Peptides and compositions for use in cosmetics - Patents.com

Abstract

The present invention refers to a peptide of 4-8 amino acids that can reduce the activity and / or signal of at least one kind of sodium channel. The peptide can also promote the production of extracellular matrix. Also disclosed herein are compositions comprising the peptide, and the use of the peptide and composition of the present invention in the field of cosmetics for the treatment of wrinkles.

Description

Detailed Description of the Invention

[0001] FIELD OF THEINVENTION The present invention relates to the field of molecular biology, more precisely to molecular biology applied to cosmetics, and even more precisely to peptides and compositions comprising said peptides that can modulate sodium channels providing a signal and / or a reduction in their expression, and thus provide muscle relaxation or a reduction in muscle contraction. Said peptides and compositions comprising said peptides can also maintain and / or synthesize or increase extracellular matrix (ECM) components. Thus, said peptides and compositions are effective in the treatment of conditions related to the modulation of the above aspects (e.g. muscle relaxation or the maintenance or synthesis of ECM) and are useful in cosmetics (anti-wrinkle activity, activity against skin laxity or slackness, improvement of skin firmness and / or elasticity).

[0002] [Background technology] In recent years, people have become increasingly interested in preventing, reducing or eliminating skin imperfections in order to maintain healthy and youthful looking skin. This has led to an increased interest in the causes that lead to the appearance of skin imperfections, and in the search for compounds and compositions that can prevent or alleviate these imperfections.

[0003] The basis or mechanism of the formation of wrinkles (e.g., expression lines) is muscle tension that pulls the skin inward. This muscle tension is the result of overactivity of the nerves that innervate the corresponding muscles. The overactivity of said nerves is then characterized by an excessive and uncontrolled release of neurotransmitters that excite the muscle fibers.

[0004] In this context, several cosmetic approaches to wrinkles and the mechanisms of their development have been developed, for example: Botulinum toxin: widely used to reduce and / or remove facial wrinkles. Especially serotype A (BOTOX® Cosmetic, Allergan Inc.). The botulinum toxin is injected locally and its paralytic effect is reversible, with an average duration of 6 months, so repeated injections are necessary. It is known that there is a risk of inducing an immune response to the botulinum preparation, resulting in loss of therapeutic efficacy. To overcome this problem, other serotypes of botulinum toxin, such as B, F, and E, have also been considered, but the serotypes also have the risk of inducing an immune response. At the molecular level, botulinum toxin is a protease that degrades neuronal proteins involved in the calcium ion-activated exocytosis mechanism. For example, botulinum toxin A cleaves the SNAP-25 protein in neurons.

[0005] Particular peptides derived from the sequences of proteins forming the SNARE complex are, for example, peptides derived from the amino and carboxyl domains of SNAP-25 (see, for example, PCT Patent Application WO97 / 34620, European Patent EP1180524B1 and European Patent EP2123673B1); peptides derived from synaptobrevin or syntaxin (see, for example, PCT Patent Application WO97 / 34620; Blanes-Mira, C., Clemente, J., Jodas, G., Gil, A., Fernandez-Ballester, G., Ponsati, B., Gutierrez, L., Perez-Paya, E., Ferrer-Montiel, A. A synthetic hexapeptide (Argireline) with antiwrinkle activity. International Journal of Cosmetic Science, (2002), 24, It is also known in the art that peptides that can inhibit neuronal exocytosis, such as peptides that compete with specific sequences located on the surface of the interaction between Munc18 and Syntaxin-1 (see PCT Patent Application PCT / EP2019 / 054479).

[0006] There is also a conotoxin in the art named Activen XEP-018™. It is a biomimetic peptide that mimics the venom of the cone snail (Conus consors) from the Indo-West Pacific region and exhibits Botox-like activity. Biologically, it acts as a modulator of neuromuscular transmission and as a potent and selective blocker of the voltage-gated sodium channel Nav1.4. Cosmetically, it acts as a fast-acting wrinkle reducer and gives the appearance of smooth skin.

[0007] However, there remains a need to find new molecules, preferably peptides, that have anti-wrinkle activity and that can aid in the maintenance and synthesis of the ECM.

[0008] Description of the Invention Voltage-gated sodium channels control nerve signaling by managing the propagation of action potentials. Inhibiting sodium channels reduces the flow of action potentials along the nerve, thus reducing the effectiveness of the signal. In skin, sodium channels such as NaV1.3 and the closely related NaV1.4 are found in the epidermis, subcutaneous adipose tissue, skeletal muscle, and nerves. These systems improve cutaneous muscle tone and thus the depth of wrinkles and creases in the skin.

[0009] In this sense, it is noteworthy that Takifugu rubripes (Nihonpufu, tiger pufferfish) is a prized food fish in Japan, despite being highly toxic due to the presence of tetrodotoxin (TTX) in its liver and ovaries. TTX is a potent toxin that specifically binds to voltage-gated sodium channels. TTX physically binds to voltage-gated sodium channels and blocks the flow of sodium ions through said channels, thereby preventing the generation and propagation of action potentials (APs). Furthermore, said pufferfish is well known for its elastic skin, which allows it to inflate its body to deter predators and recovers to its original form in a short time once it has overcome the danger. Thus, some important complementary activities could potentially be identified for said peptides on human skin, which could result in a positive impact on wrinkles, including both muscle-related or expression wrinkles, and age-related wrinkles.

[0010] After extensive and thorough research, the inventors have surprisingly found that peptides derived from Takifugu rubripes (from their protein or genome) and / or peptides derived therefrom by conservative substitutions provide suitable cosmetic anti-wrinkle activity. The peptides can provide the necessary muscle relaxant activity by modulating voltage-gated sodium channels (reducing the signal or activity provided by the channels). Furthermore, it has also been shown that the peptides of the present invention are capable of modulating the expression of voltage-gated sodium channels (downregulating or decreasing the expression of the channels) and modulating the release of noradrenaline (reducing the levels of the neurotransmitter and thus reducing the release of the neurotransmitter). Furthermore, the peptides of the present invention can also upregulate the expression of major extracellular matrix (hereinafter ECM) components such as collagen I, elastin, and hyaluronic acid at the gene and protein expression levels. These demonstrate the usefulness of the peptides of the present invention in cosmetics for the treatment, prevention and / or reduction of wrinkles (preferably both age wrinkles and muscle or expression wrinkles); treatment, prevention and / or reduction of loose or sagging skin; treatment, prevention and / or reduction of rough skin; and improvement of skin firmness and / or elasticity.

[0011] Blocking voltage-gated sodium channels (reducing signal transduction and / or their activity) for muscle relaxation is an interesting mechanism of action that has not yet been widely studied and is clearly different from that of the products currently in widespread use on the market. The peptides of the present invention reduce neurotransmission before it reaches the neuromuscular junction.

[0012] Furthermore, due to their size, the peptides of the invention have inherent advantages such as increased or prolonged stability and preferably increased or improved penetration through the skin.

[0013] In a first aspect, the present invention relates to a peptide of 4 to 8 amino acids that is capable of reducing the signal and / or activity of at least one sodium channel and thus has anti-wrinkle activity.

[0014] In a second aspect, the present invention relates to a composition comprising a peptide of the invention.

[0015] In a further aspect, the present invention relates to the cosmetic use of a peptide of the invention or a composition of the invention in a subject in need of treatment.

[0016] In a fourth aspect, the present invention relates to the cosmetic use of a peptide of the invention or a composition of the invention in a subject in need of treatment.

[0017] In a final aspect, the present invention relates to a method for reducing, preventing and / or eliminating the signs of skin aging in a subject, comprising administering to the subject a peptide of the invention or a composition of the invention.

[0018] As used herein, the term "acyclic aliphatic group" and its plurals have the ordinary meaning given to said term in the art. Thus, these terms refer to, for example, but not limited to, linear or branched alkyl, alkenyl, and alkynyl groups.

[0019] As used herein, the term "alkyl group" and its plural forms refer to a saturated, straight chain or branched group having 1 to 24, preferably 1 to 16, more preferably 1 to 14, even more preferably 1 to 12, and even more preferably 1, 2, 3, 4, 5, or 6 carbon atoms, attached to the remainder of the molecule by a single bond, for example, but not limited to, methyl, ethyl, isopropyl, n-propyl, i-propyl, isobutyl, tert-butyl, n-butyl, sec-butyl, n-pentyl, n-hexyl, heptyl, octyl, decyl, dodecyl, lauryl, hexadecyl, octadecyl, amyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl, and the like. Alkyl groups can be optionally substituted with one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0020] As used herein, the term "alkenyl group" and its plural forms refer to a linear or branched group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, even more preferably 2, 3, 4, 5 or 6 carbon atoms, conjugated or non-conjugated, one or more carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds, and bonded to the remainder of the molecule through a single bond, including, but not limited to, vinyl, oleyl, linoleyl and similar groups. The alkenyl group may be optionally substituted by one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.

[0021] As used herein, the term "alkynyl group" and its plural forms refer to a linear or branched group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, and even more preferably 2, 3, 4, 5, or 6 carbon atoms, conjugated or non-conjugated, one or more carbon-carbon triple bonds, preferably 1, 2, or 3 carbon-carbon triple bonds, and bonded to the remainder of the molecule through a single bond, including, for example, but not limited to, ethynyl groups, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, pentynyl (such as 1-pentynyl), and similar groups. Alkynyl groups may be optionally substituted with one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0022] As used herein, the term "alicyclic group" and its plural forms have the ordinary meaning given to said term in the art, and thus are used to refer to, for example, but not limited to, a cycloalkyl group or a cycloalkenyl group or a cycloalkynyl group.

