 x 104 cells) were cultured in a 24-well plate (supplemented with 10% SFB) and maintained in an oven until they reached 100% confluence. The cells were then washed with PBS (1x) and after washing, a scratch (“wound”) was made using a 1000 µL tip in the center of each well. The culture medium was replaced with 0.2% FBS, to synchronize the cell cycle of the cells in question. At this stage, HaCat cells were treated with 1% (w/v) PlateSimilis peptides 1 and 2 (separately and evaluated individually). The cells were monitored under a microscope at 0, 24 h and 48 h or until the “wound” closed. The cell migration efficiency, over 24h and 48h, was analyzed using a LumaScope microscope (Etaluma Inc, Carlsbad, CA, USA) at 10X magnification. Scale bar corresponding to 100 µm. The analysis was performed using ImageJ software (NIH, USA) and the migration area was quantified in arbitrary units (A.U.). The result of this assay was decisive for choosing the most effective sequences in the migration of human skin epithelial cells/keratinocytes.

[058] As representative results of this experiment, we show images of HaCat cultivation under the treatment of PlateSimilis 1 and 2 (both 1% w/v). Figures 4 and 5 show that normal skin keratinocytes (HaCat) treated with PlateSimilis 1 showed greater migration ability than when treated with PlateSimilis 2 (not functionalized with 5-HT and/or 5-HTP), which we used as a control. cells cultured with SFB. We observed a wound reduction/increase in wound healing of 80% ± 4.5 in keratinocytes from PlateSimilis 1-treated skin at 48h (Figure 4C and G). The wound exposed to treatment with PlateSimilis 2 led to a reduction in the wound/and an increase in wound healing of 70% ± 4.5 in skin keratinocytes (Figure 5C and G). Therefore, we conclude that PlateSimilis 1 to 1% significantly accelerates the regulated process of cell migration, a better performance than PlateSimilis 2.

PlateSimilis in its two-phase nanopolymeric delivery system, a nano-structured copolymer (Poloxamers)

[059] Reducing the size of nanometer-scale particles (nanotechnology) has been used as a platform for the development of “drug delivery” systems in innovative pharmaceutical and dermocosmetic formulations in order to obtain greater efficacy and safety. This technique allows increasing the biocompatibility and reach of active ingredients for topical use that are poorly soluble in water to structures that are difficult to permeate/penetrate. Nano suspensions (nano-polymers) have advantages, such as increased solubility, stability and dissolution speed, resulting from the increase in the surface area of the particle. In addition to these advantages, nanopolymer systems provide a strategic property to increase effectiveness, they present greater adhesion to biological membranes and cell membranes. As indicated in the initial proposal of this project, we tested two poloxamers, copolymer surfactants, with a block structure. Block copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) in the presence of glycerol [13,14], as PlateSimilis carriers, in order to make its stability and delivery abilities to the stratum corneum more effective. The surfactants/copolymers/drug delivery systems PoL-1 and PoL-2 (according to methods) were tested based on the concentration of the copolymers, thermal characteristics and compatibility with PlateSimilis active ingredients. Our data showed that obtaining poloxamers-PlateSimilis nano suspensions was possible, with more promising and effective results with PoL-2 (poloxamer 407) in the presence or absence of glycerol. Compatibility tests between the PlateSimilis incorporation concentration (5000 ng/mL) and the surfactant/co-polymer concentration range (0.5 - 10%) proved to be effective in obtaining a nanopolymeric system. The incubation time for the incorporation of PlateSimilis into the hydrophobic central core of the formed nanopolymer system varied from 12 to 48 h, under slight agitation at a controlled temperature of 4 °C, in a cold chamber. The pH range at which we observed the stability of the nano suspensions was from 4.0 to 8.0. As we mentioned, the adjustments of these parameters resulted in a two-phase nanopolymeric system for effective delivery of PlateSimilis functionalized or not with 5-HT and/or its biological precursor 5-HTP. [(13) Bodratti, A. M.; Alexandridis, P. Formulation of Poloxamers for Drug Delivery. J. Funct. Biomater. 2018, 9, 11; doi:10.3390/jfb9010011; (14) Chiappetta, D.A.; Sosnik, A. Poly(ethylene oxide)-poly(propylene oxide) block copolymer micelles as drug delivery agents: Improved hydrosolubility, stability and bioavailability of drugs. Eur. J. Pharm. Biopharma. 2007, 66, 303-317].

Determination of the mean hydrodynamic diameter (DHM) of the two-phase nanopolymer system

[060] With the aim of establishing ideal conditions for readings of mean hydrodynamic diameter (DHM) and the polydispersity index by the dynamic light scattering method, dilutions of 1:100 and 1:200 of the sample were carried out in a saturated solution of PlateSimiles peptides 1 and 2. Analyzes/measurements were carried out in triplicate at an angle of 90° using partner equipment, the Zetsizer Nano ZS90 (Malvem Instrumensts, Malvern, UK).

