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    • 专利摘要:
    Mythicin peptide and its use in cell regeneration. The present invention relates to peptides derived from myctin C and their therapeutic uses, more specifically in cellular and/or tissue regeneration. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2763349A1
    申请号:ES201831154
    申请日:2018-11-28
    公开日:2020-05-28
    发明作者:Huerta Antonio Figueras；Vega María Gasset；García Beatriz Novoa；Campos Magalí Rey；Marín Ricardo Mallavía；GALI Regla María MEDINA；Lopez Alicia Martinez
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad Miguel Hernandez de Elche；
    IPC主号:
    专利说明:

    [0002] Mythicin peptide and its use in cell regeneration
    [0004] The present invention relates to peptides comprising the sequence SEQ ID NO: 2 and their therapeutic uses, specifically in cellular and / or tissue regeneration.
    [0006] BACKGROUND OF THE INVENTION
    [0008] Antimicrobial peptides (AMP) are innate immune effectors and have a wide range of actions against many microorganisms. In marine invertebrates, living in environments with an abundance of potentially pathogenic microorganisms, AMPs are the main elements of the immune response. For example, in Mediterranean mussels ( Mytilus galloprovincialis), several types of MPA have been described, including miticin C.
    [0010] Miticin C (Myt-C) is a cysteine-rich antimicrobial peptide isolated from hemocytes of the mussel Mytilus galloprovincialis whose antibacterial and chemotactic activity has been tested in vitro (Martinez-Lopez et al., Mar Drugs. 11 (7): 2328 46 (2013)). Its antiviral effect has also been demonstrated not only against mollusc viruses, such as the oyster herpervirus OsHV-1, but also against human herpes simplex (HSV-1 and HSV-2) (Novoa B., et al., J Virol. 90 (17): 7692-702 (2016)) and against pathogenic fish rhabdoviruses (Balseiro P. et al., PLoS One.
    [0011] 6 (8): e23140 (2011)).
    [0013] During the life of any animal, wounds inevitably occur, and can be the consequence of a wide variety of events, such as contact with sharp or hot objects, or appear in certain clinical disorders, such as diabetes. Another important clinical example is the development of pressure ulcers, that is, injuries caused by unmitigated pressure on any part of the body, especially portions on bony or cartilaginous areas. Although fully treatable if discovered early, bedsores, without medical attention, like any other wound, can be fatal. In the case of a reduced general health condition, wound closure (healing) may be delayed, and may cause other problems, such as Infections, inflammation, tissue necrosis, and ineffective wound closure are, again, potentially life-threatening.
    [0015] Wounds generally occur on the skin. The skin is a vital organ that fulfills multiple functions, such as sensory functions, protective functions against external aggressions, as well as immunological, metabolic or thermoregulatory functions. These roles are possible due to the complex structure that associates various tissues. The skin consists of three distinct overlapping layers: the epidermis, the dermis, and the hypodermis. The epidermis is a lining epithelium, which constitutes the external structure of the skin and provides its protective function. This function is provided by the cohesion of the epithelial cells and by the production of a resistant and filamentous protein, keratin.
    [0017] In general, wound healing is described as consisting of three phases, namely, the inflammatory phase, the proliferative phase, and the maturation phase (termed acute inflammatory phase, synthesis of extracellular and collagen matrix, and remodeling). The sequence of the healing process begins during an acute inflammatory phase with the deposit of provisional tissue. This is followed by reepitalization, collagen synthesis and deposition, fibroblast proliferation, and neovascularization, all of which ultimately define the remodeling phase.
    [0019] The inflammatory phase is characterized by hemostasis and inflammation. The collagen exposed during wound formation activates the coagulation cascade (both the intrinsic and extrinsic pathways), initiating the inflammatory phase. Platelets, the first response cell, release multiple chemokines that help stabilize the wound by forming a clot. These mediators act to control bleeding and limit the extent of the injury. The second response cell that migrates to the wound, neutrophils, are responsible for the uptake of debris, complement-mediated opsonization of bacteria, and the destruction of bacteria by oxidative burst mechanisms (i.e., formation of superoxide and peroxide. hydrogen). Macrophages are essential for wound healing. Numerous enzymes and cytokines are secreted by macrophages, which marks the transition to the tissue reconstruction process, that is, the proliferative phase.
    [0020] During the proliferative phase, epithelialization, angiogenesis, granulation tissue formation, and collagen deposition are the main stages in wound healing. Epithelialization appears at an early stage of wound repair. Angiogenesis, stimulated, for example, by TNF-alpha, is noted by endothelial cell migration and capillary formation. The new capillaries transport nutrients to the wound and help maintain the bed of granulation tissue. The final part of the proliferative phase is the formation of granulation tissue. Fibroblasts differentiate and produce basal substance and then collagen. The basal substance is deposited in the wound bed; the collagen is then deposited as the wound undergoes the final phase of repair. Many different cytokines, including PDGF, insulin-like growth factor (IGF), and EGF, are involved in the proliferative phase of wound repair. During the maturation phase, the wound undergoes a contraction that ultimately results in less scar tissue being seen.
    [0022] From the above, it is obvious that proper wound healing involves a complex interaction of cells and substances, such as cytokines, that act in concert. Based on basic research, many drugs, substances, and treatment procedures have been proposed to stimulate wound healing.
    [0024] Many of these substances provide the patient with partial relief from wounds, require a long healing time and fail to show the optimal response to treatment. For example, patent EP0575484 discloses a pharmaceutical composition for the regeneration and repair of mammalian tissues, which includes PDGF and dexamethasone.
    [0026] Therefore, it is necessary to provide new substances and compositions with a high power in cell regeneration, leading to scarring and effectively repairing tissue damage.
    [0028] DESCRIPTION OF THE INVENTION
    [0030] The inventors of the present invention have observed that miticin-C (QSVACRSYYCSKFCGSAGCSLYGCYLLHPGKICYCLHCSR, SEQ ID NO: 1) is it expresses in response to wounds or tissue damage, thus enhancing cell regeneration and tissue repair after damage. The role of miticin-C in cell regeneration was demonstrated by an in vitro assay using human keratinocyte cells (HaCat line), and by an in vivo assay employing zebrafish ( Danio rerio) larvae . In both cases, it was seen that the wound healed faster in the presence of miticin-C than, in its absence, thus promoting healing and repairing tissue damage (see Example 1, Figures 1 and 2).
    [0032] Next, the inventors designed a library of overlapping short small peptides from the amino acid sequence of miticin-C (see Table 1), in order to identify that minimum sequence necessary for miticin-C to play its role. in cellular and / or tissue regeneration. Thus, through in vitro and in vivo tests , the inventors observed that some peptides named P1 to P4 were able to repair the wound or damage generated. These peptides present the consensus SYYCSK sequence (SEQ ID NO: 2), this being therefore the amino acid sequence responsible for carrying out the best regeneration rates after 48 hours. Particularly if amino acids F and C are added at the carboxyl terminus, they show an improvement in total wound healing at 72 h, SYYCSKFC (SEQ ID NO: 3), as can be seen in Figure 3 with the peptides called P2b, P3 and P4.
