Peptides and compositions for cosmetics

Abstract

The present invention relates to a peptide of 4 to 8 amino acids capable of reducing the activity and / or the signal of at least one type of sodium channel. The peptide is also capable of promoting the production of extracellular matrix. Compositions comprising the peptide are also disclosed as well as the use of the peptides and compositions of the invention for treating wrinkles in the field of cosmetics.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, more precisely, to molecular biology applications in cosmetics, and even more precisely, to peptides and compositions comprising said peptides, said peptides being capable of modulating sodium channels to provide reduced signaling and / or their expression, and thus providing muscle relaxation or less muscle contraction. The peptides and compositions comprising said peptides are also capable of maintaining and / or synthesizing or increasing extracellular matrix (ECM) components. Therefore, said peptides and compositions are effective in addressing conditions related to regulation in the aforementioned aspects (e.g., muscle relaxation or maintenance or synthesis of the ECM) and are useful in cosmetics (anti-wrinkle activity, activity against sagging or loose skin, improvement of skin firmness and / or elasticity). Background Technology

[0002] In recent years, there has been a growing focus on preventing, reducing, or eliminating skin defects in order to maintain healthy and youthful skin. This has led to increasing interest in understanding the causes of skin defects and in finding compounds and compositions that can prevent or mitigate them.

[0003] The formation of wrinkles (e.g., expression wrinkles) is based on the tension of muscles pulling the skin inward. This muscle tension is a result of nerve overactivity causing weakness in the corresponding muscles. Furthermore, this nerve overactivity is characterized by the uncontrolled and excessive release of neurotransmitters that stimulate muscle fibers.

[0004] In this regard, several cosmetic methods have been developed to target wrinkles and the aforementioned mechanisms of their formation, such as:

[0005] - Botulinum toxin: It has been widely used to reduce and / or eliminate expression lines, especially serum type A (botulinum toxin). Cosmetic products (Allergan Inc.). The botulinum toxin is injected locally, and its paralyzing effect (average duration of 6 months) is reversible, thus requiring repeated injections. The risk of inducing an immune response to botulinum toxin preparations is known, leading to loss of treatment efficacy. To overcome this problem, other serotypes of botulinum toxin, such as B, F, and E, have also been considered; however, these serotypes also carry the risk of inducing an immune response. At the molecular level, botulinum toxin is a protease that degrades neuronal proteins involved in exocytosis mechanisms mediated by calcium ion activation. For example, botulinum toxin A truncates the neuronal SNAP-25 protein.

[0006] - It is known in the art that certain peptides derived from the sequences of proteins forming the SNARE complex (such as: 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 synaptic vesicle-associating membrane proteins (synaptobrevin) or synaptic fusion proteins (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, AA synthetic hexapeptide (Argireline) with antiwrinkle activity). International Journal of Cosmetic Science, (2002), 24, 303-310; and peptides that compete with specific sequences on the surface that interact between Munc18 and Syntaxin-1 (see PCT patent application PCT / EP2019 / 054479) can inhibit neuronal exocytosis.

[0007] - In existing technology, there is also a technology called Activeen XEP-018 TM The cone snail toxin, namely Activeen XEP-018 TM It is a biomimetic peptide that mimics the venom of the cone snail (Conus consors) from the Indo-West Pacific region and exhibits botulinum toxin-like activity. Biologically, it acts as a regulator of neuromuscular transmission and a potent and selective blocker of the voltage-gated sodium channel Nav1.4. Cosmeticly, it functions as an immediate wrinkle relaxant, making the skin appear smoother.

[0008] However, there is still a need to find new molecules (preferably peptides) with anti-wrinkle activity that can help maintain and synthesize ECM. Summary of the Invention

[0009] Voltage-gated sodium channels control nerve signal transmission by regulating the propagation of action potentials. Inhibiting sodium channels reduces the flow of action potentials along the nerve, and thus reduces the effectiveness of the signal. Sodium channels such as NaV1.3 and the closely related NaV1.4 have been found in the skin (epidermis, subcutaneous adipose tissue, skeletal muscle, and nerves). These systems work together to regulate skin muscle tone, thereby controlling the depth of wrinkles and folds in the skin.

[0010] In this sense, it is worth noting that despite the extremely high toxicity of the liver and ovaries of the red-finned pufferfish (Takifugu rubripes, also known as the tiger pufferfish) due to the presence of tetrodotoxin (TTX, a potent toxin that specifically binds to voltage-gated sodium channels), the red-finned pufferfish remains a prized edible fish in Japan. TTX physically binds to voltage-gated sodium channels and blocks the flow of sodium ions through these channels, thereby preventing the generation and propagation of action potentials (APs). Furthermore, the pufferfish is known for its elastic skin, which allows it to inflate its body to intimidate predators and quickly revert to its original form once the danger is overcome. Therefore, several key complementary activities can potentially be identified in the peptide for use in human skin, and the peptide thus has the potential to have a positive effect on wrinkles, including muscle-related wrinkles or expression lines and chronological winkles.

[0011] Following extensive and detailed research, the inventors of this invention have surprisingly discovered that peptides derived from the red-finned pufferfish (Japanese pufferfish, tiger pufferfish) (from its proteins or genome) and / or peptides derived therefrom through conserved substitution provide appropriate cosmetic and anti-wrinkle activity. These peptides can provide the desired muscle-relaxing activity by modulating voltage-gated sodium channels (reducing the signal or activity provided by these channels). Furthermore, the peptides of this invention also exhibit the ability to modulate the expression of voltage-gated sodium channels (downregulating or reducing the expression of these channels) and regulate the release of norepinephrine (reducing levels, thereby decreasing the release of this neurotransmitter). Additionally, the peptides of this invention can upregulate the expression of key extracellular matrix (ECM) components (such as collagen I, elastin, and hyaluronic acid) at both the gene and protein expression levels. In summary, the peptides of the present invention are verified to be useful in cosmetics for treating, preventing and / or reducing wrinkles (preferably age lines and wrinkles or expression lines caused by muscle); treating, preventing and / or reducing skin sagging or looseness; treating, preventing and / or reducing skin roughness; and improving skin firmness and / or elasticity.

[0012] Blocking voltage-gated sodium channels (reducing signal transduction and / or its activity) to relax muscles is an interesting mechanism of action that has not been extensively explored and is significantly different from the mechanisms of action of products currently widely used in the market. The peptides of this invention reduce neurotransmission before reaching the neuromuscular junction.

[0013] Furthermore, the peptides of the present invention have inherent advantages due to their size, such as increased or prolonged stability and increased or improved permeability (preferably, permeation through the skin).

[0014] In a first aspect, the present invention relates to peptides of 4 to 8 amino acids that are capable of reducing the signaling and / or activity of at least one type of sodium channel and thus have anti-wrinkle activity.

[0015] In a second aspect, the present invention relates to compositions comprising the peptides of the present invention.

[0016] In another aspect, the present invention relates to the use of the peptides or compositions of the present invention as cosmetics in subjects requiring treatment.

[0017] In a fourth aspect, the present invention relates to the cosmetic use of the peptides or compositions of the present invention in subjects requiring treatment.

[0018] In a final aspect, the present invention relates to a method for reducing, preventing, and / or eliminating signs of aging on the skin of a subject, the method comprising administering to the subject a peptide of the invention or a composition of the invention.

[0019] The term “acyclic aliphatic group” and its plural forms as used herein have the general meanings given to the term in the prior art. Therefore, these terms refer to (e.g., but not limited to) straight-chain or branched alkyl, alkenyl, and alkynyl groups.

[0020] As used herein, the term "alkyl" and its plurals 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, and bonded to the remainder of the molecule by single bonds. Alkyl groups include (e.g., 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, pentyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl, and similar groups. Alkyl groups may optionally be substituted with one or more substituents (such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0021] As used herein, the term "alkenyl" and its plural form refer to a straight-chain 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, the group having one or more carbon-carbon double bonds, preferably having 1, 2, or 3 conjugated or non-conjugated carbon-carbon double bonds, the alkenyl group being bonded to the remainder of the molecule by single bonds, alkenyl groups including (e.g., but not limited to): vinyl, oleoyl, linoleoyl, and similar groups. The alkenyl group may optionally be substituted with one or more substituents (such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0022] As used herein, the term "alkynyl" and its plural form refer to a straight-chain 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, having one or more carbon-carbon triple bonds, preferably 1, 2, or 3 conjugated or non-conjugated carbon-carbon triple bonds, wherein the alkynyl group is bonded to the remainder of the molecule by single bonds. The alkynyl group includes (e.g., but not limited to): ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, pentyynyl (such as 1-pentynyl), and similar groups. The alkynyl group may optionally be substituted with one or more substituents (such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0023] The term "alicyclic group" and its plural forms as used herein have the general meanings given to the term in the prior art. Therefore, these terms are used to refer to (e.g., but not limited to) cycloalkyl, cycloalkenyl, or cycloynyl groups.

[0024] As used herein, the term "cycloalkyl" and its plurals refer to a saturated monocyclic or polycyclic aliphatic group having 3 to 24, preferably 3 to 16, more preferably 3 to 14, even more preferably 3 to 12, even more preferably 3, 4, 5 or 6 carbon atoms, and said cycloalkyl is bonded to the remainder of the molecule by a single bond. Cycloalkyl includes (for example, but not limited to): cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethylcyclohexyl, octahydroindene, decahydronaphthalene, dodecahydrophenanthrene, adamantyl, and similar groups, and may optionally be substituted by one or more groups (such as alkyl, halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0025] As used herein, the term "cycloalkenyl" and its plural form refer to a non-aromatic monocyclic or polycyclic aliphatic group having 5 to 24, preferably 5 to 16, more preferably 5 to 14, even more preferably 5 to 12, even more preferably 5 or 6 carbon atoms, having one or more carbon-carbon double bonds, preferably having 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds, said cycloalkenyl being bonded to the rest of the molecule by single bonds, cycloalkenyl including (e.g., but not limited to): cyclopent-1-en-1-yl and similar groups, and may optionally be substituted by one or more groups (such as alkyl, halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkoxythio).