[0023] As used herein, the term "cycloalkyl" and its plural forms refer to a saturated monocyclic aliphatic or polycyclic aliphatic group having 3 to 24, preferably 3 to 16, more preferably 3 to 14, even more preferably 3 to 12, and even more preferably 3, 4, 5, or 6 carbon atoms attached to the remainder of the molecule via a single bond, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethylcyclohexyl, octahydroindene, decahydronaphthalene, dodecahydro-phenalene, adamantyl, and the like. Cycloalkyls can be optionally substituted with one or more groups such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0024] As used herein, the term "cycloalkenyl" and its plural forms refer to a non-aromatic monocyclic aliphatic or polycyclic aliphatic group having 5 to 24, preferably 5 to 16, more preferably 5 to 14, even more preferably 5 to 12, and even more preferably 5 or 6 carbon atoms, conjugated or non-conjugated, one or more carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds, and bonded to the remainder of the molecule through a single bond, including, but not limited to, cyclopent-1-en-1-yl and similar groups. Cycloalkenyls can be optionally substituted with one or more groups such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0025] As used herein, the term "cycloalkynyl" and its plural forms refer to a non-aromatic monocyclic aliphatic group or a non-aromatic polycyclic aliphatic group having 8 to 24, preferably 8 to 16, more preferably 8 to 14, even more preferably 8 to 12, and even more preferably 8 or 9 carbon atoms, conjugated or non-conjugated, one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, and bonded to the remainder of the molecule via a single bond, including, for example, but not limited to, cyclooct-2-yn-1-yl groups and the like. Cycloalkynyl can be optionally substituted with one or more groups such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0026] As used herein, the term "aryl group" and its plurals refer to aromatic groups having 6 to 30, preferably 6 to 18, more preferably 6 to 10, and even more preferably 6 or 10 carbon atoms, containing 1, 2, 3 or 4 aromatic rings, bonded or fused by carbon-carbon bonds, and bonded to the remainder of the molecule through a single bond, including, but not limited to, phenyl, naphthyl, diphenyl, indenyl, phenanthryl, or anthranyl, among others. Aryl groups may be optionally substituted with one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0027] As used herein, the term "aralkyl" and its plurals refer to an alkyl group substituted with an aromatic group having 7 to 24 carbon atoms, such as, but not limited to, -(CH 2 ) 1-6-phenyl, -(CH 2 )1-6-(1-naphthyl), -(CH 2 )1-6-(2-naphthyl), -(CH 2 )1-6-CH(phenyl)2 Aralkyl groups may be optionally substituted with one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0028] As used herein, the term "heterocyclic group" and its plurals refer to a 3- to 10-membered heterocyclyl or hydrocarbon ring in which one or more of the ring atoms, preferably one, two or three of the ring atoms, are elements different from carbon, such as nitrogen, oxygen or sulfur, and may be saturated or unsaturated. For the purposes of the present invention, a heterocyclyl may be a cyclic, monocyclic, bicyclic or tricyclic system, which may include fused ring systems; the nitrogen, carbon or sulfur atoms may be optionally oxidized in the heterocyclyl radical; the nitrogen atom may be optionally quaternized; the heterocyclyl radical may be partially or fully saturated or aromatic. Preferably, the term heterocyclyl relates to a 5- or 6-membered ring. The heterocyclic group may be optionally substituted by one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.

[0029] As used herein, the term "heteroarylalkyl" and its plurals refer to an alkyl group substituted with a substituted or unsubstituted aromatic heterocyclyl group, the alkyl group having from 1 to 6 carbon atoms and the aromatic heterocyclyl group having from 2 to 24 carbon atoms and 1 to 3 non-carbon atoms, such as, but not limited to, -(CH 2 ) 1-6-imidazolyl, -(CH 2 ) 1-6-Triazolyl, -(CH 2 ) 1-6-Thienyl, -(CH 2 ) 1-6-furyl, -(CH 2) 1-6-pyrrolidinyl, and the like. Heteroarylalkyl groups can be optionally substituted with one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0030] As used herein, the terms "halo" or "halogen" refer to fluorine, chlorine, bromine, or iodine, the anions of which refer to a halide.

[0031] As used herein, the term "derivatives" and its plural forms refer to cosmetically acceptable compounds, i.e. compounds derived from the compound of interest that may be used in the preparation of a cosmetic product, and to derivatives that are not cosmetically acceptable, as they may be useful in the preparation of cosmetically acceptable derivatives.

[0032] As used herein, the term "salt" and its plural forms refer to any type of salt known in the art, such as halide salts, hydroxy acid salts (oxy acid salts, acid salts, base salts and double salts, etc.), hydroxy salts, mixed salts, oxy salts or other hydrated salts. The term includes cosmetically acceptable salts; and cosmetically unacceptable salts, since the latter may be useful in the preparation of cosmetically acceptable salts.

[0033] As used herein, the term "isomer" and its plural forms refer to optical isomers, enantiomers, stereoisomers or diastereoisomers. Individual enantiomers or diastereoisomers, as well as mixtures thereof, can be separated by conventional techniques known in the art.

[0034] As used herein, the term "solvate" and its plural forms refer to any solvate known in the art, such as polar solvate, non-polar solvate or amphiphilic solvate, including any cosmetically acceptable solvate that, when administered or applied (directly or indirectly) to a subject of interest, results in the compound of interest (the peptide or peptides of the present invention).Preferably, the solvate is a hydrate, a solvate with alcohol (such as methanol, ethanol, propanol or isopropanol), a solvate with ester (such as ethyl acetate), a solvate with ether (such as methyl ether, ethyl ether or THF (tetrahydrofuran)) or a solvate with DMF (dimethylformamide), more preferably a hydrate or a solvate with alcohol (such as ethanol).

[0035] Furthermore, as used herein, the term "amino acid" and its plural forms include not only the amino acids encoded by the genetic code, but also the non-encoded amino acids, whether naturally occurring or not, and whether D-amino acids and L-amino acids. Examples of unencoded amino acids include, but are not limited to, citrulline, ornithine, sarcosine, desmosine, norvaline, 4-aminobutyric acid, 2-aminobutyric acid, 2-aminoisobutyric acid, 6-aminohexanoic acid, 1-naphthylalanine, 2-naphthylalanine, 2-aminobenzoic acid, 4 aminobenzoic acid, 4-chlorophenylalanine, 2,3-diaminopropionic acid, 2,4 diaminobutyric acid, cycloserine, carnitine, cysteine, penicillamine, pyroglutamic acid, thienylalanine, hydroxyproline, allo-isoleucine, allo-threonine, isonipecotic acid, isoserine, phenylglycine, statins, β-alanine, norleucine, N-methyl amino acids, α-amino acids and β-amino acids, and derivatives thereof, among others. Nonetheless, additional unnatural amino acids are known in the art (see, e.g., "Unusual amino acids in peptide synthesis" by DC Roberts and F. Vellaccio, The Peptides, Vol. 5 (1983), Chapter VI, Gross E. and Meienhofer J., Eds., Academic Press, New York, USA).

[0036] As used herein, "percentage of identity" with respect to peptides, polypeptides and proteins has the meaning generally ascribed in the art and thus relates to the percentage of identical amino acids between two amino acid sequences compared after optimal alignment of these sequences, where said percentage is merely statistical and the differences between the two amino acid sequences are randomly distributed throughout the sequences. "Optimal alignment" is understood as the alignment of amino acid sequences that results in a larger percentage of identity. The percentage of identity is calculated by determining the number of identical positions in the two compared sequences where amino acids are identical, dividing the number of identical positions by the number of positions compared, and multiplying the result by 100 to obtain the percentage of identity between the two sequences. The sequence comparison between two amino acid sequences can be performed manually or by computer programs known in the art, such as the BLAST (Basic Local Alignment Search Tool) algorithm.

[0037] As described above, in a first aspect, the present invention refers to a peptide of 4 to 8 amino acids (i.e., a peptide comprising 4 to 8 amino acids, more preferably a peptide having 4 to 8 amino acids, more preferably a peptide consisting essentially of 4 to 8 amino acids, more preferably a peptide consisting of 4 to 8 amino acids), capable of reducing the signal and / or activity of at least one type of sodium channel; its acceptable isomers, salts, solvate derivatives and / or mixtures thereof.

[0038] As mentioned above, the peptide of the present invention is preferably derived from pufferfish protein and / or pufferfish genome. The pufferfish can be any pufferfish currently known or discovered in the future. More preferably, the pufferfish is Takifugu rubripes. The peptide of the present invention can also be a peptide obtained or derived from a peptide derived from pufferfish protein and / or pufferfish genome, preferably by conservative substitution.

[0039] It is contemplated that the amino acids used or present in the peptides of the invention are L-amino acids, D-amino acids, or combinations thereof. In a preferred embodiment, the amino acids used or present in the peptides of the invention are L-amino acids.

[0040] Preferably, said isomer is a stereoisomer. Said stereoisomer is intended to be an enantiomer or a diastereoisomer. Thus, in a preferred embodiment of the present invention, the peptide is a racemic mixture, a diastereomeric mixture, a pure enantiomer or a pure diastereoisomer.

[0041] Furthermore, the isomers, salts, solvates, derivatives and / or mixtures thereof are preferably cosmetically acceptable isomers, salts, solvates, derivatives and / or mixtures thereof.

[0042] As can be directly derived from the examples contained below, the peptides of the invention provide sodium channel modulation, which is manifested as a decrease in the signal and / or activity of at least one sodium channel, said decrease in the activity and / or signal of at least one sodium channel being preferably a decrease in the current provided by said at least one sodium channel, i.e. a decrease in the action potential.

[0043] The sodium channel is preferably a voltage-gated sodium channel.

[0044] At least one kind of sodium channel can be any kind of sodium channel.Examples of sodium channels are NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, NaV1.8 and NaV1.9.It is contemplated that the peptide of the present invention can reduce the activity and / or signal of one kind of sodium channel or several kinds (two or more kinds) of sodium channels.

[0045] In an embodiment, the peptide of the present invention reduces at least the activity and / or signal of the sodium channel Nav1.3, and more preferably, the peptide of the present invention reduces only the activity and / or signal of the sodium channel Nav1.3.

[0046] In a further embodiment, the peptide of the present invention reduces at least the activity and / or signal of the sodium channel NaV1.4, and more preferably, the peptide of the present invention reduces only the activity and / or signal of the sodium channel NaV1.4.

[0047] In a preferred embodiment, the peptide of the present invention reduces the signaling and / or activity of at least the sodium channels Nav1.3 and Nav1.4, and more preferably, the peptide of the present invention reduces only the activity and / or signaling of the sodium channels Nav1.3 and Nav1.4.

[0048] It should be noted that the peptides of the present invention can reduce the activity and / or signal of at least one type of sodium channel wherever sodium channels are present. Preferably, the peptides of the present invention reduce the activity and / or signal of at least one sodium channel at the presynaptic level, preferably in neurons.

[0049] In some embodiments of the present invention, reducing the signal and / or activity of at least one type of sodium channel means blocking at least one type of sodium channel, which can be partial blocking or total blocking, more preferably partial blocking.

[0050] Furthermore, in some embodiments, this embodiment can be combined with any of the above-mentioned embodiments, and the peptide of the present invention can also regulate the expression of at least one sodium channel, more preferably downregulate the expression of at least one sodium channel. The at least one sodium channel is as described above. In these embodiments, the peptide of the present invention can downregulate the expression of at least one sodium channel wherever said sodium channel is expressed. Preferably, the peptide of the present invention downregulates the expression of at least one sodium channel at the postsynaptic level, preferably in muscle cells, more preferably in skeletal muscle cells.

[0051] Moreover, in some embodiments, this embodiment can be combined with any of the above-mentioned embodiments, and the peptide of the present invention can also maintain and / or synthesize ECM, more precisely promote the maintenance of ECM and / or promote and / or increase the synthesis of ECM components.The peptide of the present invention exerts said activity by upregulating the expression of related genes and proteins of ECM; more preferably, the peptide of the present invention is capable of upregulating at least one gene and / or protein responsible for the synthesis and / or maintenance of ECM; more preferably, the peptide of the present invention is also capable of upregulating at least one gene and / or protein responsible for the synthesis and / or maintenance of ECM, selected from collagen I, elastin, hyaluronic acid or a combination thereof; even more preferably, the peptide of the present invention is also capable of upregulating collagen I, elastin and hyaluronic acid, all at the gene and / or protein level.

[0052] In a preferred embodiment, which can be combined with any of the above-mentioned embodiments, the peptides of the invention are capable of increasing the synthesis (and therefore the levels) of elastin, procollagen Iα1 and hyaluronic acid at the protein level.