    [0034] Thus, in one aspect, the present invention relates to a peptide comprising at least 80% identity with the SYYCSK sequence (SEQ ID NO: 2), preferably having a size of between 6 to 99 amino acids, and not is miticin-C with sequence SEQ ID NO: 1, preferably the peptide comprises at least 80% identity with the sequence SYYCSKFC (SEQ ID NO: 3), which preferably has a size between 6 to 99 amino acids, and that it is not miticin-C with sequence SEQ ID NO: 1. Hereinafter, this peptide will be called "peptide of the invention".
    [0036] The term "peptide", as used herein, refers to a molecule formed by the binding, in a defined order, of alpha-amino acids via a peptide bond. The terms "peptide bond", "peptide", will be understood to mean "," polypeptide "and protein are known to those skilled in the art. Hereinafter," peptide "and" polypeptide "will be used interchangeably.
    [0037] The peptide of the invention can be obtained by techniques widely known in the state of the art, such as chemical synthesis, genetic recombination, expression of the polynucleotide that encodes the polypeptide of the invention, etc. All of these techniques are routine practice for the person skilled in the art.
    [0039] Additionally, the carboxyl and amino terminal ends of the peptide of the invention can be protected against proteolysis. For example, the amino terminal end may be in the form of an acetyl group (Ac) and / or the carboxyl terminal end may be in the form of an amide group (Am). It is also possible to carry out internal modifications of the peptides so that they are resistant to proteolysis. Examples of these internal modifications include, but are not limited to, modifications in which at least one peptide bridge -CONH- is modified and replaced by a reduced bond (CH2NH), a back-reverse bond (NHCO), an oxymethylene bond (CH2-O ), a thiomethylene bond (CH2-S), a ketomethylene bond (CO-CH2), a hydroxyethylene bond (CHOH-CH2), a bond (NN), an E-alkenne bond or a -CH = CH- bond. The peptides can also be stabilized by intramolecular crossing, for example, by modifying at least two amino acid residues with olefin side chains, preferably C3-C8 alkenyl chains, preferably pentel-2-yl chains, followed by crosslinking. chains as described in technology called "staple" (Walensky et al., 2004, Science 205: 1466-1470). All of these peptides chemically modified to resist proteolysis are also contemplated within the present invention.
    [0041] Additional modifications to the peptide of the invention include covalent attachment to a polyethylene glycol (PEG) molecule by its carboxyl terminus or to a lysine residue, in order to decrease its urinary elimination and therapeutic dose, and to increase the half-life of the peptide in blood plasma. The half-life of the peptide can also be increased by including the peptide in a biodegradable and biocompatible polymeric material to form microspheres that are used as a drug delivery system. Polymers and copolymers include, without limitation, poly (D, L-lactide-co-glycolic) or PLGA. The techniques and procedures of how to manufacture lipid microspheres or nanocapsules for use in drug administration are widely known to the person skilled in the art.
    [0042] The peptide of the invention comprises at least 80% identity with the sequence SEQ ID NO: 2, preferably at least 80% identity with the sequence SEQ ID NO: 3. In the present invention, "identity" is understood or "sequence identity" to the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity will be obtained, expressed as a percentage. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST. BLAST programs, eg BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of The National Center for Biotechonology Information (NCBI). In a particular embodiment, the peptide of the invention comprises an identity of at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 2 or SEQ ID NO: 3. In another still more particular embodiment, the peptide of the invention comprises a sequence identity of 100% with the sequence SEQ ID NO: 2 or from SEQ ID NO: 3. As understood by the person skilled in the art, all peptides that have an identity of at least 80% with the sequence SEQ ID NO: 2 or SEQ ID NO: 3, and which have the same function as SEQ ID NO: 2 or SEQ ID NO: 3, that is, they have the capacity to enhance tissue regeneration and tissue damage repair, they are functionally equivalent variants of the peptide of the invention. Assays to identify peptides functionally equivalent to the peptide of the invention can be found in the Example of the present description.
    [0044] Thus, in a particular embodiment, the peptide of the invention refers to a peptide comprising the sequence SEQ ID NO: 2, preferably SEQ ID NO: 3, which can be between 6 and 99 amino acids in size, and which does not it is miticin-C with the sequence SEQ ID NO: 1.
    [0046] Another characteristic of the peptide of the invention is that it has a size of between 6 and 99 amino acids, preferably between 6 and 50, more preferably between 6 and 40. In a more particular embodiment, it has a size of between 6 and 25 amino acids, and in another still more particular embodiment, between 6 and 20 amino acids or between 6 and 15 amino acids. In another even more particular embodiment, the peptide of the invention It is 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in size.
    [0048] In another particular embodiment, the peptide of the invention refers to a peptide comprising the sequence QSVACRSYYCSK (SEQ ID NO: 4), VACRSYYCSKFC (SEQ ID NO: 5), CRSYYCSKFCGS (SEQ ID NO: 6) or SYYCSKFCGSAG (SEQ ID NO: 7). In another still more particular embodiment, the peptide of the invention refers to a peptide consisting of the sequence SYYCSK (SEQ ID NO: 2), SYYCSKFC (SEQ ID NO: 3), QSVACRSYYCSK (SEQ ID NO: 4), VACRSYYCSKFC (SEQ ID NO: 5), CRSYYCSKFCGS (SEQ ID NO: 6), or SYYCSKFCGSAG (SEQ ID NO: 7). More preferably the peptide consists of the sequence VACRSYYCSKFC (SEQ ID NO: 5), CRSYYCSKFCGS (SEQ ID NO: 6), or SYYCSKFCGSAG (SEQ ID NO: 7). As discussed above, the peptides of the invention may be protected against proteolysis, particularly the peptide of the invention refers to a peptide comprising the sequence SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO: 11 or SEQ ID NO: 12.
    [0050] The peptide of the invention can be linked to other amino acid sequences to give rise to a fusion protein. Therefore, in another aspect, the present invention relates to a fusion protein comprising the peptide of the invention, hereinafter "fusion protein of the invention".
    [0052] The term "fusion protein", as used herein, refers to an artificially designed polypeptide chain comprising two or more amino acid sequences of different origins, natural and / or artificial. Fusion protein, by definition, is never found in nature as such.
    [0054] The fusion protein of the invention can be obtained by any suitable technique that allows obtaining peptides in a single polynucleotide chain. Such techniques include recombinant techniques, where a gene construct encoding the fusion protein is introduced into a suitable vector for expression in a suitable expression system, and protein ligation techniques that involve the formation of a peptide bond between two polypeptides, such as native chemical ligation or expressed protein ligation.
