KR102607079B1 - Neuron cells penetrability enhanced peptide regulating neurotransmitter secretion - Google Patents

Abstract

The present invention relates to a neurotransmitter release-regulating peptide with improved nerve cell permeability, and more specifically, the present invention includes a nerve cell permeation-promoting peptide, 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, a functional cosmetic for skin improvement containing the cosmetic composition, and a pharmaceutical composition for external skin application containing the fusion peptide. 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, functional cosmetics, and pharmaceutical compositions for external skin use.

Description

{Neuron cells penetrability enhanced peptide regulating neurotransmitter secretion}

The present invention relates to a neurotransmitter release-regulating peptide with improved nerve cell permeability, and more specifically, the present invention relates to a nerve cell permeation-promoting peptide, a fusion peptide in which a neurotransmitter release-regulating peptide is bound to the nerve cell permeation-promoting peptide, It relates to a cosmetic composition for skin improvement containing a fusion peptide, a functional cosmetic for skin improvement containing the cosmetic composition, and a pharmaceutical composition for external skin application containing the fusion peptide.

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.

In the case of Botox, it is composed of an enzyme site at the N-terminus that cleaves SNAP-25 and a C-terminus that can penetrate into nerve cells. When the C-terminus is removed, it increases by about 10 ⁶ times when calculated using a cell activity evaluation method. It was found that activity decreased. This shows that improving nerve cell permeability is an essential process to improve the efficacy of substances that regulate the release of neurotransmitters.

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.

Therefore, in order to develop a material with excellent nerve cell permeability, the present inventors performed a phage display method combining a phage library and an elution test method of a transdermal preparation to produce a novel nerve cell permeation promoting peptide containing the amino acid sequence of SEQ ID NO: 1. was discovered. In addition, the discovered nerve cell penetration promoting peptide was combined with a peptide that regulates the release of neurotransmitters and has skin improvement effects such as wrinkle improvement, and the fusion peptide combined with the nerve cell penetration promoting peptide caused muscle contraction compared to the existing protein. The present invention was completed after confirming that the inhibition and wrinkle improvement effects were increased while the nerve cell permeability was excellent.

The purpose of the present invention is to provide a peptide that promotes nerve cell penetration containing the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a fusion peptide in which a nerve cell permeation promoting peptide containing the amino acid sequence of SEQ ID NO: 1 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 provides, as an aspect to solve the above problems and achieve the object of the present invention, a nerve cell permeation promoting peptide containing the amino acid sequence of SEQ ID NO: 1.

Nerve cell penetration promoting peptide: LRLRFITANDK (SEQ ID NO: 1)

In the present invention, “neuronal cell penetration promoting peptide” refers to a peptide that can penetrate nerves regardless of the size or nature of the molecule and has excellent neuronal retention.

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 permeation promoting peptide of the present invention has a significantly superior nerve cell permeability compared to other peptides. Indicates cell permeability.

The nerve cell permeability promoting peptide 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, and specifically, the amino acid of SEQ ID NO: 1. It may contain a peptide containing a sequence. In one example of the present invention, a peptide containing the amino acid sequence of SEQ ID NO: 1 was prepared as a peptide that promotes nerve cell penetration through a phage display method. Afterwards, as a result of confirming the nerve cell permeability of the neurotransmitter release-regulating fusion peptide to which the above-mentioned peptide was bound and the neurotransmitter release-regulating peptide (Argireline™) to which the above-mentioned peptide was not bound, the fusion peptide containing the above-mentioned nerve cell permeation-promoting peptide was confirmed. It was confirmed that nerve cell permeability increased approximately 13-fold (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.

The neurotransmitter release-regulating peptide of the present invention may include derivatives thereof. The derivative may include a peptide in which the N-terminus, C-terminus, etc. of the nerve cell penetration promoting peptide are chemically modified or amino acids are added/substituted/deleted to modify the peptide. For the purposes of the present invention, the derivative is not particularly limited as long as it exhibits excellent nerve cell permeability. For example, the nerve cell penetration-promoting peptide derivative of the present invention may be a derivative with a dansyl group attached.

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: 2 (Acetyl-EEMQRR), SEQ ID NO: 4 ([Pal]DDMQRR), and SEQ ID NO: 5 ([Pal]YPWTQRF) And, more specifically, it may be the peptide of SEQ ID NO: 2 (EEMQRR), but is not limited thereto.