[061] In the present invention, the size of the nanostructures (DHM) was decisive for the reach of PlateSimilis to the stratum corneum of the skin (proven in clinical tests). Meanwhile, PZ (Zeta potential) measurements provide practical challenges that must be taken into consideration when characterizing the interfacial properties of nanoparticles with irregular morphology, such as non-spherical colloidal particles. Several studies show that PZ is not a constant property, but depends on the morphology of the dispersion system, the physicochemical properties of the particles (pH) and the properties of the electrolyte solution in which the particles are suspended (in our case PBS). The physicochemical characterization revealed that at the beginning of the preparation of the nano suspensions (PoL-2 with PlateSimilis), a DHM of 100 nm to 200 nm and a PZ of -34.9 mV were observed, as shown in Figure 6. These values were slightly altered mainly after three months of production of the nano suspensions (PoL-2 with PlateSimilis), which we observed a DHM of 225 nm and PZ of 30.9 mV, indicating that the nano suspensions are stable for up to three (3) months after its preparation. Regarding the stability analyzes of PlateSimilis in its two-phase nanopolymeric system, through mechanical agitation tests (centrifugation), pH variation and thermal analysis (Figure 6), we observed no change in the DHM in all conditions tested, specifically the DHM if maintained between 100 nm to 200 nm. However, we observed a slight change in the PZ, which changed from -34.9 mV at the beginning of the analyzes to -25.9 mV (Figure 7). Thermal analysis constituted a critical assessment, but essential in studying the compatibility of the two-phase nanopolymeric system of PlateSimilis functionalized or not with 5-HT and/or its precursor, with instability being observed with a drastic reduction in the size of the nanostructures to 50 nm with the increase in temperature from 25^C to 75^C (ideal temperature from 0 to 6^C for incorporation). The development and validation of the PlateSimilis two-phase nanopolymeric system revealed adequate and satisfactory characteristics of linearity, specificity, precision and robustness in accordance with current regulations (RDC-166 ANVISA, 2017). It is worth mentioning that cell viability tests with human skin keratinocytes (HaCat) were also performed, showing the safety of the PlateSimilis 1 biphasic nanopolymeric system (functionalized with 5-HT and/or 5-HTP) in two different concentrations (Figure 8 ).

Determination of the zeta potential (PZ) of the two-phase nanopolymer system

[062] Analyzes to determine the zeta potential (PZ) were also conducted using the Zetasizer ZS90 equipment (Malvem Instrumensts, Malvern, UK), using the electrophoretic mobility method, which as a consequence is converted into PZ (mV) according to the Henry equation [15]. The determination of the PZ is crucial to guarantee the stability of the nanostructure, and takes into account the magnitude of the electrostatic repulsion or attraction or charges between particles. With Henry's equation, the viscosity and dielectric constant of the solution at 25 °C are taken into account. In electrophoretic mobility, the applied field strength was determined to be 20 V/cm. The conductivity range evaluated was 10 to 60 µS/cm using 0.9% NaCl solution. [(15) Cosgrove, T. Colloid Science: principles, methods and applications. 2nd ed. John Wiley & Sons Ltd, 2010].

3D Cell Culture (and 4D = 3D + time) - Human skin keratinocyte spheroids (HaCat) by NanoShuttle™ magnetic system to evaluate the Biocompatibility of PlateSimilis 1 in its biphasic nanopolymeric system

[063] Spheroids provide a 3D organizing environment with intense cell-cell interactions. As a result of their excellent regenerative properties and high production yield, cellular models in spheroids are increasingly recommended for testing in regenerative medicine. 3D models attempt to recapitulate the complex cellular organization of a tissue (even planar) such as skin. Both cell-cell adhesions [16] and cell-matrix interactions, together with the synthesis of extracellular matrix (ECM) proteins [17,18], contribute to the generation of nutrients and signal gradients typical of juxtacrine interactions, intensifying the autocrine and paracrine signaling pathways, thus offering a clinically relevant study model [19]. For these reasons, spheroids represent a useful tool for drug screening [19] and more specifically in the claim of the present invention for the research of dermocosmetic and/or biopharmaceutical active ingredients. Spheroids are useful for toxicological studies with regard to drug penetration and absorption, providing information on the diffusion of compounds [19] and exhibiting better immunomodulation, proliferation and cell activation abilities than 2D monolayer cultures [(16 ) Mingxing Lei, Linus J. Schumacher, Yung-Chih Lai, Wen-Tau Juan, Chao-Yuan Yeh, Ping Wu, Ting-Xin Jiang, Ruth E. Baker, Randall Bruce Widelitz, Li Yang, Cheng-Ming Chuong. Self-organization process in newborn skin organoid formation inspires strategies to restore hair regeneration of adult cells. Proceedings of the National Academy of Sciences, 2017, 114 (34) E7101-E7110; DOI: 10.1073/pnas.1700475114. (17) Fuchs, E. Finding one’s niche in the skin. Cell Stem Cell. 2009, 4:499-502. (18) Sasai, Y. Cytosystems dynamics in self-organization of tissue architecture. Nature, 2013, 493:318-326. (19) Laschke, M.W.; Menger, M.D. Life is 3D: Boosting Spheroid Function for Tissue Engineering. Trends in Biotechnology, 2016, 35; 2, p.133-144].

