Peptides having botulinum toxin activity and uses thereof

Abstract

This invention relates to a Trolox-peptide conjugate that exhibits activity in relieving muscle contraction and increasing extracellular matrix activity, and that the Trolox-peptide conjugate can be used as an active ingredient in pharmaceuticals, quasi-pharmaceuticals, or cosmetics for the prevention or treatment of muscle relaxation, neuromuscular disorders, or to improve skin conditions including wrinkles.

Description

TECHNICAL FIELD

[0001] The present application relates to Trolox-peptide conjugates having a structure in which Trolox is chemically conjugated with a peptide and uses thereof. BACKGROUND

[0002] To regulate muscle relaxation and contraction, neuromuscular junctions exist in the upper layer of muscle, and synaptic vesicles are carried at nerve endings. Muscles contract by receiving information transmitted from a neurotransmitter in a nerve vesicle. When the neurotransmitter is to be released, a receptor complex called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) needs to exist, which is capable of docking the neurotransmitter with the muscle. If the SNARE receptor complex is slightly unstable, the carrier cannot actively release the neurotransmitter, thus the impulse generated at the neuromuscular synapse cannot be transmitted to the muscle, the impulse is interrupted, and the muscle relaxes. In other words, there is less muscle contraction, which means that the formation of wrinkles is reduced.

[0003] Meanwhile, the cause or mechanism of the formation of facial expression wrinkles is the tension of the epidermal muscle that pulls the skin inward. This muscle tension is a result of muscle weakness and hyperactivity of the face. Hyperactivity of the nerve is a feature of uncontrolled over-release of neurotransmitters that stimulate muscle fibers. For this reason, molecules that control the release of neurotransmitters relax muscle tension to help remove facial wrinkles.

[0004] Based on this mechanism, many materials for inhibiting the formation of wrinkles have been researched and marketed.

[0005] Botulinum neurotoxin (BoNT) is a protease that cleaves SNARE proteins, which are key proteins involved in neurotransmitter release, and BoNT is most commonly used in clinical trials and cosmetics for removing facial wrinkles. BoNT cleaves SNARE proteins to inhibit the release of neurotransmitters and block nerve transmission, ultimately resulting in paralysis of muscle cells permeated by BoNT.

[0006] However, since the paralytic effect of BoNT is reversible, on average for 6 months, repeated injections of BoNT are required, and since it has a size that can be recognized by the patient's immune system, it can cause an immune response to the drug. The formation of antibodies to BoNT can cause a significant loss of therapeutic efficacy, which is a serious problem.

[0007] Therefore, there is a need to develop a new active ingredient that has the effect of treating muscle spasms and reducing or removing facial asymmetry and / or facial wrinkles, particularly facial expression wrinkles, by regulating the release of neurotransmitters, while having a simpler and more stable molecular structure without causing an immune response. SUMMARY

[0008] [Technical Problem]

[0009] An object of the present application is to provide a Trolox-peptide conjugate in which a novel peptide and Trolox are conjugated to inhibit the release of a neurotransmitter.

[0010] Another object of the present application is to provide a composition for relieving muscle contraction or relaxing a muscle.

[0011] Another object of the present application is to provide a method for muscle relaxation.

[0012] Still another object of the present application is to provide a Trolox-peptide conjugate for muscle relaxation.

[0013] Still another object of the present application is to provide a pharmaceutical composition for preventing or treating neuromuscular disorders.

[0014] Still another object of the present application is to provide a method for preventing or treating neuromuscular disorders.

[0015] Still another object of the present application is to provide a Trolox-peptide conjugate for preventing or treating neuromuscular disorders.

[0016] Still another object of the present application is to provide a composition for improving skin conditions, including wrinkle improvement.

[0017] Still another object of the present application is to provide a method for improving skin conditions.

[0018] Still another object of the present application is to provide a Trolox-peptide conjugate for improving skin conditions.

[0019] [Technical Solution]

[0020] One aspect of the present application provides a Trolox-peptide conjugate in which a peptide having an amino acid sequence of SEQ ID NO: 1 and Trolox are conjugated.

[0021] Another aspect of the present application provides a composition for muscle relaxation, which comprises a Trolox-peptide conjugate as an active ingredient.