    [0056] The fusion protein of the invention may further comprise a domain N-terminal signal peptide, which enables processing, eg, extracellular secretion, into a suitable host cell. Preferably, the N-terminal signal peptide domain comprises a protease, eg, a signal peptidase cleavage site, and therefore can be removed after or during expression to obtain the mature protein.
    [0058] Additionally, the fusion protein may comprise a recognition / purification domain, eg, an Estrep-tagged domain and / or a poly-His domain, which may be located at the N-terminus or at the C-terminus. Also, the fusion protein of the invention may further comprise a flexible C-terminal element, having a length of, for example, 1-50, preferably 10-30 amino acids that can include and / or connect to a recognition domain / purification as described in this document.
    [0060] The peptide of the invention can be linked to other amino acid sequences through a linker, which can be flexible.
    [0062] Both the peptide and the protein of the invention can be contained in a nanoparticle for better administration, conservation, etc. Thus, in another aspect, the present invention relates to a nanoparticle comprising the peptide or fusion protein of the invention.
    [0064] The term "nanoparticle", as used herein, refers to any material having dimensions in the range of from 1 to 1,000 nm. In some embodiments, the nanoparticles have dimensions in the range of 2-200 nm, preferably in the range of 2-150 nm, and even more preferably in the range of 2-100 nm. Nanoparticles can help preserve the integrity of the peptide or fusion protein in biological fluids until it reaches the target organ. Finally, nanoparticles can also be modified to target the nanoparticle to an organ of interest, by adding ligands without compromising the ability of the nanoparticles to deliver their peptide payloads. This is contemplated to allow delivery to specific cells, tissues, and organs. The targeting specificity of ligand-based delivery systems is based on the distribution of ligand receptors in different cell types. The targeting ligand may be non-covalently associated or covalent with a nanoparticle, and can be conjugated to nanoparticles by a variety of methods.
    [0066] Suitable nanoparticles that can be used in the context of the present invention include nanoscale materials such as a lipid based nanoparticle, a superparamagnetic nanoparticle, a nano helix, a semiconductor nanocrystal, a quantum dot, a polymer based nanoparticle, a silicone based nanoparticle , a silica-based nanoparticle, a metal-based nanoparticle, a fullerene, and a nanotube.
    [0068] As understood by the person skilled in the art, the peptide of the invention or the protein of the invention can also be contained within a particle. Therefore, in another aspect, the invention relates to a virus-like particle comprising the peptide or the fusion protein.
    [0070] The term "virus-like particle", also called "VLP", refers to non-infectious virus-like particles that do not contain any viral genetic material. VLPs are the result of the expression of viral structural proteins, such as capsid proteins and their self-assembly.
    [0072] In a particular embodiment, the VLP may comprise, or alternatively consist of, structural proteins of Parvovirus, Rotavirus; Norwalk virus structural proteins; alphavirus structural proteins; foot-and-mouth disease virus structural proteins; measles virus structural proteins, Sindbis virus structural proteins, structural proteins of Hepatitis B virus retrovirus structural proteins (eg, an HBcAg); structural proteins of the tobacco mosaic virus; structural proteins of the FlockHouse virus; structural proteins of human papillomavirus; polyoma virus structural proteins; Bacteriophage structural proteins, RNA phage structural proteins.
    [0074] In a particular embodiment, the peptide or fusion protein of the invention couples or binds to the capsid of the virus-like particle. The binding of the peptide or fusion protein to the capsid can be by a covalent or non-covalent bond.
    [0075] The peptide of the invention can be obtained by techniques widely known in the state of the art, such as the expression in a cell of the polynucleotide that encodes the polypeptide of the invention, or the protein of the invention, and its subsequent isolation. Therefore, in another aspect, the invention relates to a polynucleotide, hereinafter "polynucleotide of the invention", which encodes the peptide of the invention or the fusion protein of the invention.
    [0077] The terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides can contain deoxyribonucleotides, ribonucleotides and / or their analogs. Nucleotides can have any three-dimensional structure and can perform any function, known or unknown. The term "polynucleotide" includes, for example, single-stranded, double-stranded, and triple helix molecules, a gene or gene fragment, exons, introns, mRNAs, tRNAs, rRNAs, ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes and primers. In addition to a native nucleic acid molecule, a nucleic acid molecule of the present invention may also comprise modified nucleic acid molecules. As used herein, mRNA refers to an RNA that can be translated into a cell.
    [0079] In a preferred embodiment, the polynucleotide of the invention is an mRNA. The mRNA can be chemically synthesized, can be obtained by in vitro transcription, or can be synthesized in vivo in the target cell. The nucleotide sequences that make up the nucleic acid encoding the conjugate or fusion protein of the invention are in the same correct reading frame for expression thereof.
    [0081] The polynucleotide encoding the peptide or fusion protein of the invention may have various modifications made to the coding region as long as the amino acid, peptide, or fusion protein sequence does not change due to codon degeneration , or taking into consideration the codons preferred by the organism in which they are to be expressed and various modifications or alterations can be introduced even in different regions of the coding region, as long as they have no influence on the expression of the gene.
    [0082] As understood by the person skilled in the art, the polynucleotide that encodes the peptide of the invention or the fusion protein of the invention, can be included within a gene construct or within an expression vector. In general, an expression vector comprises, in addition to the polynucleotide encoding the peptide of the invention or the fusion protein of the invention, a promoter that directs its transcription (for example, pT7, plac, ptrc, ptac, pBAD, ret, etc.), to which it is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription start and end signals (tlt2, etc.). ), polyadenylation signal, origin of replication, ribosome binding sequences (RBS), coding sequences of transcriptional regulators, enhancers (enhancers), transcriptional silencers (silencers), repressors, etc. Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. they may also contain markers usable for selecting cells transfected or transformed with the gene or genes of interest. The choice of vector will depend on the host cell and the type of use to be made. For example, said vector can be a viral vector (adenovirus, adenovirus-associated virus as well as retrovirus and, in particular, lentivirus) or non-viral (pcDNA3, pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl). Said vectors can be administered directly to the subject by conventional methods. Alternatively, said vectors can be used to transform, transfect or infect cells, for example, mammalian cells, ex vivo, and subsequently implant them in the animal body to obtain the desired therapeutic effect. For administration to the recipient subject, said cells will be formulated in a suitable medium that does not adversely affect the viability of said cells. Obtaining said vector can be carried out by conventional methods known to those skilled in the art, as well as by using different widely known methods for the transformation of microorganisms and eukaryotic cells.
    [0084] In another aspect, the invention relates to a gene construct, hereinafter "gene construct of the invention", comprising the polynucleotide of the invention.