[0026] As used herein, the term "cycloalkynyl" and its plurals refer to a non-aromatic monocyclic or 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, the group having one or more carbon-carbon triple bonds, preferably having 1, 2, or 3 conjugated or non-conjugated carbon-carbon triple bonds, the cycloalkynyl group being bonded to the rest of the molecule by single bonds, the cycloalkynyl group including (e.g., but not limited to): cyclooctyl-2-yn-1-yl and similar groups, and may optionally be substituted by one or more groups (such as alkyl, halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0027] As used herein, the term "aryl" and its plural forms refer to an aromatic group having 6 to 30, preferably 6 to 18, more preferably 6 to 10, or even more preferably 6 or 10 carbon atoms, comprising 1, 2, 3, or 4 aromatic rings bonded or fused by carbon-carbon bonds and linked to the remainder of the molecule by single bonds. Aryl groups include (e.g., but not limited to): phenyl, naphthyl, diphenyl, indenyl, phenanthryl, or anthracene, etc. The aryl group may optionally be substituted with one or more substituents (such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0028] As used herein, the term "aralkyl" and its plurals refer to an alkyl group having 7 to 24 carbon atoms substituted with an aromatic group, including (e.g., but not limited to): -(CH2)1-6-phenyl, -(CH2)1-6-(1-naphthyl), -(CH2)1-6-(2-naphthyl), -(CH2)1-6-CH(phenyl)2, etc. Aralkyl groups may optionally be substituted with one or more substituents (such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0029] As used herein, the term "heterocyclic group" and its plural forms refer to 3- to 10-membered heterocyclic groups or hydrocarbon rings, wherein one or more ring atoms (preferably 1, 2, or 3 ring atoms) are elements different from carbon, such as nitrogen, oxygen, or sulfur, and may be saturated or unsaturated. For the purposes of this invention, the heterocyclic group can be a cyclic, monocyclic, bicyclic, or tricyclic system, which may include fused ring systems; and the nitrogen, carbon, or sulfur atoms may optionally be oxidized in the heterocyclic radical; the nitrogen atom may optionally be quaternized; and the heterocyclic radical may be partially or fully saturated, or may be aromatic. With increasing preference, the term heterocyclic group relates to 5- or 6-membered rings. The heterocyclic group may optionally be substituted with one or more substituents (such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0030] As used herein, the term "heteroarylalkyl" and its plural forms refer to substituted or unsubstituted aromatic heterocyclic alkyl groups having 1 to 6 carbon atoms and an aromatic heterocyclic group having 2 to 24 carbon atoms and 1 to 3 atoms other than carbon atoms, including (e.g., but not limited to): -(CH2)1-6-imidazolyl, -(CH2)1-6-triazolyl, -(CH2)1-6-thienyl, -(CH2)1-6-furanyl, -(CH2)1-6-pyrrolidinyl, and similar groups. Heteroarylalkyl groups may optionally be substituted with one or more substituents (such as halogen, hydroxyl, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio).

[0031] As used in this article, the term "halogen" or "halogen" refers to fluorine, chlorine, bromine, or iodine, and their anions refer to halides.

[0032] As used in this article, the term “derivative” and its plural form refer to cosmetically acceptable compounds (i.e., compounds of interest that can be used to prepare cosmetic products) and cosmetically unacceptable derivatives (because these derivatives can be used to prepare cosmetically acceptable derivatives).

[0033] As used herein, the term "salt" and its plural form refer to any type of salt known in the art, such as halide salts, hydroxy acid salts (such as oxyacid salts, acid salts, basic salts, and double salts), hydroxy salts, mixed salts, oxyacid salts, or other hydrated salts. The term includes cosmetically acceptable salts; and cosmetically unacceptable salts, as the latter can be used to prepare cosmetically acceptable salts.

[0034] As used herein, the term "isomer" and its plural form refer to optical isomers, enantiomers, stereoisomers, or diastereomers. Individual enantiomers or diastereomers, as well as mixtures thereof, can be separated by conventional techniques known in the art.

[0035] As used herein, the term "solvent" and its plural form refer to any solvate known in the art, such as polar, nonpolar, or amphiphilic solvates, and includes any cosmetically acceptable solvate that, when administered or applied to a subject of interest, provides (directly or indirectly) the compound of interest (one or more peptides of the present invention). Preferably, the solvate is a hydrate, a solvate having an alcohol (such as methanol, ethanol, propanol, or isopropanol), a solvate having an ester (such as ethyl acetate), a solvate having an ether (such as methyl ether, diethyl ether, or THF (tetrahydrofuran)), or a solvate having DMF (dimethylformamide), and more preferably a hydrate or a solvate having an alcohol (such as ethanol).

[0036] Furthermore, the term "amino acid" and its plural form as used herein include both amino acids encoded by the genetic code and non-coding amino acids, regardless of whether they are naturally occurring and whether they are D or L amino acids. Examples of non-coding amino acids are, but are not limited to: citrulline, ornithine, sarcosine, desmosin, valine, 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, thiophene alanine, hydroxyproline, alloleucine, allothreonine, isoperidol, isoserine, phenylglycine, statins, β-alanine, leucine, N-methyl amino acids, α-amino acids, and β-amino acids, and their derivatives. However, other non-natural amino acids are also known in the prior art (see, for example, “Unusual amino acids in peptidesynthesis”, DC Roberts and F. Vellaccio, The Peptides, Vol. 5 (1983), Chapter VI, Gross E. and Meienhofer J. eds., Academic Press, New York, USA).

[0037] As used herein, the "percentage of identity" for peptides, polypeptides, and proteins has a meaning commonly found in the art and therefore refers to the percentage of identical amino acids between two amino acid sequences (compared after optimal alignment of these sequences), where the percentage is statistically significant only and the differences between the two amino acid sequences are randomly distributed throughout the sequence. "Optimal alignment" is understood to mean that the alignment of the amino acid sequences yields a larger percentage of identity. The percentage of identity is calculated by determining the number of identical positions of amino acids in the two compared sequences, 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. Sequence comparisons 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.

[0038] As described above, in a first aspect, the present invention relates to peptides of 4 to 8 amino acids (i.e., peptides containing 4 to 8 amino acids, more preferably peptides having 4 to 8 amino acids, more preferably peptides consisting substantially of 4 to 8 amino acids, even more preferably peptides consisting of 4 to 8 amino acids), which are capable of reducing the signal and / or activity of at least one type of sodium channel; and acceptable isomers, salts, solvates, derivatives and / or mixtures thereof.

[0039] As described above, the peptides of the present invention are preferably derived from pufferfish proteins and / or from the pufferfish genome. Pufferfish can be any pufferfish now known or to be discovered in the future. More preferably, the pufferfish is the redfin pufferfish (Japanese pufferfish, tiger pufferfish). The peptides of the present invention can also be peptides obtained or derived from peptides derived from pufferfish proteins and / or the pufferfish genome, preferably through conserved substitution.

[0040] The amino acids used or present in the peptides of the present invention are envisioned to be L-amino acids, D-amino acids, or combinations thereof. In a preferred embodiment, the amino acids used or present in the peptides of the present invention are L-amino acids.

[0041] Preferably, the aforementioned isomers are stereoisomers. The stereoisomers are enantiomers or diastereomers. Therefore, in a preferred embodiment of the invention, the peptide is a racemic mixture, a diastereomer mixture, a pure enantiomer, or a pure diastereomer.

[0042] Furthermore, isomers, salts, solvates, derivatives and / or mixtures thereof are preferably cosmetically acceptable isomers, salts, solvates, derivatives and / or mixtures thereof.

[0043] As can be directly observed from the examples included below, the peptides of the present invention provide modulation of sodium channels, which manifests as a reduction in the signal and / or activity of at least one type of sodium channel. The reduction in the activity and / or signal of said at least one type of sodium channel is preferably a reduction in the current supplied by said at least one type of sodium channel, i.e., a reduction in the action potential.

[0044] The sodium channel described above is preferably a voltage-gated sodium channel.

[0045] At least one type of sodium channel can be any type 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. The peptides of the present invention are envisioned to reduce the activity and / or signaling of one type of sodium channel or several (two or more) types of sodium channels.

[0046] In one embodiment, the peptide of the present invention at least reduces the activity and / or signal of the sodium channel NaV1.3, and more preferably, the peptide of the present invention only reduces the activity and / or signal of the sodium channel NaV1.3.

[0047] In another embodiment, the peptide of the present invention at least reduces the activity and / or signal of the sodium channel NaV1.4, and more preferably, the peptide of the present invention only reduces the activity and / or signal of the sodium channel NaV1.4.

[0048] In a preferred embodiment, the peptide of the present invention at least reduces the signal and / or activity of sodium channels Nav1.3 and NaV1.4; more preferably, the peptide of the present invention reduces only the activity and / or signal of sodium channels NaV1.3 and NaV1.4.

[0049] It is noted that the peptides of the present invention can reduce the activity and / or signaling of at least one type of sodium channel, regardless of where the sodium channel is present. Preferably, the peptides of the present invention reduce the activity and / or signaling of at least one sodium channel at the presynaptic level (preferably in neurons).

[0050] In some embodiments of the 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 may be partially or completely blocked, more preferably partially blocked.