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 an amino acid sequence consisting of 3 to 40 amino acids including the amino acid sequence of SEQ ID NO: 1, and R1 is selected from the group consisting of H or C ₃ to C ₂₄ alkyl, aryl, and acyl. It may be any one, and R2 may be any one selected from the group consisting of an amino group, a hydroxyl group, and a thiol group, substituted or unsubstituted with an aliphatic or cyclic group.

Specifically, the AA is MAEDADMRNELEEMQRRADQL (SEQ ID NO: 6), ADESLESTRRMLQLVEESKDAGI (SEQ ID NO: 7), ELEEMQRRADQLA (SEQ ID NO: 8), ELEEMQRRADQL (SEQ ID NO: 9), ELEEMQRRADQ (SEQ ID NO: 10), ELEEMQRRAD (SEQ ID NO: 11), ELEEMQRRA ( SEQ ID NO: 12), ELEEMQRR (SEQ ID NO: 13), LEEMQRRADQL (SEQ ID NO: 14), LEEMQRRADQ (SEQ ID NO: 15), LEEMQRRAD (SEQ ID NO: 16), LEEMQRRA (SEQ ID NO: 17), LEEMQRR (SEQ ID NO: 18), EEMQRRADQL (SEQ ID NO: Number 19), EEMQRRADQ (SEQ ID NO: 20), EEMQRRAD (SEQ ID NO: 21), EEMQRRA (SEQ ID NO: 22), LESTRRMLQLVEE (SEQ ID NO: 23), NKDMKEAEKNLT (SEQ ID NO: 24), KNLTDL (SEQ ID NO: 25), IMEKADSNKTRIDEANQRATKMLGSG (SEQ ID NO: 26), SNKTRIDEANQRATKMLGSG (SEQ ID NO: 27), TRIDEANQRATKMLGSG (SEQ ID NO: 28), DEANQRATKMLGSG (SEQ ID NO: 29), NQRATKMLGSG (SEQ ID NO: 30), and QRATKMLGSG (SEQ ID NO: 31). 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 the amino acid sequence of SEQ ID NO: 1, and the neurotransmitter release regulating peptide may be any one or more peptides selected from the group consisting of SEQ ID NOS: 2 and 4 to 31, specifically It may include the peptide of SEQ ID NO: 2, or the peptide represented by Chemical Formula 1, an isomer thereof, a racemic compound, a cosmetically or pharmaceutically acceptable salt thereof, etc. More specifically, the fusion peptide may include the fusion peptide of SEQ ID NO: 3.

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: 2 (Argireline™, Acetyl-Glu-Glu-Met-Gln-Arg-Arg (Acetyl-EEMQRR)). A fusion peptide (SEQ ID NO: 3) was prepared by combining a nerve cell permeation promoting peptide containing the amino acid sequence of number 1. As a result of comparing the effect of the prepared fusion peptide with a conventional neurotransmitter release regulating peptide, it was found that nerve cells It was confirmed that permeability was significantly improved and the muscle contraction inhibition effect was also excellent (Tables 1 and 2 and Figure 1).

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 one embodiment of the present invention, a cream was prepared by impregnating a fusion peptide prepared by combining a neurotransmitter release regulating peptide with a nerve cell permeation promoting peptide, and as a result of confirming the skin improvement effect of the cream, it was found that the conventional neurotransmitter It was confirmed that the wrinkle improvement effect was more than two times better than the cream impregnated with the release-controlling peptide (Table 3 and Figure 2).

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 shows the results of confirming the neuronal permeability of Argireline™ (Acetyl-EEMQRR) and the neurotransmitter release regulating fusion peptide LRLRFITANDKEEMQRR (SEQ ID NO: 3).
Figure 2 is a graph showing the results of confirming the wrinkle improvement effect of a cream containing Argireline™ (Acetyl-EEMQRR) and neurotransmitter release-regulating fusion peptide LRLRFITANDKEEMQRR (SEQ ID NO: 3) on human subjects.

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 amplifying it once. Round 2 is then performed again with the phage amplified in round 1 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 obtain the amino acid sequence of a peptide showing the property of penetrating nerve cells (SEQ ID NO: 1). was selected.