[064] In the present invention, we focused on cellular organization assays in 3D spheroids, and on “time-lapse” (4D) to identify “waves” of cell proliferation and renewal promoted by PlateSimilis 1 (functionalized with 5-HT and/ or 5-HTP*, in its two-phase nanopolymeric system), and as a consequence analyze effects related to biocompatibility and toxicities of this promising active ingredient for transdermal dermocosmetic formulations. For 3D and 4D assays, we used human skin keratinocytes (HaCat) under PlateSimilis 1 treatment (1% w/v)). To achieve this, we followed two (2) treatment conditions: 1) HaCat cells were previously treated with PlateSimilis 1 (1%) for subsequent incubation of the cells with gold nanoparticles to induce spheroid formation using the NanoShuttle™ magnetic system; and 2) the spheroids of the HaCat lineage were formed by the NanoShuttle™ magnetic system for subsequent treatment with PlateSimilis 1. During the formation of the spheroid there is a movement of the cells, a process of self-organization of the formed spheroid, a critical point to be evaluated on treatment with PlateSimilis 1. Bright field optical microscopy images (Figures. 9A and 10A) show an evolution in the size and optical density of spheroids pre- and post-treatment with PlateSimilis 1. These results indicate growth progressive and cell-cell compaction (HaCat) of the spheroids mainly with post-treatment with PlateSimilis 1 (Figures 9 and 10). We observed in Figure 9A and B, within 48 h of keratinocyte spheroid formation, that cells previously treated with PlateSimilis 1 for subsequent induction of spheroid formation, demonstrated a more active cell proliferation process, triggering a regulated self-assembly of keratinocytes. of skin with a clear increase in spheroid mass (260 ± 10 µm) and its aggregation and compaction in 112 h (5th day) for morphological organization, indicating metabolic viability and cell growth rate of 10 ± 3%, in relation to control under SFB supplementation. In Figure 10A and B, we observed that the newly formed spheroids and then treated with PlateSimilis 1 (1%) showed active cell proliferation with an increase in spheroid mass (300 ± 20 µm), when compared to control spheroids (maintained only with supplementation). , untreated) (210 ± 5 µm). In 32 h and 48 h we observed a modeling/organization of its spherical structure. Indicating that up to 112 h (5th day) of maintenance and cultivation of the spheroids, the cells treated with PlateSimilis 1 were metabolically active with a tendency to stabilize. Central results in the benefits promoted by PlateSimilis 1 are highlighted in Figure 11, where we can observe the culture in spheroids in the presence of basement membrane proteins, laminin and type I collagen. The basement membrane is an ECM specialized in the adherence of (new) cells ( cell renewal process), being produced by normal epithelial cells, such as human skin keratinocytes. It is formed in a multistep process initiated by cells binding to laminin on the cell surface and leading to the accumulation of fibrillar collagen I in the nascent laminin structure [20]. (20) Rowe, R. G.; Weiss, S.J. Breaching the basement membrane: who, when and how? Trends Cell Biol. 2008 Nov;18(11):560-74. doi: 10.1016/j.tcb.2008.08.007. Bourland, J., Fradette, J. & Auger, F.A. Tissue-engineered 3D melanoma model with blood and lymphatic capillaries for drug development. Sci Rep 8, 13191 (2018). https://doi.org/10.1038/s41598-018-31502-6] .

[065] In this context, PlateSimilis 1 acted as its mirror platelet factor, TB4 and functionalized with 5-HT and/or its precursor 5-HTP, providing organization of the basement membrane and inducing the production of laminin, with a consequent increase in collagen type I [4,18]. It is worth highlighting that the functionalization of PlateSimilis 1 through a non-covalent cross-link with 5-HT and/or its precursor 5-HTP, is strategic because the peptide acts mimetic to TB-4 and 5-HT and/or 5-HTP, both active in the healing and tissue regeneration processes. We observed in Figure 11A human skin keratinocytes (HaCat) in the presence of laminin (10 µg) and treated with Platesimilis 1 in its biphasic nanopolymeric system. We noticed that the organization of spheroids induced by the NanoShuttle™ magnetic system was not able to keep the keratinocytes (+ laminin) aggregated/organized in a single spheroid structure. In 16 h we observed 3 small spheroids, where after treatment with PlateSimilis 1 there was induction of gradual cellular organization with broad cell movement (observed in 32 h and 48 h) until the unification of the spheroidal mass, where we observed a single structure in 112 h (5 th day). Quantification of total laminin showed that at 112 h (5th day) there was a 2.5-fold increase in laminin in relation to the analysis time of 32 h. On the other hand, in the control without treatment with PlateSimilis 1, we observed that the spheroids acquired a sickle shape in the presence of laminin, not leading to the same cellular organization promoted by treatment with PlateSimilis 1, an organization expected in a tissue regeneration process. In Figure 11B, we show representative microscopic images to illustrate morphological changes of 3D cell aggregates/spheroids in the presence of type I collagen (0.5 µg) and PlateSimilis 1, during an observation period of 112 h (5th day). We observed spheroids "coated" with type I collagen fibers upon treatment with PlateSimilis 1, such fibers had a gradual increase observed more intensely at 48 h and 112 h. In the graph in Figure 11C, we observed a 2.0-fold increase in collagen production in 112 h (5th day), when compared to the analysis time of 32 h. These type I collagen fibers can be associated with improvements in biomechanical properties and matrix architecture against basement membrane proteins, an important step in the tissue regeneration process [21]. (21) Sosne G, Qiu P, Kurpakus-Wheater M. Thymosin beta 4: A novel corneal wound healing and anti-inflammatory agent. Clin Ophthalmol. 2007;1(3):201-207] These results show that PlateSimilis 1 in its biphasic nanopolymeric system is a promising biomimetic peptide, demonstrating biocompatibility against cellular exposure, inducing an increase in the mass of human skin keratinocyte spheroids, with active and regulated metabolism and proliferation, inducing a coordinated cellular organization in the presence of laminin and an increase in type I collagen fibers, all results related to the processes of cell renewal and tissue regeneration.