[0051] Furthermore, in some embodiments that can be combined with any of the above embodiments, the peptides of the present invention are also capable of regulating the expression of at least one type of sodium channel, more preferably, downregulating the expression of at least one type of sodium channel. The at least one type of sodium channel is as described above. In these embodiments, the peptides of the present invention are capable of downregulating the expression of at least one type of sodium channel anywhere that at least one type of sodium channel is expressed. Preferably, the peptides of the present invention downregulate the expression of at least one sodium channel at the postsynaptic level (preferably in muscle cells, more preferably in skeletal muscle cells).

[0052] Furthermore, in some embodiments that can be combined with any of the above embodiments, the peptides of the present invention are also capable of maintaining and / or synthesizing ECM, more precisely, promoting the maintenance of ECM and / or promoting and / or increasing the synthesis of ECM components. The peptides of the present invention exert this activity by upregulating the expression of relevant genes and proteins from ECM; more preferably, the peptides of the present invention are also capable of upregulating at least one gene and / or protein responsible for the synthesis and / or maintenance of ECM; even more preferably, the peptides of the present invention are 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 combinations thereof; even more preferably, the peptides of the present invention are also capable of upregulating collagen I, elastin, and hyaluronic acid at the gene and / or protein level.

[0053] In a preferred embodiment that can be combined with any of the above embodiments, the peptides of the present invention are capable of increasing the synthesis (and thus increasing their levels) of elastin, procollagen Iα1 and hyaluronic acid at the protein level.

[0054] In another preferred embodiment that can be combined with any of the above embodiments, the peptide of the present invention is capable of downregulating the expression of genes that provide ECM degradation and / or upregulating genes that provide ECM maintenance or synthesis. More preferably, the downregulated genes are selected from MMP1, HYAL1, or combinations thereof; the upregulated genes are selected from TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1, COL7A1, or combinations thereof. Even more preferably, the downregulated genes are MMP1 and HYAL1, and the upregulated genes are TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1, and COL7A1.

[0055] The peptides of the present invention are capable of performing the above-mentioned activities, at least in fibroblasts, of maintaining and / or synthesizing ECM (more preferably, promoting the maintenance of ECM and / or promoting and / or increasing ECM synthesis).

[0056] In other embodiments that can be combined with any of the above embodiments, the peptides of the present invention reduce the level or amount of at least one neurotransmitter (more preferably, norepinephrine). The reduction in the level of said at least one neurotransmitter preferably occurs at the presynaptic level, and more preferably at the neuron level. As readily deduced, a reduction in the level or amount of said at least one neurotransmitter will result in a reduction or inhibition of the secretion of said neurotransmitter.

[0057] The above-described activities have been demonstrated in the examples included below and provide the ability of the peptides of the present invention to induce or produce muscle relaxation (or prevent muscle contraction or excessive muscle contraction), which in turn provides an anti-wrinkle effect (preferably for expression lines). Furthermore, the above-described activities also validate the usefulness of the peptides of the present invention in preventing and / or reducing signs of aging (preferably, their depth, length, and / or roughness); treating, preventing, and / or reducing skin sagging or loosening; and improving skin firmness and elasticity.

[0058] In a preferred embodiment that can be combined with any of the above embodiments, the peptide of the present invention is a peptide of 5 or 6 amino acids (i.e., a peptide containing 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, and even more preferably a peptide consisting of 5 or 6 amino acids).

[0059] Furthermore, preferably, in any of the above embodiments, the peptide of the present invention comprises at least one charged amino acid (positively or negatively charged). Even more preferably, the amino acid at the first and / or second position is a charged amino acid (positively or negatively charged). More preferably, in the peptide of the present invention, the first three amino acids are charged amino acids (positively or negatively charged).

[0060] In a preferred embodiment that can be combined with any of the above embodiments, in the peptide of the present invention, the amino acids at the first and third positions are positively charged amino acids, and the amino acid at the second position is either positively or negatively charged amino acids.

[0061] Furthermore, preferably, in any of the above embodiments, the remaining amino acids in the peptide of the present invention are polar, uncharged, nonpolar, and / or aromatic. Additionally, preferably, if present, the amino acid at the fifth position is aromatic, preferably selected from Tyr or Trp. Also preferably, if present, the amino acid at the sixth position is polar, uncharged, or nonpolar.

[0062] Therefore, in a preferred embodiment, the peptide of the present invention comprises at least one aromatic amino acid, more preferably, one or two aromatic amino acids. Even more preferably:

[0063] - If the peptide of the present invention has 5 amino acids, it has one aromatic amino acid, more preferably located at the fifth position. In this case, the aromatic amino acid is preferably Tyr.

[0064] - If the peptide of the present invention has 6 amino acids, it has 2 aromatic amino acids, one in the fourth position and the other in the fifth position. In this case, the aromatic amino acids are selected from Tyr, Trp, Phe or combinations thereof.

[0065] In any of the above embodiments, the charged amino acids (positively or negatively charged) are preferably selected from Lys, Arg, His, and Asp.

[0066] Therefore, in a preferred embodiment, the charged amino acid at the first position in the peptide of the present invention is Lys. Even more preferably, in the peptide of the present invention, the first three amino acids of the peptide are: Lys-Arg-Arg, Lys-His-Arg, Lys-Asp-Arg, or Lys-Lys-His.

[0067] In a preferred embodiment, the peptide of the present invention has the sequence of formula (I):

[0068] R1-AA1-AA2-AA3-AA4-AA5-AA6-R2

[0069] (R1-SEQ ID NO: 7-R2)

[0070] in

[0071] AA1 is selected from Lys, Arg, Ile, or His;

[0072] AA2 is selected from Lys, Arg, His, Trp, Asp, or Glu;

[0073] AA3 is selected from Lys, Arg, Gln, Pro, or His;

[0074] AA4 is selected from Gly, Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, Gln, Phe, Tyr, Lys, or Trp;

[0075] AA5 is selected from Phe, Tyr, Asn, or Trp;

[0076] AA6 does not exist or is selected from Gly, Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, or Gln;

[0077] R1 is selected from H, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, and R5-CO-, wherein R5 is selected from the group consisting of substituted or unsubstituted C1-C groups. 24 Alkyl radicals, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkyne group, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloynyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C7-C 24 Aryl groups, substituted or unsubstituted 3- to 10-membered heterocyclic rings, and substituted or unsubstituted heteroaryl groups having 1 to 6 carbon atoms and an alkyl chain of 2 to 24 carbon atoms; and

[0078] R2 is selected from H, -NR3R4-, -OR3 and -SR3, wherein R3 and R4 are independently selected from H, substituted or unsubstituted acyclic aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group and substituted or unsubstituted aralkyl group.

[0079] Alternatively, R1 and R2 are absent, and AA1 binds directly to the last amino acid of the peptide (i.e., with AA6, or with AA5 if AA6 is absent) (forming a cyclic peptide).

[0080] R1 is attached to the N-terminus of the peptide, that is, to the amino group of AA1 (replacing or replacing the H of the amino group of AA1).

[0081] R2 is attached to the C-terminus of the peptide, that is, to the carboxyl group of AA6 (replacing or replacing the OH group of the carboxyl group), or to the carboxyl group of AA5 in the absence of AA6 (replacing or replacing the OH group of the carboxyl group).

[0082] In a more preferred embodiment, the peptide is capable of downregulating the expression of at least one type of sodium channel and / or reducing the level of at least one neurotransmitter and / or maintaining and / or synthesizing ECM. Preferably, the sodium channel is a voltage-gated sodium channel, and more preferably, the voltage-gated sodium channel is Nav1.3 and / or Nav1.4. Preferably, the neurotransmitter is norepinephrine. Preferably, the ECM is selected from collagen I, elastin, hyaluronic acid, or combinations thereof.

[0083] In another preferred embodiment, the peptide has the sequence of formula (I):

[0084] R1-AA1-AA2-AA3-AA4-AA5-AA6-R2

[0085] (R1-SEQ ID NO: 7-R2)

[0086] in

[0087] AA1 is selected from Lys, Arg, or Ile;

[0088] AA2 is selected from Lys, Arg, His, Asp, Trp, or Glu;

[0089] AA3 is selected from Arg, Gln, Pro, or His;

[0090] AA4 is selected from Val, Met, Ile, Ser, Thr, Cys, Asn, Gln, Phe, Tyr, Lys, or Trp;

[0091] AA5 is selected from Tyr, Asn, or Trp;

[0092] AA6 does not exist or is selected from Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, or Gln;

[0093] R1 is selected from H, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, and R5-CO-, wherein R5 is selected from the group consisting of substituted or unsubstituted C1-C groups. 24 Alkyl radicals, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkyne group, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloynyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C7-C 24 Aryl groups, substituted or unsubstituted 3- to 10-membered heterocyclic rings, and substituted or unsubstituted heteroaryl groups having 1 to 6 carbon atoms and an alkyl chain of 2 to 24 carbon atoms; and

[0094] R2 is selected from H, -NR3R4-, -OR3 and -SR3, wherein R3 and R4 are independently selected from H, substituted or unsubstituted acyclic aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group and substituted or unsubstituted aralkyl group.

[0095] Alternatively, R1 and R2 may be absent, and AA1 may bind directly to the last amino acid of the peptide.

[0096] Preferably, in formula (I):

[0097] AA1 is Lys;

[0098] AA2 is selected from Arg, His, Lys, or Asp;

[0099] AA3 is selected from Arg or His;

[0100] AA4 is selected from Tyr, Ser, Val, or Phe;

[0101] AA5 is selected from Tyr or Trp;

[0102] AA6 does not exist or is selected from Cys or Ala.