[0022] Further, the present application provides a method for muscle relaxation, which comprises administering a Trolox-peptide conjugate to a subject, in which a peptide having an amino acid sequence of SEQ ID NO: 1 and Trolox are conjugated.

[0023] Further, the present application provides a Trolox-peptide conjugate for muscle relaxation, in which a peptide having an amino acid sequence of SEQ ID NO: 1 and Trolox are conjugated.

[0024] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of neuromuscular disorders, comprising a Trolox-peptide conjugate as an active ingredient.

[0025] Furthermore, the present invention provides a method for preventing or treating neuromuscular disorders, comprising administering a Trolox-peptide conjugate to a subject, wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox.

[0026] Furthermore, the present invention provides a Trolox-peptide conjugate wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox, the Trolox-peptide conjugate being used for the prevention or treatment of neuromuscular disorders.

[0027] Another aspect of the present invention provides a composition for improving skin condition, comprising a Trolox-peptide conjugate as an active ingredient.

[0028] Furthermore, the present invention provides a method for improving skin condition, comprising administering a Trolox-peptide conjugate to a subject, wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox.

[0029] Furthermore, the present invention provides a Trolox-peptide conjugate wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox, the Trolox-peptide conjugate being used to improve skin condition.

[0030] [Beneficial Effects]

[0031] According to the present invention, the Trolox-peptide conjugate can be used as a regulator of SNARE and neurotransmitter-mediated intracellular responses by inhibiting the release of neurotransmitters. Therefore, the Trolox-peptide conjugate of the present invention can be used as a composition for muscle relaxation, a composition for the prevention or treatment of neuromuscular disorders, or a composition for improving skin conditions (including wrinkle improvement).

[0032] However, the effects of the present invention are not limited to those described above, and those skilled in the art will clearly understand other effects not mentioned based on the following description. Attached Figure Description

[0033] Figure 1 The effect of inhibiting neurotransmitter release by treating nerve cells with the Trolox-peptide conjugate of the present invention is shown: nicotine and potassium chloride (KCl) are inducing factors that promote neurotransmitter release; tetanus toxin is a positive control group.

[0034] Figure 2Photographs to confirm the muscle relaxation effect produced by treating experimental animals with the Trolox-peptide conjugate of the present invention; BoNT, as a botulinum neurotoxin, served as a positive control group.

[0035] Figure 3 The graphs confirming the duration of the muscle relaxation effect produced by treating experimental animals with the Trolox-peptide conjugate of the present invention are as follows: the upper graph shows the DAS results when treated with the Trolox-peptide conjugate, and the lower graph shows the DAS results when treated with botulinum neurotoxin.

[0036] Figure 4 The Trolox-peptide conjugate of the present invention is shown to increase the expression of COL1A1, fibronectin and elastin, which constitute the dermis, in fibroblasts. Detailed Implementation

[0037] The present invention will now be described in detail.

[0038] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:1 and an active substance-peptide conjugate, wherein the active substance is conjugated to the peptide.

[0039] The peptides of the present invention can be synthesized using, for example, apparatus or genetic engineering techniques. When using an apparatus for synthesis, the desired peptides can be synthesized using the Fmoc solid-phase method in an automated peptide synthesizer.

[0040] In a specific exemplary embodiment, the peptide of the present invention comprising the amino acid sequence of SEQ ID NO:1 is synthesized and identified using the Fmoc solid-phase method, and selected by verifying the efficacy of the identified peptide.

[0041] In a specific exemplary implementation, in order to verify the efficacy of the identified peptide, the peptide for inhibiting neurotransmitter release is selected by verifying its inhibitory effect on SNARE complex formation and its inhibitory effect on neurotransmitter release.

[0042] Furthermore, the active substance-peptide conjugate of the present invention can be prepared by conjugating the peptide selected by this method with the active substance. In a specific exemplary embodiment, the active substance is selected by verifying the efficacy of the active substance-peptide conjugate prepared by chemically conjugating it with a peptide including the amino acid sequence of SEQ ID NO:1.

[0043] In a specific exemplary implementation, after administering 10 active substance-peptide conjugates to mice, first selecting the conjugate with a DAS analysis result of 4, the duration of the muscle relaxation effect was observed, and finally the active substance-peptide conjugate that showed the longest duration at the same dose was selected.

[0044] In this specification, the term "peptide" refers to a linear molecule composed of amino acid residues, and more specifically, the peptides of the present invention may include the amino acid sequence represented by SEQ ID NO:1.