[066] Highlighting the potential of the described invention, the effects of peptides derived from TB-4 functionalized with 5-HT and/or 5-HTP (PlateSimilis 1) on cell renewal, in 3D culture, were compared to biomimetic peptides derived from TB -4 without functionalization (PlateSimilis 2). And the results in Figure 12 show the best performance of PlateSimilis 1. Detection of p53 and ki-67 by Western blotting

[067] We used immunodetection of p53 and ki-67 by western blotting to ensure control of cell proliferation of the HaCat lineage under treatment with peptides derived from TB4, PlateSímilis 1 and 2 (1%). For western blotting experiments, cells subjected to treatment with peptides were lysed and subjected to the addition of protease and phosphatase inhibitors (1 mM EGTA, 1µg/ml aprotinin, 1µg/ml leupeptin, 1mM PMSF and phosphatase inhibitors (2 mM sodium orthovanadate 50 mM NaF, 10 mM sodium pyrophosphate)). The total protein lysate was subjected to protein measurement using the BCA kit (Merck-Sigma-Aldrich) and 60 µg of total proteins were added to each well of the 10% polyacrylamide (SDS) gel. The gels were transferred to a nitrocellulose or PVDF membrane, and the membranes were then incubated with specific monoclonal antibodies against the target proteins (Cell Signaling antibodies). After incubation with the primary antibody, the membranes are conjugated with the peroxidase-labeled secondary antibody (HRP). For the protein revelation process, we will use the ECLTM or Super SignalTM system.

[068] The results in 3D culture that demonstrate the effectiveness of PlateSimilis 1 in activating the process of cell renewal and proliferation, may raise questions regarding the proliferative behavior of keratinocytes in the face of treatment with PlateSimilis 1. In this way, we use the classic markers of cell proliferation index, Ki-67 and p53 (tumor suppressor gene). The low positivity for ki67 and the normal expression of p53 guarantee the safety in the treatment of HaCat cells by PlateSimilis 1. And the western blot results showed that HaCat cells treated with PlateSimilis 1 (1% w/v) showed very low expression of ki67 , and show normal expression of p53 (Figure 13). Reinforcing the safety of the TB-4 factor-derived peptide functionalized with 5-HT and/or 5-HTP, PlateSimilis 1. Tubule formation assay - Co-culture human skin fibroblasts (HFF-1) and endothelial cells (HUVEC) 2D model and Heterotypic culture with biomimetic matrix 3D model - under treatment with PlateSimilis 1 (1%)

[069] Human skin fibroblasts (HFF-1) were cultivated in a 96-well plate, until they reached ~80% confluence, to act as a feeder in co-culture with endothelial cells. Subsequent to the confluence of skin fibroblasts, we added endothelial cells (HUVEC) to this monolayer at a density of 3 x 104 cells/well, in RPMI medium, 10% FBS, 1% of 100 U/mL penicillin and 100 µg/mL of streptomycin for 24 hours. After the endothelial cells reached ~60% confluence, we treated the cells with TB-4-derived peptides functionalized or not with 5-HT and/or 5-HTP (see table 1), which showed the best results in the migration assays. cells and in 2D and 3D cultures (human skin keratinocyte spheroids). After treatment with PlateSimilis 1, the plate was kept in the oven at 37 °C and removed only to capture images every 2 h, the most representative being every 16 h. For this, photographs were taken under an inverted light microscope using 10x or 4x objectives. All experiments were performed with cells between passages 6 to 9. Images were captured using a Luma Scope microscope (Etaluma Inc, Carlbad, CA, USA), 10x magnification (100 µm scale) after 16 hours of cultivation. In Figure 14 we see the results with PlateSimilis 1, which was able to induce remodeling (conformational rearrangement and organization of the cytoskeleton) of endothelial cells, cells that were previously in a state of quiescence, starting to respond to cellular organization. We observed the behavior of endothelial cells under the feeder (skin fibroblasts), where we clearly noticed connection points between endothelial cells (indicated by red arrows) and the formation of tubular structures (represented by yellow circles).

