Compositions for tissue repair and hair growth containing an oligopeptide complex of the growth factor KGF-2, polyanion and nanospheres and their uses

Abstract

The invention relates to compositions containing biomimetic peptides derived from the growth factor KGF-2 (Keratinocyte Growth Factor-2 or fibroblast growth factor-10 (FGF-10)) in a highly stable complex system with a biomimetic polyanion biopolymer, preferably hyaluronic acid, in a biocompatible and hypoallergenic nano-bead transport system that allows assertive, high-performance transport of said oligopeptide / polyanion complex. The invention also involves a hair tonic and the use of these compositions to modulate the atypical changes associated with ageing hair in order to reactivate the hair growth cycle by coordinating and synchronising the anagen phase, the specific growth phase of the hair follicle structure, maintaining and stimulating hair growth, as well as preventing early hair loss, in a way that is healthy / biocompatible for hair structures, specifically for the hair cell microenvironment. In addition, the compositions presented here act to delay skin ageing through tissue repair.

Description

[0001] COMPOSITIONS FOR TISSUE REPAIR AND HAIR GROWTH CONTAINING AN OLIGOPEPTIDE COMPLEX OF GROWTH FACTOR KGF-2, POLYANION AND NANOSPHERES AND THEIR USES

[0002]

[0001] The invention concerns compositions containing biomimetic peptides derived from the growth factor KGF-2 (Keratinocyte Growth Factor-2 or fibroblast growth factor-10 (FGF-10)) in a high stability complex system with a biomimetic polyanion biopolymer, preferably hyaluronic acid in a biocompatible and hypoallergenic nanosphere transport system that allows for the assertive and high performance carrying of said oligopeptide / polyanion complex.The invention also relates to a hair tonic and the use of these compositions in modulating atypical changes associated with hair aging, reactivating the hair growth cycle by coordinating and synchronizing the anagen phase, a specific growth phase of the hair follicle structure, maintaining and stimulating hair growth, as well as preventing premature hair loss, in a manner that is healthy / biocompatible for the hair structures, specifically for the hair cellular microenvironment. Furthermore, the compositions presented herein act to delay skin aging through tissue repair.

[0003]

[0002] In the context of the hair growth cycle, it is necessary to detail the hair structure, composed of the hair follicle, whose structural organization leads to the formation of a (mini)channel that extends through the layers of the skin, from the dermis to the epidermis. It is in the hair follicle that cell division occurs, which subsequently gives rise to the shaft, the "hair strand" itself. Throughout its life, hair goes through three different phases. First, there is the growth phase, called anagen, which can last up to 7 (seven) years. Naturally, this follows the catagen phase, a transitional period (lasting up to three weeks) during which the hair stops growing and prepares to enter the "quiescence" / resting process. Finally, there is the telogen phase, during which hair loss can occur over the course of three months. Hair loss corresponds to a renewal in each (mini) cellular factory (the follicle) that produces the hair strand.It is important to highlight that a hair is eliminated from the follicle when it is pushed by the new hair that is emerging, thus completing the hair growth cycle, an (almost) never-ending cycle.

[0004]

[0003] According to medical experts, dermatologists and trichologists, it's normal and natural to lose an average of 100 to 150 strands of hair per day. This shedding is important, as it's a sign that a new strand is about to appear. It's important to note that not all follicles are simultaneously in the same phase of the growth and shedding cycle / life cycle. This means that from time to time, we'll lose all the hair on our head at once. While hair renewal, or shedding, is completely normal, there are certain situations in which excessive hair loss is noticed and prompts us to pay closer attention.

[0005] For several years, hair loss has appeared to be occurring more frequently, being one of the most common complaints made to dermatologists by both male and female patients. This is especially true after the pandemic caused by the SARS-CoV-2 virus, which leads to significant hair loss, especially in women. Recent data indicate that female hair loss has become a critical dermatological issue, rising from an incidence of 30% to 400% (Cline A, Kazemi A, Moy J, Safai B, Marmon S. A surge in the incidence of telogen effluvium in minority-predominant communities heavily impacted by COVID-19. Journal of the American Academy of Dermatology. Published online December 2020). Several studies have also begun to show that hair loss appears to occur more intensely during summer and fall and less intensely in spring, as if it were a seasonal and universal condition.Researchers at Johns Hopkins University have managed to establish a direct link between the “concept of seasonal hair loss and the human population,” proving that such a correlation does indeed exist (Hsiang EY, Semenov YR, Aguh C, Kwatra SG. Seasonality of hair loss: a time series analysis of Google Trends data 2004-2016. Br J Dermatol. 2018;178(4):978-979. doi: 10.1111 / bjd.16O75.) What seems to happen is that between summer and autumn, a large portion of the follicles are at the height of the telogen phase, causing hair loss to occur during this transition period, associated with the fact that hair growth and loss depend on variations in sunlight. The pituitary gland (responsible for regulating the activity of other glands) detects this variation and alters the levels of melatonin and prolactin (hormones) produced, and this directly reflects on the hair cycle, which is forced into the shedding phase (telogen) and results in the hair shaft falling out three months later.There's also the question of evolutionary theory, which suggests that hair's function is associated with providing warmth, a function more necessary in winter than in summer. Considering suits, special care should be taken with hair during summer and spring.

[0006]

[0005] However, you should pay attention when this fall is not so natural, and you notice that the hairs are falling drastically, or in “free fall”. You should be aware of an exaggerated fall to know how to identify adverse conditions.

[0007] Several reasons may be associated with the cause or origin of hair loss considered atypical, such as aging, illness, surgery, post-COVID-19, depression, fatigue, medications, dietary changes, fluctuations in testosterone levels, and, in women, postpartum. This hair loss can be identified and classified as alopecia areata (a multifactorial autoimmune condition) or androgenetic alopecia (common baldness). In the latter case, the thinner / less dense hairs present increasingly shorter cycles, causing continuous and excessive hair loss. Both conditions are now treatable with the supervision of specialist doctors, using solutions containing active ingredients such as growth factors and caffeine, mainly finasteride (Propecia®), administered orally, 17-α-estradiol (Avieis®), and minoxidil (Rogaine®) administered topically (Wiedemeyer K., Schill WB., Loser C.Diseases on Hair Follicles Leading to Hair Loss Part I: Nonscarring Alopecias. Dermatology for the Clinician, July • August, p. 209-214, 2004).

[0008]

[0007] However, existing treatments, despite leading to significant improvements, are not effective in curing this condition. These treatments have variable effectiveness among individuals; not everyone responds in the same way to the aforementioned treatments. Furthermore, stopping them leads to hair loss returning. Other frequently reported limitations of these treatments are the appearance of local irritations such as itching or dry skin and flaking of the scalp after chronic application of minoxidil or α-estradiol lotions and decreased libido and erectile dysfunction, in the case of finasteride (Avieis ® drug package insert, available at http: / / www.medicinanet.com.br / bula / 744 / avicis.htm, accessed on November 19, 2012; Propecia® drug package insert, available at http: / / www.medicinanet.com.br / bula / 4314 / propecia.htm, accessed on November 19, 2012; Rogaine® drug package insert, available at http: / / www.medicinanet.com.br / bula / 4444 / regaine_2.htm, accessed on August 21, 2017).

[0009]

[0009] Therefore, it is important to develop new bioactive agents and new cosmetic / medicine formulations that can be used to prevent, delay or treat alopecia.

[0010]

[0010] It's worth noting that the hair shaft (strand) is made of keratin, a fibrous protein with a structural function. To reverse any adverse conditions related to hair loss, the key is to focus on the scalp. This region provides access to the follicle's central structures and must be stimulated and adequately nourished. Consider solutions associated with the essential shampoo to maintain balanced scalp oiliness, preventing the hair follicles from clogging with excess sebum. Formulations that increase contact with the scalp, such as tonics and hair serums, are recommended.

[0011]

[0011] Seeking more effective formulations, the present invention developed dermocosmetic and biopharmaceutical compositions that present high efficacy in wound healing and hair growth through the intensification of intercellular communication to induce re-epithelialization and maintain the integrity and functionality of the hair follicle structure. The formulations comprise at least one biomimetic oligopeptide analogous to keratinocyte growth factor-2 (KGF-2), stabilized by binding to the polyanion biopolymer hyaluronic acid and carried in biocompatible and hypoallergenic nanospheres that allow the assertive and high-performance carrying / transport of said oligopeptide / polyanion complex.

[0012]

[0012] From the point of view of biophysical studies, structural characterization and thermal stability of peptides, attempts to predict the secondary structure in oligopeptide chains are not completely successful (e.g., Rost B, Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993. 20;232(2):584-99. doi:

[0013] 10.1006 / jmbi.1993.1413; Pignataro MF, Herrera MG, Dodero VI. Evaluation of Peptide / Protein Self-Assembly and Aggregation by Spectroscopic Methods. Molecules. 2020. 21 ;25(20):4854. doi: 10.3390 / molecules25204854; Silva-Lucca RA, Andrade SS, Ferreira RS, Sampaio MU, Oliva ML. Non-leafed studies of the cysteine ​​protease baupain, a papain-like enzyme from Bauhinia forficata leaves: effect of pH, guanidine hydrochloride and temperature. Molecules. 2013. 24;19(1 ):233-46. doi: 10.3390 / moleculesl 9010233.), but significant advances have been made in recent decades with powerful tools such as AlphaFold (DeepMind, Google) that uses artificial intelligence and three-dimensional augmented reality models.One possible reason that justifies this temporal “gap” in biophysical and biochemical responses is that tertiary interactions between amino acid residues that are not close to each other in the linear sequence are important in the stabilization of secondary structural elements (Bryngelson JD, Wolynes PG. Spin glasses and the statistical mechanics of protein folding. Proc Natl Acad Sci US A. 1987;84(21 ):7524-8. doi: 10.1073 / pnas.84.21.7524; Wand AJ. Deep mining of the protein energy landscape. Struct Dyn. 2023. 27;10(2):020901 . doi: 10.1063 / 4.0000180). This implies that the existence of a stable secondary structure, as observed from native coordinates, requires long-range stabilizing interactions between portions of the polypeptide chain not close to each other in linear sequence.Such a structural stability maneuver can be provided by the interaction of peptides with a dual interaction system, such as polyanion polymers such as physiological ones, hyaluronic acid, heparan sulfate and others such as sucrose octasulfate (SOS) and inositol hexaphosphate (IHP) and biocompatible and thermo-adjustable polymeric nanospheres.