[0103] In a preferred embodiment, the peptides of the present invention are selected from:

[0104] -R1-Arg-Trp-Gln-Lys-Asn-Gln-R2 (R1-SEQ ID NO: 1-R2);

[0105] -R1-Lys-Arg-Arg-Tyr-Tyr-Cys-R2 (R1-SEQ ID NO: 2-R2);

[0106] -R1-Lys-His-Arg-Ser-Tyr-R2 (R1-SEQ ID NO: 3-R2);

[0107] -R1-Lys-Asp-Arg-Val-Tyr-R2 (R1-SEQ ID NO: 4-R2);

[0108] -R1-Ile-Glu-Pro-Tyr-Tyr-Val-R2(R1-SEQ ID NO: 5-R2); or

[0109] -R1-Lys-Lys-His-Phe-Trp-Ala-R2 (R1-SEQ ID NO: 6-R2);

[0110] in:

[0111] R1 is selected from H, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroaryl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, and R5-CO-, wherein R5 is selected from the group consisting of substituted or unsubstituted C1-C groups. 24 Alkyl radicals, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkyne group, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloynyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C7-C 24 Aryl groups, substituted or unsubstituted 3- to 10-membered heterocyclic rings, and substituted or unsubstituted heteroaryl groups having 1 to 6 carbon atoms and an alkyl chain of 2 to 24 carbon atoms; and

[0112] R2 is selected from H, -NR3R4-, -OR3 and -SR3, wherein R3 and R4 are independently selected from H, substituted or unsubstituted acyclic aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group and substituted or unsubstituted aralkyl group.

[0113] Alternatively, R1 and R2 are absent, and AA1 binds directly to the last amino acid of the peptide (forming a cyclic peptide).

[0114] More preferably, the peptides of the present invention are selected from:

[0115] -R1-Lys-Asp-Arg-Val-Tyr-R2(R1-SEQ ID NO: 4-R2); or

[0116] -R1-Lys-Lys-His-Phe-Trp-Ala-R2 (R1-SEQ ID NO: 6-R2);

[0117] R1 and R2 are as disclosed above.

[0118] Even more preferably, the peptide of the present invention is:

[0119] -R1-Lys-Asp-Arg-Val-Tyr-R2 (R1-SEQ ID NO: 4-R2);

[0120] R1 and R2 are as disclosed above.

[0121] In any of the above embodiments, preferably, R1 is H or acetyl, more preferably, R1 is H.

[0122] Furthermore, in any of the above embodiments, R2 is NH2 or OH, more preferably, R2 is NH2.

[0123] Therefore, in the most preferred embodiment, the peptide of the present invention is selected from:

[0124] -Arg-Trp-Gln-Lys-Asn-Gln-NH2 (SEQ ID NO: 1-NH2);

[0125] -Lys-Arg-Arg-Tyr-Tyr-Cys-NH2 (SEQ ID NO: 2-NH2);

[0126] -Lys-His-Arg-Ser-Tyr-NH2 (SEQ ID NO: 3-NH2);

[0127] -Lys-Asp-Arg-Val-Tyr-NH2 (SEQ ID NO: 4-NH2);

[0128] -Ile-Glu-Pro-Tyr-Tyr-Val-NH2(SEQ ID NO: 5-NH2); or

[0129] -Lys-Lys-His-Phe-Trp-Ala-NH2 (SEQ ID NO: 6-NH2).

[0130] Even more preferably, the peptide of the present invention is:

[0131] -Lys-Asp-Arg-Val-Tyr-NH2(SEQ ID NO: 4-NH2); or

[0132] -Lys-Lys-His-Phe-Trp-Ala-NH2 (SEQ ID NO: 6-NH2);

[0133] Even more preferably:

[0134] -Lys-Asp-Arg-Val-Tyr-NH2 (SEQ ID NO: 4-NH2).

[0135] Note that peptides SEQ ID NO: 1 to SEQ ID NO: 3 are directly derived from the proteins and / or genome of the pufferfish *Puerca japonica* (Japanese pufferfish, tiger pufferfish). More precisely:

[0136] -SEQ ID NO: 1 is a sequence derived from the nucleolar complex protein 2 homolog of the redfin pufferfish (Uniprot reference number: H2TGB2, amino acids 88 to 93).

[0137] -SEQ ID NO: 2 is a sequence derived from the deactivated homolog 2 interacting protein-like isoform X2 (synaptic Ras GTPase activation domain) (NCBI reference sequence: XP_003976523.1, amino acids 64 to 68).

[0138] -SEQ ID NO:3 is derived from the genomic sequence predicted to be calcium-independent phospholipase A2γ-like isotype X2 (NCBI reference sequence: XP_011605802.2, amino acids 7 to 11).

[0139] The peptides of SEQ ID NO: 4 to 6 are derived from the peptides of SEQ ID NO: 1 to 3 by a conservative substitution.

[0140] Within the scope of this invention, peptides derived from or obtained from any of the above-described peptides are also included, preferably incorporating one or more conservative substitutions, provided that they retain or exhibit or improve at least one of the above-described activities (and preferably, all of the above-described activities of the peptides of this invention).

[0141] Furthermore, the scope of this invention also includes peptides having 70% identity with any of the specific sequences described above, preferably 80%, more preferably 90%, more preferably 95%, or even more preferably 99% identity, as long as they retain or exhibit or improve at least one of the above-described activities (and preferably all of the above-described activities of the peptides of this invention).

[0142] As can be directly deduced from the above, the peptides of the present invention are envisioned to be linear or cyclic peptides.

[0143] In embodiments that can be combined with any of the above embodiments, the peptide is cyclic. In this case, the first and last amino acids of the peptide of the present invention (as explained above) are bound together. Therefore, any of the above embodiments must be appropriately modified (e.g., R1 and R2 may be absent because the partially bound amino acids are now bound to each other).

[0144] In a preferred embodiment that can be combined with any of the above embodiments, the peptide is linear.

[0145] The peptides of the present invention can be synthesized and produced by any means known in the prior art. For example, they can be synthesized and produced by chemical synthesis (preferably by solid-phase peptide synthesis), by expressing the peptides in cell cultures, or by producing the peptides transgenic in plants or animals. Furthermore, the peptides of the present invention can be purified by any means known in the prior art.

[0146] In a preferred embodiment that can be combined with any of the above embodiments, the peptides of the present invention are suitable or appropriate for application via iontophoresis, more preferably applied to the face of a subject (preferably a human).

[0147] In another preferred embodiment that can be combined with any of the above embodiments, the peptides of the present invention are suitable for or adapted for subcutaneous application, more preferably for application to the face of a subject (preferably a human).

[0148] In another preferred embodiment that can be combined with any of the above embodiments, the peptides of the present invention are suitable for topical (preferably, in the form of a cream) application, more preferably for application to the face of a subject (preferably, a human).

[0149] As can be directly deduced from the examples included below, the peptides of the present invention possess the desired activity, thereby providing prevention, treatment, and / or reduction of wrinkles, at least by means of muscle relaxation and by means of maintaining and synthesizing the ECM. Furthermore, the peptides of the present invention also possess the desired activity, thereby providing prevention, treatment, and / or reduction of skin laxity or sagging and skin roughness (preferably, wrinkle roughness); and improvement of skin firmness and elasticity. Therefore, the peptides of the present invention solve the aforementioned technical problems and can be used in cosmetics.

[0150] In a second aspect, the present invention relates to compositions comprising the peptides of the present invention.

[0151] The peptides of the present invention are as disclosed above in the first aspect of the present invention.

[0152] The compositions of the present invention are preferably cosmetic compositions. Therefore, 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% (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), more preferably, 0.0005% (w / v) to 0.0015% (w / v) of the peptide of the present invention.

[0153] The proposed cosmetic composition of the present invention further comprises 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 prior art, provided that it does not affect the activity of the peptides of the present invention or does not affect the activity of the peptides of the present invention in an unacceptable manner.

[0154] In one embodiment, the composition of the present invention consists essentially of the peptides of the present invention.

[0155] In another embodiment, the composition of the present invention comprises the peptides of the present invention.

[0156] In a third aspect, the present invention relates to the use of the peptides or compositions of the present invention as cosmetics in subjects requiring treatment.

[0157] The peptides of the present invention are as disclosed above in the first aspect of the present invention.

[0158] The compositions of the present invention are as disclosed above in the second aspect of the present invention.

[0159] The peptides or compositions of the present invention are used in cosmetically effective amounts or quantities.

[0160] In a preferred embodiment, the purpose of using it as a cosmetic is to reduce, prevent, and / or eliminate signs of skin aging.

[0161] Preferably, signs of skin aging are wrinkles, roughness of the skin (preferably, wrinkle roughness), loose or sagging skin, more preferably wrinkles, roughness of the skin, non-inflammatory loose or sagging skin, even more preferably wrinkles, and even more preferably facial wrinkles. The wrinkles may be age lines or expression lines (muscle-related).

[0162] Non-inflammatory loose skin is equivalent to cosmetically loose skin, that is, non-pathological (non-medical condition).

[0163] Non-inflammatory sagging skin is equivalent to cosmetic sagging skin, that is, non-pathological (non-medical condition).

[0164] As can be directly deduced from the above, in this invention, signs of skin aging (preferably both wrinkles, age lines, and expression lines) are prevented, reduced, and / or eliminated (by the peptides or compositions of this invention) by blocking neuromuscular contraction as described above and / or by maintaining or synthesizing ECM.

[0165] The above-mentioned at least one sodium channel and at least one neurotransmitter are as described in the first aspect of the present invention.

[0166] In a third aspect of the invention, the subject to be treated is preferably a mammal, more preferably a human.