[0053] In a further preferred embodiment, which may be combined with any of the above-mentioned embodiments, the peptide of the present invention is capable of downregulating the expression of genes that lead to the degradation of ECM and / or upregulating genes that lead to the maintenance or synthesis of ECM. More preferably, the downregulated genes are selected from MMP1, HYAL1 or a combination thereof; the upregulated genes are selected from TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1, COL7A1 or a combination thereof. Even more preferably, the downregulated genes are MMP1 and HYAL1, and the upregulated genes are TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1 and COL7A1.

[0054] The peptides of the present invention are capable of exerting the above-mentioned activity of maintaining and / or synthesizing ECM (more preferably, promoting the maintenance of ECM and / or promoting and / or increasing the synthesis of ECM) at least in fibroblasts.

[0055] In a further embodiment, this embodiment can be combined with any of the above-mentioned embodiments, and the peptide of the present invention reduces the level or amount of at least one neurotransmitter, more preferably noradrenaline.The reduction of the level of at least one neurotransmitter is preferably carried out at presynaptic level, preferably in neurons.As easily inducible, the reduction of the level or amount of at least one neurotransmitter leads to the reduction or suppression of the secretion of said neurotransmitter.

[0056] The above activities are demonstrated in the examples included below, providing the ability of the peptides of the present invention to induce or produce muscle relaxation (or avoid muscle contraction or excessive muscle contraction), which in turn provides an anti-wrinkle effect (preferably against expression wrinkles). Furthermore, the above activities also verify the usefulness of the peptides of the present invention for preventing and / or reducing age-related wrinkles (preferably their depth, length and / or roughness); treating, preventing and / or reducing loose or sagging skin; and improving skin firmness and elasticity.

[0057] In a preferred embodiment, which may be combined with any of the above-mentioned embodiments, the peptide of the present invention is a peptide of 5 or 6 amino acids (i.e. a peptide comprising 5 or 6 amino acids, more preferably a peptide having 5 or 6 amino acids, more preferably a peptide consisting essentially of 5 or 6 amino acids, more preferably a peptide consisting of 5 or 6 amino acids).

[0058] Furthermore, preferably, the peptide of the present invention, in any of the above-mentioned embodiments, comprises at least one charged amino acid (positive or negative). Even more preferably, the amino acid at position 1 and / or the amino acid at position 2 are charged amino acids (positive or negative). Even more preferably, in the peptide of the present invention, the first three amino acids are charged amino acids (positive or negative).

[0059] In a preferred embodiment, which can be combined with any of the above-mentioned embodiments, in the peptide of the present invention, the amino acids at positions 1 and 3 are positively charged amino acids and the amino acid at position 2 is a positively or negatively charged amino acid.

[0060] Furthermore, preferably, in any of the above embodiments, in the peptide of the present invention, the remaining amino acids are uncharged polar, apolar and / or aromatic.Furthermore, preferably, if present, the amino acid at position 5 is aromatic, preferably selected from Tyr or Trp.Also preferably, if present, the amino acid at position 6 is uncharged polar or apolar.

[0061] Thus, in a preferred embodiment, the peptide of the invention comprises at least one aromatic amino acid, more preferably one or two aromatic amino acids. Even more preferably: When the peptide of the invention has 5 amino acids, it has one aromatic amino acid, more preferably at position 5. In this case, the aromatic amino acid is preferably Tyr.

[0062] When the peptide of the invention has six amino acids, it has two aromatic amino acids, one at position 4 and one at position 5. In this case, the aromatic amino acids are selected from Tyr, Trp, Phe or a combination thereof.

[0063] In any of the above embodiments, the charged amino acids (positive or negative) are preferably selected from Lys, Arg, His, Asp.

[0064] Thus, in a preferred embodiment, in the peptide of the invention, the charged amino acid at position 1 is Lys. Even more preferably, in the peptide of the invention, the first three amino acids of the peptide are Lys-Arg-Arg, Lys-His-Arg, Lys-Asp-Arg or Lys-Lys-His.

[0065] In a preferred embodiment, the peptide of the invention has the sequence of formula (I): R 1 -AA1-AA2-AA3-AA4-AA5-AA6-R 2 (R 1 -SEQ ID NO:7-R 2 ) Where: AA1 is selected from Lys, Arg, Ile, or His; AA2 is selected from Lys, Arg, His, Trp, Asp or Glu; AA3 is selected from Lys, Arg, Gln, Pro, or His; AA4 is selected from Gly, Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, Gln, Phe, Tyr, Lys, or Trp; AA5 is selected from Phe, Tyr, Asn, or Trp; AA6 is absent or selected from Gly, Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, or Gln; R 1 is H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R 5 -CO-, where R 5 is a substituted or unsubstituted C 1 -C 24 Alkyl radicals, substituted or unsubstituted C 2 -C 24 Alkenyl, substituted or unsubstituted C 2 -C 24 Alkynyl, substituted or unsubstituted C 3 -C 24 Cycloalkyl, substituted or unsubstituted C 5 -C 24 Cycloalkenyl, substituted or unsubstituted C 8 -C 24 Cycloalkynyl, substituted or unsubstituted C 6 -C 30 Aryl, substituted or unsubstituted C 7 -C 24aralkyl, 3- to 10-membered substituted or unsubstituted heterocyclyl rings, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms and an alkyl chain of 1 to 6 carbon atoms; and R 2 -H, -NR 3 R 4 -, -OR 3 and -SR 3 where R 3 and R 4 is independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl; Or, R 1 and R 2 is absent and AA1 is directly linked to the last amino acid of the peptide, ie, AA6 or AA5 if AA6 is absent (forming a cyclic peptide).

[0066] R 1 is linked to the N-terminus of the peptide, i.e., the amino group of AA1 (substitutes or replaces the H from the amino group of AA1).

[0067] R 2 is linked to the C-terminus of the peptide, i.e., to the carboxy group of AA6, or, if AA6 is absent, to the carboxy group of AA5 (substitutes or replaces the OH of the carboxy group).

[0068] In a more preferred embodiment, said peptide is capable of downregulating the expression of at least one sodium channel, and / or reducing the level of at least one neurotransmitter, and / or maintaining and / or synthesizing ECM.Preferably, said sodium channel is a voltage-gated sodium channel, more preferably, said voltage-gated sodium channel is Nav1.3 and / or Nav1.4.Preferably, said neurotransmitter is noradrenaline.Preferably, said ECM is selected from collagen I, elastin, hyaluronic acid, or combinations thereof.

[0069] In another preferred embodiment, the peptide has the sequence of formula (I): R 1 -AA1-AA2-AA3-AA4-AA5-AA6-R 2 (R 1 -SEQ ID NO:7-R 2 ) Where: AA1 is selected from Lys, Arg, or Ile; AA2 is selected from Lys, Arg, His, Asp, Trp or Glu; AA3 is selected from Arg, Gln, Pro, or His; AA4 is selected from Val, Met, Ile, Ser, Thr, Cys, Asn, Gln, Phe, Tyr, Lys, or Trp; AA5 is selected from Tyr, Asn, or Trp; AA6 is absent or selected from Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, or Gln; R 1 is H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R 5 -CO-, where R5 is a substituted or unsubstituted C 1 -C 24 Alkyl radicals, substituted or unsubstituted C 2 -C 24 Alkenyl, substituted or unsubstituted C 2 -C 24 Alkynyl, substituted or unsubstituted C 3 -C 24 Cycloalkyl, substituted or unsubstituted C 5 -C 24 Cycloalkenyl, substituted or unsubstituted C 8 -C 24 Cycloalkynyl, substituted or unsubstituted C 6 -C 30 Aryl, substituted or unsubstituted C 7 -C 24 aralkyl, 3- to 10-membered substituted or unsubstituted heterocyclyl rings, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms and an alkyl chain of 1 to 6 carbon atoms; and R 2 -H, -NR 3 R 4 -, -OR 3 and -SR 3 where R 3 and R 4 is independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl; Or, R 1 and R 2 is absent and AA1 is directly attached to the last amino acid of the peptide.

[0070] Preferably, in formula (I): AA1 is Lys AA2 is selected from Arg, His, Lys, or Asp; AA3 is selected from Arg or His; AA4 is selected from Tyr, Ser, Val, or Phe; AA5 is selected from Tyr or Trp; AA6 is absent, selected from Cys or Ala.

[0071] In a preferred embodiment, the peptide of the invention is selected from: R 1 -Arg-Trp-Gln-Lys-Asn-Gln-R 2 (R 1 -SEQ ID NO:1-R 2 ); R 1 -Lys-Arg-Arg-Tyr-Tyr-Cys-R 2 (R 1 -Sequence number 2-R 2 ); R 1 -Lys-His-Arg-Ser-Tyr-R 2 (R 1 -SEQ ID NO:3-R 2 ); R 1 -Lys-Asp-Arg-Val-Tyr-R 2 (R 1 -SEQ ID NO:4-R 2 ); R 1 -Ile-Glu-Pro-Tyr-Tyr-Val-R 2 (R 1 -SEQ ID NO:5-R 2 );or R 1 -Lys-Lys-His-Phe-Trp-Ala-R 2 (R 1 -SEQ ID NO:6-R 2 ); Where: R 1 is H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R 5-CO-, where R 5 is a substituted or unsubstituted C 1 -C 24 Alkyl radicals, substituted or unsubstituted C 2 -C 24 Alkenyl, substituted or unsubstituted C 2 -C 24 Alkynyl, substituted or unsubstituted C 3 -C 24 Cycloalkyl, substituted or unsubstituted C 5 -C 24 Cycloalkenyl, substituted or unsubstituted C 8 -C 24 Cycloalkynyl, substituted or unsubstituted C 6 -C 30 Aryl, substituted or unsubstituted C 7 -C 24 aralkyl, 3- to 10-membered substituted or unsubstituted heterocyclyl rings, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms and an alkyl chain of 1 to 6 carbon atoms; and R 2 -H, -NR 3 R 4 -, -OR 3 and -SR 3 where R 3 and R 4 is independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl; Or, R 1 and R 2 is absent and AA1 is directly attached to the last amino acid of the peptide (forming a cyclic peptide).

[0072] More preferably, the peptide of the invention is selected from: R 1 -Lys-Asp-Arg-Val-Tyr-R2 (R 1 -SEQ ID NO:4-R 2 );or R 1 -Lys-Lys-His-Phe-Trp-Ala-R 2 (R 1 -SEQ ID NO:6-R 2 ); R 1 and R 2 is disclosed above.

[0073] Even more preferably, the peptide of the invention is: R 1 -Lys-Asp-Arg-Val-Tyr-R 2 (R 1 -SEQ ID NO:4-R 2 ); R 1 and R 2 is disclosed above.

[0074] In any of the above embodiments, preferably, R 1 is H or acetyl, more preferably R 1 is H.

[0075] In any of the above embodiments, R 2 NH 2 or OH, more preferably R 2 NH 2 It is.

[0076] Thus, in a most preferred embodiment, the peptide of the invention is selected from: Arg-Trp-Gln-Lys-Asn-Gln-NH 2 (SEQ ID NO:1-NH 2 ); Lys-Arg-Arg-Tyr-Tyr-Cys-NH 2 (SEQ ID NO:2-NH 2 ); Lys-His-Arg-Ser-Tyr-NH 2 (SEQ ID NO:3-NH 2 ); Lys-Asp-Arg-Val-Tyr-NH 2 (SEQ ID NO:4-NH 2 ); Ile-Glu-Pro-Tyr-Tyr-Val-NH 2 (SEQ ID NO:5-NH 2 );or Lys-Lys-His-Phe-Trp-Ala-NH 2 (SEQ ID NO:6-NH) 2 .