    [0085] Preferably, the gene construct of the invention comprises the polynucleotide of the invention operatively linked to expression regulatory sequences of the polynucleotide of the invention. In principle, any promoter can be used in the gene constructs of the present invention, as long as said promoter is compatible with the cells in which it is desired to express the polynucleotide.
    [0087] A promoter, or promoter region, is a nucleotide sequence that controls the transcription of a given gene (nucleotide sequences). In the present invention it refers to a nucleotide sequence that controls the transcription of the polynucleotide of the invention. The promoter sequences can be unidirectional or bidirectional. A unidirectional promoter is one that controls the transcription of one gene or more genes that are in tandem with the first one. "Tandem" refers to the 3 'end of the first gene being followed, either consecutively or separated by a certain nucleotide sequence, by the 5' end of the second gene. A bidirectional promoter refers to the promoter region that controls transcription in two opposite directions, that is, that a bidirectional promoter directs the transcription of two genes located in a divergent way, that is to say in the opposite direction, the 5 'end of both nucleotide sequences being closer to each other than the 3' end. The terms "promoter" and "promoter region" are used interchangeably with the present invention. Furthermore, the promoters in the present invention can be either constitutive or inducible. The term "inducible", as used in the present description, refers to the possibility that the promoter has a control element that allows to activate or deactivate (repress) the transcription of the gene that it regulates, in the presence of a factor external to the developer.
    [0089] Additionally, the gene construct of the invention may contain labels or tags that allow the isolation of the peptide of the invention once it is synthesized in the cell.
    [0091] On the other hand, the polynucleotide or the gene construct of the invention may be part of a vector. Thus, in another aspect, the invention relates to a vector, hereinafter "vector of the invention", comprising the polynucleotide or the gene construct of the invention.
    [0092] The term "vector", as used herein, refers to a nucleic acid sequence that comprises the necessary sequences so that upon transcription and translation of those sequences into a cell, a polypeptide encoded by the nucleic acid of the invention. Said sequence is operatively linked to additional segments that provide its autonomous replication in a host cell of interest. Preferably, the vector is an expression vector, which is defined as a vector that, in addition to the regions of autonomous replication in a host cell, contains regions operatively linked to the nucleic acid of the invention and that are capable of enhancing the expression of the nucleic acid products according to the invention. The vectors of the invention can be obtained by means of techniques widely known in the state of the art.
    [0094] The person skilled in the art will appreciate that there is no limitation as to the type of vector that can be used, since said vector can be a cloning vector suitable for propagation or an expression vector. Thus, suitable vectors according to the present invention include, without limitation, (i) prokaryotic expression vectors such as pUC18, pUC19, Bluescript and its derivatives, mp18, mp19, pBR322, pMB9, CoIEl, pCRl, RP4, phage and shuttle vectors such as pSA3 and pAT28, (ii) yeast expression vectors such as 2 micron plasmid type vectors, integration plasmids, YEP vectors, centromeric plasmids, and the like, (iii) cell expression vectors from insects such as pAC series and pVL series vectors, (iv) plant expression vectors such as pIBI series vectors, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like and (v) superior eukaryotic cell expression vectors well based on viral vectors, including, but not limited to, adenoviruses, adenovirus-associated viruses as well as retroviruses and lentiviruses, as well as non-viral vectors including, not limited to, pSilencer 4 .1-CMV (Ambion), pcDNA3, pcDNA3.1 / hygpHCMV / Zeo, pCR3.1, pEFVHis, pIND / GS, pRc / HCMV2, pSV40 / Ze02, pTRACER HCMV, pUB6N5-His, pVAXl, pZeoSV2, pCl, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl.
    [0096] The vector of the invention can be used to transform, transfect or infect cells capable of being transformed, transfected or infected by said vector.
    [0098] Said cells can be prokaryotes or eukaryotes. As an example, the vector Where said DNA sequence is introduced, it can be a plasmid or a vector that, when introduced into a host cell, is integrated into the genome of said cell and replicates together with the chromosome / s in which it has been integrated. Obtaining said vector can be carried out by conventional methods known to those skilled in the art.
    [0100] Therefore, in another aspect, the invention relates to a cell, hereinafter "cell of the invention", comprising a peptide, a fusion protein, a polynucleotide, a gene construct, or a vector according to the present invention , for which said cell has been able to be transformed, transfected or infected with the construct or vector provided by this invention. Transformed, transfected or infected cells can be obtained by conventional methods known to those skilled in the art, including, without limitation a, electroporation, protoplast fusion, co-precipitation with calcium phosphate (CaPO4) and precipitation with calcium chloride (CaCl2).
    [0102] In a particular embodiment, said host cell is an animal cell, preferably a mammalian cell and more preferably a human cell, transfected or infected with an appropriate vector.
    [0104] Suitable host cells for expression of the polypeptide of the invention include, but are not limited to, mammalian cells, plant cells, insect cells, fungal cells, and bacterial cells. Mammalian cells suitable for in the present invention include epithelial cell lines (porcine, human, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.). .), glial cells (murine, human, etc.), liver cell lines (monkey, etc.), CHO cells (ChineseHamsterOvary), COS cells, BHK cells, HeLa cells, 911, ATI080, A549, 293 or PER. C6, human ECCs 5 NTERA-2 cells, mESC line D3 cells, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3, 293T, REH and MCF-7 cells and hMSCs cells (human mesenchymalstemcells ), and GliNS2 cells (glioma stem cells).
    [0106] In another aspect, the invention relates to a composition, preferably a pharmaceutical or cosmetic composition, comprising a quantity therapeutically or cosmetically effective of the peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector or cell of the invention, and optionally a pharmaceutically or cosmetically acceptable carrier or excipient.
    [0108] The peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, and cell of the invention have been previously defined.
    [0110] As used in the present invention, the term "pharmaceutical composition" refers to a formulation that has been adapted to administer a predetermined dose of one or more useful therapeutic agents to a cell, a group of cells, an organ, a tissue or an animal in which there is damage or injury and requires tissue and / or cell regeneration.
    [0112] As used in the present invention, the term "cosmetic composition" refers to a formulation that has been adapted to administer a predetermined dose of one or more useful cosmetic agents to a cell, a group of cells, an organ, a tissue or an animal in which there is aesthetic damage or injury and requires tissue and / or cell regeneration.
    [0114] The term "therapeutically or cosmetically effective amount", as used herein, is understood as an amount capable of providing a therapeutic or cosmetic effect, and which can be determined by the person skilled in the art by commonly used means. The amount of the peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector or cell of the invention that can be included in the pharmaceutical or cosmetic compositions according to the invention will vary depending on the subject and in the particular way of the administration.