[070] For the 3D culture assay for visualization by fluorescence microscopy, the HUVEC line was maintained in culture at a density of 3 x 104 cells/well, in RPMI medium, 10% SFB, 1% 100 U/mL penicillin and 100 µg/mL of streptomycin for 24 hours. After this period, the biomimetic matrix CHEMICON Fibrin Gel (Merck, Darmstadt, Germany) was added to each well and incubated with the cells for 24 h. Subsequently, PlateSimilis 1 (1%) was added as treatment and incubated for another 24 h. In the next step, the culture medium was removed, and the HUVECs cells were fixed with PFA (4% PBS) for 10 min, washed with PBS (1x), and permeabilized with Triton X-100 (0.1% PBS). and blocked with BSA (bovine albumin, 3% in 1x PBS) for 1 hour. Subsequently, cells were washed 3 times with PBS (1x) and incubated for 16 h at 4°C in a humid chamber with the appropriate antibodies, ActinGreen 488, ReadyProbes Reagent (AlexaFluor 488 phalloidin) and DAPI-Invitrogen nucleus marker, both from Thermo Fisher Scientific (MA, USA) at a dilution of 1:1000. Images were captured using a Luma Scope microscope (Etaluma Inc, Carlbad, CA, USA), 10x magnification (100 µm scale) after 72 hours of cultivation.

[071] Following the same “Biotech-educated platelets” context (literal translation: platelets educated by biotechnology), we invested in the execution of a 3D model for ex vivo angiogenesis assays, where we used a biomimetic matrix (Merck) rich in fibrinogen, fibronectin and vitronectin, to analyze by fluorescence microscopy the cellular organization of endothelial cells and the reorganization of the cytoskeleton (F-actin bundles, in ActinGreen 488 green) after treatment with PlateSimilis 1. This solution acts on the formation of tubular microstructures ex vivo, which can improve microvascularization in the region stimulated by PlateSimilis 1. Acting on the biomimetic support/scalffold, PlateSimilis 1 was able to guarantee the proliferation of HUVECs in 3D cultures and the formation of a microvascular network (structures) ex vivo without requiring the addition of additional recombinant growth factors (Figure 15).

[072] Still in Figures 15 and 16, PlateSimilis 1 was able to induce remodeling (conformational rearrangement and organization of the cytoskeleton) of endothelial cells, cells that were previously in a state of quiescence, starting to respond to cellular organization. We observed the behavior of endothelial cells under the feeder (skin fibroblasts), where we clearly noticed connection points between endothelial cells (indicated by red arrows) and the formation of tubular structures (represented by yellow circles). Figure 15 shows the set of images of the organization of the cytoskeleton of endothelial cells activated by PlateSimilis 1, from the perspective of the gel/matrix (Fibrin Merck) by confocal microscopy through the detection of F-actin (bundles in the cellular cytoskeleton) by labeling the phalloidin (Alexa-green), cell nucleus labeled blue (DAPI). Cytation - “time lapse” - Multi-modal Imaging System for monitoring the Adhesion and Proliferation of human skin keratinocytes (HaCat) treated with PlateSimilis 1 (peptide derived from TB-4 functionalized with 5-HT and/or 5-HTP) and PlateSimilis 2 (non-functionalized TB-4-derived peptide)

[073] The human skin keratinocyte lineage, HaCat, was thawed and then placed in culture in an appropriate count and culture medium for “time-lapse” monitoring on a Cytation 5 microscope (Biotek), in order to monitor its sedimentation, adherence and proliferation initiated by contact, via juxtacrine signaling. The treatment was followed as follows: i) HaCat control with medium supplemented with 10% FBS; ii) HaCat cells were treated with 1% PlateSimilis 1 and PlateSimilis 2; iii) HaCat cells were treated with 1% PlateSimilis 1 + 5% ascorbic acid (Nano Vit C, BioVital) the same for PlateSimilis 2 and iv) skin keratinocytes were treated with 1% PlateSimilis 1 + Niacinamide PC (DMS ) 4%, the same for PlateSimilis 2. Images were acquired every 10 minutes for 24 h.

[074] In Figure 16 we can observe recently thawed human skin keratinocytes (HaCat) initiating cellular processes/events, such as the dynamics of cell adhesion and proliferation, with two well-defined regions in this process; i) filopodia or "microspikes" (a process that cells use to recognize their environment, and involves actin-rich plasma membrane protrusions that function as environmental sensors) and ii) the lamellipodia (motility) process well identified in keratinocytes. With the treatment established with PlateSimilis 1, we identified in the videos the intensification of nascent adhesion formations, with the resulting formation of protrusions accelerated in cell proliferation under the treatment with PlateSimilis 1, and enhanced in the presence of ascorbic acid (5%) and niacinamide (4 %), compared to the treatment of active ingredients individually introduced into the culture system (Figure 16 A-F). We can conclude from these results that PlateSimilis 1 is an active ingredient in a two-phase nanopolymeric system, inducing the cell renewal process, the synthesis of laminin and type I collagen, having a synergistic effect with the ingredients ascorbic acid and niacinamide.