[0077] Even more preferably, the peptide of the invention is: Lys-Asp-Arg-Val-Tyr-NH 2 (SEQ ID NO:4-NH 2 );or Lys-Lys-His-Phe-Trp-Ala-NH 2 (SEQ ID NO:6-NH) 2 Even more preferably Lys-Asp-Arg-Val-Tyr-NH 2 (SEQ ID NO:4-NH 2 ).

[0078] It should be noted that peptides SEQ ID NO:1 to SEQ ID NO:3 are derived directly from the protein and / or genome of the pufferfish Takifugu rubripes. More precisely, SEQ ID NO: 1 is derived from the sequence of nucleolar complex protein 2 homologue of the pufferfish Takifugu rubripes (Uniprot reference number: H2TGB2, amino acids 88 to 93).

[0079] Sequence number 2 is derived from the sequence of Disabled homolog 2-interacting protein-like isoform X2 (Synaptic Ras-GTPase activating domain) (NCBI Reference Sequence: XP_003976523.1, amino acids 64 to 68).

[0080] SEQ ID NO:3 is derived from a genomic sequence predicted for calcium-independent phospholipase A2-γ-like isoform X2 (NCBI Reference Sequence: XP_011605802.2, amino acids 7 to 11).

[0081] The peptides of SEQ ID NOs: 4 to 6 are derived from the peptides of SEQ ID NOs: 1 to 3 by conservative substitutions.

[0082] Also included within the scope of the present invention are peptides derived or obtained from any of the above disclosed peptides, preferably incorporating one or more conservative substitutions, so long as they retain, exhibit or improve at least one of the aforementioned activities (preferably all of the aforementioned activities for the peptides of the present invention).

[0083] Additionally, within the scope of the present invention are peptides having a percentage of identity of 70%, preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 99% to any of the above-mentioned specific sequences, so long as they retain, exhibit or improve at least one of the above-mentioned activities (preferably all of the above-mentioned activities for the peptides of the present invention).

[0084] As follows directly from the above, it is contemplated that the peptides of the present invention are linear or cyclic peptides.

[0085] In an embodiment, this embodiment can be combined with any of the above embodiments, where the peptide is cyclic. In this case, the first and last amino acids of the peptide of the invention are linked (according to the above explanation). Therefore, appropriate modifications to any of the above embodiments must be made (e.g., R 1 and R 2 may not be present because the amino acids to which the site is attached are now bound to each other).

[0086] In a preferred embodiment, which may be combined with any of the previous embodiments, said peptide is linear.

[0087] The peptides of the present invention can be synthesized and produced by any means known in the art. For example, they can be synthesized and produced by chemical synthesis (preferably by means of solid-phase peptide synthesis), by expression of said peptides in cell culture, or by means of transgenic production of peptides in plants or animals. Furthermore, the peptides of the present invention can be purified by any means known in the art.

[0088] In a preferred embodiment, which may be combined with any of the embodiments disclosed above, the peptides of the present invention are suitable or adapted for application by iontophoresis, more preferably to the face of a subject, preferably a human.

[0089] In a further preferred embodiment, which may be combined with any of the embodiments disclosed above, the peptide of the present invention is suitable or adapted to be applied subcutaneously, more preferably to the face of a subject, preferably a human.

[0090] In another preferred embodiment, which may be combined with any of the embodiments disclosed above, the peptide of the present invention is suitable or adapted to be applied topically, preferably in the form of a cream, more preferably to the face of a subject, preferably a human.

[0091] As can be directly derived from the examples included below, the peptide of the present invention has the activity required to provide the prevention, treatment and / or reduction of wrinkles by at least muscle relaxation and ECM maintenance and synthesis.Furthermore, the peptide of the present invention also has the activity required to provide the prevention, treatment and / or reduction of loose or sagging skin and rough skin (preferably rough wrinkles); and improvement of skin firmness and elasticity.Therefore, the peptide of the present invention solves the above-mentioned technical problems and is useful in cosmetics.

[0092] In a second aspect, the present invention refers to a composition comprising a peptide of the invention.

[0093] The peptide of the invention is as disclosed above in the first aspect of the invention.

[0094] The composition of the present invention is preferably a cosmetic composition.Accordingly, in this embodiment, the cosmetic composition of the present invention comprises a cosmetically effective amount of at least one peptide of the present invention, more preferably, the cosmetic composition of the present invention comprises 0.0001% (weight / volume, g / 100mL, hereinafter, w / v) to 0.05% (w / v) of the peptide of the present invention.In the most preferred embodiment, the cosmetic composition of the present invention comprises 0.0001% (w / v) to 0.005% (w / v) of the peptide of the present invention, more preferably, 0.0005% (w / v) to 0.0015% (w / v).

[0095] The cosmetic composition of the present invention is also intended to include at least one additional cosmetic ingredient. The additional cosmetic ingredient may be at least one excipient and / or at least one additional cosmetic active ingredient. The additional cosmetic ingredient may be any cosmetic ingredient known in the art, as long as it does not affect or does not affect in an unacceptable manner the activity of the peptide of the present invention.

[0096] In an embodiment, a composition of the invention consists essentially of a peptide of the invention.

[0097] In another embodiment, a composition of the invention consists of a peptide of the invention.

[0098] In a third aspect, the present invention refers to the use of a peptide of the invention or a composition of the invention as a cosmetic in a subject in need of treatment.

[0099] The peptide of the invention is as disclosed above in the first aspect of the invention.

[0100] The composition of the present invention is as disclosed above in the second aspect of the invention.

[0101] The peptides or compositions of the present invention are used in a cosmetically effective amount or quantity.

[0102] In a preferred embodiment, the cosmetic use is to reduce, prevent and / or eliminate the signs of aging of the skin.

[0103] Preferably, the signs of skin aging are wrinkles, rough skin (preferably rough wrinkles), loose skin or sagging skin, more preferably wrinkles, rough skin, non-inflammatory loose skin or non-inflammatory loose skin, more preferably wrinkles, even more preferably facial wrinkles. Said wrinkles may be chronological wrinkles or expression (muscle-derived) wrinkles.

[0104] Non-inflamed loose skin corresponds to cosmetic loose skin, ie non-pathological (non-medical) loose skin.

[0105] Non-inflammatory loose skin corresponds to cosmetic loose skin, ie non-pathological (non-medical condition) loose skin.

[0106] As directly follows from the above, in the present invention, the signs of skin ageing (preferably both chronological and expression wrinkles) are prevented, reduced and / or eliminated (by the peptides of the invention or by the compositions of the invention) by blocking neuromuscular contractions and / or by maintaining or synthesizing ECM, as already explained above.

[0107] The at least one sodium channel and the at least one neurotransmitter are as described above in the first aspect of the invention.

[0108] In this third aspect of the invention, the subject in need of treatment is preferably a mammal, more preferably a human.

[0109] In this third aspect of the invention, it is also contemplated that the cosmetic use is for improving skin firmness and / or elasticity.

[0110] In a fourth aspect, as mentioned above, the present invention relates to the cosmetic use of a peptide of the invention or a composition of the invention in a subject in need of treatment.

[0111] Preferably, in this fourth aspect of the invention, the cosmetic use is to reduce, prevent and / or eliminate the signs of aging of the skin.

[0112] In this fourth aspect of the invention, it is also contemplated that the cosmetic use is for improving skin firmness and / or elasticity.

[0113] The peptide of the invention is as disclosed above in the first aspect of the invention.

[0114] The composition of the present invention is as disclosed above in the second aspect of the invention.

[0115] The signs of skin ageing and the subject in need of treatment are as described above in the third aspect of the invention.

[0116] Furthermore, all of the embodiments discussed above in relation to the third aspect of the invention are directly applicable to this fourth aspect of the invention, with appropriate adaptations.

[0117] In a fifth and final aspect, the present invention relates to a method for reducing, preventing and / or eliminating the signs of skin ageing in a subject, comprising administering to the subject a peptide of the invention or a composition of the invention.

[0118] The peptide of the invention is as disclosed above in the first aspect of the invention.

[0119] The composition of the present invention is as disclosed above in the second aspect of the invention.

[0120] The signs and targets of skin ageing are as described above in the third aspect of the invention.

[0121] Furthermore, all of the embodiments discussed above in the third and fourth aspects of the invention are directly applicable to this fifth aspect of the invention, with appropriate adaptations.

[0122] Sequence Listing Free Text SEQ ID NO: 1: Peptide derived from the sequence of the nucleolar complex protein 2 homologue of the pufferfish Takifugu rubripes (Uniprot reference number: H2TGB2, amino acids 88 to 93).

[0123] Sequence number 2: Peptide derived from the sequence of Disabled homolog 2-interacting protein-like isoform X2 (Synaptic Ras-GTPase activating domain) (NCBI Reference Sequence: XP_003976523.1, amino acids 64 to 68).

[0124] SEQ ID NO: 3: Peptide derived from genomic sequence predicted to be calcium-independent phospholipase A2-γ-like isoform X2 (NCBI Reference Sequence: XP_011605802.2, amino acids 7 to 11) SEQ ID NO: 4: Artificially synthesized sequence SEQ ID NO: 5: Artificially synthesized sequence SEQ ID NO: 6: Artificially synthesized sequence SEQ ID NO: 7: Artificially synthesized sequence Xaa is selected from Lys, Arg, or His. Xaa is selected from Lys, Arg, His, Asp, or Glu. Xaa is selected from Lys, Arg, or His. Xaa is selected from Gly, Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, Gln, Phe, Tyr, or Trp. Xaa is selected from Phe, Tyr or Trp Xaa is absent or is selected from Gly, Ala, Val, Leu, Met, lie, Ser, Thr, Cys, Pro, Asn, or Gln.

[0125] BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding, the invention will now be described in more detail below with reference to the enclosed drawings, which are given by way of example and have reference to illustrative and non-limiting examples.

[0126] FIG. 1 is a schematic representation of peptide SEQ ID NO:4-NH 21A and 1B show cell viability results obtained for 0.01 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, or 1 mg / mL of peptide SEQ ID NO:4-NH. More specifically, FIG. 1A shows viability in HEKa cells and FIG. 1B shows viability in HDFa cells. In both figures, the Y-axis refers to the percentage of cell viability, with the viability of the negative control (untreated cells) being taken as 100%. In both figures, the columns from left to right on the x-axis represent the percentage of cell viability obtained for 0.01 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, or 1 mg / mL of peptide SEQ ID NO:4-NH. 2 This refers to cells treated with

[0127] FIG. 2 shows the results obtained in Example 3, i.e., the time course current (area under the response curve) of the samples tested, and thus the activity of the voltage-gated sodium channel (NaV1.3). The y-axis refers to the inward current (Amp). The x-axis refers to the different experimental groups tested, with the left side referring to the negative control and the right side referring to the treated sample for each set of columns. On the x-axis, the set of columns from left to right refers to the following experimental treatments or groups: SEQ ID NO:1-NH 2 , SEQ ID NO:2-NH 2 , SEQ ID NO:3-NH 2 and amitriptyline.