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

A fusion peptide containing the amino acid sequence of SEQ ID NO: 3 as a fusion peptide combining the nerve cell permeation promoting peptide having the amino acid sequence of SEQ ID NO: 1 obtained in Example 1 and the neurotransmitter release regulating peptide having the amino acid sequence of SEQ ID NO: 2 The peptide was synthesized, isolated, and purified to prepare a fusion peptide that regulates neurotransmitter release.

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

The neurotransmitter release-regulating fusion peptide was 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 neurotransmitter release-regulating fusion peptide synthesized in Example 2-1 was isolated 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, a highly purified fusion peptide (SEQ ID NO: 3) was obtained, and the synthesis yield was about 30 ± 5%.

Experiment example : Neurotransmitter release control fusion of peptides Effect verification

In order to confirm the use of the neurotransmitter release-regulating fusion peptide prepared in Example 2 for improving skin, an experiment was performed to check nerve cell permeability, muscle contraction inhibition effect, and wrinkle improvement effect as follows.

Experiment example 1: Fusion to regulate neurotransmitter release of peptides neuronal permeability

The neuronal cell permeability of the neurotransmitter release-regulating fusion peptide 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), cells were lysed, and the amount of peptide that penetrated nerve cells was calculated. The results are shown in Table 1 below.

Neuronal permeability of fusion peptides that regulate neurotransmitter release. processed peptides Relative transmittance (%) Argireline™ 100±14 Neurotransmitter release-regulating fusion peptide 1360±55

As shown in Table 1 above, when treated with a fusion peptide that regulates neurotransmitter release, it was confirmed that nerve cell permeability increased about 13 times compared to Argireline™.

Therefore, it was found that when the neurotransmitter release-regulating fusion peptide of the present invention was used, the neurotransmitter release-regulating peptide's neuronal permeability was significantly increased.

Experiment example 2: Fusion to regulate neurotransmitter release of peptides Muscle contraction inhibitory effect

To confirm the muscle contraction inhibitory effect of the neurotransmitter release-regulating fusion peptide prepared in Example 2, first, C2C12 cells were plated with 10% (v/v) fetal calf serum and 1% (v/v) antibiotics. After culture in DMEM medium containing this, neuroblasts were additionally co-cultured on the same plate. Then, when cell contraction of C2C12 cells started, the number of contractions of C2C12 cells was measured for 30 seconds, all medium was removed, washed three times with PBS, and control peptide and fusion peptide were 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, and the results of the concentration that inhibits 50% activity are shown in Table 2 below.

Muscle contraction inhibitory activity of neurotransmitter release-regulating fusion peptides processed peptides Concentration that inhibits muscle contraction by 50% (ppm) Argireline™ 5±2 Fusion peptide that regulates neurotransmitter release 0.4±0.08

As shown in Table 2 above, it was confirmed that Argireline™ and the 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.

Experiment example 3: Wrinkle improvement effect

In order to confirm the wrinkle-improving effect of the neurotransmitter release-regulating fusion peptide prepared in Example 2, Argireline™ and the neurotransmitter release-regulating fusion peptide were added to a general oil-in-water emulsion cream, respectively. The wrinkle improvement effect was compared by impregnation, and the ingredients and contents contained in each containing cream are shown in Table 3 below.

ingredient Argireline™
cream containing Cream containing fusion peptide that regulates neurotransmitter release C14-22 alcohol, C12-20 alkyl glucoside
(Mixture C14-22 alcohol: C12-20 alkyl glucoside
= 80:20 weight ratio) 1.5 1.5 Glyceryl Stearate, PEG-100 Stearate
(Mixture 50:50 weight ratio) 1.2 1.2 Glyceryl Stearate 0.9 0.9 Cetearyl alcohol 1.5 1.5 Polyglyceryl-3methylglucose distearate 1.5 1.5 Hydrogenated polydecene 4.5 4.5 Cyclohexasiloxane 3.5 3.5 carbomer 0.2 0.2 Tromethamine 0.2 0.2 glycerin 3 3 DPG 5 5 1,2-hexanediol 2 2 Argireline™ 0.1 Fusion peptide that regulates neurotransmitter release 0.1 Purified water Remaining amount (To 100) Remaining amount (To 100)

Cream containing Argireline™ and cream containing fusion peptide that regulates neurotransmitter release were treated daily for 12 weeks on wrinkles around the eyes, and the degree of wrinkle improvement was confirmed using a silicone replica and wrinkle image analysis method (N=21).