[0045] A “peptide” can be an amino acid variant or fragment with a different sequence obtained by the deletion, insertion, substitution, or combination thereof of amino acid residues within a range that does not affect function. Amino acid exchanges that do not completely alter the activity of the peptide are known in the art. In some cases, amino acid exchanges can be modified by phosphorylation, sulfation, acrylate esterification, glycosylation, methylation, farnesylation, etc. Therefore, the present invention includes peptides having an amino acid sequence substantially identical to that of the peptide having the amino acid sequence of SEQ ID NO:1, as well as variants or active fragments of that peptide. Substantially identical protein refers to an amino acid sequence with at least 75%, preferably at least 80%, for example at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence homology to the amino acid sequence of SEQ ID NO:1, but is not limited thereto, and amino acid sequences with more than 75% homology and having the same activity are included within the scope of the present invention. Furthermore, the peptides of the present invention may also include amino acid sequences prepared for specific purposes of increasing the target sequence, tag, labeled residues, and half-life or peptide stability.

[0046] Furthermore, to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), modified specificity (e.g., broad bioactivity spectrum), and reduced antigenicity, a protecting group can be attached to the N-terminus or C-terminus of the peptide. For example, the protecting group can be acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, or polyethylene glycol (PEG). However, the protecting group can include any component that enhances the modification of the peptide, particularly enhancing the stability of the peptide, without limitation. "Stability" is used to mean not only the in vivo stability that protects the peptides of the present invention from attack by proteolytic enzymes in vivo, but also storage stability (e.g., room temperature storage stability).

[0047] As used herein, the term "active substance" refers to a substance having at least one carboxyl group that can chemically conjugate to the N-terminus of the peptide of the present invention and synergistically induce the activity of the peptide. Specifically, the active substance may be Trolox.

[0048] The "Trolox" of this invention is 6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-carboxylic acid. Trolox is a water-soluble analog of vitamin E and is used as an antioxidant. The structure of Trolox is shown in the following chemical formula 1.

[0049] [Chemical Formula 1]

[0050]

[0051] In the active substance-peptide conjugate of the present invention, when the active substance is Trolox, the peptide conjugated with Trolox is called "Trolox-peptide conjugate".

[0052] Specifically, the present invention provides a Trolox-peptide conjugate, wherein Trolox is conjugated with a peptide having the amino acid sequence of SEQ ID NO:1.

[0053] Trolox can conjugate to the N-terminus, C-terminus, or side chain of a peptide. Specifically, the carboxyl group of Trolox can bind to the N-terminus of a peptide.

[0054] An embodiment of the Trolox-peptide conjugate can be represented by the following chemical formula 2.

[0055] [Chemical Formula 2]

[0056]

[0057] In specific exemplary embodiments of the present invention, Trolox-peptide conjugates have been shown to have excellent effects in inhibiting the release of neurotransmitters from nerve cell terminals and relaxing contracted muscles in experimental animals.

[0058] Furthermore, in specific exemplary embodiments of the present invention, it has been confirmed that the Trolox-peptide conjugate of the present invention has an excellent effect in relieving muscle contraction, and also has a significant effect in improving skin wrinkles by increasing the expression of proteins constituting the dermis.

[0059] On the other hand, the present invention provides a composition for muscle relaxation comprising a Trolox-peptide conjugate, wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox.

[0060] Furthermore, the present invention provides a method for muscle relaxation, comprising administering a Trolox-peptide conjugate to a subject, wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox.

[0061] Furthermore, the present invention provides a Trolox-peptide conjugate wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox, the Trolox-peptide conjugate being used for muscle relaxation.

[0062] Details about peptides, Trolox, and Trolox-peptide conjugates are as described above.

[0063] Trolox-peptide conjugates can alleviate muscle contraction or relax muscles by inhibiting the release of neurotransmitters from nerve cell terminals.

[0064] Compositions for muscle relaxation can be prepared in any formulation commonly prepared in the art, for example, in the form of oral formulations, topical formulations, suppositories and sterile injections such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, according to general methods.

[0065] "Subject" can include a person. Furthermore, the term "subject" can refer to an object requiring administration of the Trolox-peptide conjugate of the present invention, and the object requiring administration can be an object requiring muscle relaxation.