[0014]

[0013] Folding / organization studies of immunogenic peptide fragments of proteins in aqueous solution and related peptide models with interactions with polyanions (Krittanai C, Johnson WC. Correcting the circular dichroism spectra of peptides for contributions of absorbing side chains. Anal Biochem. 1997. 1 ;253(1 ):57-64. doi:

[0015] 10.1006 / abio.1997.2366; Pellegrini L, Burke DF, von Delft F, Mulloy B, Blundell TL. 2000. Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature 407:1029-1034; Derrick T, Grillo AO, Vitharana SN, Jones L, Rexroad J, Shah A, Perkins M, Spitznagel TM, Middaugh CR. Effect of polyanions on the structure and stability of repifermin (keratinocyte growth factor-2). J Pharm Sci. 2007;96(4):761 -76. doi: 10.1002 / jps.20797.) also showed that peptide fragments can have significant induced stability. Quantitative determinations of secondary structure equilibrium constants for peptide fragments can be used to assess the contribution to beta (|3) organization, with the folding dynamics of peptides and proteins established by the probability of microdomain-microdomain coalescence used to determine a likely rate of collision-diffusion folding / organization.

[0016]

[0014] In a more detailed analysis of biophysical methods for structural characterization of proteins and peptides, already mentioned previously, circular dichroism (CD) spectroscopy has become the method of choice for monitoring the secondary structure of peptides and proteins as a function of solvent and changes in pH and temperature. Researchers measure CD spectra and analyze secondary structure using several available computer programs (Yang, JT, Wu, CC, and Martinez, HM (1986) Methods Enzymol. 130, 208-269; Krittanai C, Johnson WC. Correcting the circular dichroism spectra of peptides for contributions of absorbing side chains. Anal Biochem. 1997. 1;253(1):57-64. doi: 10.1006 / abio.1997.2366; Wadhwani P, Sekaran S, Strandberg E, Bürck J, Chugh A, Ulrich AS. Membrane Interactions of Latarcins: Antimicrobial Peptides from Spider Venom. Int J Mol Sci. 2021, 21;22(18):10156. doi: 10.3390 / ijms221810156.).The analyses require measurement of a far-UV CD spectrum, where the CD (under incident circularly polarized light) is dominated by amide chromophores. However, some amino acid side chains are known to absorb light in the far-UV region, causing interference with the amide. Far-UV CD studies of several proteins and peptides have demonstrated unusual CD spectra due to aromatic side chains. In most cases, tyrosine side chains are the sources of interference. Some researchers have performed theoretical studies and calculated the rotational force resulting from the interaction of these side chains with the electronic transitions of the peptide bond (Krittanai C, Johnson WC. Correcting the circular dichroism spectra of peptides for contributions of absorbing side chains. Anal Biochem. 1997. 1;253(1):57-64. doi: 10.1006 / abio.1997.2366).

[0017]

[0015] Another technique complementary to CD and widely used for the biophysical characterization of proteins and peptides is the steady-state fluorescence acquisition method. Fluorescence spectroscopy is a versatile tool for evaluating the self-assembly of peptides and proteins. Its high sensitivity, preservation of the entire sample during analysis, and the possibility of real-time measurements allow for the evaluation of the process of monomer aggregation to the formation of large assembled structures. Different approaches can be used to evaluate protein and peptide aggregation, such as the presence of intrinsic fluorophore groups like the aromatic amino acids tyrosine, tryptophan, and phenylalanine. Tryptophan and tyrosine can be excited between 280 and 285 nm, depending on the polarity or pH of the solvent, which can alter their ionic state.The maximum emission wavelength in water is 350 nm for tryptophan and 304 nm for tyrosine (Karplus M, Weaver DL. Protein folding dynamics: the diffusion-collision model and experimental data. Protein Sci. 1994;3(4):650-68. doi: 10.1002 / pro.5560030413; Nassar R, Dignon GL, Razban RM, Dill KA. The Protein Folding Problem: The Role of Theory. J Mol Biol. 2021. 1;433(20):167126. doi: 10.1016 / j.jmb.2021 .167126.).

[0018]

[0016] Furthermore, to avoid tyrosine emission, tryptophan can be selectively excited at 295 nm. Phenylalanine has the shortest absorption and emission wavelengths (Àex = 260 nm and Àem = 282) and the lowest quantum yield. Tryptophan has the highest quantum yield (0.14 over 0.13 for tyrosine and 0.02 for phenylalanine), which is the photon emission efficiency defined by the ratio of the number of photons emitted to the number of photons absorbed. Importantly, tryptophan is highly sensitive to the emission spectra of the local environment. In this sense, its emission spectrum is blue-shifted when the solvent polarity or solvent exposure is decreased, as happens when it is “buried” hidden in a protein inner core.One of the most classic examples is tryptophan 48 of azurin, which is fully buried (Δem = 308 nm) and shows red shifts to Δem = 350 nm when the polarity of the environment increases, as occurs in denaturation. The magnitude of the shift depends not only on the water molecules surrounding the tryptophan but also on the orientation of the indole ring (Karplus M, Weaver DL. Protein folding dynamics: the collision-diffusion model and experimental data. Protein Sci. 1994;3(4):650-68. doi: 10.1002 / pro.5560030413; Nassar R, Dignon GL, Razban RM, Dill KA. The Protein Folding Problem: The Role of Theory.

[0019]

[0017] In summary, tryptophan-containing proteins and peptides exhibit a fluorescence emission that is a linear combination of the emission of each tryptophan present in their sequence, and the maximum spectrum can vary from 308 to 350 nm, depending on the environment and orientation of the indole ring. Because of this, many algorithms have been designed to decompose the emission spectra of tryptophan proteins into their different components. In the case of tyrosine, the change from a polar to a nonpolar environment produces an increase in its fluorescence intensity. It is relevant to mention that when the tyrosine anion is formed, the maximum emission occurs at 340 nm (Karplus M, Weaver DL. Protein folding dynamics: the diffusion-collision model and experimental data. Protein Sci. 1994;3(4):650-68. doi: 10.1002 / pro.5560030413; Nassar R, Dignon GL, Razban RM, Dill KA. The Protein Folding Problem: The Role of Theory. J Mol Biol. 2021 ;433(20): 167126. doi: 10.1016 / j.jmb.2021 .167126; Fowler, WCIntrinsic Fluorescence in Peptide Amphiphile Micelles with Protein-Inspired Phosphate Sensing. Biomacromolecules 2022, 23, 11 , 4804-4813. https: / / doi.Org / 10.1021 / acs. biomac.2c00960).

[0020]

[0018] Interestingly, tryptophan fluorescence can be quenched by intrinsic quenchers present in the protein, such as amine, carboxylic acid, disulfide, and histidine groups, or external ones by the addition of iodide and acrylamide, which are electron-rich species. Additionally, the time a fluorophore spends in the excited state before emitting a photon and returning to the ground state, called its lifetime, is sensitive to the local environment, varying in the range between 1 and 6 ns. Tyrosine fluorescence is useful for studying protein self-assembly when there is no tryptophan in the protein sequence. Furthermore, tyrosine is susceptible to quenching by energy transfer to nearby tryptophan residues or by tyrosine / tyrosine homotransfer (Fowler, WC Intrinsic Fluorescence in Peptide Amphiphile Micelles with Protein-Inspired Phosphate Sensing. Biomacromolecules 2022, 23, 11 , 4804-4813. https: / / doi.Org / 10.1021 / acs.biomac.2c00960).

[0021]

[0019] Rescuing the state of the art of the conformation or “folding” adopted by a protein, it is clear that this organization is governed by its amino acid sequence, reinforcing an underlying stereochemical code for protein folding. There are extensive scientific studies that describe the loss of folding and refolding of many globular proteins in terms of the two-state model (native^denatured) (Silva-Lucca RA, Andrade SS, Ferreira RS, Sampaio MU, Oliva ML. Unfolding studies of the cysteine ​​protease baupain, a papain-like enzyme from Bauhinia forficata leaves: effect of pH, guanidine hydrochloride and temperature. Molecules. 2013. 24;19(1):233-46. doi:

[0022] 10.3390 / moleculesl 9010233).

