KR102622462B1 - Peptide for accelerating delivery to nerve cells - Google Patents

Abstract

The present invention relates to a neurotransmitter release-regulating peptide with improved nerve cell permeability. More specifically, the present invention relates to a neurotransmitter release-regulating peptide with improved nerve cell permeability, a fusion peptide in which a neurotransmitter release-regulating peptide is bound to the peptide, and the fusion peptide. It relates to a cosmetic composition for skin improvement comprising: The fusion peptide of the present invention combines a peptide capable of controlling neurotransmitter release with a peptide that promotes nerve cell permeability, thereby maintaining or improving the ability of the peptide to produce useful effects such as wrinkle improvement, while at the same time significantly improving nerve cell permeability. Therefore, it can be widely used as an active ingredient in cosmetic compositions for skin improvement and functional cosmetics.

Description

Peptide for accelerating delivery to nerve cells}

The present invention relates to a peptide for skin improvement, or a cosmetic composition containing the same, and specifically relates to a functional cosmetic for skin improvement and a pharmaceutical composition for external skin application containing the peptide or a cosmetic composition.

The release of neurotransmitters is caused by the fusion of synaptic vesicles located at nerve terminals containing neurotransmitters and the presynaptic membrane, and then a passage is formed between the two boundaries. SNARE protein, a three-protein complex consisting of the vesicle protein VAMP (synaptobrevin) and the plasma membrane-binding proteins Syntaxin 1a and SNAP-25, provides the fundamental force for the fusion of synaptic vesicles and the presynaptic membrane. Here, the opening of the neurotransmitter release channel by membrane fusion between synaptic vesicles and the presynaptic membrane involves t-SNARE and vesicles (a complex of two types of syntaxin 1a protein and SNAP-25 protein attached to the target membrane). It is the result of the action of v-SNARE attached to the vesicle. During membrane fusion, rearrangement of the lipid bilayer occurs. Since biological membranes strongly repel each other, these membranes do not fuse spontaneously and a strong external force must be applied to overcome the repulsive force between the membranes. It is known that SNARE proteins provide this force. As such, the formation of the SNARE complex is a key phenomenon in extracellular exocytosis, including the release of neurotransmitters.

Recently, there has been development of peptides that have a role similar to Botox, reducing muscle contraction in a non-paralytic manner without the side effects of toxins. These peptides play a role in alleviating the appearance of wrinkles by temporarily blocking the secretion of neurotransmitters such as acetylcholine, which are responsible for the contraction of muscle cells.

However, despite the development of peptides that play a role similar to Botox, there was a problem in that the effect was insufficient when applied to the skin in a formulation such as a cream, and thus the original efficacy could not be exerted.

Meanwhile, another example in which the effect as a cell penetrating peptide (CPP) has been confirmed is a peptide having the amino acid sequence from positions 267 to 300 of the VP22 protein of HSV-1 (herpes simplex virus type 1) (Elliott, G et al., Cell , 88:223, 1997), a peptide having the amino acid sequence from the 84th to the 92nd of the UL-56 protein of HSV-2 (GeneBank code: D1047[gi:221784]) and Drosophila Peptides having the amino acid sequence from positions 339 to 355 of the antennapedia (ANTP) protein of Drosophila sp. (Schwarze, SR et al., Trends. Pharmacol . Sci. , 21:45, 2000), etc. The effect was also confirmed in the case of an artificial peptide listing electrically positive amino acids (Laus, R. et al., Nature. Biotechnol. , 18:1269, 2000).

Since it was discovered that a fusion protein prepared by linking CPP with another peptide or protein can be efficiently transported into cells, various applications using CPP have been attempted (Korean Patent Registration No. 10-0568457), but the penetrating functional peptide is Because it originated from a virus, there were problems in terms of safety.

Accordingly, the present inventors confirmed that improvement in nerve cell permeability is necessary to improve the efficacy of substances that regulate the release of neurotransmitters.

In order to develop a material with excellent nerve cell permeability, a phage display method combining a phage library and an elution test method of a transdermal preparation was performed to produce a novel nerve cell permeation promoting peptide containing one of the following amino acid sequences: Nev 1 or Nev 4. It was excavated. In addition, the discovered nerve cell penetration promoting peptide was combined with a peptide that regulates neurotransmitter release, and the fusion peptide combined with the nerve cell penetration promoting peptide was found to have superior nerve cell permeability and muscle contraction inhibition effects compared to existing proteins. This was confirmed and the present invention was completed.

The purpose of the present invention is to provide a nerve cell penetration promoting peptide or a derivative thereof consisting of one of the following amino acid sequences: Nev 1 or Nev 4.

Another object of the present invention is to provide a fusion peptide or a derivative thereof in which a nerve cell permeation promoting peptide containing one of the following amino acid sequences, Nev 1 or Nev 4, is bound to a neurotransmitter release regulating peptide.

Another object of the present invention is to provide a cosmetic composition for skin improvement containing the fusion peptide as an active ingredient.