    [0116] The appropriate dose of the active ingredient (s) within the pharmaceutical or cosmetic composition will depend on the type of tissue damage to be treated, the severity and course of the disease or damage, whether the composition is administered for cosmetic, preventive or therapeutic purposes, prior therapy , patient's medical history and response to the peptide or polypeptide, and the discretion of the treating physician. The amount of peptide, fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector or cell of the invention is suitably administered to the patient or subject at one time or during a series of treatments. Depending on the type and severity of the disease or damage, an appropriate dose level will generally be from about 0.1 to about 10 mg / kg; more preferably from about 0.5 to about 5 mg / kg, even more preferably from about 0.75 to about 2.5 mg / kg, even more preferably about 1 mg / kg, which can be administered in single or multiple doses. The compounds can be administered on a regimen of 1 to several times per day or for two, three, four, five, six or seven days, preferably once every other day. The pharmaceutical or cosmetic composition can be administered for at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 or more days, preferably for 14 days. The pharmaceutical or cosmetic composition can preferably be administered once every other day for 14 days.
    [0118] The pharmaceutical compositions of the invention also contain one or more additional pharmaceutically acceptable excipients. By "pharmaceutically acceptable excipient" is meant a therapeutically inactive substance that is used to incorporate the active ingredient and that is acceptable to the patient from a pharmacological / toxicological point of view and to the pharmaceutical chemist who manufactures it from a physical / chemical point of view. regarding composition, formulation, stability, patient acceptance and bioavailability. The excipient or vehicle also includes any substance that serves to improve the supply and efficacy of the active ingredient within the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include one or more of the list comprising: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, that improve the shelf life or efficacy of the fusion protein or of the compositions that are part of the pharmaceutical compositions. Examples of suitable vehicles are well known in the literature. Examples of vehicles without limitation are a series of saccharides such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; a series of starches such as corn starch, wheat starch, rice starch and potato starch; a series of cellulose such as cellulose, methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl cellulose; and a series of fillers such as gelatin and polyvinylpyrrolidone. In some cases, a disintegrant such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate can be added.
    [0120] The cosmetic composition may further comprise adjuvants, excipients and other usual additives in the cosmetic and dermatological field, such as, for example and without limitation, stabilizers, preservatives, antioxidants, solvents, perfumes, chelating agents, odor absorbers, chemical or mineral filters. , mineral pigments, organic solvents, silicones, thickeners, softeners, emulsifiers, antifoaming agents, moisturizers, fragrances, surfactants, anionic, cationic, nonionic or amphoteric polymers or their mixtures, sequestering agents, propellants, acidifying or basifying agents, dyes, colorants , pigments or nanopigments, or any other ingredient normally used in cosmetic products and compositions.
    [0122] The composition of the invention may further comprise any substance that can be administered to a wound, tissue injury, or site of inflammation. For example, the disclosed composition may further comprise one or more classes of antibiotics (eg aminoglycosides, cephalosporins, chloramphenicol, clindamycin, erythromycin, fluoroquinolones, macrolides, azolides, metronidazole, penicillins, tetracyclines, trimethoprim-sulfamethoxazole, vancomins) for example andrans (for example testosterone), cholestanes (for example cholesterol), colic acids (for example colic acid), corticosteroids (for example dexamethasone), estranes (for example estradiol), pregnanes (for example progesterone), narcotic and non-narcotic analgesics (eg morphine, codema, heroma, hydromorphone, levorphanol, meperidine, methadone, oxycodone, propoxyphene, fentanyl, methadone, naloxone, buprenorphine, butorphanol, nalbuphine, pentazocine), chemotherapy (eg anti-cancer drugs such as, but not limited to, altretamine, asparaginase, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, diethylstilbesterol, ethinyl estradiol, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, goserelin, hydroxyurea, idarubicin, ifosfamide, leuprolide, levamisole, lomustine, mecloretamine megestrol, melfalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, paclitaxel, pentostatin, pipobroman, plicamycin, prednisone, procarbazine, streptozocin, tamoxifen, diphostenate, vinblastine, alkenacloflutane; alpha amylase; amcinafal; amcinafide; sodium amfenac; amiprilose hydrochloride; anakinra; anirolaco; anitrazaphene; apazone; balsalazide disodium; bendazac; benoxaprofen; benzydamine hydrochloride; bromelamine; taperoprofen; cyclopropene; ; Clobetasone Butyrate; Clopyrazone; Cloticasone Propionate; Cormetasone Acetate; Cortodoxone; Decanoate; Deflazacort; Delatestril; Depo-Testosterone; Deonide; Deoxymethasone; Dexamethasone Dichlorophenal Potassium; Diclofenac Sodium Dichlorophenal; difluprednate, diftalone, dimethyl sulfoxide; drocinonide; endrisone; enlimomab; enolicam sodium; epirizole; etodolac; ethofenamate; felbinaco; fenamol; fenbufen; fenclofenac; phenchloracus; fendosal; fenpipalone; fentiazaco; flazalone; fluazacort; flufenamic acid; flumizole; flunisolide acetate; flunixin; flunixin meglumine; fluocortibutyl; fluorometolone acetate; flucuazone; flurbiprofen; fluretophen; fluticasone propionate; furaprofen; furobufen; halcinonide; halobetasol propionate; halopredone acetate; ibufenaco; ibuprofen; aluminum ibuprofen; ibuprofen piconol; ilonidap; indomethacin; sodium indomethacin; indoprofen; indoxol; intrazole; isoflupredone acetate; isoxepaque; isoxicam; ketoprofen; lofemizole hydrochloride; lomoxicam; loteprednol etabonate; sodium meclofenamate; meclofenamic acid; mechlorisone dibutyrate; mefenamic acid; mesalamine; meseclazone; mesterolone; methandrostenolone; methenolone; methenolone acetate; methylprednisolone suptanate; momiflumate; nabumetone; nandrolone; naproxen; sodium naproxen; naproxol; nimazone; olsalazine sodium; orgotem; orpanoxine; oxandrolone; oxaprozine; oxyphenbutazone; oxymetholone; paraniline hydrochloride; sodium pentosan polysulfate; sodium fenbutazone glycerate; pirfenidone; piroxicam; piroxicam cinnamate; piroxicam olamine; pirprofen; prednazate; priphelon; prodolic acid; provuazone; proxazole; proxazole citrate; rimexolone; romazarit; salcolex; salnacedine; salsalate; bloody chloride; seclazone; sermetacin; stanozolol; sudoxicam; sulindac; suprofen; talmetacin; talniflumate; talosalate; tebufelone; tenidap; sodic tenidap; tenoxicam; tesicam; tesimida; testosterone; testosterone combinations; tetridamine; thiopinac; thixocortol pivalate; tolmetin; tolmetin sodium; triclonide; triflumidate; zidomethacin; sodium zomepirac), or antihistammic agents (for example ethanolamines (such as diphenhydramine carbinoxamine), ethylenediamine (such as tripelenamine pyrilamine), alkylamine (such as chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine), other antihistamines such as astemizole, triphenanedin, phenapyrinine, clepheniramine, clepheniramine, clepheniramine.