[0023]

[0020] Explaining how a protein synthesized as a linear polypeptide rapidly assembles into its complex, biologically active structure, even with a large number of potential conformational states, remains an intriguing and unsolved problem that has stimulated research in this area over the years. Indeed, knowledge of the properties of protein folding and structural rearrangements in peptides, as well as the properties of intermediate species, is important for understanding the role played by intermediate states in protein folding and indicates how dramatic consequences can be generated by mutant proteins and lack of peptide (or oligopeptide) stability, leading to genetic diseases, biotechnological problems, and difficulties in perfecting the in silico design of biomimetic peptides, potential candidates for dermocosmetic active ingredients and biopharmaceutical drugs.One of the intermediate structural states is the molten globule (MG) state, a non-native compact state of a protein or peptide, considered the most typical intermediate folding and has been considered as a general intermediate of protein and peptide (oligopeptide and polypeptide) folding. This state is characterized by the presence of a significant amount of secondary structure, lack of more specific tertiary structures associated with the folding / tight packing of amino acid side chains, and the presence of a loosely folded / packed hydrophobic core that is accessible to the molecules of the involved solvent (Silva-Lucca RA, Andrade SS, Ferreira RS, Sampaio MU, Oliva ML. Unfolding studies of the cysteine ​​protease baupain, a papain-like enzyme from leaves of Bauhinia forficata: effect of pH, guanidine hydrochloride and temperature. Molecules. 2013. 24; 19(1 ):233-46. doi:.

[0024] 10.3390 / moleculesl 9010233; Pignataro MF, Herrera MG, Dodero VI. Evaluation of Peptide / Protein Self-Assembly and Aggregation by Spectroscopic Methods. Molecules. 2020 Oct 21 ;25(20):4854. doi: 10.3390 / molecules25204854).

[0025]

[0021] From a biochemical point of view, MG structures have been observed in different proteins and peptides under different conditions, such as temperature variations, low and high pH, ​​or low and high concentrations of denaturing agents such as urea and guanidine hydrochloride (GdnHCI). The most widely studied protein, and representative of this functional intermediate folding state “molten globule” (MG), is α-lactalbumin (Silva-Lucca RA, Andrade SS, Ferreira RS, Sampaio MU, Oliva ML. Unfolding studies of the cysteine ​​protease baupain, a papain-like enzyme from leaves of Bauhinia forficata: effect of pH, guanidine hydrochloride, and temperature. (keratinocyte growth factor-2).

[0026]

[0022] Many proteins can exist in alternative conformational forms with each distinguishable state exhibiting unique functional properties. One class of proteins in which some members exhibit this property are the fibroblast growth factors (FGFs). The FGF family contains 23 currently recognized homologs, most of which exhibit more conventional stability properties with well-defined native states under physiological conditions (Xie Y, Su N, Yang J, Tan Q, Huang S, Jin M, Ni Z, Zhang B, Zhang D, Luo F, Chen H, Sun X, Feng JQ, Qi H, Chen L. FGF / FGFR signaling in health and disease. Signal Transduct Target Then 2020 Sep 2;5(1 ): 181 . doi: 10.1038 / S41392-020-00222-7).

[0027]

[0023] Keratinocyte growth factor-1 (KGF-1 or FGF-7), however, exhibits only moderate stability somewhat similar to that of FGF-1. FGF-1 exists under physiological conditions (pH 5-8, 0-0.2 M salt, and temperature 30-50°C) in a molten globule state with near-native secondary structure content, disrupted tertiary contacts, and the ability to bind nonpolar dyes. This form of the protein binds to biological membranes and may be involved in the protein's unusual transport properties (biotrafficking), which include exit from cells without the use of a conventional signal peptide. Many FGFs bind to various polyanions that may be involved in their functional presentation to cell surface protein kinase receptors; their ability to move into cells when fused to some toxin polyanions leads to their inactivation, suggesting alternative roles for polyanion binding.FGF-1 also exhibits increased resistance to proteolysis, oxidation, and thermal unfolding (loss of folding) when bound to various polyanions (such as heparin) (Rosengart TK, Johnson WV, Friesel R, Clark R, Maciag T. 1988. Heparin protects heparin-binding growth factor-1 from proteolytic inactivation in vitro. Biochem Biophys Res Co. 152:432-440; Chen BL and Arakawa T. 1996. Stabilization of recombinant human keratinocyte growth factor by osmolytes and salt. H, Chen L. FGF / FGFR signaling in health and disease.

[0028]

[0024] Another member of the FGF family, FGF-10, also widely known as KGF-2 (keratinocyte growth factor 2), exhibits significant structural changes under mild conditions that are modulated by polyanions, but these changes are distinct from those observed in FGF-1, but suggest that the dynamic structural behavior of KGF-2 may also have functional significance. KGF-2 has been reported to show similar biological and biochemical behavior to KGF-1 (Igarashi M, Finch PW, Aaronson SA. 1998. Characterization of recombinant human growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7). J Biol Chem 273:13230-13235; Xie Y. Su N, Yang J, Tan Q, Huang S, Jin M. Ni Z, Zhang B, Zhang D, Luo F, Chen H, Sun X, Feng JQ, Qi H, Chen L. FGF / FGFR signaling in health and disease.The structures of KGF-1 and KGF-2 show 57% homology / identity; both act in paracrine signaling (signaling involving neighboring cells / signals transmitted over short distances); they are naturally produced by fibroblasts / dermis with exclusive action on their receptors in keratinocytes / epidermis (inside / out action); both factors are recruited and active in the process of cell renewal, remodeling of the extracellular matrix and cytoskeleton (providing support and protection, respectively), and tissue repair. KGF-1 has a site of interaction with heparan sulfate and is produced mainly in the initial period of cellular renewal and regeneration processes, promoting the proliferation, migration, and differentiation of keratinocytes. It also acts indirectly on angiogenesis and cellular remodeling, inducing the production of extracellular matrix (mainly collagen) in the late period of tissue repair, contributing to tissue reorganization.KGF-2 has preventive and curative effects on the tissue repair process, with strong mitogenic activity in epidermal cells (promoting proliferation, migration, and differentiation of keratinocytes) and activating the cell granulation process, stimulating essential communication / connection between keratinocytes and fibroblasts, stimulating hair growth (Jang JH. Stimulation of human hair growth by the recombinant human keratinocyte growth factor-2 (KGF-2). Biotechnol Lett. 2005 Jun;27(11 )749-52. doi: 10.1007 / s10529-005-5624-y; Chen K, Rao Z, Dong S, Chen Y, Wang X, Luo Y, Gong F, Li X. Roles of the fibroblast growth factor signal transduction system in tissue injury repair. Bums Trauma. 2022, 23;10:tkac005. doi: 10.1093 / burnst / tkac005.).

[0029]

[0025] In addition to their direct and indirect physiological roles in angiogenesis, wound healing, and embryonic development, these growth factors have been postulated to have potential therapeutic significance, especially due to their involvement in the processes of wound healing and hair growth. As important as it is to note the similarities of KGF-2 with many other members of the FGF family, it is even more important and strategic to recognize the differences between them. In particular, the lack of a MG state as observed in some other FGF proteins may be significant for their interactions with unique receptors and biological activities. However, such an absence of the intermediate MG structural state in biomimetic peptides derived from these growth factors has not yet been confirmed (Jang JH. Stimulation of human hair growth by the recombinant human keratinocyte growth factor-2 (KGF-2). Biotechnol Lett. 2005 Jun;27(1 1 )749-52.doi: 10.1007 / s10529-005-5624-y; Xie Y, Su N, Yang J, Tan Q, Huang S, Jin M, Ni Z, Zhang B, Zhang D, Luo F, Chen H, Sun X, Feng JQ, Qi H, Chen L. FGF / FGFR signaling in health and disease. Signal Transduct Target Then 2020 Sep 2;5(1 ):181. doi: 10.1038 / s41392-020-00222-7; Chen K, Rao Z, Dong S, Chen Y, Wang X, Luo Y, Gong F, Li X. Roles of the fibroblast growth factor signal transduction system in tissue injury repair. Bums Trauma. 2022, 23;10:tkac005. doi: 10.1093 / burnst / tkac005).

[0026] Given the broad and strategic physiological functions of KGF-2, biomimetic peptides derived from KGF-2 have been designed and synthesized in order to recapitulate the full potential of the biological activity of the original protein, rationalizing the peptide size, structural stabilization to guarantee biological function through interaction with polyanions and an intelligent delivery system capable of delivering the stabilized active directly to the target, in a sustained and bio-guided manner.

[0030]

[0027] The present invention shows results of the complex formed by a biomimetic peptide derived from KGF-2 structurally stabilized in a polyanion (bio)polymer (hyaluronic acid) inserted in a delivery system based on biocompatible polymeric nanospheres, for use in multifunctional dermocosmetics, intended for tissue repair and hair growth applications. The invention also provides a hair tonic containing these peptides and capable of acting in the induction of hair growth.

[0031]

[0028] Thus, the present invention deals with a solution capable of modulating atypical changes associated with hair aging in order to reactivate the hair growth cycle by coordinating and synchronizing the anagen phase, a specific phase of hair growth in the structure of the hair follicle, maintaining and stimulating hair growth and also preventing premature hair loss, and in a healthy / biocompatible manner for the hair and the hair cellular microenvironment.

[0032]

[0029] Using advanced technologies for biomimetic peptide design, with alignment of in silico analysis using molecular docking techniques, molecular dynamics and high-performance delivery system, biomimetic peptides derived from KGF-2 structurally stabilized in a polyanion biopolymer, hyaluronic acid, in a nanosphere transport system, showed prominent and surprising results of high performance and biocompatibility in in vitro efficacy tests.