Development of dermocosmetic formulations for the incorporation of PlateSimilis 1 (peptide derived from TB-4 functionalized with 5-HT and/or 5-HTP)

[075] We describe below the components of the spray, cream (oil-free) and gel formulations, which were selected according to stability and compatibility with PlateSimilis 1 in its two-phase nanopolymeric system (Tables 2, 3, and 4). PlateSimilis 1 provides an anti-oxidant scenario through synergistic action with active antioxidants such as vitamin C (Nano vit C), niacinamide PC and glutathione. The objective at this stage was to develop minimalist and stable preparations for the incorporation of PlateSimilis 1. Table 2. Spray formulation with PlateSimilis 1 in its two-phase nanopolymeric system. Table 3. Cream formulation (oil-free) with PlateSimilis 1 in its two-phase nanopolymeric system. Table 4 Gel

Stability tests of formulations with the incorporation of PlateSimilis 1 (peptide derived from TB-4 functionalized with 5-HT and/or 5-HTP) - organoleptic characteristics, pH value and viscosity in relation to time and temperature

[076] For the tests, factors that affect stability were taken into account, such as physical and physicochemical changes, such as: appearance, odor, color, pH changes and apparent viscosity.

[077] We adopted the following criteria to evaluate possible changes after the incorporation of PlateSimilis 1 (PlateSimilis Éris) (peptide derived from TB-4 functionalized with 5-HT and/or 5-HTP) in the formulations:

[078] Appearance: the appearance and integrity of the formulations maintaining the initial appearance under storage conditions, with the exception of high temperatures, where the perception of small modification was accepted;

[079] Odor and color: these characteristics were monitored during storage in indirect sunlight, where normality is expected. However, at high temperatures slight changes are expected;

[080] pH changes: variations greater than ± 10% were not accepted, compared to the initial value of each formulation, under all storage conditions;

[081] Apparent viscosity: modifications were permitted, as long as they did not alter the visual perception of the samples (ANVISA, 2004).

[082] Such normal stability analyzes were monitored for 90 days, and the formulations were kept in various temperature conditions: room temperature and indirect sunlight (25 °C ± 0.5); refrigerator (5.0 °C ± 0.5); and oven at two temperatures (37.0 °C ± 0.5 and 45 °C ± 0.5). The results obtained can determine the shelf life of the product/formulation and evaluate the incorporation and compatibility of the formulation in relation to PlateSimilis 1 (peptide derived from TB-4 functionalized with 5-HT and/or 5-HTP). The periodicity of readings and observations in the storage conditions of the formulations with PlateSimilis 1 were time 0 (zero = t0) after 48 h of preparation rest, 1st, 7th, 15th, 30th and 90th days ( ANVISA, 2012). We use the following classification to evaluate formulations with PlateSimilis 1 incorporated in terms of appearance, color and odor: Normal - N; LM/A - slightly modified/altered; modified - M and intensely modified - IM. pH measurements were carried out using a pH meter (Fisherbrand, USA). The apparent viscosity was evaluated on a spindle 10 viscometer (Fungilab). The analyzes were carried out at room temperature, 30 minutes after being removed from their storage conditions. We follow the criteria for organoleptic and physical-chemical specifications of raw materials, according to the Cosmetic Products Stability Guide, ANVISA, 2004.

[083] The results of the incorporation of PlateSimilis 1 reflect the stability of the preparations with uniformity and absence of aggregate formation. We also observed the action of PlateSimilis 1 as a modifier of sensorial properties in the cream formulation (oil-free emulsion) with Polawax wax as the base formula. PlateSimilis 1 in its two-phase nanopolymeric delivery system can provide sensory changes such as increased shine and creaminess. Below we detail the stability tests of the spray, cream and gel formulations (Figure 17).

[084] The preliminary stability assessment demonstrated that the spray formulations (for example: for micellar water), cream (oil-free emulsion) and gel maintained normal appearances under the imposed conditions. Changes in rheological properties (physiochemical properties) represent a critical factor that indicate a predisposition for the occurrence of problems, especially in the case of emulsions, which are thermodynamically unstable systems. Such changes can interfere with the homogeneity of the cream and the coalescence of the oily droplets. Therefore, stability tests must be a priority in the development of formulations, as well as in the development of new active ingredients (nanostructured) for their efficient incorporation. In Figure 17 and Table 5 we observe the cream and gel formulations during the stability test due to pH fluctuations (without apparent and significant changes in the homogeneity of the formulations, without phase separation). Still in Table 5 we have the finest results of the analyzes, indicating the evaluation of organoleptic characteristics, viscosity and fluctuations in pH. At the end of 60 and 90 days, the gel and cream formulations presented a slightly modified/altered appearance when subjected to high temperatures in an oven (37 °C to 45 °C), and at the end of 90 days they presented a slightly modified/altered appearance to sunlight. In the analysis of apparent viscosity (cP), no changes were detected (perceptible or when reading on the viscometer) in the four (4) storage conditions, from the beginning of the analyzes at T0 until the end of the study time (90 days). We therefore conclude that the incorporation of PlateSimilis 1 (in a two-phase nanostructured system) in the formulations was effective, without altering the primary stability of the formulations (gel, cream/emulsion and spray) under the conditions evaluated. Table 5. Assessment of Organoleptic Characteristics, Viscosity and pH of Formulations with PlateSimilis Éris (Spray, oil-free cream emulsion and gel) under Different Storage Conditions.