[0167] In a third aspect of the invention, cosmetic uses for improving skin firmness and / or elasticity are also envisioned.

[0168] In a fourth aspect, as described above, the present invention relates to the cosmetic use of the peptides or compositions of the present invention in subjects requiring treatment.

[0169] Preferably, in a fourth aspect of the invention, the cosmetic use is to reduce, prevent, and / or eliminate signs of skin aging.

[0170] In a fourth aspect of the invention, cosmetic uses for improving skin firmness and / or elasticity are also envisioned.

[0171] The peptides of the present invention are as disclosed above in the first aspect of the present invention.

[0172] The compositions of the present invention are as disclosed above in the second aspect of the present invention.

[0173] Signs of skin aging and subjects requiring treatment are as described above in the third aspect of the invention.

[0174] Furthermore, all embodiments described in the third aspect of the invention are directly applicable to the fourth aspect of the invention with appropriate modifications.

[0175] In the fifth and final aspects, the present invention relates to methods for reducing, preventing and / or eliminating signs of skin aging in a subject, said methods comprising administering to the subject a peptide of the invention or a composition of the invention.

[0176] The peptides of the present invention are as disclosed above in the first aspect of the present invention.

[0177] The compositions of the present invention are as disclosed above in the second aspect of the present invention.

[0178] Signs of skin aging and subjects are as described above in the third aspect of the invention.

[0179] Furthermore, all embodiments described in the third and fourth aspects of the present invention are directly applicable to the fifth aspect of the present invention with appropriate modifications.

[0180] Sequence List Free Text

[0181] SEQ ID NO:1: Peptide derived from the sequence of a nucleolar complex protein 2 homolog of the pufferfish redfin pufferfish (Uniprot reference number: H2TGB2, amino acids 88 to 93).

[0182] SEQ ID NO:2: Peptide derived from the sequence of the deactivated homolog 2 interacting protein-like isoform X2 (synaptic Ras GTPase activation domain) (NCBI reference sequence: XP_003976523.1, amino acids 64 to 68).

[0183] SEQ ID NO:3: Peptide derived from the genomic sequence of a calcium-independent phospholipase A2γ-like isotype X2 (NCBI reference sequence: XP_011605802.2, amino acids 7 to 11).

[0184] SEQ ID NO:4: Artificially synthesized sequence.

[0185] SEQ ID NO:5: Artificially synthesized sequence.

[0186] SEQ ID NO:6: Artificially synthesized sequence.

[0187] SEQ ID NO:7: Artificially synthesized sequence,

[0188] Xaa is selected from Lys, Arg, or His.

[0189] Xaa is selected from Lys, Arg, His, Asp, or Glu.

[0190] Xaa is selected from Lys, Arg, or His.

[0191] Xaa is selected from Gly, Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, Gln, Phe, Tyr, or Trp.

[0192] Xaa is selected from Phe, Tyr, or Trp.

[0193] Xaa does not exist or is selected from Gly, Ala, Val, Leu, Met, Ile, Ser, Thr, Cys, Pro, Asn, or Gln. Attached Figure Description

[0194] For a better understanding, the invention will now be described in more detail with reference to the accompanying drawings (which are presented by way of example) and illustrative and non-limiting embodiments.

[0195] Figure 1 shows the cell viability results obtained for peptide SEQ ID NO:4-NH2 in Example 2 included below. More specifically, Figure 1A This demonstrates the viability of HEKa cells. Figure 1B This graph shows the viability of HDFa cells. In both graphs, the y-axis represents the percentage of cell viability, with the viability of the negative control (untreated cells) set at 100%. In both graphs, the bars on the x-axis, from left to right, represent: the negative control (untreated cells); and cells treated with peptide SEQ ID NO:4-NH2 at concentrations of 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.

[0196] Figure 2 The results obtained in Example 3 are shown. Specifically, the current (area under the response curve) obtained by the tested sample over time is, therefore, the activity of the voltage-gated sodium channel (NaV1.3). The y-axis represents the inward current in Amp. The x-axis represents the different experimental groups tested; for each pair of columns, the left column represents the negative control, and the right column represents the treated sample. On the x-axis, the paired columns from left to right represent the following experimental treatments or groups: SEQ ID NO:1-NH2, SEQ ID NO:2-NH2, SEQ ID NO:3-NH2, and amitriptyline.

[0197] Figure 3 The results obtained in Example 4 are shown. The graph shows the percentage inhibition of the voltage-gated sodium channel. The y-axis reflects the percentage inhibition of the voltage-gated sodium channel relative to the negative control (0%), and the x-axis represents the logarithm of the concentrations (in M) of the different peptides tested. The line with a triangle is SEQ ID NO:2-NH2; the line with a square is SEQ ID NO:3-NH2; the line with a cross is SEQ ID NO:4-NH2; the line with dots is SEQ ID NO:5-NH2; and the line with a diamond is SEQ ID NO:6-NH2.

[0198] Figure 4 The results show the levels of norepinephrine obtained in Example 5 after treatment with SEQ ID NO:6-NH2. The y-axis shows the percentage of norepinephrine relative to the positive control (untreated depolarized cells, set at 100%). The x-axis shows the different treatment groups, with bars from left to right: baseline; positive control (untreated depolarized cells); depolarized cells treated with 0.05 mg / mL peptide SEQ ID NO:6-NH2; and depolarized cells treated with 0.1 mg / mL peptide SEQ ID NO:6-NH2.

[0199] Figure 5 The results show the levels of norepinephrine obtained in Example 5 after treatment with SEQ ID NO:4-NH2. The y-axis shows the percentage of norepinephrine relative to the positive control (untreated depolarized cells, set at 100%). The x-axis shows the different treatment groups, with bars from left to right: baseline; positive control (untreated depolarized cells); depolarized cells treated with 0.05 mg / mL peptide SEQ ID NO:4-NH2; and depolarized cells treated with 0.1 mg / mL peptide SEQ ID NO:4-NH2.

[0200] Figure 6The results show the gene expression outcomes obtained in Example 6 after treatment with the peptide SEQ ID NO:4-NH2 at a concentration of 0.01 mg / mL for 24 hours. Each bar, from top to bottom, represents one of the following genes: SCN1B, SCN4A, SCN3A, and CALM3. Bars extending to the left (i.e., negative values) indicate gene downregulation.

[0201] Figure 7 The results show gene expression obtained after treatment with peptide SEQ ID NO:4-NH2 at a concentration of 0.005 mg / mL for 24 hours in Example 6. Each bar, from top to bottom, represents one of the following genes: RAPSN, SCN3A, and CALM3. Bars extending to the left (i.e., negative values) indicate gene downregulation.

[0202] Figure 8 The results of elastin production or synthesis obtained by treating HDFa cells with peptide SEQ ID NO:4-NH2 at concentrations of 0.005 mg / mL, 0.01 mg / mL, or 0.015 mg / mL in Example 7 for 48 hours are shown. Elastin production or synthesis is expressed as a percentage relative to a baseline condition (untreated cells), which is set at 100%. The y-axis shows the percentage of elastin relative to the baseline condition. The x-axis shows the different treatment groups, with bars from left to right: baseline condition (untreated cells); cells treated with 0.005 mg / mL peptide SEQ ID NO:4-NH2; cells treated with 0.01 mg / mL peptide SEQ ID NO:4-NH2; and cells treated with 0.015 mg / mL peptide SEQ ID NO:4-NH2.

[0203] Figure 9 The results of procollagen Iα1 release obtained by treating HDFa cells with peptide SEQ ID NO:4-NH2 at 0.01 mg / mL, 0.015 mg / mL, or 0.05 mg / mL for 48 hours in Example 8 are shown. Procollagen Iα1 release is expressed as a percentage relative to the baseline condition (untreated cells), which is set at 100%. The y-axis shows the percentage of procollagen Iα1 release relative to the baseline condition. The x-axis shows the different treatment groups, with bars from left to right: baseline condition (untreated cells); cells treated with peptide SEQ ID NO:4-NH2 at 0.01 mg / mL; cells treated with peptide SEQ ID NO:4-NH2 at 0.015 mg / mL; and cells treated with peptide SEQ ID NO:4-NH2 at 0.05 mg / mL.

[0204] Figure 10The gene expression results obtained by treating HDFa cells with peptide SEQ ID NO:4-NH2 in Example 10 are shown: 0.01 mg / mL peptide for 24 hours (MMP1, COL3A1, and COL4A1), 0.05 mg / mL peptide for 24 hours (TIMP2, FBLN5, LOXL1, and COL7A1), 0.005 mg / mL peptide for 6 hours (HYAL1), and 0.05 mg / mL peptide for 6 hours (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 gene downregulation. Bars extending to the right (i.e., positive values) indicate gene upregulation. Detailed Implementation

[0205] Example

[0206] abbreviation:

[0207] The abbreviations for amino acids follow the biochemical nomenclature recommendations made by the IUPAC-IUB Joint Committee in Eur. J. Biochem. (1984) 138: 937.

[0208] Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Boc, tert-butoxycarbonyl; cDNA, complementary DNA; C-terminus, carboxyl terminus; Cys, cysteine; DCM, dichloromethane; DIPCDI, N,N'-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; Glu, glutamic acid; Gln, glutamine; HBSS, Hank's balanced salt solution; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; His, histidine; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; Ile, isoleucine; LSGS, low serum growth supplement; Ly s, lysine; MBHA, p-methyldiphenylamine; Me, methyl; MeCN, acetonitrile; MEM, essential medium; MeOH, methanol; Met, methionine; N-terminus, amino terminus; 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 growth medium; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TPA, 12-O-tetradecanoylphorbol-13-acetate; Trp, tryptophan; Trt, triphenylmethyl or triphenylmethyl; Tyr, tyrosine; Val, valine.