[0033] Increasingly elaborate scientific and clinical studies have shown that if the follicular structure is viable, hair has regenerative potential and can reactivate the hair growth cycle, initiating the anagen phase (the hair follicle growth phase), even after experiencing drastic hair loss. Deep within the hair follicle structure is a reservoir of stem cells that maintain the intense cycle of cell proliferation and hair shaft production. We are born with all the hair follicles we will ever have, but aging, disease, changes in testosterone levels, genetic misfortune, and even COVID-19 can kill the stem cells in their reservoir at the base of the follicle structure (where hair strands are produced).Once these stem cells are gone, your hair will also begin the shedding cycle, because there will be no way to start the growth cycle without the necessary cellular machinery.

[0034]

[0031] However, if there is still a minimal follicular structure with viable stem cells, there are solutions that can trigger the regeneration process, which is the innate ability of living organisms to repair themselves through the beneficial use of their own resources or processes. Preserving the regeneration properties of the skin, a complex integumentary system, is a fundamental strategy for overcoming the irregular processes that begin with aging. With advancing age and depending on lifestyle, the metabolism and function of cells decrease or change, and their ability to interact with each other deteriorates, weakening the regeneration process. In hair, this regeneration cycle is inevitably reflected in color (appearance of gray hair), hair loss, and thinning. Andrade, SA; Faria, AVS; Fuhler, GM; Peppelenbosch, MP; Ferreira-Halder, CV. Biotech-educated Platelets: beyond tissue regeneration 2.0. in press. 2020.

[0035]

[0032] Given this scenario between hair growth and loss cycles, the present invention surprisingly highlights the stabilization process of the biomimetic peptide derived from KGF-2 (a factor closely related to the innate capacity to activate the tissue regeneration process, with consequent participation in the wound healing process, acting as a biostimulator of hair growth), its coordinated thermal stability through the interaction with hyaluronic acid (polyanion polymer) and its insertion into thermo-adjustable polymeric nanospheres. Some precision nanopolymeric delivery systems are developed to ensure dermatological safety and to overcome limitations of topical application, allowing greater penetration through the cutaneous biological barriers - microenvironmental / microbiota and cellular - which are heterogeneous between populations and change due to skin types and the aging process (Gould, J. Superpowered skin. Nature, v.563 (2018)). The results regarding the biophysical characterization of the stability of the KGF-2-derived peptide demonstrated in this invention surprisingly demonstrate the presence of the intermediate state MG with the interaction with the hyaluronic acid polyanion that provides greater structural and thermal stability with the amplification of the benefits related to the KGF-2 protein of origin, which are superior in relation to the solutions widely used in classic dermocosmetic formulations intended for hair growth.

[0036]

[0033] We can cite here, as an example, the study of the structural stability of KGF-2 (original protein) provided by the interaction with other polyanions such as heparin, which, contrary to what was demonstrated in the aforementioned invention, the study did not indicate the presence of the intermediate structural form MG, thus demonstrating the differential of the present invention, highlighting the promising use of the oligopeptide complex derived from the KGF-2 growth factor and the hyaluronic acid polyanion inserted in the high-performance intelligent delivery system as a potential biomimetic active ingredient intended to induce the tissue regeneration and repair process, with consequent induction of the hair growth cycle.

[0037]

[0034] In searches carried out in patent databases, a document (US 201 1 / 0160131 A1) was found that describes the use of growth factor-mimetic peptides for their cosmetic applications and in “nanosomosis” systems, but there is no description of interaction with polyanions to increase the structural and / or thermal stability of the described peptide sequences. In the searches, patents were found that cite the use of the peptide described here in treatments for eye disorders (WO2022 / 204331) and coating of medical devices (US20060024347). However, none of the documents describe the use of the peptide of the present invention in hair growth or its stabilization through the use of polyanion.

[0035] In view of the above, this invention provides, in a novel manner, a peptide derived from the growth factor KGF-2, which mimics its biological activity and which comprises the amino acid sequence SEQ ID No. 1 and SEQ ID No. 2.

[0038]

[0036] In order to develop peptides with actions identical to the naturally occurring growth factor KGF-2 and with more significant characteristics, such as activity, skin penetration and stability than the naturally occurring KGF-2, the inventors carried out intensive research. As a result, the present invention describes two peptides that mimic the growth factor KGF-2 with excellent characteristics based on the amino acid sequence of naturally occurring growth factors.

[0039]

[0037] The peptides of the present invention include amino acid sequences derived from human KGF-2 represented by SEQ ID No. 1 and SEQ ID No. 2, as well as their derivatives containing Acetyl, Palmitoyl or Myristoyl groups in their N-terminal portions.

[0040]

[0038] The peptides of the invention can be prepared by conventional chemical synthesis processes known as solid phase synthesis techniques (Merrifield, J. Amer: Chem. Soc. 85: 2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)).

[0041]

[0039] .With the identified biomimetic peptide sequences, added to the interaction with the hyaluronic acid polyanion to increase the structural stability of the oligopeptide and the encapsulation in a nanopolymeric system (100 ± 200 nm) for effective and high-performance transport, the present invention provides an oligopeptide / polyanion / nanosphere complex* that provides: (1) biocompatibility (low toxicity / dermatological safety); (2) design versatility; (3) stability; (4) high encapsulation efficiency; (5) high permeation, capacity for interaction with biological systems in terms of affinity and flexibility; (6) assertive biomolecular signaling, specific target and (7) biological kinetics / controlled release

[0042]

[0040] The integration of this biocompatible delivery system with the carrier of the biomimetic peptide complex* derived from KGF-2 and polyanion accelerates epithelial cell division, contributing to rapid and coordinated cell proliferation. Like its parent protein / mirror, KGF-2, the complex* plays a fundamental role in the cell adhesion process, essentially functioning as a bridge (increasing intercellular crosstalk) between cell "generations" (stem cells and differentiated cells) in the initial stage of the hair growth cycle. In addition to improving skin elasticity, promoting hair growth, and accelerating wound healing, the complex* can be used with a variety of dermocosmetic applications, but is not limited to these.

[0043]

[0041] Furthermore, surprisingly, in the present invention, the complex* (peptide / polyanion / nanospheres) proved to be indisputably superior in terms of biocompatibility and toxicological safety than the market benchmark solution (Minoxidil) widely used in hair growth formulations.

[0044]

[0042] Therefore, in addition to identifying the amino acid sequences of the biomimetic oligopeptide to the growth factor KGF-2 (SEQ ID NO: 1 and SEQ ID NO: 2), the peptide / polyanion complex* and the combination with polymeric nanocarriers (nanospheres) as transport vehicles can be understood as an inventive activity, since they promote an increase in the structural and thermal stability of the biomimetic oligopeptide and increase its permeation into the skin, providing high performance in a sustained and coordinated manner in the processes of cell proliferation and activation of the hair growth process.

[0045] BRIEF DESCRIPTION OF THE FIGURES

[0046]

[0043] Figure 1. Temperature-dependent conformational changes of the growth factor-derived oligopeptide KGF-2 in 10 mM phosphate / borate / acetate (PBA), pH 7.4. (A) Far-UV CD spectra of the oligopeptide (1%) alone and in complex with the polyanion hyaluronic acid (10-fold concentrated) over temperature variations from 37°C to 70°C. (B) Relative changes of the mean residual ellipticity (MRE) values ​​at 210 nm (squares) and 222 nm (circles) as a function of temperature. (C) Sigmoidal fitting of these points gives a midpoint around 70°C for this transition.

[0047]

[0044] Figure 2. Structure loss / unfolding of the complex* containing KGF-2-derived oligopeptide induced by GdnHCI in 10 mM phosphate / borate / acetate (PBA), pH 7.0, 25°C, as a function of GdnHCI concentration. (A) Intrinsic fluorescence emission spectra were measured in the absence and presence of chaotropic agent. (B) Transition curves normalized to GdnHCI-induced transition. The fractions with structure loss (“unfolded”) were calculated by equation 1 using the fluorescence emission maxima (Àmax, squares) and the mean residual ellipticity values ​​at 222 nm (circles). Sigmoidal fits to CD ( ) and fluorescence data ( — ) determined the midpoints at 4.1 ± 0.2 M and 4.3 ± 0.2 M GdnHCI. Equation 1 **DRONE® KGF-2 = peptide / polyanion / nanosphere complex

[0048]

[0045] Figure 3. Analysis of cellular biocompatibility by MTT reduction (A) and incorporation of neutral red dye (B) under treatment with a biomimetic polyanion biopolymer complex (analyses at 24h and 48h). Analysis of Cell Viability in keratinocytes (HaCat / epidermis) and fibroblasts (HFF-1 / dermis) from human skin. Treatment with **DRONE® KGF-2 (0.5% and 1%) at two different concentrations was performed separately and in isolation according to the conditions of each cell type. The tests were performed simultaneously in comparison with the market benchmark solution (minoxidil (5%)). The analyses were monitored for 24h and 48h, where **DRONE® KGF-2 proved to be safe and biocompatible superior to the market benchmark solution, presenting +25% biocompatibility. For the MTT reduction test, values ​​below 70% cell viability are considered toxic. Experiments performed in triplicate. The statistical test used was t-test (*<0.001, **DRONE® KGF-224h and 48h versus control) GraphPad PRISM5.0 (La Jolla, CA).

[0049]

[0046] Figure 4. Analysis of organization, compaction, proliferation and cell renewal of skin cells (epidermal keratinocytes, HaCat lineage, 5 x 10 3 cells) in a 3D / spheroid system on Nunclon Sphera 3D culture system plates (ThermoFisher Scientific, USA). Newly formed spheroids (Oh) were treated with 2% **DRONE® KGF-2. Control group spheroids were not exposed to treatment and were maintained with appropriate medium (DMEN) and supplementation. 3D culture was monitored for 120 h, observed under an optical microscope, and the spheroid compaction capacity was measured using ImageJ (scale 100 pm - magnification 10x). Results are representative of n=4. Statistical significance was assessed by one-way ANOVA and Student's t-test.