[0066] "Administration" means providing a patient with a predetermined substance by any suitable method, and the composition of the present invention can be administered orally or parenterally via any of the commonly used routes, as long as the composition can reach the target tissue. Furthermore, the composition can be administered via any device capable of transporting the active substance to target cells.

[0067] In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of neuromuscular disorders, comprising a Trolox-peptide conjugate as an active ingredient.

[0068] Furthermore, the present invention provides a method for preventing or treating neuromuscular disorders, comprising administering a Trolox-peptide conjugate to a subject, wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox.

[0069] Subjects may be those who need to be administered Trolox-peptide conjugates, or those who need to prevent or treat neuromuscular disorders.

[0070] Furthermore, the present invention provides a Trolox-peptide conjugate wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox, the Trolox-peptide conjugate being used for the prevention or treatment of neuromuscular disorders.

[0071] Trolox-peptide conjugates can be used to inhibit neurotransmitters by suppressing the formation of the SNARE complex, and Trolox-peptide conjugates can be used to prevent or treat neuromuscular disorders to relieve muscle contraction or relax muscles.

[0072] Neuromuscular disorders are diseases caused by excessive release of neurotransmitters, resulting in muscle contraction or stiffness. For example, neuromuscular disorders can be categorized into the following groups: myoclonus, hemifacial spasm, spasmodic torticollis, anal fissure, blepharospasm, hemifacial spasm, cerebral palsy, strabismus, temporomandibular joint disorder, neuralgia, myalgia, overactive bladder, urge incontinence, dystonia, hyperhidrosis, and diseases caused by excessive release of neurotransmitters from glands controlled by the cholinergic nervous system.

[0073] "Prevention" refers to the entire effect of inhibiting or delaying the onset of disease by applying Trolox-peptide conjugates or compositions containing Trolox-peptide conjugates.

[0074] "Treatment" or "improvement" refers to the overall effect of improving or beneficially altering the symptoms of a disease by applying Trolox-peptide conjugates or compositions containing Trolox-peptide conjugates.

[0075] Compositions for the prevention or treatment of neuromuscular disorders may be formulated and used in the form of oral formulations, topical formulations, suppositories, and sterile injections, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, by conventional methods, and may include suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions for formulation.

[0076] Pharmaceutically acceptable carriers may include lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, which are commonly used in formulations.

[0077] In addition to the above-mentioned components, the pharmaceutical composition may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0078] The pharmaceutical composition may be administered orally or parenterally according to the desired method (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, or local administration), and its dosage may vary depending on the patient's condition and weight, the severity of the disease, the form of the medicine, and the route and time of administration, and may be appropriately selected by those skilled in the art.

[0079] The pharmaceutical composition is administered at a pharmaceutically effective dose. In this invention, a "pharmaceutically effective dose" refers to an adequate amount for treating a disease with a reasonable benefit / risk ratio suitable for medical treatment, and can be determined based on factors including the patient's disease type and severity, drug activity, drug sensitivity, timing of administration, route of administration, elimination rate, duration of treatment, and concurrently used drugs, as well as other factors known in the medical field. The pharmaceutical composition can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered alone or multiple times. Importantly, the dosage taken into account to achieve the maximum effect with the minimum amount without side effects can be readily determined by those skilled in the art.

[0080] The effective dose of the pharmaceutical composition can vary depending on the patient's age, sex, condition, weight, the absorption, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and the combination of drugs used. It can also be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc. For example, the pharmaceutical composition can be administered at a dose of approximately 0.0001 μg to 500 mg per kg of patient body weight per day, preferably 0.01 μg to 100 mg.

[0081] In another aspect, the present invention provides a composition for improving skin condition, comprising a Trolox-peptide conjugate as an active ingredient, wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox.

[0082] Furthermore, the present invention provides a method for improving skin condition, comprising administering a Trolox-peptide conjugate to a subject, wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox.

[0083] Subjects may be those who need to be treated with Trolox-peptide conjugates or those who need to improve their skin condition.

[0084] Furthermore, the present invention provides a Trolox-peptide conjugate wherein a peptide having the amino acid sequence of SEQ ID NO:1 is conjugated with Trolox, the Trolox-peptide conjugate being used to improve skin condition.

[0085] Details about peptides, Trolox, and Trolox-peptide conjugates are as described above.