In vitro tests and clinical tests of PlateSimilis 1 (PlateSimilis Éris - peptide derived from TB-4 functionalized with 5-HT and/or 5-HTP) following the OECD and the ANVISA Cosmetics Safety Guide

[085] Toxicity and safety tests were carried out to classify PlateSimilis 1 (PlateSimilis Éris, peptide derived from TB-4 functionalized with 5-HT and/or 5-HTP) as a safe and effective dermocosmetic ingredient, as provided for by RDC n. ° 48/2013 and Guide for Safety Assessment of Cosmetic Products, 2nd edition, ANVISA (2012). The tests carried out were as follows: permeability and opacity in bovine cornea (BCOP) - test carried out in a specialized company (InvitroCell - company specific method) in accordance with OECD - 437; phototoxicity (OECD 432) in vitro; Human epidermis reconstituted in vitro (SkinEthic RHE) EpiSkin in accordance with OECD No. 439. In addition to these safety and toxicity tests, a perceived efficacy test was carried out on volunteers to prove its action on cell renewal and migration, and support of “claim”/anti-aging, anti-wrinkle and anti-aging attributes. This item represents a succession of positive results, a promising evolution in the development of PlateSimilis 1, since the initial in vitro tests, such as: i) BigData analysis of platelet content omics tests; ii) with the selection of the sequence of the first PlateSimilis (in silico) through molecular docking and molecular dynamics, until its synthesis; iii) initial cell viability tests, first safety tests; iv) the choice of a drug delivery system to increase performance as an active ingredient for topical use, a biphasic nanopolymeric system, giving a platelet signature; v) in vitro tests in 3D and 4D culture, on human skin keratinocyte spheroids indicating the effects of PlateSimilis 1 and its delivery system (biphasic nanopolymeric) on cell renewal and regeneration processes, including the benefits in increasing the production of laminin and type I collagen; vi) testing the incorporation of PlateSimilis 1 into cosmetic formulations (gel, cream (oil-free emulsion) and spray); vii) stability tests of formulations containing PlateSimilis 1; viii) for the then in vitro safety and toxicity assessment in accordance with OECD and ANVISA. The results obtained in these 8 evaluation stages served as the basis for claiming the present invention with PlateSimilis 1 - PlateSimilis Éris (with biomimetic peptides to the Thymosin-B4 polypeptide functionalized with 5-HT and/or its biological precursor 5-HTP), for the market dermocosmetics industry.

[086] For the in vitro tests to evaluate the safety and toxicity of PlateSimilis 1, we followed the primary tests indicated in the Guide for the safety evaluation of cosmetic products and raw materials by ANVISA and OECD, which are: i) evaluation of the potential eye irritation using the BCOP method (bovine corneal opacity and permeability test); ii) in vitro phototoxicity test using the method described by OECD 432; and iii) evaluation of dermal irritation in vitro in a SKINETHIC™ RHE model (reconstructed human epidermis, EpiSkin Brazil) according to the OECD 439 method (2020). All of these tests were carried out at the Investiga Group's InVitroCell company, located in Campinas, SP. The methods used are the property of the contracted company, as mentioned in methods. We sent a 1% solution of PlateSimilis 1 in its two-phase nanopolymer system to InVitroCell for analysis. The results of the analysis of the three tests indicated the safety of PlateSimilis 1. Figure 18 shows the results of the safety reports issued by the contracted company.

[087] After the in vitro safety tests, we proceeded to clinical safety tests on volunteers, also using the services of a specialized company, Allergisa from Grupo Investiga. The following tests were carried out: i) evaluation of the potential for primary, accumulated irritability and skin sensitization of a product under controlled and maximized conditions (RIPT) with 69 volunteers/participants and the test ii) evaluation of the potential for photoallergy and skin phototoxicity of a product under controlled and maximized conditions (PHOTO) with 35 volunteers/participants. The test results indicated the safety of PlateSimilis 1, there were no reports of reactions or discomfort by participants, supporting the “claims”/attributes of dermatologically tested and hypoallergenic (Figure 19).