    [0124] The pharmaceutical or cosmetic compositions of the invention can be administered by any suitable type of route, such as orally, nasal, ocular, topical, intradermal, intracranial or intravenous. The preferred route of administration of such pharmaceutical compositions is the oral, nasal, ocular, topical, intracranial or intradermal route.
    [0126] "Oral route" is understood as the pharmaceutical composition incorporated into the body after swallowing.
    [0128] By "nasal route" is meant the administration of the pharmaceutical composition blown into the nose.
    [0130] "Ocular route" is understood as the topical administration of the pharmaceutical composition by instillation directly into the eye.
    [0132] The "topical route" is understood as application to the exterior of the body, such as, for example, the skin, scalp and nails, and also application to mucosa such as, without limitation, oral, nasal or rectal mucosa.
    [0133] "Intradermally" is understood as the administration of the pharmaceutical composition by injection into the dermis.
    [0135] "Intracranial route" is understood as the administration of the pharmaceutical composition into the skull.
    [0137] "Intravenous route" is understood as the administration of the pharmaceutical composition by injection into the blood stream.
    [0139] Preferably, the cosmetic composition of the present invention may be presented, for example and without limitation, in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water or water-in-oil emulsion, or multiple, of an aqueous or oily gel, of a liquid anhydrous product, of a dispersion of oil in an aqueous phase with the help of spheroids (such as nanospheres, nanocapsules and lipid vesicles), silicone-in-water emulsions, microemulsions, pastes , milks, balms, foams, lotions, creams, soaps, hydroalcoholic solutions, hydroglycolic solutions, liniments, serums, polysaccharide films, ointments, fatty ointments, mousses, ointments, gels, cream gels, foam gels, emulsion-gels and solutions.
    [0141] Materials comprising the compositions disclosed herein are also contemplated by the present invention (eg, peptides, fusion proteins, nucleic acids, vectors, cells, polynucleotides, etc.). For example, materials used to treat wounds are disclosed, in which the materials are coated with the peptide, fusion protein, nanoparticle, the virus-like particle, polynucleotide, vector, cell, or composition of the invention. Illustrative, non-limiting examples of materials used to treat wounds include bandages, surgical tape, sutures, staples, or grafts (eg, skin grafts).
    [0143] For example, the material (bandage, surgical tape, suture, staple, graft) can be soaked in the disclosed peptide at a suitable concentration. The material can then be dried and sealed in a sterile container. The peptide can also be incorporated into a crosslinkable hydrogel system, such as poly (lactic-coglycolic acid) (PLGA) or polyurethane, which can then be designed as materials to treat wounds (eg, bandage, surgical tape, suture, staple, graft). Thus, hydrogel-peptide composites are disclosed.
    [0145] Medical implants coated with the peptide of the invention are also disclosed prior to implantation in a subject. For example, a common problem in such implant surgeries is the formation of a contraction capsule around the implant from the formation of scar tissue leading to undue hardening, contraction, and ultimately deformation of the tissue of interest. The use of the present polypeptides in or on the implant can reduce or prevent this deformation. Non-limiting examples of medical implants include: limb prostheses, breast implants, penile implants, testicular implants, artificial eyes, facial implants, artificial joints, heart valve prostheses, vascular prostheses, dental prostheses, facial prostheses, inclined disc valve , valve caged ball, ear prostheses, nose prostheses, pacemakers, cochlear implants, and skin substitutes (eg, pig / pig skin heterograft, BIOBRANE, cultured keratinocytes).
    [0147] The peptide according to the invention is coated on a medical implant or wound treatment material. Therefore, another aspect of the invention relates to a material or implant comprising the peptide, the fusion protein, the nanoparticle, the virus-like particle, the polynucleotide, the gene construct, the vector, the cell or the composition. described in the present invention.
    [0149] Another aspect of the present invention relates to the peptide, the fusion protein, the nanoparticle, the virus-like particle, the polynucleotide, the gene construct, the vector, the cell, the composition, the material or the implant of the present invention for use as a medicine. Alternatively, this aspect of the invention relates to the use of the peptide, the fusion protein, the nanoparticle, the virus-like particle, the polynucleotide, the gene construct, the vector, the cell, the composition, the material or the implant for the manufacture of a medicine.
    [0151] The present invention further provides a method of promoting wound healing after tissue injury in a subject, comprising administering to the subject one or more of the compositions of the present invention (eg, peptides, polynucleotides, or vectors) in a vehicle. pharmaceutically acceptable. Further disclosed is a method of treating a subject with tissue injury, comprising administering to the subject one or more of the compositions disclosed herein (eg, peptides, polynucleotides, or vectors) in a pharmaceutically acceptable vehicle.
    [0153] "Promote,""promotion" and "promoting" refer to an increase in an activity, response, status, disease, or other biological parameter. This may include but is not limited to the onset of the activity, response, status, or disease. This may also include, for example, a 10% increase in activity, response, status, or disease compared to the native or control level, therefore the increase may be 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100%, or any amount of increase in between compared to native or control levels.
    [0154] By "treating" or "treating" is meant a method of reducing the effects of a disease or condition. Treatment may also refer to a method of reducing the underlying cause of the disease or condition itself, rather than just the symptoms. Treatment may be any reduction in native levels and may be but is not limited to complete suppression of the disease, condition or symptoms of the disease or condition. For example, a disclosed method of promoting wound healing is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject with the disease compared to native levels in the same subject or control subjects. . Therefore, the reduction can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between compared to native levels or control.
    [0156] As used herein, "subject" includes, but is not limited to, animals or plants. The subject may be a vertebrate, more specifically a mammal (eg, a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The subject may be an invertebrate, more specifically an arthropod (eg insects and crustaceans). The term does not indicate a particular age or sex, therefore, adult and newborn subjects, as well as fetuses, both male and female, are intended to be covered.
    [0158] Another aspect of the present invention relates to the fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, cell, composition, material, or implant of the present invention for its use in cellular and / or tissue regeneration, in particular for use in the treatment of a wound, preferably the wound is selected from the group consisting of a skin wound, a pressure ulcer and / or an external wound or internal.
    [0160] Another aspect of the present invention relates to cosmetic use of the fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, cell, composition, material, or implant of the present invention as a healing and / or skin repairing agent, preferably the use It would be in dermocosmetic treatments as a healing and skin repairing agent or in topically applied dermocosmetic compositions.