Another object of the present invention is to provide a functional cosmetic for skin improvement containing the cosmetic composition as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for external skin application containing the fusion peptide as an active ingredient.

The present invention, as an aspect to solve the above problems and achieve the object of the present invention, provides a nerve cell permeation promoting peptide or a derivative thereof consisting of the amino acid sequence of Nev 1 or Nev 4 below. Another example provides a fusion peptide or a derivative thereof that is fused with the amino acid sequence of Nev 1 or Nev 4 below to promote neuronal penetration of a neurotransmitter release-regulating peptide.

amino acid sequence

Nev 1: ALKEVGA (SEQ ID NO: 1)

Nev 4: VPMKVAA (SEQ ID NO: 2)

In the present invention, “neuronal cell-penetrating peptide” refers to a peptide that can penetrate nerves regardless of the size or nature of the molecule and has excellent neuronal retention. In addition, it refers to a peptide that not only can penetrate nerve cells, but also enhances the penetration of other peptides into nerve cells. Nerve cell-penetrating peptides can be used to include peptides that can promote the penetration of other peptides into nerve cells.

In the present invention, “neural cell permeability” refers to the ability or property of a peptide to penetrate nerve cells and penetrate into the inner part of nerve cells, and the nerve cell penetration promoting peptide of the present invention is better than other peptides. Not only does it have excellent nerve cell permeability, but it also shows the effect of promoting nerve cell penetration of other peptides.

The nerve cell permeability promoting peptide (Nev 1 and/or Nev 4) of the present invention may include a peptide with excellent nerve cell permeability discovered by performing a phage display method combining a phage library and an elution test method of a transdermal preparation. , specifically, may include a peptide consisting of one amino acid sequence among Nev 1 and/or Nev 4. In one embodiment of the present invention, peptides containing Nev 1 and Nev 4 amino acid sequences, respectively, were prepared as nerve cell penetration promoting peptides through a phage display method. Afterwards, as a result of comparing neurotransmitter release regulating peptide (Argireline™) and nerve cell permeability, it was confirmed that the nerve cell permeability promoting peptide increased nerve cell permeability by about 35 to 100 times (Table 1).

In the present invention, "neurotransmitters" are a series of substances that are released from nerve cells in the body, including the brain, and transmit information to adjacent nerve cells, etc., crossing the synapse from one neuron to another 'target' neuron. It is an endogenous chemical that transmits signals. Neurotransmitters are packaged into synaptic vesicles clustered under the membrane of the axon terminal in the presynaptic part of the synapse, then released within the synaptic gap and released across the synaptic gap. At this time, the neurotransmitter is released from the membrane in the postsynaptic part of the synapse. Binds to a specific receptor. Neurotransmitters of the present invention may include, but are not limited to, dopamine, serotonin, histamine, acetylcholine, adrenaline, noradrenaline, gamma aminobutyric acid (GABA), L-glutamic acid, glycine, etc.

In the present invention, “neurotransmitter release-regulating peptide” refers to a peptide that can inhibit muscle contraction by blocking the transmission of neurotransmitters to neurotransmitter receptors and can exhibit wrinkle-improving effects. Specifically, in order to move a muscle, a neurotransmitter must be transmitted from a nerve cell to a muscle cell through a synapse. In order for the neurotransmitter acetylcholine to be secreted from the synapse, a SNARE complex is formed at the terminal of the nerve cell. A process of release to the synapse is required. Botox, as commonly known, destroys the component that forms the snare complex (SNAP-25), preventing acetylcholine from being released into the synapse. On the other hand, Botox-like peptide has a structure similar to some of the components of the Snare complex (SNAP-25), and plays a role in preventing the release of acetylcholine by participating in the composition process of the complex instead of SNAP-25.

Peptides used herein may include not only peptides but also derivatives thereof. The derivative may include a peptide in which the N-terminus, C-terminus, etc. of the nerve cell penetration-promoting peptide or the neurotransmitter release-regulating peptide are chemically modified or amino acids are added/substituted/deleted, and there are special restrictions. It doesn't work.

The neurotransmitter release-regulating peptide of the present invention may be one widely known in the industry, and is not particularly limited to the type of peptide.

The neurotransmitter release-regulating peptide of the present invention may include not only natural peptides but also chemically synthesized peptides, and may also include peptide derivatives of peptides known to have anti-wrinkle effects. Specifically, the neurotransmitter release regulating peptide may be any one or more peptides selected from the group consisting of SEQ ID NO: 5 (EEMQRR), SEQ ID NO: 6 (DDMQRR), and SEQ ID NO: 7 (YPWTQRF), and more specifically, SEQ ID NO: 5 It may be a peptide of (EEMQRR), but is not limited thereto.

In another embodiment, the neurotransmitter release regulating peptide may be one or more peptides selected from the group consisting of Acetyl-EEMQRR, Palmityl-DDMQRR, and Palmityl-YPWTQRF.

Additionally, the neurotransmitter release-regulating peptide of the present invention may include a peptide represented by the following formula (1), an isomer thereof, a racemic compound, a cosmetically or pharmaceutically acceptable salt thereof, etc.