[0209] Regarding the chemical synthesis procedures included in the embodiments, it is noted that all synthesis processes are performed in a polypropylene syringe equipped with a porous polyethylene disc or a porous plate. The synthesis was carried out in a reactor. All reagents and solvents were of synthetic quality and ready for use without any additional 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)). Washings were performed with DMF (3 × 1 min) and DCM (3 × 1 min) between deprotection, coupling, and re-deprotection stages, each time using 10 mL solvent / g resin. Coupling was carried out with 3 mL solvent / g resin. Coupling was controlled by ninhydrin testing (Kaiser E. et al., Anal. Biochem., 1970, 34:595598). All synthetic reactions and washes were performed at room temperature (RT).

[0210] Example 1. Synthesis and preparation of peptides.

[0211] a) Obtain Fmoc-AA1-AA2-AA3-AA4-AA5-AA6-Rink-MBHA-resin, wherein AA1 is L-Lys; AA2 It is L-Lys; AA3 is L-His; AA4 is L-Phe; AA5 is L-Trp; and AA6 is L-Ala.

[0212] Weights were standardized. Following the general procedure known in the prior art, 4.8 g (2.5 mmol) of Fmoc-Rink-MBHA resin functionalized to 0.52 mmol / g was treated with piperidine-DMF to remove Fmoc groups. Using DMF as a solvent, 2.33 g of Fmoc-L-Ala-OH (7.5 mmol; 3 equivalents) was incorporated into the deprotected resin for 1 hour in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equivalents) and HOBt (1.01 g; 7.5 mmol; 3 equivalents).

[0213] The resin was then washed using general methods known in the art, and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocol, 3.94 g of Fmoc-L-Trp(Boc)-OH (7.5 mmol; 3 equivalences) was coupled sequentially; followed by 2.90 g of Fmoc-L-Phe-OH (7.5 mmol; 3 equivalences); followed by 4.64 g of Fmoc-L-His(Trt)-OH (7.5 mmol; 3 equivalences); followed by 3.51 g of Fmoc-L-Lys(Boc)-OH (7.5 mmol; 3 equivalences) and then another 3.51 g of Fmoc-L-Lys(Boc)-OH (7.5 mmol; 3 equivalences), each coupling carried out in the presence of 1.01 g of HOBt (7.5 mmol; 3 equivalences) and 1.17 mL of DIPCDI (7.5 mmol; 3 equivalences). As described above, the Fmoc group is deprotected between each amino acid addition step.

[0214] After synthesis, the peptide resin was washed with DCM (5 times each, 3 minutes each time) and dried under vacuum.

[0215] b) Obtain Fmoc-AA1-AA2-AA3-AA4-AA5-Rink-MBHA-resin, wherein AA1 is L-Lys; AA2 is L- Asp; AA3 is L-Arg; AA4 is L-Val and AA5 is L-Tyr.

[0216] Weights were standardized. Following the general procedure known in the prior art, 4.8 g (2.5 mmol) of Fmoc-Rink-MBHA resin functionalized to 0.52 mmol / g was treated with piperidine-DMF to remove Fmoc groups. Using DMF as a solvent, 3.44 g of Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 equivalents) was incorporated into the deprotected resin for 1 hour in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equivalents) and HOBt (1.01 g; 7.5 mmol; 3 equivalents).

[0217] The resin was then washed using general methods known in the art, and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocol, 2.55 g of Fmoc-L-Val-OH (7.5 mmol; 3 equivalences) was coupled sequentially; followed by 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalences); followed by 3.08 g of Fmoc-L-Asp(tBu)-OH (7.5 mmol; 3 equivalences) and then 3.51 g of Fmoc-L-Lys(Boc)-OH (7.5 mmol; 3 equivalences), each coupling being carried out in the presence of 1.01 g of HOBt (7.5 mmol; 3 equivalences) and 1.17 mL of DIPCDI (7.5 mmol; 3 equivalences). As described above, the Fmoc group was deprotected between each amino acid addition step.

[0218] After synthesis, the peptide resin was washed with DCM (5 times each, 3 minutes each time) and dried under vacuum.

[0219] Using the above synthetic procedure, the following sequence was synthesized by selecting the required amino acids:

[0220] Arg-Trp-Gln-Lys-Asn-Gln-NH2 (SEQ ID NO: 1-NH2);

[0221] Lys-Arg-Arg-Tyr-Tyr-Cys-NH2 (SEQ ID NO: 2-NH2);

[0222] Lys-His-Arg-Ser-Tyr-NH2 (SEQ ID NO: 3-NH2);

[0223] Lys-Asp-Arg-Val-Tyr-NH2 (SEQ ID NO: 4-NH2);

[0224] Ile-Glu-Pro-Tyr-Tyr-Val-NH2(SEQ ID NO:5-NH2); and

[0225] Lys-Lys-His-Phe-Trp-Ala-NH2 (SEQ ID NO: 6-NH2).

[0226] c) Remove the Fmoc N-terminal protecting group from the peptide synthesized according to a) or b).

[0227] The N-terminal Fmoc groups of the peptide resins obtained in a) or b) above were deprotected with 20% (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 peptide resins were washed with DMF (5 × 1 min) and DCM (4 × 1 min) and dried under vacuum.

[0228] d) The pyrolysis process of the polymer carrier of the peptide-based resin obtained according to c).

[0229] Weights were standardized. 200 mg of the dried peptide resin obtained in c) was treated with 5 mL of TFA / TIS / H2O (90:5:5 v / v) at room temperature with stirring for 2 hours. The filtrate was collected and precipitated with 50 mL (8 to 10 times) of cold diethyl ether. The ether solution was evaporated to dryness under reduced pressure at room temperature, the precipitate was redissolved in 50% (v / v) MeCN in H2O and lyophilized.

[0230] e) Characterize the peptides synthesized and prepared according to d).

[0231] The peptides obtained according to d) were analyzed by HPLC using a Shimadzu instrument (Kyoto, Japan) with a reversed-phase column (150 x 4.6 mm, XBridge Peptide BEHC18, 3.5 μm, Waters, USA) at a flow rate of 1.25 mL / min using a gradient of H2O (+0.045% (v / v) TFA) and MeCN (+0.036% (v / v) TFA), and detected at 220 nm. All peptides showed a purity exceeding 80%. The identity of the obtained peptides was confirmed by ESI-MS using MeOH as the mobile phase at a flow rate of 0.2 mL / min in a Water ZQ 4000 detector. The results indicate that peptides SEQ ID NO:1-NH2, SEQ ID NO:2-NH2, SEQ ID NO:3-NH2, SEQ ID NO:4-NH2, SEQ ID NO:5-NH2 and SEQ ID NO:6-NH2 were correctly and efficiently synthesized.

[0232] Example 2. Toxicity analysis of the peptides of the present invention

[0233] In this context, the MTT assay (3-[4,5-dimethylthiazolyl-2-yl]-2,5-diphenyltetrazolium bromide) was used to evaluate the cytotoxic potential of the peptides of the present invention against primary HEKa (human epidermal keratinocytes of adult skin) and HDFa (human dermal fibroblasts of adult skin).

[0234] For this embodiment, peptide SEQ ID NO:4-NH2 was used and prepared according to Example 1.

[0235] A stock solution of the peptide (25 mg / mL) was prepared in distilled water. The stock solution was further diluted to obtain the final working concentrations in the corresponding culture media (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).

[0236] Untreated cells were used as a negative control sample (benchmark).

[0237] In short, HEKa and HDFa cells were arranged at 1x10 5 Cells were seeded at a concentration of 100 cells / mL in 96-well plates to a final volume of 200 μL (20,000 cells / well). HEKa cells were incubated in dermal cell basal medium supplemented with a keratinocyte growth kit; and HDFa cells were incubated in medium 106 supplemented with 2% LSGS. Cells were incubated at 37°C, 5% CO2, and saturated humidity.

[0238] After 24 hours of incubation, cells were treated with peptides at different concentrations (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) in triplicate for MTT assay. After 24 hours of peptide treatment, the culture medium was completely removed, and 100 μL of 5 mg / mL MTT solution in replenished medium was added to each well. The cells were then incubated at 37°C for 3 hours. After MTT incubation, the culture medium from each well was aspirated, and 150 μL of DMSO was added to dissolve the formed methyl groups. Crystals. Place the plate on a shaker for 5 minutes to completely dissolve the crystals, and then use a Multiskan FC microplate reader to determine the absorbance of each well at 545 nm, which is proportional to the number of live cells in the culture.

[0239] Absorbance values ​​were standardized using data obtained from untreated cells (control) to obtain... Figure 1A and 1B The percentage of vitality shown. In 3 independent experiments (N=3), each condition was performed in triplicate (n=3).

[0240] Peptides with an activity level above 80% are considered safe for efficacy testing. Conversely, concentrations with an activity level below 80% are considered potentially toxic and are not used for efficacy testing purposes.

[0241] The results are summarized in Figure 1A (HEKa cell viability) and 1B (HDFa cell viability). It can be directly concluded from the figures that after treatment with the test peptide at concentrations of 0.001 mg / mL to 0.5 mg / mL, the HEKa cell viability was higher than 80%; after treatment with the test peptide at concentrations of 0.001 mg / mL to 1 mg / mL, the HDFa cell viability was higher than 80%, demonstrating the safety of the peptides of the present invention.

[0242] Example 3. Analysis of the regulation of sodium channel activity or signal transduction

[0243] For this embodiment, peptides SEQ ID NO:1-NH2, SEQ ID NO:2-NH2 and SEQ ID NO:3-NH2 were prepared according to Example 1.