As a result, as shown in Figure 2, compared to Argireline™ cream, the neurotransmitter release-regulating fusion peptide cream showed an improvement effect that was more than twice as good, and as a result, the neurotransmitter release-regulating fusion peptide cream was more effective than the neurotransmitter release-regulating peptide. It was found that it effectively penetrates into nerve cells and has an excellent wrinkle improvement effect.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

<110> LG HOUSEHOLD & HEALTH CARE LTD. <120> Neuron cells penetrability enhancing peptide regulating neurotransmitter secretion <130> KPA160709-KR <160> 31 <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 <400> 1 Leu Arg Leu Arg Phe Ile Thr Ala Asn Asp Lys 1 5 10 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 <400> 2 Glu Glu Met Gln Arg Arg 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 <400> 3 Leu Arg Leu Arg Phe Ile Thr Ala Asn Asp Lys Glu Glu Met Gln Arg 1 5 10 15 Arg <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptide 4 <400> 4 Asp Asp Met Gln Arg Arg 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 5 <400> 5 Tyr Pro Trp Thr Gln Arg Phe 1 5 <210> 6 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> peptide 6 <400> 6 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> 7 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> peptide 7 <400> 7 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> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide 8 <400> 8 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp Gln Leu Ala 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 9 <400> 9 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp Gln Leu 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide 10 <400> 10 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp Gln 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 11 <400> 11 Glu Leu Glu Glu Met Gln Arg Arg Ala Asp 1 5 10 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 12 <400> 12 Glu Leu Glu Glu Met Gln Arg Arg Ala 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide 13 <400> 13 Glu Leu Glu Glu Met Gln Arg Arg 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide 14 <400> 14 Leu Glu Glu Met Gln Arg Arg Ala Asp Gln Leu 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 15 <400> 15 Leu Glu Glu Met Gln Arg Arg Ala Asp Gln 1 5 10 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 16 <400> 16 Leu Glu Glu Met Gln Arg Arg Ala Asp 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide 17 <400> 17 Leu Glu Glu Met Gln Arg Arg Ala 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 18 <400> 18 Leu Glu Glu Met Gln Arg Arg 1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 19 <400> 19 Glu Glu Met Gln Arg Arg Ala Asp Gln Leu 1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 20 <400> 20 Glu Glu Met Gln Arg Arg Ala Asp Gln 1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide 21 <400> 21 Glu Glu Met Gln Arg Arg Ala Asp 1 5 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 22 <400> 22 Glu Glu Met Gln Arg Arg Ala 1 5 <210> 23 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide 23 <400> 23 Leu Glu Ser Thr Arg Arg Met Leu Gln Leu Val Glu Glu 1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 24 <400> 24 Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr 1 5 10 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptide 25 <400> 25 Lys Asn Leu Thr Asp Leu 1 5 <210> 26 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> peptide 26 <400> 26 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> 27 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> peptide 27 <400> 27 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> 28 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> peptide 28 <400> 28 Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser 1 5 10 15 Gly <210> 29 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide 29 <400> 29 Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 1 5 10 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide 30 <400>30 Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 1 5 10 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 31 <400> 31 Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 1 5 10

Claims (12)

A peptide that promotes nerve cell penetration and consists of the amino acid sequence of SEQ ID NO: 1.
A fusion peptide in which a nerve cell permeation promoting peptide consisting of the amino acid sequence of SEQ ID NO. 1 is bound to a neurotransmitter release regulating peptide.
The fusion peptide according to claim 2, 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 fusion peptide according to claim 2, wherein the neurotransmitter release regulating peptide is a peptide containing one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 2 and 4 to 31.
The fusion peptide according to claim 2, wherein the neurotransmitter release regulating peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 2.
The fusion peptide according to claim 2, wherein the fusion peptide includes a linker peptide between a peptide that promotes nerve cell penetration and a peptide that regulates neurotransmitter release.
The fusion peptide according to claim 2, wherein the fusion peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 3.
A cosmetic composition for improving wrinkles comprising the fusion peptide of any one of claims 2 to 7 as an active ingredient.
delete A functional cosmetic for wrinkle improvement comprising the cosmetic composition of claim 8 as an active ingredient.
delete delete