[Formula 1]

R1-AA-R2

In Formula 1, AA may be the amino acid sequence of Nev 1 or Nev 4, or may be an amino acid sequence consisting of 3 to 40 amino acids. R1 may be H or any one selected from the group consisting of C ₃ to C ₂₄ alkyl, aryl, and acyl, and R2 may be an amino group substituted or unsubstituted with an aliphatic or cyclic group, It may be any one selected from the group consisting of a hydroxyl group and a thiol group.

Specifically, the AA is MAEDADMRNELEEMQRRADQL (SEQ ID NO: 8), ADESLESTRRMLQLVEESKDAGI (SEQ ID NO: 9), ELEEMQRRADQLA (SEQ ID NO: 10), ELEEMQRRADQL (SEQ ID NO: 11), ELEEMQRRADQ (SEQ ID NO: 12), ELEEMQRRAD (SEQ ID NO: 13), ELEEMQRRA ( SEQ ID NO: 14), ELEEMQRR (SEQ ID NO: 15), LEEMQRRADQL (SEQ ID NO: 16), LEEMQRRADQ (SEQ ID NO: 17), LEEMQRRAD (SEQ ID NO: 18), LEEMQRRA (SEQ ID NO: 19), LEEMQRR (SEQ ID NO: 20), EEMQRRADQL (SEQ ID NO: Number 21), EEMQRRADQ (SEQ ID NO: 22), EEMQRRAD (SEQ ID NO: 23), EEMQRRA (SEQ ID NO: 24), LESTRRMLQLVEE (SEQ ID NO: 25), NKDMKEAEKNLT (SEQ ID NO: 26), KNLTDL (SEQ ID NO: 27), IMEKADSNKTRIDEANQRATKMLGSG (SEQ ID NO: 28), SNKTRIDEANQRATKMLGSG (SEQ ID NO: 29), TRIDEANQRATKMLGSG (SEQ ID NO: 30), DEANQRATKMLGSG (SEQ ID NO: 31), NQRATKMLGSG (SEQ ID NO: 32), and QRATKMLGSG (SEQ ID NO: 33). there is. Additionally, the AA may include an amino acid sequence derived from the amino acid sequence of the SNAP-25 protein.

In the present invention, a “fusion peptide” is a peptide artificially synthesized such that the nerve cell penetration promoting peptide is bound to another protein or peptide, and may specifically include the nerve cell penetration promoting peptide and the neurotransmitter release regulating peptide. You can. More specifically, the nerve cell permeation promoting peptide may be a peptide containing one amino acid sequence of Nev 1 (SEQ ID NO: 1) or Nev 4 (SEQ ID NO: 2), and the neurotransmitter release regulating peptide is SEQ ID NO: 5 to It may be any one or more peptides selected from the group consisting of 33, specifically the peptide of SEQ ID NO: 5, or the peptide represented by Formula 1, an isomer thereof, a racemic compound, a cosmetically or pharmaceutically acceptable salt thereof, etc. You can. More specifically, the fusion peptide may be a fusion peptide of SEQ ID NO: 3 or SEQ ID NO: 4.

The fusion peptide may include a peptide having a sequence that is different in one or more amino acid residues from the wild-type amino acid sequence of each domain included therein. Amino acid exchanges in peptides that do not overall alter the activity of the molecule are known in the art. The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. In addition, it may include proteins with increased structural stability against heat, pH, etc. or increased protein activity due to mutations or modifications in the amino acid sequence.

The fusion peptide or the protein constituting the fusion peptide is manufactured by a chemical protein synthesis method known in the art, or the gene encoding the fusion peptide is amplified by PCR (polymerase chain reaction) or synthesized by a known method. It can be manufactured by cloning into an expression vector and expressing it.

The fusion peptide of the present invention may be a peptide with a short amino acid sequence consisting of 17 to 40 amino acids, but is not limited thereto.

The fusion peptide of the present invention may include a linker peptide between the nerve cell penetration promoting peptide and the neurotransmitter release regulating peptide. Specifically, in the fusion peptide, the nerve cell penetration promoting peptide may be directly linked to the N-terminus of the neurotransmitter release regulating peptide, or may be fused to the N-terminus through a linker.

The linker can specifically connect using amino acids such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, and arginic acid, and more specifically Multiple amino acids such as valine, leucine, aspartic acid, glycine, alanine, and proline can be used to connect them. More specifically, considering the ease of genetic manipulation, amino acids such as glycine, valine, leucine, and aspartic acid can be linked. Can be used by connecting 1 to 5 pieces. For example, a fusion peptide can be prepared by connecting the C-terminus of the nerve cell penetration promoting peptide and the N-terminus of the neurotransmitter release regulating peptide through a linker composed of two peptides (GG).