Clinical Efficacy Tests of PlateSimilis 1 (PlateSimilis Éris) by a specialized company - Analysis of Anti-aging effects by volunteers (25 participants)

[088] Following the clinical safety tests, we carried out clinical tests on the effectiveness of PlateSimilis 1, tests carried out at the company Allergisa of Grupo Investiga. The test chosen to attest to the effect of PlateSimilis 1 as an inducer of cell renewal was “evaluation of the effectiveness perceived by the research participant of a product under normal conditions of use”, where the following benefits/effects that PlateSimilis 1 (in biphasic nanopolymeric system) can provide according to laboratory tests in 3D and 4D culture: 1) hydration; 2) brightness and luminosity; 3) vitality; 4) signs of aging and 5) general appearance of the skin. And to support the effects of PlateSimilis 1 in in vitro tests in 3D and 4D cellular models, we correlated it with the effects found by participants in clinical tests for the anti-wrinkle, anti-aging and anti-aging attributes/”claims”, where 87% of (25) clinical test participants indicated increased hydration, which corroborates the effects of PlateSimilis 1 in stimulating the production of extracellular matrix proteins and actin filaments (a barrier effect to prevent water loss, with greater adsorption of water in the extracellular matrix); 87% of participants also indicated increased brightness/luminosity effects connected to the PlateSimilis 1 two-phase nanopolymeric system; 82% of test subjects indicated an increase in skin vitality. It is interesting to highlight that this effect perceived by the volunteers led us to validate it from a molecular point of view, and actually points out that it can be correlated to the process of cell renewal and increase in support structures such as laminin, type I collagen and f-actin ; the increase in extracellular matrix proteins, the increase in actin filaments, as well as the increase in cell proliferation and renewal justify that 70% of participants noticed a decrease in signs of aging, and 92% of participants confirmed an improvement in the general appearance of the skin . Results that support the anti-wrinkle, anti-aging and anti-aging claims/attributes for PlateSimilis 1 (Figure 20).

Statistical analysis

[089] The results obtained were analyzed using GraphPad Prism 8.0 software (Graphpad Software Inc., San Diego, CA). Statistical analysis was performed using Anova followed by t-test, when comparing only 2 different groups, and Turkey's test for multiple comparisons. All experiments were repeated at least three times and expressed as mean ± standard deviation. For all statistical tests used in the present study, the significance level established was 5% (p<0.05).

Claims (9)

1. Biomimetic Peptides characterized by consisting of the sequences represented by SEQ ID NO. 1 to SEQ ID NO. 17. 2. Dermocosmetic and bio-pharmaceutical compositions characterized by comprising at least one of the sequences represented by SEQ ID NO. 1 to SEQ ID NO. 17 and acceptable excipients for pharmaceutical and/or cosmetic use. 3. Composition according to claim 2, characterized in that it consists of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 from 0.2 to 3%, panthenol 0.2 to 3%, vegetable glycerin from 0.1 to 1.0%, phenoxyethanol from 0.1 to 1.0%, deionized water (qsp). 4. Composition according to claims 2 and 3 characterized in that it consists of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 at 1.0%, panthenol 1.0%, vegetable glycerin of 1 .0%, 0.5% phenoxyethanol, deionized water (qsp) and in spray form. 5. Composition according to claim 2, characterized in that it consists of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 from 0.2 to 3%, polawax wax from 5 to 20%, TCM from 1 to 10%, Vegetable Glycerin 1 to 10%, Vitamin C 1 to 10% or Niacinamide PC 1 to 10%, Liquid Vitamin E 0.1 to 3%, EDTA 0.01 to 2%, Chloromethylisothiazolinone /Methylisothiazolinone from 0.1 to 3%, Phenoxyethanol from 0.1 to 3% and deionized water (qsp). 6. Composition according to claims 2 and 5, characterized in that it consists of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 at 1.0%, polawax wax 12%, TCM 5%, glycerin 5% vegetable, Vitamin C 5% or Niacinamide PC 4%, liquid vitamin E 0.5%, EDTA 0.05%, Chloromethylisothiazolinone/Methylisothiazolinone 0.5%, Phenoxyethanol 0.5% and deionized water (qsp) and be in the form of a cream. 7. Composition according to claim 2, characterized in that it consists of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 from 0.2 to 3%, natrosol from 0.5 to 5%, panthenol from 0.5 to 5%, vegetable glycerin from 0.5 to 6%, Chloromethylisothiazolinone/Methylisothiazolinone from 0.1 to 3%, Phenoxyethanol from 0.1 to 3% and deionized water (qsp). 8. Composition according to claims 2 and 7, characterized in that it consists of at least one of the biomimetic peptides represented by SEQ ID NO. 1 to SEQ ID NO. 17 at 2%, natrosol 1%, panthenol 1%, vegetable glycerin 0 .5%, Chloromethylisothiazolinone/Methylisothiazolinone 0.5%, Phenoxyethanol 0.5% and deionized water (qsp) and be in gel form. 9. Use of biomimetic peptides characterized by being in the production of dermocosmetic and bio-pharmaceutical compositions and by the peptides being selected from the group comprising SEQ SEQ ID N° 1 to SEQ ID N° 17.