[0086] "Skin condition" refers to the decrease in skin elasticity, the formation of wrinkles or fine lines on the face caused by skin aging. Aging includes endogenous aging and exogenous aging caused by the passage of time and external environment.

[0087] In addition, wrinkles refer to wrinkles that appear on all parts of the body, including the face, and can include both static and dynamic wrinkles.

[0088] "Improving skin condition" refers to maintaining or enhancing the ability to maintain or enhance skin wrinkles and elasticity. For example, it can be any of the following groups: reducing the appearance of fine lines, reducing the appearance of wrinkles, making the eyes appear wider (winding), lifting the corners of the mouth, reducing muscle mass, reducing facial asymmetry, and flattening the line extending from the upper lip.

[0089] Trolox-peptide conjugates inhibit the formation of the SNARE complex, preventing the release of neurotransmitters, thereby relieving facial muscle contractions and showing an effect of improving skin condition.

[0090] In addition, Trolox-peptide conjugates induce i) an increase in the expression of ColIα1 (Iα1 type collagen), ii) an increase in the expression of fibronectin gene, or iii) an increase in the expression of elastin gene to prevent or improve skin wrinkles, thereby showing an effect of improving skin condition.

[0091] Specifically, collagen is the main protein constituting the skin. It is synthesized in the form of procollagen in fibroblasts located in the dermis, then released into the extracellular matrix and converted into active collagen. However, the Trolox-peptide conjugate of the present invention can inhibit wrinkle formation by increasing the synthesis of active collagen in the dermis. Furthermore, the dermis is composed of collagen as collagen fibers and elastin as elastic fibers. In this regard, collagen fibers contribute to the formation of skin tension and structural integrity, while elastin fibers participate in skin elasticity, thereby enhancing skin elasticity. Specifically, the Trolox-peptide conjugate according to the present invention promotes elastin expression, thereby exhibiting an effect of enhancing skin elasticity.

[0092] Compositions used to improve skin condition can be cosmetic compositions.

[0093] Cosmetic compositions can be prepared in any formulation commonly prepared in the art, and can be formulated, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, sprays, etc., but are not limited thereto.

[0094] Cosmetic compositions can be prepared in various forms, such as toners, nourishing lotions, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, masks, sprays, powders, hair growth oils, hair lotions, shampoos, hair dyes, conditioners, hair sprays, hair aerosols, hair oils, solutions such as gels, sol-gels, lotions, oils, waxes, aerosols, etc., but not limited to these.

[0095] In addition to Trolox-peptide conjugates, cosmetic compositions may also contain other additives, such as excipients and carriers, and may be applied and mixed with commonly used ingredients in general skin cosmetics as needed.

[0096] When the cosmetic composition is formulated as a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone resin, bentonite, silica, talc, zinc oxide, etc., can be used as carrier ingredients.

[0097] When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, the formulation may also contain propellants such as chlorofluorocarbons, propane / butane, or dimethyl ether, but is not limited thereto.

[0098] When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component. For example, the carrier component may be water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid esters of sorbitol.

[0099] When the cosmetic composition is formulated as a suspension, liquid diluents such as water, ethanol or propylene glycol, suspensions such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth gum, etc., can be used as carrier components.

[0100] When the formulation of a cosmetic composition is a cleanser containing surfactants, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurate, sarcosinates, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, etc., can be used as carrier components.

[0101] When the cosmetic composition is formulated as a shampoo, the base formulation of the cosmetic composition is a shampoo. Basic ingredients used to form the shampoo, such as thickeners, surfactants, viscosity modifiers, humectants, pH adjusters, preservatives, and essential oils, can be mixed with the Trolox-peptide conjugate of this invention. CDE can be used as a thickener, LES as an anionic surfactant and cocobetaine as an amphoteric surfactant can be used as surfactants, polyquaternium salts can be used as viscosity modifiers, glycerin can be used as a humectant, and citric acid and sodium hydroxide can be used as pH adjusters. Grapefruit extract and the like can be used as preservatives, and essential oils such as cedarwood, peppermint, and rosemary, serine amino acids, pentanol, or vitamin E can also be added.

[0102] In addition to the Trolox-peptide conjugate and carrier components of the present invention as active ingredients, the ingredients contained in the cosmetic composition may also include ingredients commonly used in cosmetic compositions, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances.