[0128] Figure 3 shows the results obtained in Example 4. The figure shows the inhibition rate of voltage-gated sodium channels. The Y-axis represents the inhibition rate of voltage-gated sodium channels relative to the negative control (0%), and the X-axis represents the logarithm of the concentration in M ​​of the different peptides tested. The triangular line represents SEQ ID NO: 2-NH 2 and the square line represents SEQ ID NO: 3-NH 2 and the crosses represent SEQ ID NO: 4-NH 2 and the circled line represents SEQ ID NO:5-NH 2 and the diamond line represents SEQ ID NO: 6-NH 2 It is.

[0129] FIG. 4 shows the sequence of SEQ ID NO: 6-NH 2The results obtained for the levels of noradrenaline when treated with 0.05 mg / mL of peptide SEQ ID NO:6-NH 2 and 0.1 mg / mL of peptide SEQ ID NO:6-NH 2 Depolarized cells treated with

[0130] FIG. 5 shows SEQ ID NO: 4-NH 2 4 shows the results obtained for the levels of noradrenaline in Example 5, treated with 0.05 mg / mL of peptide SEQ ID NO:4-NH. The y-axis shows the percentage of noradrenaline relative to the positive control (untreated depolarized cells, taken as 100%). The x-axis shows the different treatment groups, represented by bars from left to right: basal state; positive control (depolarized cells without treatment); 0.05 mg / mL of peptide SEQ ID NO:4-NH 2 and 0.1 mg / mL of peptide SEQ ID NO:4-NH 2 Depolarized cells treated with

[0131] FIG. 6 shows peptide SEQ ID NO:4-NH 2 1 shows gene expression results obtained in Example 6 when treated with 500 mg / kg / day for 24 hours. Each bar represents one gene from top to bottom: SCN1B, SCN4A, SCN3A and CALM3. Bars extending to the left (i.e., negative values) indicate downregulation of genes.

[0132] FIG. 7 shows peptide SEQ ID NO:4-NH 2 1 shows gene expression results obtained in Example 6 when treated with 500 mg / kg / day for 24 hours. Each bar represents one gene from top to bottom: RAPSN, SCN3A, and CALM3. Bars extending to the left (i.e., negative values) indicate downregulation of genes.

[0133] FIG. 8 shows peptide SEQ ID NO:4-NH at concentrations of 0.005 mg / mL, 0.01 mg / mL or 0.015 mg / mL.2 4 shows the results of elastin production or synthesis obtained in Example 7, when HDFa cells were treated with 0.005 mg / mL of peptide SEQ ID NO:4-NH. Elastin production or synthesis is expressed as a percentage relative to the basal state (untreated cells), which is established as 100%. The y-axis shows the percentage of elastin relative to the basal state. The x-axis shows the different treatment groups, represented by bars from left to right: basal state (untreated cells); 0.005 mg / mL of peptide SEQ ID NO:4-NH 2 Cells treated with 0.01 mg / mL of peptide SEQ ID NO:4-NH 2 and 0.015 mg / mL of peptide SEQ ID NO:4-NH 2 Cells treated with.

[0134] FIG. 9 shows peptide SEQ ID NO:4-NH at 0.01 mg / mL, 0.015 mg / mL or 0.05 mg / mL. 2 FIG. 1 shows the results obtained for the release of procollagen Iα1 in Example 8, when HDFa cells were treated for 48 hours with 0.01 mg / mL of peptide SEQ ID NO:4-NH. The release of procollagen Iα1 is expressed as a percentage relative to the basal state (untreated cells), established as 100%. The y-axis shows the percentage of procollagen Iα1 release relative to the basal state. The x-axis shows the different treatment groups, indicated by bars from left to right: basal state (untreated cells); 0.01 mg / mL of peptide SEQ ID NO:4-NH 2 Cells treated with 0.015 mg / mL of peptide SEQ ID NO:4-NH 2 and 0.05 mg / mL of peptide SEQ ID NO:4-NH 2 Cells treated with.

[0135] FIG. 10 shows how HDFa cells were treated with peptide SEQ ID NO:4-NH 21 shows gene expression results obtained in Example 10 when treated with 0.01 mg / mL peptides (MMP1, COL3A1 and COL4A1) for 24 hours, 0.05 mg / mL peptides (TIMP2, FBLN5, LOXL1 and COL7A1) for 24 hours, 0.005 mg / mL peptide (HYAL1) for 6 hours, and 0.05 mg / mL peptide (HAS1) for 6 hours using 100% ethanol. Each bar represents one gene from top to bottom: MMP1, HYAL1, TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1 and COL7A1. Bars extending to the left (i.e., negative values) indicate downregulation of the gene. Bars extending to the right (i.e., positive values) indicate upregulation of the gene.

[0136] [Example] Abbreviation: The abbreviations used for amino acids follow the 1983 IUPAC-IUB Joint Commission on Biochemical Nomenclature recommendations outlined in Eur. J. Biochem. (1984) 138:937.

[0137] Ala, alanine;Arg, arginine;Asn, asparagine;Asp, aspartic acid;Boc, tert-butyloxycarbonyl;cDNA, complementary DNA;C-terminus, carboxy-terminus;Cys, cysteine;DCM, dichloromethane;DIPCDI, N,N'-diisopropylcarbodiimide;DMF, N,N-dimethylformamide;equiv, equivalent;ESI-MS, electrospray ionization mass spectrometry;Fmoc, 9-fluorenylmethyloxycarbonyl;Glu, glutamic acid;Gln, glutamine;HBSS, Hanks' Balanced Salt Solution;HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;His, histidine;HOBt, 1-hydroxybenzotriazole;HPLC, high performance liquid chromatography;HRP, horseradish peroxidase;Ile, isoleucine;LSGS, Low Serum Growth Supplement;Lys, lysine;MBHA, p-methylbenzhydrylamine;Me, methyl;MeCN, acetonitrile;MEM, minimum essential medium;MeOH, methanol;Met, methionine;N-terminal, amino-terminal;Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;Phe, phenylalanine;Pro, proline;RNA, ribonucleic acid;RT, room temperature;RT-qPCR, quantitative reverse transcription polymerase chain reaction;Ser, serine;SKM-D, skeletal muscle cell differentiation medium;SKM-M, skeletal muscle cell proliferation medium;tBu, tert-butyl;TFA, trifluoroacetic acid;TIS, triisopropylsilane;TPA, 12-O-tetradecanoylphorbol-13-acetate;Trp, tryptophan;Trt, triphenylmethyl or trityl;Tyr, tyrosine;Val, valine.

[0138] Regarding the chemical synthesis procedures included in the examples, it should be noted that all synthesis steps were carried out in polypropylene syringes fitted with porous polyethylene disks or in Pyrex® reactors fitted with porous plates. All reagents and solvents were of synthesis quality and were used without further treatment. Solvents and soluble reagents were removed by aspiration. Fmoc groups were removed with piperidine-DMF (2:8, volume / volume, v / v) (at least 1×1 min, 2×10 min, 5 mL / g resin) (Lloyd Williams P. et al., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC, 1997, Boca Raton (Fla., USA)). Washing between deprotection, coupling and deprotection again steps was done with DMF (3×1 min) and DCM (3×1 min), using 10 ml solvent / g resin each time. Coupling reactions were carried out with 3 ml solvent / g resin. Coupling control was performed by carrying out the ninhydrin test (Kaiser E. et al., Anal. Biochem., 1970, 34:595-598). All synthesis reactions and washings were carried out at RT.

[0139] Example 1. Synthesis and preparation of peptides

[0140] [ka]

[0141] Weight was normalized. 4.8 g (2.5 mmol) of Fmoc-Rink-MBHA resin with 0.52 mmol / g functionalization was treated with piperidine-DMF according to the general protocol described in the art to remove the Fmoc group. 2.33 g of Fmoc-L-Ala-OH (7.5 mmol; 3 equiv) was loaded onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as solvent for 1 hour.

[0142] The resin was then washed and the Fmoc group deprotection procedure was repeated to couple the next amino acid as described in common methods known in the art. Following the protocol described above, 3.94 g of Fmoc-L-Trp(Boc)-OH (7.5 mmol; 3 equiv), followed by 2.90 g of Fmoc-L-Phe-OH (7.5 mmol; 3 equiv), followed by 4.64 g of Fmoc-L-His(Trt)-OH (7.5 mmol; 3 equiv), followed by 3.51 g of Fmoc-L-Lys(Boc)-OH (7.5 mmol; 3 equiv) and followed by 3.51 g of Fmoc-L-Lys(Boc)-OH (7.5 mmol; 3 equiv), were coupled in sequence, respectively, in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already mentioned above, a deprotection treatment of the Fmoc group was carried out between each amino acid addition step.

[0143] After synthesis, the peptide resin was washed with DCM (5 times, 3 min each) and dried under vacuum.

[0144] [ka]

[0145] Weight was normalized. 4.8 g (2.5 mmol) of Fmoc-Rink-MBHA resin with 0.52 mmol / g functionalization was treated with piperidine-DMF according to the general protocol described in the art to remove the Fmoc group. 3.44 g of Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 equiv) was loaded onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as solvent for 1 hour.

[0146] The resin was then washed as described in the general methods known in the art and the Fmoc group deprotection was repeated to attach the next amino acid. Following the protocol described above, 2.55 g of Fmoc-L-Val-OH (7.5 mmol; 3 equiv) was coupled, followed by 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv), followed by 3.08 g of Fmoc-L-Asp(tBu)-OH (7.5 mmol; 3 equiv), followed by 3.51 g of Fmoc-L-Lys(Boc)-OH (7.5 mmol; 3 equiv), in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). Between each amino acid addition step, the Fmoc group deprotection was performed as already described above.

[0147] After synthesis, the peptide resin was washed with DCM (5 times, 3 min each) and dried under vacuum.

[0148] Using the synthesis procedure described above, the required amino acids were selected and the following sequences were synthesized: Arg-Trp-Gln-Lys-Asn-Gln-NH 2 (SEQ ID NO:1-NH 2 ); Lys-Arg-Arg-Tyr-Tyr-Cys-NH 2 (SEQ ID NO:2-NH 2 ); Lys-His-Arg-Ser-Tyr-NH 2 (SEQ ID NO:3-NH 2 ); Lys-Asp-Arg-Val-Tyr-NH 2 (SEQ ID NO:4-NH 2 ); Ile-Glu-Pro-Tyr-Tyr-Val-NH 2 (SEQ ID NO:5-NH 2 ); and Lys-Lys-His-Phe-Trp-Ala-NH 2 (SEQ ID NO:6-NH 2 ).

[0149] [ka]

[0150] The N-terminal Fmoc group of the peptidyl resin obtained in a) or b) above was deprotected with 20% (volume / volume, hereafter v / v) piperidine in DMF (1×1 min+2×10 min) ((Lloyd Williams P. et al. (1997) Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, Boca Raton (Fla., USA)). The peptidyl resin was washed with DMF (5×1 min) and DCM (4×1 min) and dried under vacuum.