    [0162] Another aspect of the invention relates to a peptide comprising the sequence SEQ ID NO: 2, for use in cell regeneration, in particular for use in treating a wound, preferably the wound is selected from the group consisting of a skin wound, a pressure ulcer, and / or an external or internal wound. More preferably the peptide comprises the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. In a more preferred embodiment the peptide It consists of the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, most preferably the peptide It consists of the sequence SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
    [0164] In the present invention, the term "regeneration" refers to the process by which the structure and function of damaged organs or body parts are recovered. The regeneration process can occur at multiple states, levels of biological organization, and the ability of different organisms to regenerate missing parts is highly variable. Regeneration can take place at the cellular (cellular regeneration), tissue (tissue regeneration), organ, structure and even whole body levels.
    [0165] Non-limiting examples of injuries are:
    [0167] - a burn wound, which is the injury resulting from exposure to heat, electricity, radiation (for example, sunburn and laser surgery), or caustic chemicals,
    [0169] - ulcers,
    [0171] - wounds in diabetes mellitus, which are generally foot injuries due to numbness caused by nerve damage (diabetic neuropathy) and decreased blood flow to the legs and feet. The most serious injury is a foot ulcer. Diabetic foot ulcers have a very high risk of being infected, and sometimes cannot be cured. Unhealed foot ulcers are a common cause of amputation in people with diabetes,
    [0172] decubitus wounds, that is, injuries caused by unmitigated pressure on any part of the body, especially portions on bony or cartilaginous areas,
    [0174] - wounds due to an external force that damages the tissue,
    [0176] - skin wounds due to aging or the environment. This includes, for example, cracks, dry skin, roughness of the skin, and the like.
    [0178] The wounds can be an internal wound, an oral wound, a skin wound, an external wound, an ulcer and / or a pressure ulcer, damage to the eye, an eye injury, for example, conjunctiva. Other conditions that can be treated with the peptides according to the disclosure include oral ulcers, oral aphthous lesions, glossodynia, burning tongue, psoriasis, eczema, and hair loss.
    [0180] These types of skin damage and injuries are known to those skilled in the art. An internal wound is a wound present in the body, for example, due to a surgical incision. An oral wound is a wound present in the oral cavity. A skin wound is a wound on the skin. An external wound should be understood as a wound that is visible and accessible from outside the body. An ulcer is an injury to the skin surface or a mucous surface. A pressure ulcer is a wound or ulcer caused by prolonged pressure on the skin and tissues when in a position for a long period of time, such as lying in bed. The bony areas of the body are the most frequently affected sites, which become ischemic under sustained and constant pressure. Skin aging is the change in the appearance of the skin due to time or exposure to the environment or the state of health of an individual. Cellulite is the definition used in cosmetics in relation to a flabby or wavy appearance of the skin (orange peel in Dutch).
    [0182] In a particular embodiment of the invention, the peptide of the invention is used for the treatment of a skin wound. Skin wounds can be wounds to the epidermis or dermis of the skin. There are several types of wounds in which the skin or tissue may need repair: abrasions, lacerations, incisions, perforations, and avulsions and burns. The use of a peptide according to the invention can improve the general health of the skin. In another particular embodiment of the Invention, the method is used for the treatment of an oral wound. Oral wounds are wounds in any part of the oral cavity in which the oral mucosa is damaged. In another particular embodiment of the invention, the peptide of the invention is used for the treatment of an internal wound. Internal wounds are wounds in which cell layers of endodermal or mesodermal origin are damaged. Examples are wounds in arteries or veins, peritoneum or pericardium.
    [0184] Within the present invention, the therapeutic or cosmetic use of all those embodiments that, directly or indirectly, comprise the peptide comprising the sequence SEQ ID NO: 2, for the treatment of a skin wound as previously defined, is also contemplated. in the present description. Examples of such embodiments relate to the fusion protein, nanoparticle, virus-like particle, polynucleotide, gene construct, vector, cell, composition, material or implant comprising the peptide comprising the sequence SEQ ID NO: 2. All of these terms have also been previously defined in the present description.
    [0186] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages, and features of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0188] BRIEF DESCRIPTION OF THE FIGURES
    [0190] Figure 1 (A and B) Effect of the entire peptide of miticin C on wound healing in HaCat cells.
    [0192] Figure 2. (A) Appearance of the tail of zebrafish larvae with the tail intact. (B) Fish after sectioning off a piece of tail. (C) Evolution over time of tail tissue regeneration. (D) Comparison of tissue regeneration in fish treated and not treated with miticin after 4 days.
    [0194] Figure 3. Evolution of wound healing in HaCat cells treated with the miticin-C peptides.
    [0196] Figure 4. Effect of the complete peptide of miticin on the proliferation of human keratinocyte cells (HaCat).
    [0198] Figure 5. Effect of peptides on cell viability of HaCat cells.
    [0200] Figure 6. Comparison of regeneration in fish treated and untreated with miticin-C peptides after 4 days.
    [0202] EXAMPLES
    [0204] Next, the invention will be illustrated by tests carried out by the inventors, which shows the effectiveness of the product of the invention.
    [0206] Example 1: Peptides and their synthesis
    [0207] The mature miticin-C chain (Myt-C), composed of 40 amino acids with the sequence QSVACRSYYCSKFCGSAGCSLYGCYLLHPGKICYCLHCSR (SEQ ID NO: 1) was taken as a model for the synthesis of 14 dodecapeptides with a sequence shift of two amino acid residues and amidated at the terminal carboxyl and acetylated at the amino. The complete chain and the peptides thus designed were synthesized and purified by high-performance liquid chromatography (HPLC) at the Proteomics service of the National Center for Biotechnology of the CSIC with a purity of> 95%. Its sequences were verified by mass spectrometry.
    [0209] Possible traces of trifluoroacetic acid (TFA) from the synthesis process were removed with three freeze-lyophilization cycles using 10 mM HCl as solvent. The contents of the vial (3 mg of each peptide) were dissolved in 1 mL of serum-free DMEM aqueous culture medium supplemented with gentamicin and a commercial protease inhibitor cocktail (Protease Inhibitor Tablets, Thermo Scientific, Ref.
    [0210] 88265) leaving a final concentration of the stock solution of 3 mg / mL (676.8 pM) that was stored at -80 ° C before use.
    [0213] Table 1: Synthetic peptides designed from the miticin-C sequence with its molecular weight (P.M.).
    [0215] Example 2: Determination of the cytotoxicity of Miticin C in vitro.
    [0217] In vitro cytotoxicity determination of the mature miticin C chain (SEQ ID NO: 1) and its fragments (Table 1) was evaluated in the HaCat line of keratinocytes human, by means of the MTT test in the concentration range 0-40 pM (Figures 4 and 5). As seen in Figures 4 and 5, treatment with the peptides did not affect the viability of HaCat cells.
    [0219] Example 3: Determination of Mythicin C wound repair activity in vitro.
    [0221] The determination of the wound repair activity in vitro was carried out using monolayers of the HaCat line of human keratinocytes.