[0050]

[0047] Figure 5. **DRONE® KGF-2 induces the expression of keratin 5, a keratin intermediate filament that induces connection and uniformity in the epidermis. Analysis in epidermal keratinocytes (HaCat) (coculture cell model). HaCat cells were exposed to **DRONE® KGF-2 (1%) for 48h. Then, the cells were lysed, and the monoclonal antibody marker keratin 5 was evaluated by western blot. GAPDH was used as a normalizer and endogenous control. The tests were performed simultaneously in comparison with the market benchmark solution (minoxidil (5%)), where **DRONE® KGF-2 induced an increase in keratin 5 expression 40% more than the market benchmark solution. GraphPad ANOVA statistics. Representative images of three independent experiments. Experiments performed in triplicate. Statistical testing used t-test (*<0.001 ) GraphPad PRISM5.0 (La Jolla, CA).

[0051]

[0048] Figure 6. **DRONE® KGF-2 - Analysis of cell proliferation and renewal of dermal fibroblasts (HFF-1) and epidermal keratinocytes (HaCat) in a 3D / spheroid co-culture system, under treatment with 1% DRONE® KGF-2, control with medium supplemented without treatment, with laminin coat. The tests were performed simultaneously in comparison with the market benchmark solution (minoxidil (5%)). (A) DRONE® KGF-2 induces downregulation of E-cadherin and upregulation of β-cadherin in epidermal keratinocytes. (B) ERK had its proliferation controlled in keratinocytes. (C) DRONE® KGF-2 induces the expression of P-38 in cellular maintenance in keratinocytes. (D) DRONE® KGF-2 in the coordinated control of p53 in keratinocytes. The analyses were monitored for 6 days, where **DRONE® KGF-2 proved to be more effective in stimulating the production of type I collagen by +140% more than the market benchmark solution.The images also reinforce the biocompatibility and safety provided by **DRONE® KGF-2, unlike the market benchmark. Formation of skin keratinocyte spheroids in a feeder system with skin fibroblasts (support and feeding bed), 2D and 3D co-culture monitored for 4 days.

[0052]

[0049] Figure 7. **DRONE® KGF-2 is effective in activating the MEK-ERK and p38 signaling cascade resulting from the interaction with its tyrosine kinase receptor, in epidermal keratinocytes (feeder / coat co-culture cellular model with laminin). This pathway is essential for activating the processes of cell proliferation, migration and renewal. HaCat cells were exposed to the polyanion complex for high structural and thermal stability of **DRONE® KGF-2 inserted in nanospheres (1%) for 48h. Then, the cells were lysed, and the markers E-cadherin, N-cadherin, [3-Catenin, p38 and p53 (monoclonal antibodies) were evaluated by western blot. GAPDH was used as a normalizer and endogenous control. GraphPad ANOVA statistics. Representative images of three independent experiments. GraphPad ANOVA statistics. Representative images of three independent experiments. Experiments performed in triplicate. The statistical test used was t-test (*<0.001) GraphPad PRISM5.0 (La Jolla, CA).

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054]

[0050] The peptides derived from keratinocyte growth factor 2, KGF-2 of the present invention are:

[0055] (I) SEQ ID NO. 2 [Acetyl]- (X)YASFNWQHNGRQMYVAL(X)-CONH2

[0056] (II) SEQ ID NO. 3 [Acetyl]- (X)YKSKKGGWTHG(X)-CONH2

[0057] (III) SEQ ID NO. 2 [Palmitoyl]- (X)YASFNWQHNGRQMYVAL(X)-CONH2

[0058] (IV) SEQ ID NO. 3 [Palmitoyl]-(X)YKSKKGGWTHG(X)-CONH2

[0059] (V) SEQ ID NO. 2 [Miristoyl]- (X)YASFNWQHNGRQMYVAL(X)-CONH2

[0060] (VI) SEQ ID NO. 2 [Miristoyl]- (X)YKSKKGGWTHG (X)-CONH2

[0061] (X) may be one or more Gly and / or Arg residues and may be absent in the sequences.

[0062]

[0051] The present invention relates to highly effective dermocosmetic and biopharmaceutical compositions in strengthening and reactivating the intercellular / inter-layer interaction of the skin (including the structure of the hair bulb / hair follicle), properties affected in the processes of hair loss and aging.More specifically, the invention relates to a highly stable system / complex* for transporting a biomimetic oligopeptide analogous to growth factor KGF-2, thermally stabilized by a physiological polyanion biopolymer and transported by highly biocompatible polymeric nanospheres. This system ensures the thermal stability of the oligopeptide and, consequently, ensures an effective and prolonged biological effect. This system successfully promotes wound healing and hair growth by enhancing intercellular communication to induce re-epithelialization and maintain the integrity and functionality of the hair follicle structure, activating the anagen phase. The invention also provides a hair tonic for hair growth. The invention also describes the use of these biopolymers (SEQ ID NOs. 1 and 2) in tissue regeneration and hair growth processes.

[0052] The polyanion of the present invention may be hyaluronic acid and / or heparan sulfate and / or heparin.

[0063]

[0053] The biopolymers of SEQ ID NO: 1 and SEQ ID NO: 2 may present an Acetyl, Palmitoyl or Myristoyl group at their N terminal ends.

[0064]

[0054] The polymeric nanospheres may be selected from the group consisting of variations of poloxamers block copolymers, poly(D-L-lactide-co-glycolide), polylactic acids, alginate, dextran, chitosan, hydroxyapatite, collagen, fibrin, hyaluronic acid, carbomers, poly(amino acid), poly(propylene fumarate-co-ethylene glycol) P(PF co-EG) block copolymer, polyanhydride poly(fumaric-co-sebacic) anhydride, poly(ethylene oxide)-poly(lactideZglycolide), poly(amino acid), polyvinyl alcohol, and poly(ethylene glycol).

[0065]

[0055] The polymeric nanospheres may be microcapsules or lipid bilayer nanocapsules, preferably specialized transport liposomes. More specifically, to the polypeptides of SEQ ID NO: 1 and / or SEQ ID NO: 2 at a concentration of 0.00001% to 3% is added hyaluronic acid 0.1 to 1% and carried in polymeric nanospheres of Poloxamer block copolymer (PEO-PPO-PEO) 5 to 40%. This oligopeptide / hyaluronic acid / nanospheres complex is added at a concentration of 0.1 to 5% in phosphate buffer (PBS).

[0066]

[0056] The tonic for stimulating hair growth comprises a mixture of phase A components consisting of qsp water, at least one peptide represented by Seq ID No. 1 and / or Seq ID No. 2 (0.1 to 5%), a chelating agent (0.01 to 2%), glycerin (0.1 to 4%), jaborandi extract (0.1 to 2.0%), D-panthenol (0.1 to 2.0%) and transcutol (0.5 to 6%);Phase B components consisting of water qsp, 1,2 Hexanediol (0.1 to 3.0%), hyaluronic acid (0.1 to 5.0%), sorbic acid (0.1 to 10.0%), alginate siloxanetriol (0.1 to 3.0%), biotin (1.0 to 10%), butylene glycol (1.0 to 10%), caffeine (1.0 to 10%), caprylyl glycol (1.0 to 10%), copper tripeptide-1 (1.0 to 10%), copernicia cerifera wax (0.1 to 3.0%), copper tripeptide-1 (0.1 to 3.0%), phenoxyethanol (0.05 to 3.0%), sodium phosphate (1.0 to 10%), glycerin (0.05 to 3.0%), glycerol (0.1 to 5.0%), lecithin (1.0 to 10%), sorbitan oleate (0.1 to 3.0%), arnica montana flower oil (0.1 to 3.0%), Simmondsia chinensis seed oil (1.0 to 10%), panthenol (1.0 to 10%), pentylene glycol (1.0 to 10%), Pilocarpus microphyllus leaf extract (1.0 to 10%), poloxamer 407 (0.05 to 5.0%), quercetin (0.05 to 3.0%), phosphate buffered saline (0.05 to 3.0%), siloxanetriol alginate (1.0 to 10%), sodium methylparaben (0.1 to 3.0%), steareth-21 (0.5 to 10%) and phosphate buffer (0.1 to 5.0%);phase C component consisting of water-soluble fragrance and phase D components a preservative (optifen).;

[0067]

[0057] The chelating agent may be selected from the group comprising disodium EDTA, tetrasodium EDTA and / or citrates.

[0068]

[0058] Glycerin may be of vegetable and / or synthetic origin.

[0069]

[0059] The preservative preferably comprises phenoxyethane (0.1 to 2%) and caprylyl glycol (0.1 to 2%).

[0070]

[0060] This wide range of functionalities of the present invention is directly linked to the design advantages of smart, (bio)tunable, biomimetic and biocompatible (bio)materials, both for the area of ​​dermocosmetic active ingredients and for the area of ​​cell-based medicine / regenerative medicine. The invention studied: i) interaction / binding of the hyaluronic acid polyanion with the biomimetic oligopeptide derived from KGF-2, including encapsulation in polymeric nanospheres; ii) the biophysical structural properties of these interactions and iii) effects on the stability of the oligopeptide in cellular safety and efficacy assays (in vitro), results that reinforce the great potential for application of said complex in dermocosmetic and (bio)pharmaceutical formulations.Employing a variety of biophysical and cellular methods sensitive to the structure of the KGF-2-derived oligopeptide, including 1) circular dichroism (CD); 2) intrinsic fluorescence of the peptide / complex; 3) biocompatibility tests and 4) efficacy tests in three-dimensional (3D) cellular models between dermal fibroblasts and epidermal keratinocytes, greater efficacy was observed in strengthening and reactivating the intercellular / inter-layer interaction of the skin, a property impaired / affected in the processes of hair loss and aging.