[0151] [ka]

[0152] The weight was normalized. 200 mg of the dried peptidyl resin obtained in c) was added to 5 mL of TFA / TIS / H 2 The mixture was treated with 50 mL (volume ratio 90:5:5) of cold diethyl ether for 2 h under stirring at room temperature. The filtrate was collected and precipitated with 50 mL (8-10 times) of cold diethyl ether. The ether solution was evaporated to dryness at room temperature under reduced pressure, and the precipitate was collected with H 2 It was redissolved in 50% (v / v) MeCN in O and lyophilized.

[0153] [ka]

[0154] d) HPLC analysis of the peptides obtained according to the above procedure was carried out using a reversed-phase column (150×4.6 mm, XBridge Peptide BEH C18, 3.5 μm, Waters, USA). 2A gradient of MeCN (+0.036% (v / v) TFA) in 0 (+0.045% (v / v) TFA) was performed at a flow rate of 1.25 mL / min with detection at 220 nm. All peptides showed a purity of more than 80%. The identity of the obtained peptides was confirmed by ESI-MS using a Water ZQ 4000 detector at a flow rate of 0.2 mL / min with MeOH as mobile phase. The obtained results showed that peptide SEQ ID NO:1-NH 2 , SEQ ID NO:2-NH 2 , SEQ ID NO:3-NH 2 , SEQ ID NO:4-NH 2 , SEQ ID NO:5-NH 2 and SEQ ID NO:6-NH 2 was shown to be correctly and efficiently synthesized.

[0155] Example 2. Analysis of toxicity of the peptides of the present invention In this case, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate the cytotoxic potential of the peptides of the invention against HEKa (human epidermal keratinocytes from adult skin) and HDFa (human dermal fibroblasts from adult skin) primary cells.

[0156] In this example, the peptide SEQ ID NO:4-NH 2 Prepared according to Example 1 using:

[0157] Stock solutions of peptides were prepared at 25 mg / mL in distilled water, which were further diluted to obtain the corresponding final working concentrations in culture medium (0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL).

[0158] Untreated cells were used as a negative control sample (Basal).

[0159] Briefly, HEKa and HDFa cells were plated at 1 × 10 in a final volume of 200 μL of medium (20,000 cells / well). 5The cells were seeded in 96-well plates at a concentration of 10 cells / mL. HEKa cells were cultured in dermal cell basal medium supplemented with Keratinocyte Proliferation Kit; HDFa cells were cultured in 106 medium supplemented with 2% LSGS. Cells were incubated at 37°C and CO 2 The cultures were incubated at 5% and saturated humidity.

[0160] After 24 h of incubation, cells were treated in triplicate with different concentrations of peptides (0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL) and MTT assessment was performed. After 24 h of peptide treatment, the medium was completely removed and 100 μL of 5 mg / mL MTT solution in supplemented medium was added to each well and cells were incubated at 37 °C for 3 h. After incubation with MTT, the medium in each well was aspirated and 150 μL of DMSO was added to solubilize the formed formazan crystals. To completely solubilize the crystals, the plate was placed on a shaker for 5 min and then the absorbance at 545 nm of each well, which is directly proportional to the number of viable cells in the medium, was measured using a microplate reader Multiskan FC.

[0161] Absorbance values ​​were normalized using data obtained with untreated cells (control) to obtain the % viability shown in Figures 1A and 1B. Each condition was performed in triplicate (n=3) in three independent experiments (N=3).

[0162] Concentrations of peptides that result in greater than 80% viability are considered safe for efficacy testing. Conversely, concentrations that result in less than 80% viability are considered potentially toxic and will not be used for efficacy testing purposes.

[0163] The obtained results are summarized in Figure 1A (viability in HEKa cells) and Figure 1B (viability in HDFa cells). As can be directly derived from the figures, the viability in HEKa cells after treatment with 0.001mg / mL-0.5mg / mL of the test peptide is more than 80%; the viability in HDFa cells after treatment with 0.001mg / mL-1mg / mL of the test peptide is more than 80%, which demonstrates the safety of the peptide of the present invention.

[0164] Example 3. Analysis of modulation of sodium channel activity or signal transduction In this example, the peptide SEQ ID NO:1-NH 2 , SEQ ID NO:2-NH 2 and SEQ ID NO:3-NH 2 was prepared according to Example 1.

[0165] Human NaV1.3 stably transfected into Chinese hamster ovary (CHO) cells was used to measure ion channel activity electrophysiologically using a QPatch48 HT (Sophion, Denmark). Experiments were performed in triplicate and a peptide concentration of 10 μM was used. Negative controls were performed separately for each peptide. Amitriptyline was used as a positive control.

[0166] The results obtained in this example are summarized in Figure 2. As can be derived from said figure, all three peptides showed inhibition of NaV1.3 inward current, resulting in less negative current (Amp). Thus, all three peptides were able to block or modulate (reduce activity or signal) the sodium channel Nav1.3. Moreover, surprisingly, SEQ ID NO:2-NH 2 and SEQ ID NO:3-NH 2 were able to demonstrate an increased and statistically significant effect (p=0.018 and p=0.039, respectively).

[0167] Example 4. Analysis of modulation of sodium channel activity or signal transduction In this example, peptide SEQ ID NO:2-NH 2 , SEQ ID NO:3-NH 2 , SEQ ID NO:4-NH 2 , SEQ ID NO:5-NH 2 and SEQ ID NO:6-NH 2 was prepared.

[0168] Ion channel activity was measured electrophysiologically using SHSY-5Y cells with a QPatch48 HT (Sophion, Denmark). The samples tested were as follows: Negative control: 0.05% (v / v) bovine serum albumin Positive control: Tetradotoxin (TTX) at concentrations of 3 nM, 10 nM and 30 nM SEQ ID NO:2-NH 2 Concentrations of 0.005mg / mL, 0.01mg / mL and 0.05mg / mL SEQ ID NO:3-NH 2 Concentrations of 0.005mg / mL, 0.01mg / mL and 0.05mg / mL SEQ ID NO:4-NH 2 Concentrations of 0.005mg / mL, 0.01mg / mL and 0.05mg / mL SEQ ID NO:5-NH 2 Concentrations of 0.005mg / mL, 0.01mg / mL and 0.05mg / mL SEQ ID NO:6-NH 2 Concentrations of 0.005mg / mL, 0.01mg / mL and 0.05mg / mL.

[0169] Percent inhibition for each test dose application period was calculated as the decrease in mean cursor value (peak current) relative to the cursor value measured at the end of the vehicle control (pre-drug administration) and normalized to the maximum inhibition recorded with the add-on addition of TTX.

[0170] The results obtained are summarized in FIG. 3, which shows the percentage of inhibition (or blockage) of the sodium channel (inactivated state). It can be seen that all peptides tested effectively modulated (reduce activity or signal) the sodium channel in a dose-dependent manner. The peptide with the best results at the two highest concentrations tested was SEQ ID NO: 4-NH 2 On the other hand, the peptide with the best results at low concentrations tested was SEQ ID NO:5-NH 2 It was.

[0171] As a result, the peptides of the present invention showed a modulatory effect similar to that achieved with low concentrations of TTX (high concentrations of TTX produced a complete blocking effect).

[0172] Example 5. Analysis of noradrenaline levels In this example, the peptide SEQ ID NO:4-NH 2 and SEQ ID NO:6-NH 2 was prepared according to Example 1.

[0173] In this case, noradrenaline (NA) levels were measured in differentiated SHSY5Y cells (a human neuronal model). This test was used to determine whether candidate substances could modulate neurotransmitter release.

[0174] Human SH-SY5Y cells were seeded in triplicate in 24-well plates at a cell density of 250,000 cells / well. SH-SY5Y were differentiated into a neuronal phenotype with retinoic acid for 1 week and then treated with the above peptides at 0.05 mg / mL and 0.1 mg / mL for 30 min.

[0175] Afterwards, the cells were washed and first treated with 100 nM 12-0-tetradecanoyl-phorbol-13-acetate (TPA) for 8 min for enhanced release, and then with 100 mM KCl in HBSS with 20 mM HEPES buffer for 5 min to achieve evoked release of neurotransmitters. Finally, the cells were lysed and homogenized directly on the wells 10 times with a 0.8 mm gauge needle. The homogenized cell suspension was centrifuged at maximum speed for 15 min at 4 °C. The supernatant was collected and stored at -80 °C until total noradrenaline was quantified by enzyme-linked immunosorbent assay (ELISA) using a human noradrenaline ELISA kit according to the manufacturer's instructions. The total protein content of each sample was quantified using a Micro BCA Protein Assay kit to normalize the data.

[0176] Each condition was performed at least three times in three independent experiments (n = 3), and samples were normalized with respect to the total protein with respect to the noradrenaline obtained in the positive control (samples without peptide but stimulated) to obtain a percentage with respect to the positive control.

[0177] The results obtained are summarized in Figures 4 and 5. As can be directly derived from said figures, the tested peptides (representative or exemplary of the peptides of the present invention) reduced the level of noradrenaline (more than 30% reduction), thus providing a Botox-like effect. Thus, this treatment is shown to regulate the release of neurotransmitters in stimulated differentiated SH-SY5Y cells, demonstrating that the peptides of the present invention provide anti-wrinkle activity.

[0178] Example 6. Analysis of expressed muscle contraction-related genes In this example, peptide SEQ ID NO:4-NH 2 was prepared.

[0179] Stock solutions of peptides were prepared in Milli-Q water at 25 mg / mL. This stock was further diluted with the corresponding supplemented medium to obtain final working concentrations of 0.01 mg / mL and 0.005 mg / mL. Untreated cells were used as negative control samples.

[0180] The relevant genes whose expression was measured were: SCN1B (sodium voltage-gated channel beta subunit 1), SCN4A (sodium voltage-gated channel alpha subunit 4), SCN3A (sodium voltage-gated channel alpha subunit 3), CALM3 (calmodulin 3), and RAPSN (synaptic receptor-associated protein). The primers used for each gene were commercially available TaqMan Gene Expression Assay probes. The experiment was performed in vitro with muscle cells, and two concentrations of peptide (0.01 mg / mL and 0.005 mg / mL) were tested for 24 hours.

[0181] Briefly, human skeletal myoblasts were cultured in 12-well plates at 1 × 10 5 Seed at a density of cells / well and culture under standard culture conditions (SKM-M medium supplemented with 1% antibiotic / antimycotic; 37 °C, 95% relative humidity, CO) for 48 h to 72 h until the seeded myoblasts reach 80%–90% confluence. 25%). To induce myotube differentiation (differentiation into muscle cells), the growth medium was then replaced with SKM-D medium, and fresh medium was added every 2–3 days. Six to seven days after the start of the differentiation process, myocytes were treated with 0.01 mg / mL or 0.005 mg / mL of the above peptides for an additional 24 h. Untreated cells served as basal controls. Finally, cells were lysed and RNA extraction was performed using the Qiagen RNeasy Mini kit according to the manufacturer's instructions. The purified RNA was used to generate the corresponding cDNA by reverse transcription, which served as a template for amplification. RT-qPCR was performed using a StepOne plus Real-Time PCR machine with a panel of TaqMan assay probes specified above and 2x gene expression Master Mix. Amplification included 40 cycles of: 95°C for 15 s (denaturation), and 60°C for 1 min (annealing and extension).