[0103] Compositions used to improve skin condition can be quasi-pharmaceutical compositions.

[0104] "Quasi-medicines" refer to products used for the diagnosis, treatment, improvement, mitigation, treatment, or prevention of diseases in humans or animals that have a milder effect than pharmaceuticals. For example, under the Pharmaceutical Affairs Act, quasi-medicines include products used to treat or prevent diseases in humans / animals, products that have a mild or no direct effect on humans, and products used for pharmaceutical purposes, excluding products intended for pharmaceutical purposes.

[0105] The quasi-pharmaceutical composition can be prepared by selecting one or more of the following groups: body cleansers, foaming agents, soaps, face masks, ointments, creams, lotions, serums, and sprays, but is not limited thereto. Furthermore, the composition can be prepared in the form of strips, sanitary napkins, etc., but is not limited thereto.

[0106] When Trolox-peptide conjugates are used as quasi-pharmaceutical additives, they can be added as is or used together with other quasi-pharmaceutical or quasi-pharmaceutical ingredients, and can be used appropriately according to conventional methods.

[0107] As described above, the Trolox-peptide conjugate of the present invention inhibits the release of neurotransmitters from the nerve cell terminals, thereby having the effect of relieving muscle contraction or relaxing muscles. Therefore, it can be used as a raw material for pharmaceutical, quasi-pharmaceutical or cosmetic compositions to relieve muscle contraction.

[0108] The present invention will now be described in detail through examples and experimental cases.

[0109] However, the following embodiments and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following embodiments and experimental examples.

[0110] [Example 1]

[0111] Synthesis and confirmation of the physical properties of new peptides

[0112] 1-1. Synthesis of a peptide comprising the amino acid sequence of SEQ ID NO: 1

[0113] A novel peptide sequence “KFLIK” consisting of the amino acid sequence represented by SEQ ID NO:1 was prepared using a known method. The molecular weight of the peptide including the amino acid sequence of SEQ ID NO:1 was determined using a molecular weight analyzer, and the results confirmed that the molecular weight of the peptide including the amino acid sequence of SEQ ID NO:1 is equivalent to 647.4 Da.

[0114] 1-2. Evaluation of high-temperature stability over a long period of storage

[0115] The peptide of the present invention, consisting of the amino acid sequence of SEQ ID NO:1, was dissolved in sterile distilled water at a concentration of 1000 ppm and stored at 45°C for 7, 14, 28, 60 and 75 days, and then analyzed by HPLC.

[0116] As a result, such Figure 1 As shown in A, it was confirmed that the stability of the peptide of the present invention was maintained within the longest observation period of 75 days at 45°C.

[0117] 1-3. Evaluation of high-temperature stability

[0118] The peptide of the present invention was dissolved in sterile distilled water at a concentration of 1000 ppm, heated at 121°C for 15 minutes and 30 minutes, and then analyzed by HPLC.

[0119] As a result, such Figure 1 As shown in B, it was confirmed that the stability of the peptides of the present invention was maintained at 121°C for a maximum heating time of 30 minutes.

[0120] [Example 2]

[0121] Preparation of Trolox-peptide conjugates

[0122] In a peptide reactor, 1 mole of peptide-based resin and 10 ml of 1-methyl-2-pyrrolidone (NMP) were added, along with 270 mg (2.0 equivalents) of 1-hydroxybenzotriazole (1-HOBt) and 759 mg (2.0 equivalents) of N,N,N',N'-tetramethyl-O-(1H-benzotriazole-1-yl)ureonium hexafluorophosphate (HBTU), and the reaction was carried out for 30 minutes. Then, 388 mg (3.0 equivalents) of N,N'-diisopropylethylamine (DIEA) and 500 mg (2.0 equivalents) of Trolox were added to the reaction product, and the reaction was carried out at room temperature for 72 hours, followed by filtration. The filtrate and lysis solution were then reacted at room temperature for 2 hours to remove resin and protecting groups. Finally, 10 ml of diethyl ether was added to the filtrate and crystallization was performed to prepare the Trolox-peptide conjugate (i.e., the conjugate of the peptide of SEQ ID NO:1 with Trolox). The reaction formula for preparing Trolox-peptide conjugates is shown in the following reaction formula 1.