In one embodiment of the present invention, the neurotransmitter release regulating peptide is a peptide containing the amino acid sequence of SEQ ID NO: 5 (Argireline™, Acetyl-Glu-Glu-Met-Gln-Arg-Arg (Acetyl-EEMQRR)) and Nev A fusion peptide (ALKEVGAEEMQRR or VPMKVAAEEMQRR) was prepared by combining a nerve cell penetration promoting peptide containing the amino acid sequence 1 (SEQ ID NO: 1) or Nev 4 (SEQ ID NO: 2), and the effect of the prepared fusion peptide was tested on conventional neurotransmission. As a result of comparison with the substance release regulating peptide, it was confirmed that nerve cell permeability was significantly improved and the muscle contraction inhibition effect was also excellent (Table 2 and Figures 1 and 2).

In another aspect of the present invention, a cosmetic composition for skin improvement containing the fusion peptide as an active ingredient is provided.

Additionally, specifically, a cosmetic composition for improving wrinkles containing the fusion peptide of the present invention as an active ingredient can be provided.

In the present invention, “wrinkle improvement” refers to suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already formed.

The fusion peptide of the present invention may be included in an amount of 0.0001 to 50% by weight based on the weight of the total cosmetic composition. If the content of the fusion peptide is less than 0.0001% by weight relative to the total weight of the cosmetic composition, it is difficult to expect a substantial skin improvement effect, and if it is more than 50% by weight, problems such as instability of the formulation may occur.

The cosmetic composition according to the present invention includes solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath agent, sunscreen cream, sun oil, suspension, Can be prepared in a formulation selected from the group consisting of, but not limited to, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays. No.

In addition, the cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, Thickeners, chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto.

Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier ingredient may be animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, or mixtures thereof may be used as the carrier ingredient, and especially in the case of a spray, chloro May contain propellants such as fluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. there is.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is soap, alkali metal salts of fatty acids, hemiester salts of fatty acids, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier ingredients. You can.

In another aspect of the present invention, a functional cosmetic for skin improvement comprising the cosmetic composition as an active ingredient is provided.

Additionally, specifically, it is possible to provide a functional cosmetic for wrinkle improvement containing the cosmetic composition of the present invention as an active ingredient. The cosmetic composition and wrinkle improvement are as described above.

In the present invention, "functional cosmetics (cosmedical, cosmeceutical)" refers to a product that has the professional therapeutic function of a pharmaceutical introduced into cosmetics and has specialized functionality that emphasizes bioactive effects and effects, unlike general cosmetics, and whitens the skin. This refers to cosmetics prescribed by Ordinance of the Ministry of Health and Welfare, among products that help with skin wrinkles, products that help improve skin wrinkles, and products that help tan the skin beautifully or protect the skin from ultraviolet rays.

The functional cosmetics of the present invention can be manufactured by adding an appropriate carrier used in the production of cosmetics for general skin to the cosmetic composition. At this time, the carrier used is not particularly limited, but specifically, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. can be used alone or in appropriate combination.

The functional cosmetic of the present invention exhibits a wrinkle-improving effect, and its formulation is not particularly limited, but includes, for example, solution, emulsion, suspension, paste, cream, lotion, gel, powder, spray, surfactant-containing cleansing, It can be manufactured in formulations such as oil, soap, liquid cleansing agent, bath salt, foundation, makeup base, essence, lotion, foam, pack, softening water, sunscreen cream, sun oil, etc. Specifically, external skin ointment, softening lotion, It can be manufactured in the form of nourishing lotion, nourishing cream, massage cream, essence, pack, emulsion or oil gel. In this case, the carrier used can be selectively used depending on the formulation of the cosmetic. For example, when manufacturing cosmetics in the form of ointments, pastes, creams or gels, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier ingredients. Can be used alone or in combination; When manufacturing cosmetics in the form of powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, chlorofluorohydrocarbon, propane/butane, dimethyl ether, etc. are used as carrier ingredients. Can be used as or in combination; When manufacturing cosmetics in the form of a solution or emulsion, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, cottonseed oil, and peanut are used as carrier ingredients. oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol fatty ester, polyethylene glycol, or sorbitan fatty acid ester, etc. can be used alone or in combination; When manufacturing suspension-type cosmetics, water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, microcrystalline cellulose, and aluminum metahydroxide are used as carrier ingredients. , bentonite, agar, tracant, etc. can be used alone or in combination; When manufacturing cosmetics in the form of toilet soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. are used as carrier ingredients. It can be used alone or in combination.