[0244] Electrophysiological studies were performed on human NaV1.3 stably transfected into Chinese hamster ovary (CHO) cells using QPatch48 HT (Sophion, Denmark) to measure ion channel activity. Experiments were performed in triplicate, using 10 μM peptides. Individual negative controls were provided for each peptide. Amitriptyline was used as a positive control.

[0245] The results obtained in this embodiment are summarized in Figure 2 The figure shows that all three peptides exhibited inhibition of the inward current of NaV1.3, and therefore possessed smaller negative currents (Amp). Thus, all three peptides were able to block or modulate (reduce activity or signaling) the sodium channel Nav1.3. Furthermore, surprisingly, SEQ ID NO:2-NH2 and SEQ ID NO:3-NH2 produced statistically significant increases (p = 0.018 and p = 0.039, respectively).

[0246] Example 4. Analysis of the regulation of sodium channel activity or signal transduction

[0247] For this embodiment, peptides SEQ ID NO:2-NH2, SEQ ID NO:3-NH2, SEQ ID NO:4-NH2, SEQ ID NO:5-NH2 and SEQ ID NO:6-NH2 were prepared according to Example 1.

[0248] Electrophysiological studies were performed on SHSY-5Y cells using a QPatch48 HT (Sophion, Denmark) to measure ion channel activity. The samples tested were:

[0249] - Negative control: 0.05% (v / v) bovine serum albumin.

[0250] - Positive controls: Tetrodotoxin (TTX) at concentrations of 3 nM, 10 nM and 30 nM.

[0251] -SEQ ID NO:2-NH2, with concentrations of 0.005 mg / mL, 0.01 mg / mL and 0.05 mg / mL.

[0252] -SEQ ID NO:3-NH2, with concentrations of 0.005 mg / mL, 0.01 mg / mL and 0.05 mg / mL.

[0253] -SEQ ID NO:4-NH2, with concentrations of 0.005 mg / mL, 0.01 mg / mL and 0.05 mg / mL.

[0254] -SEQ ID NO:5-NH2, with concentrations of 0.005 mg / mL, 0.01 mg / mL and 0.05 mg / mL.

[0255] -SEQ ID NO:6-NH2, with concentrations of 0.005 mg / mL, 0.01 mg / mL and 0.05 mg / mL.

[0256] The percentage of inhibition for each test dose period was calculated as a reduction in the average vernier value (peak current) relative to the vernier value measured at the end of the mediator control (before administration), and normalized for the maximum inhibition recorded with super-maximum added TTX.

[0257] The results are summarized in Figure 3 The values ​​show the percentage of inhibition (or blockage) of sodium channels (in the inactivated state). It can be seen that all tested peptides effectively modulated (reduced activity or signaling) sodium channels in a dose-dependent manner. The peptide that yielded the best results at the two higher concentrations tested was SEQ ID NO:4-NH2. On the other hand, the peptide that performed best at the lower concentration tested was SEQ ID NO:5-NH2.

[0258] The results showed that the peptides of the present invention exhibited a similar regulatory effect to low concentrations of TTX (higher concentrations of TTX produced a complete blocking effect).

[0259] Example 5. Norepinephrine Level Analysis

[0260] In this embodiment, peptides SEQ ID NO:4-NH2 and SEQ ID NO:6-NH2 were prepared according to Example 1.

[0261] In this context, norepinephrine (NA) levels were measured in differentiated SHSY5Y cells (a human neuron model). This test was used to determine whether the candidate could regulate neurotransmitter release.

[0262] Human SH-SY5Y cells were seeded in triplicate in 24-well plates at a cell density of 250,000 cells / well. SH-SY5Y cells were induced to differentiate into neuronal phenotypes by continuous administration of retinoic acid for one week, followed by treatment with the aforementioned peptide at concentrations of 0.05 mg / mL and 0.1 mg / mL for 30 minutes.

[0263] Cells were then washed and treated first with 100 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 8 min to enhance release, followed by treatment with 100 mM KCl for 5 min to induce neurotransmitter release, all in HBSS with 20 mM HEPES buffer. Finally, cells were lysed and homogenized directly onto wells through a 0.8 mm needle 10 times. The homogenized cell suspension was centrifuged at maximum speed for 15 min at 4 °C. The supernatant was collected and held at -80 °C until total norepinephrine was quantified by enzyme-linked immunosorbent assay (ELISA) using a human norepinephrine ELISA kit according to the manufacturer's instructions. Total protein content for each sample was quantified using a Micro BCA protein assay kit to standardize the data.

[0264] In three independent experiments (N=3), each condition was performed at least in triplicate (n=3), and the total protein in the samples and the norepinephrine obtained for the positive control (a peptide-free but stimulating sample) were standardized to obtain the percentage for the positive control.

[0265] The results are summarized in Figure 4 and Figure 5 As can be directly seen from the figures, the tested peptides (representative or examples of the peptides of the present invention) exhibited lower levels of norepinephrine (reduced by more than 30%), and thus provided botulinum toxin-like effects. Therefore, the treatment showed regulation of neurotransmitter release in stimulated and differentiated SH-SY5Y cells, indicating that the peptides of the present invention provide anti-wrinkle activity.

[0266] Example 6. Analysis of the expression of muscle contraction-related genes

[0267] For this embodiment, peptide SEQ ID NO:4-NH2 was prepared according to Example 1.

[0268] A stock solution of the peptide at 25 mg / mL was prepared in milli-Q water. This stock solution was further diluted in the appropriate supplemental medium to obtain final working concentrations of 0.01 mg / mL and 0.005 mg / mL. Untreated cells were used as negative control samples.

[0269] The genes whose expression was measured were: SCN1B (sodium voltage-gated channel β subunit 1), SCN4A (sodium voltage-gated channel α subunit 4), SCN3A (sodium voltage-gated channel α subunit 3), CALM3 (calmodulin 3), and RAPSN (synaptic receptor-associated protein). Primers used for each gene were commercially available TaqMan gene expression assay probes. The assay was performed in vitro in muscle cells, and two peptide concentrations (0.01 mg / mL and 0.005 mg / mL) were tested at 24 hours.

[0270] In short, human skeletal muscle myoblasts were divided into groups of 1×102 5 Cells were seeded at a density of 100 cells / well in 12-well plates and maintained under standard culture conditions (SKM-M medium supplemented with 1% antibiotic / antifungal agent; 37°C, 95% relative humidity, 5% CO2) for 48-72 hours until the myoblasts reached 80%-90% confluence. The growth medium was then replaced with SKM-D medium to induce myotube differentiation (differentiation into myocytes), with fresh medium added every 2-3 days. Six to seven days after the start of differentiation, cardiomyocytes were treated with the aforementioned peptide at 0.01 mg / mL or 0.005 mg / mL for an additional 24 hours. Untreated cells served as a baseline control. Cells were finally lysed, and RNA was extracted using the Qiagen RNeasy Mini kit according to the manufacturer's instructions. The purified RNA was used to generate the corresponding cDNA via reverse transcription as a template for amplification. RT-qPCR was performed using a StepOneplus real-time PCR instrument with the aforementioned TaqMan assay probe set and 2x gene expression premix (Master Mix). The amplification consisted of fewer than 40 cycles: 15 seconds at 95°C (denaturation) and 1 minute at 60°C (annealing and extension).

[0271] The data were analyzed using the ΔΔCt method, which provides target gene expression values ​​as fold changes between the treated sample and the untreated baseline. Both samples were normalized using the relative expression of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

[0272] The analysis steps include:

[0273] 1. Calculate the average Ct for each case.

[0274] 2. Calculate the ΔCT test sample and ΔCT calibration sample.

[0275] 3. Calculate ΔΔCT: ΔΔCT = ΔCT test sample – ΔCT calibration sample

[0276] 4. Through 2 -ΔΔCT Acquisition rate.

[0277] The results are summarized in Figure 6 and Figure 7 The peptide SEQ ID NO:4-NH2 (as an example of the peptide of the present invention) is shown to regulate the expression of the relevant genes tested in muscle cells. Therefore, the peptide can regulate (downregulate) the expression of sodium channels and muscle contraction-related genes in muscle cells.

[0278] Example 7. Analysis of elastin synthesis

[0279] For this embodiment, peptide SEQ ID NO:4-NH2 was prepared according to Example 1.

[0280] A stock solution of the peptide (25 mg / mL) was prepared in water. This stock solution was further diluted to obtain final working concentrations of 0.005 mg / mL, 0.01 mg / mL, or 0.015 mg / mL in complete culture medium (medium 10⁶ supplemented with 2% LSGS). Untreated cells were used as the baseline condition.

[0281] For this study, HDFa cells were seeded at a density of 120,000 cells / well in 12-well plates and maintained under standard culture conditions (medium 10⁶ + 2% LSGS; 37°C, 95% RH, 5% CO₂) for 24 hours. Then, fibroblasts were treated with the aforementioned peptides at concentrations of 0.005 mg / mL, 0.01 mg / mL, or 0.015 mg / mL for 48 hours. After treatment, cells were harvested, and elastin was quantified using the Fastin Elastin Assay Kit according to the manufacturer's instructions. Each condition was performed in at least triplicate in three independent experiments (N=3).

[0282] The results are summarized in Figure 8 In comparison with untreated samples, HDFa treated with the peptides of the present invention at concentrations of 0.01 mg / mL and 0.015 mg / mL showed increased elastin levels. These results indicate that the peptides of the present invention have the potential to increase skin elasticity and improve skin structural integrity, as well as improve (prevent, treat, or reduce) loose and / or sagging skin. Furthermore, the increased elastin synthesis or production also suggests that the peptides of the present invention can improve skin firmness and wound healing ability.