    [0223] A "wound" was performed and photographed at different times to determine the degree of its closure, in the presence and absence of miticin C peptides. First, the capacity of the miticin C peptide was confirmed (SEQ ID NO: 1) to stimulate wound healing in keratinocytes in vitro (Figure 1). Cells treated with a concentration of 5 pM showed faster wound healing than control cells. At 48 h the closure of the wound was significantly higher in the treated cells and at 72 h the wound was totally repaired unlike the control cells.
    [0225] This assay was repeated again with the overlapping peptides based on the mature miticin C chain in Table 1. As seen in Figure 3, the P1, P2a, P2b, P3 and P4 peptides induced cell proliferation. When cells were wounded, significantly more healing was observed in those that had been treated with peptides P1, P2a, P2b, P3, and P4. 48 hours after treatment, it is observed, as shown in Figure 3, that the peptides P1, P2a, P2b, P3 and P4, unlike the control, show a lower percentage of wound opening and, therefore, have increased wound healing and damage repair. At 72 hours, the peptides P2b, P3 and P4 had managed to completely close the wound (Figure 3). The P6, P7 and P9 peptides due to problems in the synthesis process showed poor solubility, and were not taken into account in the tests.
    [0227] Example 4: Determination of miticin C wound repair activity in vivo.
    [0228] After the experiments on the HaCat line of human keratinocytes, an in vivo test was designed using zebrafish ( Danium rhenium ) larvae. Larvae 3 days post-fertilization were used and a tail cut was performed. Subsequently, tissue regeneration was studied in larvae treated with the peptides of Table 1 of miticin C and in the larvae not treated with miticin C, by means of photographs under the magnifying glass and image analysis using the imageJ program. Figures 2 and 6 show the results showing a significantly higher tissue regeneration of the tail in those larvae treated with the complete miticin C peptide and with the overlapping peptides P2a, P2b, P3 and P4 of the mature miticin peptide. C. The presence of neutrophils in the wound area was evaluated using for this the line of transgenic fish Tg (Mpx: GFP) i114, which have neutrophils labeled with GFP. In this way, the ability of miticin C in the resolution of inflammation associated with the wound was determined.
    权利要求:
    Claims (25)
    [1]
    1. A peptide comprising sequence SEQ ID NO: 2 and which is not miticin-C with sequence SEQ ID NO: 1.
    [2]
    2. The peptide according to claim 1, wherein the peptide is between 6 and 40 amino acids in size.
    [3]
    3. The peptide according to any of claims 1 or 2, comprising the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
    [4]
    4. The peptide according to claim 1, consisting of the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
    [5]
    5. The peptide according to claim 4, consisting of the sequence SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7
    [6]
    6. A fusion protein comprising the peptide according to any of claims 1 to 5.
    [7]
    7. A nanoparticle comprising the peptide according to any of claims 1 to 5, or the fusion protein according to claim 6.
    [8]
    8. A virus-like particle comprising the peptide according to any of claims 1 to 5, or the fusion protein according to claim 6.
    [9]
    9. A polynucleotide encoding the peptide according to any of claims 1 to 5, or the fusion protein according to claim 6.
    [10]
    10. A gene construct comprising the polynucleotide according to claim 9.
    [11]
    11. A vector comprising the polynucleotide according to claim 9 or the gene construct according to claim 10.
    [12]
    12. A cell comprising the peptide according to any of claims 1 to 5, the fusion protein according to claim 6, the polynucleotide according to claim 9, the gene construct according to claim 10, or the vector according to claim 11.
    [13]
    13. A composition comprising the peptide according to any of claims 1 to 5, the fusion protein according to claim 6, the nanoparticle according to claim 7, the virus-like particle according to claim 8, the polynucleotide according to claim 9, the gene construct according to claim 10, the vector according to claim 11, or the cell according to claim 12.
    [14]
    14. The composition according to claim 13, wherein the composition is a pharmaceutical composition or cosmetic composition.
    [15]
    15. The composition according to claim 14, further comprising a pharmaceutically or cosmetically acceptable carrier or excipient.
    [16]
    16. Material or implant comprising the peptide according to any of claims 1 to 5, the fusion protein according to claim 6, the nanoparticle according to claim 7, the virus-like particle according to claim 8, the polynucleotide according to claim 9, the construction gene according to claim 10, the vector according to claim 11, the cell according to claim 12 or the composition according to any of claims 13 to 15.
    [17]
    17. A fusion protein according to claim 6, a nanoparticle according to claim 7, a virus-like particle according to claim 8, a polynucleotide according to claim 9, a gene construct according to claim 10, a vector according to claim 11, a cell according to claim 12, a composition according to any of claims 13 to 15 or a material according to claim 16, for use as a medicine.
    [18]
    18. A fusion protein according to claim 6, a nanoparticle according to claim 7, a virus-like particle according to claim 8, a polynucleotide according to claim 9, a gene construct according to claim 10, a vector according to claim 11, a cell according to claim 12, a composition according to any one of claims 13 to 15 or a material according to claim 16, for use in the treatment of wounds, preferably the wound is selected from the group that It consists of a skin wound, a pressure ulcer, and / or an external or internal wound.
    [19]
    19. Cosmetic use of a fusion protein according to claim 6, a nanoparticle according to claim 7, a virus-like particle according to claim 8, a polynucleotide according to claim 9, a gene construct according to claim 10, a vector according to claim 11, a cell according to claim 12, a composition according to any of claims 13 to 15 or a material according to claim 16, as a healing and / or skin repairing agent.
    [20]
    20. Peptide comprising the sequence SEQ ID NO: 2, for use in treating a wound, preferably the wound is selected from the group consisting of a skin wound, a pressure ulcer and / or an external wound or internal.
    [21]
    21. Peptide for use according to claim 20, comprising the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
    [22]
    22. Peptide for use according to claim 21, consisting of the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
    [23]
    23. Cosmetic use of the peptide comprising the sequence SEQ ID NO: 2 as a healing and / or skin repairing agent.
    [24]
    24. Use according to claim 23, wherein the peptide comprises the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
    [25]
    25. Use according to claim 24, where the peptide consists of the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7
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    同族专利:
    公开号 | 公开日
    WO2020109642A1|2020-06-04|
    ES2763349B2|2021-12-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    FR2796072A1|1999-07-08|2001-01-12|Centre Nat Rech Scient|MOLLUSC ANTI-MICROBIAL PEPTIDES|
    ES2334083A1|2007-11-28|2010-03-04|Consejo Superior De Investigaciones Cientificas|Method for the identification of antibacterial and antiviral polypeptides obtained from mytilus edulis |
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    PCT/ES2019/070815| WO2020109642A1|2018-11-28|2019-11-28|Myticin peptide and its use in cell regeneration|
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