Specifically, the external skin ointment may be prepared to contain 50 to 97% by weight of petrolatum and 0.1 to 5% by weight of polyoxyethylene oleyl-ether phosphate in addition to the fusion peptide of the present invention; In addition to the fusion peptide of the present invention, the softening lotion can be prepared to contain 1 to 10% by weight of polyhydric alcohols such as propylene glycol or glycerin and 0.05 to 2% by weight of surfactants such as polyethylene oleyl ether or polyoxyethylene hydrogenated castor oil. ; Nutrient lotions and nourishing creams are manufactured to contain, in addition to the fusion peptide of the present invention, 5 to 20% by weight of oils such as squalane, vaseline or octyldodecanol, and 3 to 15% by weight of wax components such as cetanol, stearyl alcohol or beeswax. can be; The essence may be prepared to contain 5 to 30% by weight of polyhydric alcohols such as glycerin or propylene glycol in addition to the fusion peptide of the present invention; The massage cream may be prepared to contain 30 to 70% by weight of oils such as liquid paraffin, petrolatum, or isononyl isononanoate in addition to the fusion peptide of the present invention; The pack is manufactured as a peel-off pack containing 5 to 20% by weight of polyvinyl alcohol in addition to the fusion peptide of the present invention, or 5 to 5% of pigments such as kaolin, talc, zinc oxide, or titanium dioxide in general emulsified cosmetics. It can be made into a wash off pack containing 30% by weight.

In another aspect of the present invention, a pharmaceutical composition for external skin application containing the fusion peptide as an active ingredient is provided. The fusion peptide is as described above.

The fusion peptide of the present invention can regulate the release of neurotransmitters. Specifically, it can block the transmission of neurotransmitters to neurotransmitter receptors, thereby suppressing the muscle contraction effect and showing a wrinkle improvement effect. The fusion peptide provided by the present invention maintains or improves the ability of the substance to exhibit useful effects such as improving skin wrinkles, while significantly improving nerve cell permeability, so it is a pharmaceutical composition for external application for skin that exhibits the effects of inhibiting muscle contraction or improving wrinkles. It can be used as an active ingredient.

In the present invention, “external skin preparation” refers to a solid, semi-solid or liquid external preparation made by mixing a medicine with various bases such as fats and oils, petroleum jelly, lanolin, glycerol, etc. so that it can be easily applied to the skin. The external dosage form is not particularly limited, but powder, gel, ointment, cream, liquid or aerosol dosage forms are preferred.

For the purpose of the present invention, the external skin preparation may be interpreted as a preparation containing the fusion peptide of the present invention and a base that is an appropriate carrier for external skin preparation, but is not particularly limited thereto.

The nerve cell permeation promoting peptide of the present invention is a peptide with improved nerve cell permeability, and the fusion peptide in which the nerve cell permeation promoting peptide is bound to a peptide capable of controlling neurotransmitter release has the ability of the peptide to exhibit useful effects such as wrinkle improvement. Since this is maintained or improved while at the same time significantly improving nerve cell permeability, it can be widely used as an active ingredient in cosmetic compositions for skin improvement, functional cosmetics, and pharmaceutical compositions for external skin use.

The following drawings attached to this specification illustrate preferred embodiments of the present invention, and serve to further understand the technical idea of the present invention along with the contents of the above-described invention. Therefore, the present invention is limited to the matters described in such drawings. It should not be interpreted in a limited way.
Figure 1 is a cell penetration image obtained through confocal microscopy of Argireline™ and neurotransmitter release regulating fusion peptides ALKEVGAEEMQRR (SEQ ID NO: 3, fusion peptide 1) and VPMKVAAEEMQRR (SEQ ID NO: 4, fusion peptide 4). Shows neuronal permeability.
Figure 2 is a graph showing the muscle contraction reduction ratio of Argireline™ and neurotransmitter release regulating fusion peptides ALKEVGAEEMQRR (SEQ ID NO: 3, fusion peptide 1) and VPMKVAAEEMQRR (SEQ ID NO: 4, fusion peptide 4).

Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples and experimental examples are for illustrative purposes only and the scope of the present invention is not limited to these examples and experimental examples.

Example 1: Promote nerve cell penetration of peptides Selection

To select peptides that promote nerve cell penetration, a phage display method was performed, combining a phage library and an elution test method for transdermal preparations.

First, 10 ⁹ phages derived from the Ph.D-12 phage library kit (New England Biolab) were added to the cell culture medium.

Neuroblasts, keratinocytes, and fibroblasts were each cultured on a plate in DMEM medium containing 10% (v/v) fetal calf serum and 1% (v/v) antibiotics. The phage library was diluted in cell medium and treated with keratinocytes. Phages that did not penetrate the keratinocytes were collected and treated with fibroblasts again, and the same process was repeated. After excluding the phages that penetrate keratinocytes and fibroblasts, the remaining phages are treated with nerve cells and cultured for a certain period of time. The phages remaining outside the cells are washed, and the nerve cells are lysed to remove the phages inside. was selected.

The amplification was performed using E. coli ER2738 (New England Biolab) as a host cell. Specifically, 5 mL of the phage solution was added to the E. coli ER2738 strain cultured with shaking in 25 mL of LB medium and cultured for 4 hours. Next, the culture medium was centrifuged at 8,000G to obtain a supernatant containing the phage fraction. The supernatant was treated with 6 mL of precipitation solution (20% PEG6000, 2.5M NaCl) and reacted to precipitate the phages. The reaction solution was centrifuged at 8,000 G to precipitate the phages, and the precipitate was suspended in TBS solution. Thus, an amplified phage solution was obtained.