[0071]

[0061] The present invention was designed to intervene and reactivate the hair growth cycle and act in cell renewal, tissue regeneration, and wound healing processes in an effective, biocompatible, and dermatologically safe manner. Using as a strategy a natural and important physiological maneuver of fibroblast growth factors (FGFs) the interaction with polyanions such as heparan sulfate and hyaluronic acid. Regarding the aforementioned invention, the interaction capacity of a biomimetic oligopeptide derived from factor KGF-2 (FGF10) with the polyanion hyaluronic acid was explored to create more stable dermocosmetic and pharmaceutical formulations with high added biological value due to the use of a high-performance bio-guided nanopolymeric delivery system.

[0072]

[0062] The nanospheres or nanomicelles in the following examples comprise self-assembling copolymer surfactants / surfactants that favor the assertive delivery of peptide or non-peptide actives in complete biocompatibility with biological structures that make up human skin. Said nanomicelle / nanosphere compositions (composed of a mix of glycerol and poloxamer) are acquired under controlled temperature conditions and under a hypertonic saline medium that provide a high molar ratio of the peptide or non-peptide agent (to be carried) and surfactant, for transport through the tortuous, complex and congested pathways of the skin's integumentary system.

[0073]

[0063] The present invention may be better understood, in a non-limiting manner, from the following examinations:

[0074] Example 1- CD (Circular dichroism), estimation of secondary structure and temperature-dependent conformational changes

[0075]

[0064] In this invention, we detail the biophysical characterization of the isolated oligopeptide derived from KGF-2 and the interaction between the oligopeptide derived from KGF-2 and the polyanion hyaluronic acid, which is distinct from the classic polyanion heparan sulfate. In this sense, we proceeded to the circular dichroism (CD) analysis of the oligopeptide in comparison with the protein of origin KGF-2. Detailing the analysis of KGF-2 (protein with 194 amino acids, MM 22,509 kDa) in solution by the Far-llV CD technique, generally used to analyze the secondary structure of a protein, in this case, it was employed to detect a combination of secondary and tertiary structural alterations.Typical CD signals for the FGF family of proteins are observed with extrema near 206 nm (negative) and 227–229 nm (positive) and are presumed to reflect the presence of the β-trefoil structure and particularly optically active Trp residues, respectively (Copeland RA, Ji H, Halfpenny AJ, Willaims RW, Thompson KC, Herber WK, Thomas KA, Bruner MW, Ryan JA, Marquis-Omer D, Sanyal G, Sitrin RD, Yamazaki S, Middaugh CR. 1991. The structure of human fibroblast growth factor and its interaction with heparin. These features of the CD spectrum are also typical of proteins such as human interleukin a and b, which share extensive β-trefoil structure.The far-UV CD spectrum of KGF-2 as a function of temperature is illustrated in Figure 1A, where the expected positive peak at 229 nm and the negative signal at 206 nm are clearly evident, as demonstrated in other scientific works (Derrick T, Grillo AO, Vitharana SN, Jones L, Rexroad J, Shah A, Perkins M, Spitznagel TM, Middaugh CR. Effect of polyions on the structure and stability of repifermin (keratinocyte growth factor-2). Temperature-induced conformational changes were monitored by following the CD spectrum signal at these two wavelengths as a function of temperature (Figure 1A and B).Regarding the aforementioned analysis of structural stability with monitoring of the CD spectra of the oligopeptide (± 17 amino acids) derived from the growth factor KGF-2, spectra distinct from that of the parent protein KGF-2 were observed when in the presence of the biopolymer polyanion hyaluronic acid (Figure 1 B). The CD spectrum of the oligopeptide / polyanion complex in 10 mM PBA solution, pH 7.0 (Figure 1 B, red line) presented two minima, one at 222 nm, related to the strong hydrogen bonding environment of the α-helices, and another at 210 nm, characteristic of the α-helix structure (NELSON, David L; COX, Michael M.. Lehninger's Principles of Biochemistry. 7 Porto Alegre: Artmed, 2019, 1278 p.). The analysis of the secondary structure content using the CDPro software with the programs SELCON3, CONTINLL, CDSSTR estimated the predominant presence of a-helix, in minority [3-sheet, [3-tum and of unordered structures.As expected, there are changes in the CD spectra between the original KGF-2 protein and the oligopeptide, as well as differences with the spectra of the complex* with increasing temperature, demonstrating a transient spectrum, without the characteristic secondary structures, but partially maintaining biological activity, indicative of the MG state. Reinforcing this, the biological activity confirms the similarity to KGF-2, as it is an oligopeptide with similar primary sequence and some secondary characteristics in relation to the original / mirror protein.It is worth highlighting that the present invention also seeks to demonstrate the surprising result of the mix between the Poloxamer block copolymer (5%-40%) of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) in the presence of glycerol (5%-20%), which are amenable to processing at low temperature and in hypertonic conditions (saline solution to avoid aggregates) to remove all free and weakly bound surfactant, forming nanospheres / nano-micelles (a micellar nanopolymer system), self-assembling thermosensitive / thermore reversible copolymers with high loading yield enabling a controlled release of its content, dispersion plasticity and high delivery performance of the KGF-2-derived oligopeptide in a site-specific manner.

[0076]

[0065] Figure 1 C shows the CD spectra of the thermal denaturation conditions, or thermal stability of the oligopeptide and complex*. This spectral region monitors the conformational changes of the oligopeptide backbone. Thermal assays were performed in the range of 25°C to 90°C (with emphasis on 70°C), with 5°C intervals. The spectra in Figures 1A and 1C show thermal scans at different temperatures, clearly demonstrating the significant differences in thermal stability, as the CD spectra show the loss of two typical bands with increasing temperature. In contrast, the temperature-induced unfolding of the complex* containing the KGF-2-derived oligopeptide was incomplete, as the spectrum at 70°C still shows a band centered at 215 nm, characteristic of the beta-sheet structure, although less intense.The mean residual ellipticities [0] at 210 nm and 222 nm (MRE210 and MRE222) were further analyzed to detect conformational change and were plotted against temperature, as shown in Figure 1C. In this case, the complex containing the oligopeptide / polyanion / nanospheres complex* presents pronounced structural stability up to 70°C, with slight fluctuations in the MRE values ​​(mean residual ellipticities [0]). Sigmoidal fits of these points provide a midpoint around 80°C for this curve. Corroborating the spectral changes, a significant variation in the secondary estimate occurs at the transition temperature, from 80 to 85°C, since at 80°C the oligopeptide / polyanion / nanospheres complex* already presents a complete loss of structural stability, with significant loss of the characteristic beta-sheet and beta-trefoil structure.Furthermore, the data show greater structural stability of the oligopeptide / polyanion / nanospheres complex*, possibly attributed to 3-sheet structures, since they are more resistant and less susceptible to unfolding (Tarrahimofrad H, Meimandipour A, Arjmand S, Beigi Nassiri M, Jahangirian E, Tavana H, Zamani J, Rahimnahal S, Aminzadeh S. Structural and biochemical characterization of a novel thermophilic Coh01 147 protease. PLoS One. 2020. 23; 15(6):e0234958. Doi: 10.1371 / jornal.pone.0234958.). The transition was irreversible, since an attempt at refolding, by decreasing the temperature under the same unfolding conditions, did not lead to the recovery of the structural characteristics of the oligopeptide / polyanion / nanospheres complex* (data not presented).

[0077] Example 2. Structural loss / unfolding of the complex* containing KGF-2-derived oligopeptide induced by GdnHCI (denaturing agent)

[0078]

[0066] Chaotropic agents such as GdnHCI or urea are commonly used in unfolding / refolding or folding / unfolding studies, although the unfolding mechanism is unclear. These studies were followed by far-CD and intrinsic fluorescence. The intrinsic fluorescence emission spectrum provides a sensitive probe for characterizing proteins by monitoring tryptophan residues, which are very sensitive to environmental polarity. The emission maximum (Àmax) and fluorescence intensity can be used to characterize proteins and determine conformational changes (Silva-Lucca RA, Andrade SS, Ferreira RS, Sampaio MU, Oliva ML. Unfolding studies of the cysteine ​​protease baupain, a papain-like enzyme from leaves of Bauhinia forficata: effect of pH, guanidine hydrochloride and temperature. Molecules. 2013. 24;19(1 ):233-46. doi: 10.3390 / moleculesl 9010233.). The effects of GdnHCI on the fluorescence emission spectra are shown in Figure 2A and 2B.The intrinsic fluorescence emission of the oligopeptide / polyanion / nanospheres complex* at pH 7.0 showed an emission maximum (Àmax) at 343 nm, showing exposure of tryptophan residues due to the presence of solvent in this microenvironment (Figure 2A, black line). The fluorescence emission spectra of the oligopeptide / polyanion / nanospheres complex* showed no significant change in intensity or emission maximum at 0-3.5 M GdnHCI (Figure 2B). However, the fluorescence intensity increased by 13% at 6.0 M GdnHCI, along with a 7 nm red-shift in the emission maximum from 343 to 350 nm (red line) at 6.0 M GdnHCI, indicating a polar environment of tryptophan residues.The sigmoidal curves suggested the presence of cooperative conformational changes induced by unfolding in the presence of GdnHCI in a single step from the native form of the oligopeptide / polyanion / nanosphere complex* to the unfolding form (N— >U ), with no detectable intermediates measured by CD and fluorescence (Figure 2B).