[0283] Example 8. Analysis of procollagen Iα1 release

[0284] For this embodiment, peptide SEQ ID NO:4-NH2 was prepared according to Example 1.

[0285] A stock solution of the peptide (25 mg / mL) was prepared in water. This stock solution was further diluted to obtain final working concentrations of 0.01 mg / mL, 0.015 mg / mL, or 0.05 mg / mL in complete culture medium (medium 10⁶ supplemented with 2% LSGS). Untreated cells were used as the baseline condition.

[0286] In this study, HDFa cells were seeded at a density of 100,000 cells / well in 24-well plates and maintained under standard culture conditions (medium supplemented with 2% LSGS 10⁶; 37°C, 95% relative humidity, 5% CO₂) for 24 hours.

[0287] Twenty-four hours later, cells were treated with the peptides of the present invention (0.01 mg / mL, 0.015 mg / mL, and 0.05 mg / mL) for 48 hours. After treatment, the supernatant was collected and centrifuged to remove cell debris, and kept at -80°C until procollagen Iα1 was quantified using the Human Procollagen Iα1 DuoSet ELISA Kit according to the manufacturer's instructions. In three independent experiments (N=3), each condition was performed in at least triplicate (n=3).

[0288] The results are summarized in Figure 9 As can be directly seen from the figure, at all tested concentrations, the release of procollagen Iα1 protein increased in a dose-dependent manner, with a significant increase of up to 53% at the highest tested concentration (0.05 mg / mL). These results indicate that the peptides of the present invention provide type I collagen release.

[0289] Example 9. Analysis of the synthesis of hyaluronic acid.

[0290] For this embodiment, peptide SEQ ID NO:4-NH2 was prepared according to Example 1.

[0291] In this context, the aim of the experiment was to evaluate in vitro the ability of the peptides of the present invention to increase the synthesis or production of hyaluronic acid (HA) in human skin fibroblasts. This evaluation was performed by determining the amount of HA after treating cells with the peptides (at different concentrations described below) for 48 hours.

[0292] in short:

[0293] The experimental group is:

[0294] - Untreated cell cultures (negative control).

[0295] - Cells treated with the peptides of the present invention (0.005 mg / mL, 0.01 mg / mL or 0.05 mg / mL).

[0296] The treatment lasted 48 hours. Three copies of the treatment were prepared and conducted in two different experimental phases.

[0297] The experimental model used in this study was human fibroblasts (SF-TY JCRB) cultured in MEM containing 10% fetal bovine serum and 1% antibiotics at 37°C and 5% CO2.

[0298] Cells were loaded at a rate of 1×10 4 Cells / well were seeded in 96-well plates and kept in standard culture conditions (37°C, 95% relative humidity, 5% CO2) for 24 hours before treatment was applied.

[0299] At the end of the experimental phase, hyaluronic acid synthesis was determined by ELISA. A commercial kit was used for this purpose. This test kit employs a two-site sandwich ELISA to quantify HA in the sample. Briefly, an antibody specific to HA is pre-coated on a microplate; standards and samples are pipetted into the wells, and any HA present is bound by the immobilized antibody; after removing any unbound material, a biotin-conjugated antibody specific to HA is added to the wells; after washing, streptavidin-conjugated horseradish peroxidase (HRP) is added to the wells; after washing to remove any unbound avidin-enzyme reagent, the substrate solution is added to the wells, and color development is performed proportionally to the amount of HA bound in the initial step. Color development is stopped, and the color intensity is measured. A calibration curve of known concentrations of hyaluronic acid standards is used to determine the composition of the tested samples.

[0300] The results are summarized in Table 1. Results are expressed as % of HA content in cell culture medium and cell lysates, calculated by setting the HA content in the negative control to 100% and appropriately comparing it with the remaining samples. The % change in HA content between the negative control and the samples demonstrates the efficacy of the peptides tested in this invention in increasing hyaluronic acid synthesis or production.

[0301] Table 1. Results of HA content obtained in the first experimental phase of 48 hours. ** It was statistically significant (p<0.01); *** It was statistically significant (p < 0.001).

[0302]

[0303] It can be readily concluded from Table 1 that the peptides of the present invention (such as those exemplified by SEQ ID NO:4-NH2) provide for an increase in the synthesis or production of HA.

[0304] Example 10. Analysis of ECM-related gene expression

[0305] For this embodiment, peptide SEQ ID NO:4-NH2 was prepared according to Example 1.

[0306] A peptide stock solution of 25 mg / mL was prepared in milli-Q water. This stock solution was further diluted in the appropriate supplemental 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.

[0307] The genes whose expression was measured were: MMP1 (matrix metallopeptidase 1), HYAL1 (hyaluronidase 1), TIMP2 (TIMP metallopeptidase inhibitor 2), HAS1 (hyaluronic acid synthase 1), FBLN5 (fibrin 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). Primers used for each gene were commercially available TaqMan gene expression assay probes.

[0308] In short, HDFa cells were placed at a density of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 10⁶ cells / well in 12-well plates and incubated for 24 hours under standard culture conditions (10⁶ medium supplemented with 1% LSGS; 37°C, 95% RH, 5% CO₂). Cells were then treated with the aforementioned peptide at 0.005 mg / mL, 0.01 mg / mL, or 0.05 mg / mL for an additional 6 or 24 hours.

[0309] Cells were finally lysed, and RNA was extracted using the Qiagen RNeasy Mini kit according to the manufacturer's instructions. The purified RNA was used to generate the corresponding cDNA via reverse transcription, which served as a template for amplification. RT-qPCR was performed using a StepOne plus real-time PCR instrument with the TaqMan assay probe set and 2x gene expression premix (Master Mix) described above. Amplification consisted of fewer than 40 cycles: 95°C (denaturation) for 15 seconds and 60°C (annealing and extension) for 1 minute.

[0310] The data were analyzed using the ΔΔCt method, which provides target gene expression values ​​as fold changes between the treated sample and the untreated baseline. Both samples were normalized using the relative expression of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

[0311] The analysis steps include:

[0312] 1. Calculate the average Ct for each case.

[0313] 2. Calculate the ΔCT test sample and ΔCT calibration sample.

[0314] 3. Calculate ΔΔCT: ΔΔCT = ΔCT test sample – ΔCT calibration sample

[0315] 4. Through 2 -ΔΔCT Acquisition rate.

[0316] The results are summarized in Figure 10 The study showed that peptide SEQ ID NO:4-NH2 (as an example of the peptide of the present invention) could regulate the expression of the tested ECM-related genes in fibroblasts in the following ways: MMP1 was downregulated and COL3A1 and COL4A1 were upregulated after 24 hours of treatment with 0.01 mg / mL peptide; TIMP2, FBLN5, LOXL1, and COL7A1 were upregulated after 24 hours of treatment with 0.05 mg / mL peptide; HYAL1 was downregulated after 6 hours of treatment with 0.005 mg / mL peptide; and HAS1 was upregulated after 6 hours of treatment with 0.05 mg / mL peptide. MMP1 and HYAL1 are genes associated with ECM and HA degradation, respectively. TIMP2, HAS1, FBLN5, LOXL1, COL4A1, COL3A1, and COL7A1 are genes associated with ECM synthesis and efficient maintenance. Therefore, the expression profiles obtained with the peptides of the present invention (such as those exemplified by SEQ ID NO:4-NH2) demonstrate their usefulness in the maintenance, synthesis, or increase of the ECM.

[0317] Therefore, as demonstrated in the above embodiments, the peptides of the present invention are efficiently produced, and they exhibit the activities mentioned throughout the description:

[0318] - To regulate or reduce the activity or signal transduction of voltage-gated sodium channels.

[0319] -Regulate or reduce the expression of voltage-gated sodium channels.

[0320] - Reduces norepinephrine levels (and thus inhibits norepinephrine release).

[0321] - Increases the production of elastin.

[0322] -Increases the release of procollagen Iα1.

[0323] - Increase hyaluronic acid levels.

[0324] - Favorable regulation of ECM-related genes.

[0325] Therefore, the peptides of the present invention can prevent, treat or reduce wrinkles (both age lines and expression lines), roughness, loose or sagging skin; and improve skin firmness and / or elasticity, thereby solving the above-mentioned technical problems existing in the prior art.

Claims

A peptide of 1.5 to 8 amino acids or an acceptable salt thereof, said peptide being capable of reducing the activity and / or signaling of at least one voltage-gated sodium channel type in human cells, wherein said peptide comprises the sequence of formula (I): The peptides mentioned therein are selected from: - R1-Arg-Trp-Gln-Lys-Asn-Gln-R2; - R1-Lys-Arg-Arg-Tyr-Tyr-Cys-R2; - R1-Lys-His-Arg-Ser-Tyr-R2; - R1-Ile-Glu-Pro-Tyr-Tyr-Val-R2; or - R1-Lys-Lys-His-Phe-Trp-Ala-R2; R1 is H; and R2 is selected from H, NH2, or -OH.

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 of reducing the level of at least one neurotransmitter, wherein the neurotransmitter is norepinephrine.

3. The peptide according to claim 1 or 2, characterized in that... R2 is NH2.

4. A composition comprising the peptide according to any one of claims 1 to 3.

5. Use of a peptide consisting of the sequence R1-Lys-Asp-Arg-Val-Tyr-R2 as a cosmetic in subjects in need, wherein R1 is H and R2 is selected from H, NH2 or -OH.

6. The use as a cosmetic product according to claim 5, characterized in that... The intended use is to reduce, prevent, and / or eliminate signs of skin aging.

7. The use as a cosmetic product according to claim 6, characterized in that... The sign of skin aging is wrinkles.

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