In this way, 1 round is defined as adding phage to a nerve cell to obtain a phage that has penetrated the nerve cell and then proceeding with the amplification process once. Then, the phage amplified in round 1 is used to perform another 2 rounds to amplify the nerve cell. Phages with good nerve cell penetration ability were selected by competing for penetration ability, and a total of 3 rounds were performed.

In order to confirm the sequence of the peptide contained in the phage finally obtained as a result of performing the 3 rounds, the TBS solution containing the phage was added to E. coli ER2738 strain and suspended, TOP agar was added to the suspension and mixed, and then the mixture was LB/X-gal/IPTG was added to the top of the plate medium and solidified. Then, the solidified medium was cultured for 16 hours, and blue colonies were selected. The strain derived from the selected colony was cultured for 6 hours, DNA was obtained therefrom, and then the base sequence derived from the phage was analyzed to determine the Nev 1 and Nev 4 amino acids of the peptide showing the property of penetrating nerve cells. Sequences were selected.

Example 2: Regulation of neurotransmitter release To peptides Promotes nerve cell penetration peptide go combined fusion of peptides manufacturing

A fusion peptide in which the nerve cell permeation promoting peptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 obtained in Example 1 and the neurotransmitter release regulating peptide having the amino acid sequence of SEQ ID NO: 5 are combined were synthesized, separated, and By purification, fusion peptides that regulate neurotransmitter release were prepared (hereinafter referred to as fusion peptide 1 and fusion peptide 4).

Example 2-1: Neurotransmitter release control fusion of peptides synthesis

The neurotransmitter release-regulating fusion peptides were each synthesized using a solid phase peptide synthesis method using an automatic peptide synthesizer (Applied Biosystems Model 431A).

Specifically, 0.25 mmol of parahydroxy methylphenyloxymethyl polystyrene (HMP) resin was placed in a standard reaction vessel (38 mL), and the Fmoc-amino acid at the carboxy terminus of the peptide to be synthesized was added and synthesis was started. A cartridge containing 1 mmol of Fmoc-amino acid is arranged on the guideway in the sequence from the carboxyl terminal amino acid to the amino acid terminal. At this time, the metal cap of the cartridge was removed, and empty cartridges containing no amino acids were placed at the beginning and end.

Peptide synthesis was performed according to the standard scale Fmoc coupling protocol developed by ABI, editing parameters before implementation and following the automatic synthesis menu (see ABI User's Manual. Jan, 1992). When using standard scale Fmoc, deprotection was performed using 20% piperidine diluted in N-methylpyrrolidine (NMP) for 21 min, followed by a 9 min wash with NMP and coupling for 71 min. It was carried out for a while. 1-Hydroxy-benzotriazole (HOBT) was used for coupling, and a system of washing with NMP for 7 minutes was used.

Example 2- 2: Fusion of peptides Separation and purification

The two types of neurotransmitter release-regulating fusion peptides synthesized in Example 2-1 were separated and purified through the following process.

First, the fusion peptide synthesized in Example 2-1 was separated from the solid support using trifluoroacetic acid (TFA), and ABI's manual (Introduction to Cleavage Techniques, P6-19 (1990)) was used. For reference, the fusion peptide was isolated. Specifically, the resin with the fusion peptide synthesized in Example 2-1 was placed in a round flask and cooled, then 0.75 g of crystalline phenol, 0.25 mL of 1,2-ethanedithiol (EDT), 0.5 mL of thioanisole, and distilled water. Add 0.5 mL and 10 mL of TFA, close the lid, and react at room temperature for 1 to 2 hours. After the reaction was completed, the resin and the reaction solution were filtered under low vacuum through a sintered glass funnel to separate the resin and the fusion peptide solution. The flask and glass funnel were washed with 5 to 10 mL of dichloromethane (DCM) to mix the dichloromethane with the fusion peptide solution, and more than 50 mL of cold diethyl ethyl was added to obtain a precipitate of the peptide. This precipitate was filtered through a funnel under low vacuum, the precipitate collected on the funnel was dried, and then dissolved in 30% acetic acid and freeze-dried. The fusion peptide thus obtained was purified by HPLC (High Performance Liquid Chromatography). At this time, the column was used as a C18 analytical column (Pharmacia), buffer solution A was equilibrated using 10% acetonitrile + 0.05% TFA, and buffer solution B was equilibrated using 80% acetonitrile + 0.05% TFA to produce fusion peptide. was eluted. As a result, two types of highly purified fusion peptides were obtained, and the synthesis yield was about 30 ± 5%.

Experiment example : Promotes selected nerve cell penetration peptide and neurotransmitter release-controlled fusion. of peptides Effect verification

In order to confirm the skin improvement use of the nerve cell permeation promoting peptide and the neurotransmitter release regulating fusion peptide selected and prepared in Examples 1 and 2, an experiment was conducted to confirm the nerve cell permeability and muscle contraction inhibition effects as follows. .