[0182] The data obtained were analyzed using the ΔΔCt method, which provides expression values ​​of the target genes as fold changes in treated samples compared to untreated basal samples. Both samples were normalized with the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0183] The analytical steps included: 1. Calculate the average Ct for each condition 2. Calculate the ΔCT test sample and the ΔCT calibration sample.

[0184] 3. Calculate ΔΔCT: ΔΔCT = ΔCT test sample - ΔCT calibration sample 4.2 -ΔΔCT The ratio is calculated by:

[0185] The results obtained are summarized in Figures 6 and 7. 2(as an example of a peptide of the present invention) is able to regulate the expression in muscle cells of the relevant genes tested, thus indicating that this peptide is able to regulate (downregulate) the expression of sodium channels and muscle contraction related genes in muscle cells.

[0186] Example 7. Analysis of elastin synthesis In this example, peptide SEQ ID NO:4-NH 2 was prepared.

[0187] Stock solutions of peptides were prepared in water at 25 mg / mL. This stock was further diluted to give final working concentrations of 0.005 mg / mL, 0.01 mg / mL or 0.015 mg / mL in complete medium (106 medium supplemented with 2% LSGS). Untreated cells were used as basal conditions.

[0188] For this study, HDFa cells were seeded in 12-well plates at a density of 120,000 cells / well and cultured under standard culture conditions (106 medium + 2% LSGS; 37 °C, 95% relative humidity, CO 2 Fibroblasts were maintained at 0.005mg / mL, 0.01mg / mL or 0.015mg / mL of the above peptides for 48 hours. After treatment, cells were harvested for elastin quantification using the Fastin Elastin Assay Kit according to the manufacturer's instructions. Each condition was performed at least three times (n=3) in three independent experiments.

[0189] The results obtained are summarized in FIG. 8. HDFa treated with 0.01 mg / mL and 0.015 mg / mL of the peptide of the present invention showed increased elastin levels compared to untreated samples. These results suggest that the peptide of the present invention may not only increase skin elasticity, but also improve the structural integrity of the skin and play a role in improving (preventing, treating or reducing) skin looseness and / or sagging. Furthermore, the increase in elastin synthesis or production also demonstrates that the peptide of the present invention can improve skin firmness and wound healing ability.

[0190] Example 8. Analysis of procollagen Iα1 release In this example, peptide SEQ ID NO:4-NH 2 was prepared.

[0191] Stock solutions of peptides were prepared at 25 mg / mL in water. This stock was further diluted to obtain final working concentrations of 0.01 mg / mL, 0.015 mg / mL or 0.05 mg / mL in complete medium (106 medium supplemented with 2% LSGS). Untreated cells were used as basal conditions.

[0192] In this study, HDFa cells were seeded in 24-well plates at a density of 100,000 cells / well and cultured under standard culture conditions (106 medium supplemented with 2% LSGS; 37 °C, 95% relative humidity, CO 2 5%) for 24 hours.

[0193] After 24 hours, the cells were treated with the above-mentioned peptides of the invention (0.01 mg / mL, 0.015 mg / mL and 0.05 mg / mL) for 48 hours. After treatment, the supernatants were collected, centrifuged to remove cell debris and stored at -80°C until used for quantification of procollagen Iα1 using the Human Procollagen Iα1 DuoSet ELISA Kit according to the manufacturer's instructions. Each condition was performed at least three times (n=3) in three independent experiments (N=3).

[0194] The results obtained are summarized in Figure 9. As can be directly derived from said figure, the release of procollagen Iα1 protein increased in a dose-dependent manner at all tested concentrations, and significantly increased by 53% at the highest tested concentration (0.05 mg / mL). These results demonstrate that the peptides of the invention lead to the release of type I collagen.

[0195] Example 9. Analysis of hyaluronic acid synthesis. In this example, peptide SEQ ID NO:4-NH 2 was prepared.

[0196] In this case, the purpose of the experiment was to evaluate in vitro the ability of the peptides of the invention to increase hyaluronic acid (HA) synthesis or production in human dermal fibroblasts by measuring the amount of HA after treating the cells with the peptides (at different concentrations as described below) for 48 hours.

[0197] In brief: The experimental groups were as follows: Untreated cell cultures (negative control).

[0198] Cells treated with the peptides of the invention (0.005 mg / mL, 0.01 mg / mL or 0.05 mg / mL).

[0199] Treatments were performed for 48 hours and were done in triplicate in two different experimental sessions.

[0200] The experimental model used in this study was cultured in MEM supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C and CO 2 The cells were human fibroblasts (SF-TY JCRB) cultured at 5%.

[0201] Cells were plated in a 96-well plate at 1 x 10 4 Cells were seeded at 100 cells / well and cultured under standard culture conditions (37°C, 95% relative humidity, CO 2 5%) for 24 hours before treatment.

[0202] At the end of the experimental period, hyaluronan synthesis was measured by ELISA. A commercially available kit was used for this purpose. The test kit employed a two-site sandwich ELISA to quantify HA in the samples. Briefly, an antibody specific for HA was precoated on a microplate; standards and samples were pipetted into the wells and any HA present was bound to the immobilized antibody; after removing unbound material, a biotin-conjugated antibody specific for HA was added to the wells; after washing, streptavidin-conjugated horseradish peroxidase (HRP) was added to the wells; after washing to remove unbound avidin-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of HA bound in the first step. Color development was stopped and the color intensity was measured. Quantification of the test samples was performed using a calibration curve of standards of hyaluronan with known concentrations.

[0203] The results obtained are summarized in Table 1. The results are expressed as % of HA content in the cell culture medium and in the cell culture fluid, calculated by taking the HA content in the negative control as 100% and making appropriate comparisons with the remaining samples. The percent change in HA content between the negative control and the samples indicates the effectiveness of the test peptides of the invention to increase the synthesis or production of hyaluronic acid.

[0204] [Table 1]

[0205] As can be easily derived from Table 1, the peptide of the present invention (SEQ ID NO: 4-NH 2 (exemplified by: ) result in increased synthesis or production of hyaluronan.

[0206] Example 10. Analysis of ECM-related gene expression. In this example, peptide SEQ ID NO:4-NH 2 was prepared.

[0207] Stock solutions of peptides were prepared in Milli-Q water at 25 mg / mL. This stock was further diluted with the corresponding supplemented medium to obtain final working concentrations of 0.05 mg / mL, 0.01 mg / mL and 0.005 mg / mL. Untreated cells were used as negative control samples.

[0208] The expression of the relevant genes measured were as follows: MMP1 (matrix metallopeptidase 1), HYAL1 (hyaluronidase 1), TIMP2 (TIMP metallopeptidase inhibitor 2), HAS1 (hyaluronan synthase 1), FBLN5 (fibulin 5), LOXL1 (lysyl oxidase-like 1), COL4A1 (type IV collagen α1 chain), COL3A1 (type III collagen α1 chain), and COL7A1 (type VII collagen α1 chain). Commercially available TaqMan Gene Expression Assay probes were used as primers for each gene.

[0209] Briefly, HDFa cells were plated in 12-well plates at 1.5 × 10 5 Cells were seeded at a density of 10 cells / well and cultured under standard culture conditions (10 medium supplemented with 1% LSGS; 37 °C, 95% relative humidity, CO 2 5%) for 24 hours, after which the cells were treated with 0.005 mg / mL, 0.01 mg / mL, or 0.05 mg / mL of the above peptides for an additional 6 or 24 hours.

[0210] Finally, cells were lysed and RNA extraction was performed using the Qiagen RNeasy Mini kit according to the manufacturer's instructions. The purified RNA was used to generate the corresponding cDNA by reverse transcription, which served as a template for amplification. RT-qPCR was performed using a panel of TaqMan assay probes specified above and 2x gene expression Master Mix on a StepOne plus Real-Time PCR machine. Amplification included 40 cycles of: 95°C for 15 s (denaturation) and 60°C for 1 min (annealing and extension).

[0211] The data obtained were analyzed using the ΔΔCt method, which provides expression values ​​of the target genes as fold changes in treated samples compared to untreated basal samples. Both samples were normalized with the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0212] The analytical steps included: 1. Calculate the average Ct for each condition 2. Calculate ΔCT Test Sample and ΔCT Calibration Sample 3. Calculate ΔΔCT: ΔΔCT = ΔCT test sample - ΔCT calibration sample 4.2 -ΔΔCT The ratio is calculated by:

[0213] The results obtained are summarized in Figure 10. 2 (as an example of the peptide of the present invention) can regulate the expression in fibroblasts of the tested ECM-related genes: 0.01 mg / mL peptide, downregulation of MMP1 and upregulation of COL3A1 and COL4A1 for 24 hours; 0.05 mg / mL peptide, upregulation of TIMP2, FBLN5, LOXL1 and COL7A1 for 24 hours; 0.005 mg / mL peptide, downregulation of HYAL1 for 6 hours; 0.05 mg / mL peptide, upregulation of HAS1 for 6 hours. MMP1 and HYAL1 are genes related to the degradation of ECM and HA, respectively. TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1 and COL7A1 are genes involved in the synthesis and effective maintenance of ECM. Thus, the peptide of the present invention (SEQ ID NO: 4-NH 2 Expression profiles obtained using the IL-1 ...

[0214] Thus, as demonstrated in the Examples contained above, the peptides of the present invention were successfully produced and exhibited the activities mentioned throughout this specification: Modulating or decreasing voltage-gated sodium channel activity or signaling Regulating or decreasing the expression of voltage-gated sodium channels Decreasing noradrenaline levels (thus inhibiting the release of noradrenaline) Increases elastin production Increases release of procollagen Iα1 Increases hyaluronic acid levels Favorable regulation of ECM-associated genes.