[0079]

[0067] In this invention we concluded that due to the alteration of the “folding”Vunfolding / incomplete thermal unfolding, the biological activity of the oligopeptide / polyanion / nanospheres complex* is maintained fully up to 40°C, and presents low activity at extreme temperatures (60, 80 and 100°C), similar to that observed with the protein of origin. The data show that although the secondary structure was maintained up to 70°C (Figure 1), the oligopeptide has its biological action affected (data not shown).

[0080] Example 3- In vitro Biocompatibility Tests *the* oligopeptide / polyanion / nanospheres complex will be called Drone System KGF-2.

[0068] The tests used the oligopeptide of SEQ ID No. 1, in the form YASFNWQHNGRQMYVAL(X)I-CONH2, complexed to hyaluronic acid in polymeric nanospheres. The oligopeptide / polyanion / nanospheres complex was called DRONE® KGF-2.

[0081]

[0069] Cell viability assays by MTT Reduction Analysis, by the neutral red method and morphological analysis.

[0082]

[0070] Biocompatibility and efficacy analyses are the pillars that govern the safety of active ingredients intended for dermocosmetic formulations. Following ANVISA's Guide for the Safety Assessment of Cosmetic Products, DRONE® KGF-2 plus hyaluronic acid was subjected to in vitro safety tests in cell culture in a 2D monolayer system, such as the traditional tests of i) analysis of MTT reduction, by detection of formazan salts, a test widely used in the areas of cell biology and the cosmetics market; ii) the other assay is based on neutral red uptake, which provides a quantitative estimate of the number of viable cells in a cell culture. Neutral red uptake is one of the most widely used cytotoxicity tests with many biomedical applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in lysosomes (Repetto, G., del Peso, A. & Zurita, J.Neutral red uptake assay for the estimation of cell viability / cytotoxicity. Nat Protoc 3, 1125-1131 (2008). https: / / doi.org / 10.1038 / nprot.2008.75.). It is important to mention that in parallel with the quantitative analysis of cell viability, cell morphology was monitored by optical microscopy.

[0083]

[0071] For the two cellular in vitro safety and cytotoxicity assessment assays of DRONE® KGF-2 plus hyaluronic acid, HaCat and HFF-1 cells (~4 x 10 4cells / well), derived from human keratinocytes (epidermis) and fibroblasts (dermis), respectively, were cultured in 96-well plates. After 24 hours, the different cell lines were treated with DRONE® KGF-2 plus hyaluronic acid (1%), and incubated at 37°C, 5% CO2 for 24 and 48 hours. Subsequently, the MTT reduction capacity and the incorporation of neutral red dye by human skin keratinocytes and fibroblasts were analyzed by a colorimetric method. In both cellular biocompatibility assays, we compared the results obtained with the complex* called DRONE® KGF-2 plus hyaluronic acid with the market benchmark used as the “gold standard” in solutions to combat hair loss, Minoxidil, an active ingredient composed of a piperidine ring.

[0084]

[0072] The graphs in Figure 3 show the safety of DRONE® KGF-2 plus hyaluronic acid regarding the metabolic activity and viability of skin cells. In all cell lines tested, DRONE® KGF-2 plus hyaluronic acid (2%) did not affect metabolic activity, without compromising cell viability, proving biocompatible in the 24- and 48-hour analyses. In contrast, cells treated with the market benchmark Minoxidil solution were sensitive to concentrations of 2% to 5%, with a drastic drop in cell viability to 69±6% when the cells, keratinocytes / epidermis and fibroblasts / dermis, were treated with 5% minoxidil solution, a dose commonly used in hair formulations. The cell viability results highlight the biocompatibility of DRONE® KGF-2 plus hyaluronic acid with the cells that make up the skin structures (Figure 3).The biocompatibility of DRONE® KGF-2 plus hyaluronic acid stands out compared to minoxidil, a remarkable result, since minoxidil is an active ingredient used as a first-line treatment for androgenetic alopecia. Our results corroborate reports of adverse effects from topical minoxidil, such as contact dermatitis, tachycardia, and hypertrichosis. Skin irritation has also been reported with the use of 2% minoxidil (Tai, T., Kochhar, A. Physiology and Medical Treatments for Alopecia, Facial Plastic Surgery Clinics of North America, v.28, Issue 2, 2020; 149-159. https: / / doi.org / 10.1016 / j.fsc.2020.01.004.).

[0085] Example 4- The DRONE® KGF-2 plus hyaluronic acid complex* activates the cell signaling pathway in keratinocytes that induces the production / expression of keratin 5, providing more strength to the hair structures

[0086]

[0073] Keratins 5 and 14 are proteins expressed primarily in basal keratinocytes in the epidermis, including the hair bulb / hair follicle, but are specifically present in the stratified epithelium lining the skin (Bejaoui M, Villareal MO, Isoda H. [3-catenin-mediated hair growth induction effect of 3,4,5-tri-O-caffeoylquinic acid. Aging (Albany NY). 2019 Jun 29; 11 (12):4216-4237. doi: 10.18632 / aging.102048; Driskell RR, Clavel C, Rendl M, Watt FM. Hair follicle dermal papilla cells at a glance. J Cell Sci. 2011 ; 124:1179-82. https: / / doi.org / 10.1242 / jcs.082446.). Keratin intermediate filaments compose the cytoskeletal framework within epithelial cells, which contributes to cellular architecture and provides cells with the ability to withstand mechanical and non-mechanical stresses, as in the case of the hair shaft.These keratins are structurally organized by dimerization among themselves and are involved in the formation of epithelial hair “buds”, actively acting in the phase transition of the hair growth cycle, with indirect regulation of cell proliferation in the dermal papilla (mesenchymal cells, fibroblasts)* (Bejaoui M, Villareal MO, Isoda H. [3-catenin-mediated hair growth induction effect of 3,4,5-tri-O-caffeoylquinic acid. Aging (Albany NY). 2019 Jun 29;11 (12):4216-4237. doi: 10.18632 / aging.102048; Driskell RR, Clavel C, Rendl M, Watt FM. Hair follicle dermal papilla cells at a glance. J Cell Sci. 2011 ; 124: 1179-82. https: / / doi.org / 10.1242 / jcs.082446.). Cytokeratins, such as keratin 5, form strong networks that help bind keratinocytes together and anchor the epidermis to the underlying layers of the skin. Acting on the cytoskeleton, they form intermediate filaments within epithelial cells and help maintain the strength of capillary structures.It is an important protein found in hair that contributes to its mechanical strength. In this scenario of providing structure and hair strength by increasing the expression of strategic keratins, the DRONE® KGF-2 plus hyaluronic acid complex* at a concentration of 2% was more effective than the benchmark active ingredient, 5% minoxidil, activating keratin 5 expression 40% more than the control and 20% more than 5% minoxidil. Therefore, DRONE® KGF-2 plus 2% hyaluronic acid was more effective in providing strength to the hair shaft than the market benchmark (Figure 4).

[0087]

[0074] Highlight the images of keratinocyte spheroids (3D cellular model) confirming the biocompatibility of DRONE® KGF-2 plus hyaluronic acid, and the cellular sensitivity when keratinocytes were exposed to 5% Minoxidil (Figure 4).

[0088]

[0075] *Note: It is important to highlight that the hair follicle / hair bulb is composed of two compartments, the epidermal (epithelial) and the dermal (mesenchymal), and its formation and structuring occurs through coordinated and complex communication / crosstalk between dermal cells that play the role of inducers / stimulators of epithelial cells. The mesenchymal compartment is composed of specialized fibroblasts divided into dermal papilla (DP), located at the proximal end of the hair follicle surrounded by matrix cells, and dermal sheath (DS), considered as a reservoir of dermal / mesenchymal cells (Bejaoui M, Villareal MO, Isoda H. [3-catenin-mediated hair growth induction effect of 3,4,5-tri-O-caffeoylquinic acid. Aging (Albany NY). 2019 Jun 29; 11 (12):4216-4237. doi: 10.18632 / aging.102048.].

[0089] Example 5. The DRONE® KGF-2 plus hyaluronic acid complex* activates the β-catenin and ERK cell signaling pathway with induction of special abilities with the increase of N-cadherin, promoting hair growth

[0090]

[0076] [3-catenin and its interaction with the p38 (cell maintenance) and ERK (cell proliferation) pathways promotes hair growth (Albany NY). 2019 Jun 29; 11(12):4216-4237. doi: 10.18632 / aging.102048.). The DRONE® KGF-2 complex* plus hyaluronic acid derived from FGF-10 or KGF-2 is a natural hair growth inducer, and its action involves upregulating the [3-catenin] pathway to promote the prolongation of the anagen phase (growth phase) of the hair growth cycle. And the activation of complementary pathways also completes and supports the promotion of effective hair growth. In addition to these multiple pathways and signals, crosstalk between the epidermis and dermis is essential to initiate hair follicle regeneration, and epithelial [3-catenin] signaling is the first key pathway in hair follicle morphogenesis.Another "fine" regulation of key proteins related to this proliferative phase of growth is the reduction of E-cadherin expression to increase N-cadherin, a process that DRONE® KGF-2 plus hyaluronic acid (2%) is able to promote effectively and biocompatiblely. The detected expression level of the cell cycle protein p53 indicates that DRONE® KGF-2 plus hyaluronic acid is BioSafe (Figure 5).