Experiment example 1: Promote nerve cell penetration of peptides Verification of neuron selective permeability

Peptides were synthesized by binding fluorescein isothiocyanate (FITC), a fluorescent substance, to the ends of two peptides (N1 (SEQ ID NO: 1) and N4 (SEQ ID NO: 2) selected from the phage library. As controls, peptides combining FITC with Argireline™ and TAT-argifeline (TAT-argi) were used. Argireline™ was used as one of the substances that act on nerve cells, and TAT combined with Argireline™ is a trans-activating transcriptional activator, or a type of CPP, and is known to promote the uptake of substances by cells. The fluorescence intensity was measured when the four types of peptides were absorbed for a certain period of time in the keratinocytes and nerve cells that make up the stratum corneum of the skin, and the ratio (fold) of the fluorescence intensity of the peptides compared to Argireline™ is shown in Table 1 below.

Degree of penetration of neuron-penetrating peptides into keratinocytes and neurons

(Ratio (fold) compared to Argireline™)

As shown in Table 1 above, when N1, N4, and TAT-argi were treated with keratinocytes, the degree of penetration was 26 to 31 times higher than that of Argireline™, and the degree was that TAT-argi and the neuron-penetrating peptide It was similar. However, when it was treated with nerve cells, the nerve cell permeable peptide was superior to TAT-argi, and from this, it could be said that the two types of nerve cell permeable peptides had excellent nerve cell selective permeability.

Experiment example 2: Fusion to regulate neurotransmitter release of peptides neuronal permeability

The neuronal permeability of the two neurotransmitter release-regulating fusion peptides prepared in Example 2 was confirmed using neuroblast cells. First, neuroblast cells were cultured on a plate in DMEM medium containing 10% (v/v) fetal calf serum and 1% (v/v) antibiotics. Then, Argireline™ and a neurotransmitter release-regulating fusion peptide containing fluorescein isothiocyanate (fitc) bound to the C-terminus were added, reacted for 1 hour, and then washed with PBS. Afterwards, cell penetration images were acquired through a confocal microscope (Figure 1). Through this, it was found that the nerve cell permeability of the fusion peptide combined with the nerve cell permeable peptide was significantly improved compared to the existing Argireline™.

Experiment example 3: Fusion to regulate neurotransmitter release of peptides Muscle contraction inhibitory effect

To confirm the muscle contraction inhibitory effect of the two fusion peptides prepared in Example 2, first, C2C12 cells were plated with DMEM containing 10% (v/v) fetal calf serum and 1% (v/v) antibiotics. After culturing in medium, neuroblasts were additionally co-cultured on the same plate. Then, when cell contraction of C2C12 cells begins, the number of contractions of C2C12 cells is measured for 30 seconds, all medium is removed, washed three times with PBS, and control peptide and fusion peptide are added at each concentration in medium without calf serum. A medium containing stars was added and reacted for 2 hours. Afterwards, the number of contractions of C2C12 cells was measured again for 30 seconds to confirm the degree of inhibition of muscle contraction. These results and the results of the concentration that inhibits 50% activity are shown in Figure 2 and Table 2.

Neurotransmitter release-controlled fusion of peptides Muscle contraction inhibitory activity processed peptides Concentration that inhibits muscle contraction by 50% (ppm) Argireline™ 3±1.7 Fusion peptide 1 0.32±0.05 Fusion peptide 4 0.4±0.07

As shown in Table 2 above, it was confirmed that Argireline™ and neurotransmitter release regulating fusion peptide inhibited the release of neurotransmitters from nerve cells and reduced the number of contractions of C2C12 cells. In addition, it was confirmed that the fusion peptide that regulates neurotransmitter release combined with a peptide that promotes nerve cell penetration increases the degree of muscle contraction inhibition compared to Argireline™.

Therefore, it was found that the use of the neurotransmitter release-regulating fusion peptide of the present invention had an inhibitory effect on muscle contraction at a lower concentration than the neurotransmitter release-regulating peptide.