[0215] Thus, the peptides of the present invention can prevent, treat or reduce wrinkles (both age and expression wrinkles), roughness, loose or sagging skin; as well as improve skin firmness and / or elasticity, solving the above-mentioned technical problems existing in the art. [Brief description of the drawings]

[0216] [Figure 1A] FIG. 1 shows cell viability results obtained for peptide SEQ ID NO:4-NH2 in Example 2 included below. More specifically, FIG. 1A shows viability in HEKa cells and FIG. 1B shows viability in HDFa cells. In both figures, the Y-axis refers to the percentage of cell viability, with the viability of the negative control (untreated cells) being taken as 100%. In both figures, the columns from left to right on the x-axis refer to the negative control (untreated cells); and cells treated with peptide SEQ ID NO:4-NH2 at 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL or 1 mg / mL. [Figure 1B] Same as Figure 1A. [Diagram 2]Figure 2 shows the results obtained in Example 3, i.e., the time course current (area under the response curve) of the samples tested, and thus the activity of the voltage-gated sodium channel (NaV1.3). The Y-axis refers to the inward current (Amp). The x-axis refers to the different experimental groups tested, with the left side referring to the negative control and the right side referring to the treated samples for each set of columns. On the x-axis, the set of columns from left to right refers to the following experimental treatments or groups: SEQ ID NO:1-NH2, SEQ ID NO:2-NH2, SEQ ID NO:3-NH2 and amitriptyline. [Diagram 3] Figure 3 shows the results obtained in Example 4. This figure shows the inhibition rate of voltage-gated sodium channels. The Y-axis represents the inhibition rate of voltage-gated sodium channels relative to the negative control (0%), and the X-axis refers to the logarithm of the concentration in M ​​of the different peptides tested. The triangle line is SEQ ID NO:2-NH2; the square line is SEQ ID NO:3-NH2; the cross line is SEQ ID NO:4-NH2; the circle line is SEQ ID NO:5-NH2; the diamond line is SEQ ID NO:6-NH2. [Figure 4] Figure 4 shows the results obtained in Example 5 for the levels of noradrenaline when treated with SEQ ID NO:6-NH2. The y-axis shows the percentage of noradrenaline relative to the positive control (untreated depolarized cells, taken as 100%). The x-axis shows the different treatment groups, represented by bars from left to right: basal state; positive control (depolarized cells without treatment); depolarized cells treated with 0.05 mg / mL of peptide SEQ ID NO:6-NH2; and depolarized cells treated with 0.1 mg / mL of peptide SEQ ID NO:6-NH2. [Diagram 5] Figure 5 shows the results obtained for the levels of noradrenaline in Example 5 treated with SEQ ID NO: 4-NH2. The y-axis shows the percentage of noradrenaline relative to the positive control (untreated depolarized cells, taken as 100%). The x-axis shows the different treatment groups, represented by bars from left to right: basal state; positive control (depolarized cells without treatment); depolarized cells treated with 0.05 mg / mL of peptide SEQ ID NO: 4-NH2; and depolarized cells treated with 0.1 mg / mL of peptide SEQ ID NO: 4-NH2. [Figure 6]Figure 6 shows the gene expression results obtained in Example 6 when treated with peptide SEQ ID NO: 4-NH2 at a concentration of 0.01 mg / mL for 24 hours. Each bar represents one gene from top to bottom: SCN1B, SCN4A, SCN3A and CALM3. Bars extending to the left (i.e. negative values) indicate downregulation of genes. [Figure 7] Figure 7 shows the gene expression results obtained in Example 6 when treated with peptide SEQ ID NO: 4-NH2 at a concentration of 0.005 mg / mL for 24 hours. Each bar represents one gene from top to bottom: RAPSN, SCN3A and CALM3. Bars extending to the left (i.e., negative values) indicate downregulation of the gene. [Figure 8] Figure 8 shows the results of elastin production or synthesis obtained in Example 7 when HDFa cells are treated with peptide SEQ ID NO: 4-NH2 at concentrations of 0.005 mg / mL, 0.01 mg / mL or 0.015 mg / mL for 48 hours. Elastin production or synthesis is expressed as a percentage relative to the basal state (untreated cells), established as 100%. The Y-axis shows the percentage of elastin relative to the basal state. The x-axis shows the different treatment groups, represented by bars from left to right: basal state (untreated cells); cells treated with peptide SEQ ID NO: 4-NH2 at 0.005 mg / mL; cells treated with peptide SEQ ID NO: 4-NH2 at 0.01 mg / mL; and cells treated with peptide SEQ ID NO: 4-NH2 at 0.015 mg / mL. [Figure 9]Figure 9 shows the results obtained for the release of procollagen Iα1 in Example 8, when HDFa cells are treated with 0.01 mg / mL, 0.015 mg / mL or 0.05 mg / mL of peptide SEQ ID NO:4-NH2 for 48 hours. The release of procollagen Iα1 is expressed as a percentage relative to the basal state (untreated cells), established as 100%. The y-axis shows the percentage of the release of procollagen Iα1 relative to the basal state. The x-axis shows the different treatment groups, represented by bars from left to right: basal state (untreated cells); cells treated with 0.01 mg / mL of peptide SEQ ID NO:4-NH2; cells treated with 0.015 mg / mL of peptide SEQ ID NO:4-NH2; and cells treated with 0.05 mg / mL of peptide SEQ ID NO:4-NH2. [Figure 10] Figure 10 shows gene expression results obtained in Example 10 when HDFa cells were treated with peptide SEQ ID NO: 4-NH2 for 24 hours with 0.01 mg / mL peptides (MMP1, COL3A1 and COL4A1), 24 hours with 0.05 mg / mL peptides (TIMP2, FBLN5, LOXL1 and COL7A1), 6 hours with 0.005 mg / mL peptide (HYAL1) and 6 hours with 0.05 mg / mL peptide (HAS1). Each bar represents one gene from top to bottom: MMP1, HYAL1, TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1 and COL7A1. Bars extending to the left (i.e. negative values) indicate downregulation of the gene. Bars extending to the right (i.e. positive values) indicate upregulation of the gene.

Claims

1. A peptide of 5 to 8 amino acids capable of decreasing the activity and / or signaling of at least one type of sodium channel; its permitted isomers, salts, solvates, derivatives and / or mixtures thereof, wherein said peptide comprises a sequence of formula (I): <h2 style=";text-align:left;direction:ltr">1<h2 style=";text-align:left;direction:ltr"> １ <h2 style=";text-align:left;direction:ltr"> I am sorry, I am not sure<h2 style=";text-align:left;direction:ltr"> ２ (R １ -SEQ ID NO:7-R ２ ) wherein AA1 is selected from Lys, Arg, or Ile; AA2 is selected from Lys, Arg, His, Asp, Trp or Glu; AA3 is selected from Arg, Gln, Pro, or His; AA4 is selected from Val, Met, lie, Ser, Thr, Cys, Asn, Gin, Phe, Tyr, Lys, or Trp; AA5 is selected from Tyr, Asn, or Trp; AA6 is absent or selected from Ala, Val, Leu, Met, He, Ser, Thr, Cys, Pro, Asn, or Gln; R １ is H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R ５ —CO—, where R ５ is a substituted or unsubstituted C １ -C ２４ Alkyl radical, substituted or unsubstituted C ２ -C ２４ Alkenyl, substituted or unsubstituted C ２ -C ２４ Alkynyl, substituted or unsubstituted C ３ -C ２４ Cycloalkyl, substituted or unsubstituted C ５ -C ２４ Cycloalkenyl, substituted or unsubstituted C ８ -C ２４ Cycloalkynyl, substituted or unsubstituted C ６ -C ３０ Aryl, substituted or unsubstituted C ７ -C ２４ is selected from the group formed by aralkyl, 3- to 10-membered substituted or unsubstituted heterocyclyl rings, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms and an alkyl chain of 1 to 6 carbon atoms; and R ２ is H, -NR ３ R ４ -, -OR ３ and -SR ３ where R ３ and R ４ is independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl; Or, R １ and R ２ is not present and AA1 is directly attached to the last amino acid of said peptide; its permitted isomers, salts, solvates, derivatives and / or mixtures thereof.

2. The peptide according to claim 1, characterized in that the peptide is capable of downregulating the expression of at least one type of sodium channel and / or capable of reducing the level of at least one neurotransmitter and / or capable of maintaining and / or synthesizing ECM.

3. The peptide of claim 2, wherein the sodium channel is a voltage-gated sodium channel.

4. The peptide of claim 3, wherein the voltage-gated sodium channel is Nav1.3 and / or Nav1.

4.

5. The peptide of claim 2 , wherein the neurotransmitter is noradrenaline.

6. 3. The peptide of claim 2, wherein the ECM is selected from collagen I, elastin, hyaluronic acid, or a combination thereof.

7. Having the sequence of formula (I): <h2 style=";text-align:left;direction:ltr">1<h2 style=";text-align:left;direction:ltr"> １ <h2 style=";text-align:left;direction:ltr"> I am sorry, I am not sure<h2 style=";text-align:left;direction:ltr"> ２ (R １ -SEQ ID NO:7-R ２ ) Where: AA1 is selected from Lys, Arg, or Ile; AA2 is selected from Lys, Arg, His, Asp, Trp or Glu; AA3 is selected from Arg, Gln, Pro, or His; AA4 is selected from Val, Met, lie, Ser, Thr, Cys, Asn, Gin, Phe, Tyr, Lys, or Trp; AA5 is selected from Tyr, Asn, or Trp; AA6 is absent or selected from Ala, Val, Leu, Met, He, Ser, Thr, Cys, Pro, Asn, or Gln; R １ is H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R ５ —CO—, where R ５ is a substituted or unsubstituted C １ -C ２４ Alkyl radical, substituted or unsubstituted C ２ -C ２４ Alkenyl, substituted or unsubstituted C ２ -C ２４ Alkynyl, substituted or unsubstituted C ３ -C ２４ Cycloalkyl, substituted or unsubstituted C ５ -C ２４ Cycloalkenyl, substituted or unsubstituted C ８ -C ２４ Cycloalkynyl, substituted or unsubstituted C ６ -C ３０ Aryl, substituted or unsubstituted C ７ -C ２４ is selected from the group formed by aralkyl, 3- to 10-membered substituted or unsubstituted heterocyclyl rings, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms and an alkyl chain of 1 to 6 carbon atoms; and R ２ is H, -NR ３ R ４ -, -OR ３ and -SR ３ where R ３ and R ４ is independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl; Or, R １ and R ２ The peptide according to any one of claims 1 to 6, characterized in that: is absent and AA1 is directly bonded to the last amino acid of the peptide.

8. The peptide is selected from: R １ -Arg-Trp-Gln-Lys-Asn-Gln-R ２ ; ( １ ____________________________________________________ ２ または ( １ ____________________________________________________ ２ R １ -Lys-Asp-Ar-Val-Tyr-R ２ ; R １ -Ile-Glu-Pro-Tyr-Tyr-Val-R ２ ;or R １ -Lys-Lys-His-Phe-Trp-Ala-R ２ Where: R １ is H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R ５ —CO—, where R ５ is a substituted or unsubstituted C １ -C ２４ Alkyl radical, substituted or unsubstituted C ２ -C ２４ Alkenyl, substituted or unsubstituted C ２ -C ２４ Alkynyl, substituted or unsubstituted C ３ -C ２４ Cycloalkyl, substituted or unsubstituted C ５ -C ２４ Cycloalkenyl, substituted or unsubstituted C ８ -C ２４ Cycloalkynyl, substituted or unsubstituted C ６ -C ３０ Aryl, substituted or unsubstituted C ７ -C ２４ is selected from the group formed by aralkyl, 3- to 10-membered substituted or unsubstituted heterocyclyl rings, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms and an alkyl chain of 1 to 6 carbon atoms; and R ２ is H, -NR ３ R ４ -, -OR ３ and -SR ３ where R ３ and R ４ is independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl; Or, R １ and R ２ The peptide according to any one of claims 1 to 7, characterized in that: is absent and AA1 is directly bonded to the last amino acid of the peptide.

9. R １ is H, and / or R ２ NH ２ 9. The peptide according to claim 7 or 8, characterized in that it is

10. A composition comprising a peptide according to any one of claims 1 to 9.

11. Use of a peptide according to any one of claims 1 to 9 or a composition according to claim 10 as a cosmetic in a subject in need thereof.

12. 12. Cosmetic use according to claim 11, characterized in that the cosmetic use consists in reducing, preventing and / or eliminating the signs of skin ageing.

13. 13. The cosmetic use according to claim 12, characterized in that the signs of skin aging are wrinkles.

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