[0091] Example 6 - DRONE® KGF-2 plus hyaluronic acid as a tissue repair enhancer

[0092] Our cellular processes are incredibly ingenious, dynamic, and simultaneous, and we observe this in our daily lives when we witness the cellular healing process of a simple "cut" in our skin. This healing process is considered one of the most complex and multifaceted processes in our body, where the skin's biomechanics are tested and demanded in different ways. Wound compaction / contraction is a critical process for achieving effective, scar-free tissue repair. And in this dynamic and complex process, DRONE® KGF-2 plus hyaluronic acid acts by stimulating functional cellular contraction in the re-epithelialization process. DRONE® KGF-2 plus hyaluronic acid effectively induces this contraction / compaction, within 100 pm, of skin epithelial cells / keratinocytes. Mechanical compaction of keratinocytes is an event described in the tissue repair process, re-epithelialization (Figure 6).

[0093] Example 7 - DRONE® KGF-2 plus hyaluronic acid activates processes that lead to skin support with increased collagen synthesis from the perspective of keratinocytes (epidermal spheroids) in a co-culture system on a dermal fibroblast feeder.

[0094]

[0078] DRONE® KGF-2 plus Hyaluronic Acid presents HIGH structural and thermal stability favoring the Interaction between Epidermal Keratinocytes and Dermal Fibroblasts with Extracellular Matrix production induced by **DRONE® KGF-2 plus Hyaluronic Acid.

[0095] Example 8 - HAIR GROWTH TONIC

[0096]

[0079] A hair tonic type formulation was prepared comprising the following components:

[0097] PHASE A

[0098] PHASE B

[0099] PHASE C

[0100] PHASE D

[0101] PROCEDURE:

[0102]

[0080] Using a propeller homogenizer, solubilize the active ingredients present in phase A. Then, add the inputs from phase B item by item and wait for the materials to completely solubilize. After that, solubilize the fragrance present in phase C and finish the product by adding the preservative in phase D. This annex presents the control code for the biological sequence listing.

[0103] Control Code

[0104] Field 1

[0105] Field 2

[0106] Other Information:

[0107] - File Name: Untitledl_ST25.txt

[0108] - Code Generation Date: 07 / 17 / 2023

[0109] - Code Generation Time: 14:08:51

[0110] - Control Code:

[0111] - Field 1: 7642BDA2923FEA14

[0112] - Field 2: 7C62661C95695642

Claims

CLAIMS 1- Compositions for hair growth and tissue regeneration characterized by comprising a complex of at least one polypeptide with SEQ ID No. 1 and / or SEQ ID No. 2 with a polyanion, carried in biocompatible polymeric nanospheres, at a concentration of 0.1 to 5% in buffer. 2- Compositions for hair growth and tissue regeneration according to claim 1, characterized by the polyanion consisting of hyaluronic acid and / or heparan sulfate and / or heparin. 3- Compositions for hair growth and tissue regeneration according to claim 1, characterized in that SEQ ID No. 1 and SEQ ID No. 2 may have an Acetyl, Palmitoyl or Myristoyl group at their N-terminal ends. 4- Compositions for hair growth and tissue regeneration according to claim 1, characterized in that the polymeric nanospheres are selected from the group consisting of variations of poloxamer block copolymers, poly(D. L-lactide-co-glycolide), polylactic acids, alginate, dextran, chitosan, hydroxyapatite, collagen, fibrin, hyaluronic acid, carbomers, poly(amino acid), poly(propylene fumarate-co-ethylene glycol) P(PF co-EG) block copolymer, polyanhydride poly(fumaric-co-sebacic) anhydride, poly(ethylene oxide)-poly(lactide / glycolide), poly(amino acid), polyvinyl alcohol and poly(ethylene glycol). 5- Compositions for hair growth and tissue regeneration according to claim 4, characterized in that the polymeric nanospheres can be microcapsules or nanocapsules of lipid bilayer, preferably specialized transport liposomes. 6- Compositions for hair growth and tissue regeneration according to claim 1, characterized in that the buffer is preferably phosphate buffer (PBS). 7- Compositions for hair growth and tissue regeneration according to claims 1 to 6, characterized by comprising at least one polypeptide of SEQ ID No. 1 and / or SEQ ID No. 2 complexed with hyaluronic acid and carried in Poloxamer block copolymer (PEO-PPO-PEO) polymer nanospheres, at a concentration of 0.1 to 5% in phosphate buffer (PBS). 8- Compositions for hair growth and tissue regeneration according to claims 1 to 7, characterized by comprising at least one polypeptide of SEQ ID No. 1 and / or SEQ ID No. 2 (0.00001% to 3%) complexed with hyaluronic acid (0.1 to 1%) and carried in Poloxamer block copolymer (PEO-PPO-PEO) polymer nanospheres (5 to 40%), at a concentration of 0.1 to 5% in phosphate buffer (PBS). 9- Tonic for stimulating hair growth characterized by comprising a mixture of phase A components consisting of water qsp, at least one peptide represented by Seq ID No. 1 and / or Seq ID No. 2 complexed with polyanion in polymeric nanospheres (0.1 to 5%), a chelating agent (0.01 to 2%), glycerin (0.1 to 4%), jaborandi extract (0.1 to 2.0%), D-panthenol (0.1 to 2.0%) and transcutol (0.5 to 6%);Phase B components consisting of water q.s.p., 1,2-hexanediol (0.1 to 3.0%), hyaluronic acid (0.1 to 5.0%), sorbic acid (0.1 to 10.0%), siloxanetriol alginate (0.1 to 3.0%), biotin (1.0 to 10%), butylene glycol (1.0 to 10%), caffeine (1.0 to 10%), caprylyl glycol (1.0 to 10%), copper tripeptide-1 (1.0 to 10%), copernicia cerifera cera (0.1 to 3.0%), COPPER tripeptide-1 (0.1 to 3.0%), phenoxyethanol (0.05 to 3.0%), sodium phosphate (1.0 to 10%), glycerin (0.05 to 3.0%), glycerol (0.1 to 5.0%), lecithin (1 ,0 to 10%), sorbitan oleate (0.1 to 3.0%), arnica montana flower oil (0.1 to 3.0%), Simmondsia chinensis seed oil (1.0 to 10%), panthenol (1.0 to 10%), pentylene glycol (1.0 to 10%), Pilocarpus microphyllus leaf extract (1.0 to 10%), poloxamer 407 (0.05 to 5.0%), quercetin (0.05 to 3.0%), phosphate-buffered saline (0.05 to 3.0%), siloxanetriol alginate (1.0 to 10%), sodium methylparaben (0.1 to 3.0%), steareth-21 (0.5 to 10%) and buffer; phosphate (0.1 to 5.0%); phase C component consisting of a water-soluble fragrance and phase D components consisting of a preservative (optifen). 10-Tonic for stimulating hair growth according to claim 9, characterized in that the chelating agent can be selected from the group comprising disodium EDTA, tetrasodium EDTA and / or citrates. 11 - Hair growth stimulating tonic according to claim 9, characterized in that the glycerin may be of vegetable and / or synthetic origin. 12- Tonic for stimulating hair growth according to claim 9, characterized by the polyanion consisting of hyaluronic acid and / or heparan sulfate and / or heparin. 13-Tonic for stimulating hair growth according to claim 9, characterized in that SEQ ID No. 1 and SEQ ID No. 2 may have an Acetyl, Palmitoyl or Myristoyl group at their N-terminal ends. 14-Tonic for stimulating hair growth according to claim 9, characterized by the preservative preferably comprising phenoxyethane 0.1 to 2% and caprylyl glycol 0.1 to 2%. 15- Use of peptides SEQ ID No. 1 and / or SEQ ID No. 2 characterized by being in the preparation of compositions for tissue regeneration and hair growth. 16- Use of peptides with SEQ ID No. 1 and / or SEQ ID No. 2 according to claim 15, characterized by being complexed to a polyanion and carried by biocompatible polymeric nanospheres. 17- Use of peptides with SEQ ID No. 1 and / or SEQ ID No. 2 according to claims 15 and 16, characterized by the polyanion consisting of hyaluronic acid and / or heparan sulfate and / or heparin. 18-Use of the peptides of SEQ ID No. 1 and / or SEQ ID No. 2 according to claims 15 to 17, characterized in that SEQ ID No. 1 and SEQ ID No. 2 may present an Acetyl, Palmitoyl or Myristoyl group in their N-terminal ends. 19- Use of peptides of SEQ ID No. 1 and / or SEQ ID No. 2 according to claims 15 to 18, characterized in that the polymeric nanospheres are selected from the group consisting of variations of poloxamer block copolymers, poly(D.L-lactide-co-glycolide), polylactic acids, alginate, dextran, chitosan, hydroxyapatite, collagen, fibrin, hyaluronic acid, carbomers, poly(amino acid), poly(propylene fumarate-co-ethylene glycol) P(PF co-EG) block copolymer, polyanhydride poly(fumaric-co-sebacic) anhydride, poly(ethylene oxide)-poly(lactideZ glycolide), poly(amino acid), polyvinyl alcohol and poly(ethylene glycol). 20- Use of peptides with SEQ ID No. 1 and / or SEQ ID No. 2 according to claims 15 to 19, characterized in that the polymeric nanospheres can be microcapsules or nanocapsules of lipid bilayer, preferably specialized transport liposomes.

Images