<110> LG HOUSEHOLD & HEALTH CARE LTD. <120> Peptide for accelerating delivery to nerve cells <130> LH16-214 <150> KR 10-2016-0006037 <151> 2016-01-18 <160> 33 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Nev 1 <400> 1 Ala Leu Lys Glu Val Gly Ala 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Nev 4 <400> 2 Val Pro Met Lys Val Ala Ala 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Fusion peptide 1 <400> 3 Ala Leu Lys Glu Val Gly Ala Glu Glu Met Gln Arg Arg 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Fusion peptide 4 <400> 4 Val Pro Met Lys Val Ala Ala Glu Glu Met Gln Arg Arg 1 5 10 <210> 5 <211> 6 <212> PRT <213> Homo sapiens <400> 5 Glu Glu Met Gln Arg Arg 1 5 <210> 6 <211> 6 <212> PRT <213> Homo sapiens <400> 6 Asp Asp Met Gln Arg Arg 1 5 <210> 7 <211> 7 <212> PRT <213> Homo sapiens <400> 7 Tyr Pro Trp Thr Gln Arg Phe 1 5 <210> 8 <211> 21 <212> PRT <213> Homo sapiens <400> 8 Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg 1 5 10 15 Arg Ala Asp Gln Leu 20 <210> 9 <211> 23 <212> PRT <213> Homo sapiens <400> 9 Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met Leu Gln Leu Val Glu 1 5 10 15 Glu Ser Lys Asp Ala Gly Ile 20 <210> 10 <211> 13 <212> PRT <213> Homo sapiens <400> 10 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp Gln Leu Ala 1 5 10 <210> 11 <211> 12 <212> PRT <213> Homo sapiens <400> 11 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp Gln Leu 1 5 10 <210> 12 <211> 11 <212> PRT <213> Homo sapiens <400> 12 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp Gln 1 5 10 <210> 13 <211> 10 <212> PRT <213> Homo sapiens <400> 13 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp 1 5 10 <210> 14 <211> 9 <212> PRT <213> Homo sapiens <400> 14 Glu Leu Glu Glu Met Gln Arg Arg Ala 1 5 <210> 15 <211> 8 <212> PRT <213> Homo sapiens <400> 15 Glu Leu Glu Glu Met Gln Arg Arg 1 5 <210> 16 <211> 11 <212> PRT <213> Homo sapiens <400> 16 Leu Glu Glu Met Gln Arg Arg Ala Asp Gln Leu 1 5 10 <210> 17 <211> 10 <212> PRT <213> Homo sapiens <400> 17 Leu Glu Glu Met Gln Arg Arg Ala Asp Gln 1 5 10 <210> 18 <211> 9 <212> PRT <213> Homo sapiens <400> 18 Leu Glu Glu Met Gln Arg Arg Ala Asp 1 5 <210> 19 <211> 8 <212> PRT <213> Homo sapiens <400> 19 Leu Glu Glu Met Gln Arg Arg Ala 1 5 <210> 20 <211> 7 <212> PRT <213> Homo sapiens <400> 20 Leu Glu Glu Met Gln Arg Arg 1 5 <210> 21 <211> 10 <212> PRT <213> Homo sapiens <400> 21 Glu Glu Met Gln Arg Arg Ala Asp Gln Leu 1 5 10 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> 22 Glu Glu Met Gln Arg Arg Ala Asp Gln 1 5 <210> 23 <211> 8 <212> PRT <213> Homo sapiens <400> 23 Glu Glu Met Gln Arg Arg Ala Asp 1 5 <210> 24 <211> 7 <212> PRT <213> Homo sapiens <400> 24 Glu Glu Met Gln Arg Arg Ala 1 5 <210> 25 <211> 13 <212> PRT <213> Homo sapiens <400> 25 Leu Glu Ser Thr Arg Arg Met Leu Gln Leu Val Glu Glu 1 5 10 <210> 26 <211> 12 <212> PRT <213> Homo sapiens <400> 26 Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr 1 5 10 <210> 27 <211> 6 <212> PRT <213> Homo sapiens <400> 27 Lys Asn Leu Thr Asp Leu 1 5 <210> 28 <211> 26 <212> PRT <213> Homo sapiens <400> 28 Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn 1 5 10 15 Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 20 25 <210> 29 <211> 20 <212> PRT <213> Homo sapiens <400> 29 Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met 1 5 10 15 Leu Gly Ser Gly 20 <210> 30 <211> 17 <212> PRT <213> Homo sapiens <400>30 Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser 1 5 10 15 Gly <210> 31 <211> 14 <212> PRT <213> Homo sapiens <400> 31 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 1 5 10 <210> 32 <211> 11 <212> PRT <213> Homo sapiens <400> 32 Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 1 5 10 <210> 33 <211> 6 <212> PRT <213> Homo sapiens <400> 33 Lys Asn Leu Thr Asp Leu 1 5

Claims (10)

Nerve cell penetrating peptide consisting of SEQ ID NO: 1 or SEQ ID NO: 2. A composition for promoting nerve cell permeability comprising a nerve cell permeable peptide consisting of SEQ ID NO: 1 or SEQ ID NO: 2 of claim 1. It is a fusion peptide in which a neuron-penetrating peptide consisting of SEQ ID NO: 1 or SEQ ID NO: 2 is bound to a neurotransmitter release-regulating peptide,
The neurotransmitter release regulating peptide is
A fusion peptide characterized in that it is a peptide consisting of the amino acid sequence of SEQ ID NO: 5. The fusion peptide according to claim 3, wherein the neurotransmitter is at least one selected from the group consisting of dopamine, serotonin, histamine, acetylcholine, adrenaline, noradrenaline, gamma aminobutyric acid, L-glutamic acid, and glycine. The composition for promoting nerve cell penetration according to claim 2, wherein the peptide is combined with a peptide consisting of the amino acid sequence of SEQ ID NO: 5 to promote nerve cell penetration. delete delete The fusion peptide according to claim 3, wherein the fusion peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. A cosmetic composition for improving skin wrinkles comprising the fusion peptide of any one of claims 3, 4, and 8 as an active ingredient. delete