[0123] [Reaction Formula 1]

[0124]

[0125] Mass spectrometry analysis of the prepared Trolox-peptide conjugate confirmed the synthesis of a Trolox-peptide conjugate with a molecular weight of 880.1 Da.

[0126] [Experimental Example 1]

[0127] The inhibitory effect of Trolox-peptide conjugates on neurotransmitter release in nerve cells

[0128] To confirm the inhibitory effect of Trolox-peptide conjugates on neurotransmitter release in nerve cells, the release of acetylcholine, a neurotransmitter, was measured using a neuroblastoma-derived cell line (SH-SY5Y).

[0129] Specifically, SH-SY5Y cells were injected into culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 hours, after which the culture medium was replaced with serum-free medium. Subsequently, the culture dishes were treated with Trolox-peptide conjugates at concentrations of 1 μM, 10 μM, and 50 μM, while the positive control group was treated with 50 nM tetanus toxin and cultured for 48 hours. Acetylcholine release was then induced by treatment with nicotinic acid (NIC) and potassium chloride (KCl) for 30 minutes. The amount of acetylcholine released from the culture medium was then measured using an acetylcholine assay kit (Abcam).

[0130] As a result, such Figure 1As shown, in the groups treated with only nicotinic acid (NIC) and potassium chloride (KCl), the release of acetylcholine was at least three times that of the untreated control group, but in all groups treated with Trolox-peptide conjugates, the release of acetylcholine was significantly inhibited.

[0131] [Experimental Example 2]

[0132] Muscle relaxation effect of Trolox-peptide conjugates in laboratory animals

[0133] 2-1. Preparation of an animal model

[0134] Specifically, in the animal experiments, 7-week-old female Sprague-Dawley rats (Sam Taco BIOKOREA) were used, and the rats were divided into groups treated with 1U and 3U of botulinum neurotoxin (BoNT), respectively, and groups treated with the Trolox-peptide conjugate of the present invention at doses of 2.5 mg / rat and 5 mg / rat.

[0135] Botulinum neurotoxin and the Trolox-peptide conjugate of the present invention were respectively applied to the right calf muscle of experimental animals.

[0136] 2-2. Confirmation of muscle contraction alleviating effect

[0137] To confirm the effect of Trolox-peptide conjugates on muscle relaxation, the time taken to relax the paralyzed toe muscles in each treatment group of Example 2-1 above was compared by visual observation analysis.

[0138] Specifically, the right foot of rats in each treatment group was observed, and muscle paralysis was assessed using the Toe Abduction Score (DAS). In the DAS analysis, rats were floated by their tails for a short period to induce a characteristic startle response, in which the hind limbs extended and the hind toes opened. The degree of the startle response was evaluated using a 5-point scale (0 to 4). Here, 0 points represents a normal startle response, and 4 points represents the maximum reduction in toe abduction and leg extension (0 points: all five toes are separated; 1 point: one outward-pointing toe is brought together during abduction; 2 points: three toes are brought together; 3 points: four toes are brought together; 4 points: all five toes are brought together).

[0139] As a result, see Table 2 and Figure 2 and 3 As shown, the muscle relaxation effect was the highest in all groups after 3 days, and the effect decreased to some extent over time. Compared with the group injected with Botox, the muscle relaxation effect of the group injected with the Trolox-peptide conjugate of the present invention lasted longer.

[0140] [Table 1]

[0141]

[0142] [Experiment Example 3]

[0143] Trolox peptide conjugate improves skin wrinkles

[0144] Mouse fibroblasts (NIH3T3) were used to confirm the expression patterns of proteins that make up the dermis in order to confirm whether the Trolox-peptide conjugate of the present invention has the effect of improving skin wrinkles.

[0145] Specifically, NIH3T3 cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 μM / well into 6-well plates and incubated at 37°C in a 5% CO2 incubator for 16 hours. Next, the plates were treated with Trolox-peptide conjugates at concentrations of 1 μM, 10 μM, and 50 μM, and incubated again for 16 hours. RNA was then isolated from the cultured cells. cDNA was synthesized from the isolated RNA using a cDNA synthesis kit (Intron, Korea). The primer base sequences used at this time are shown in Table 3. The amplified PCR products were electrophoresed on an agarose gel to confirm the DNA bands.

[0146] [Table 2]

[0147]

[0148] As a result, such Figure 4 As shown, compared with the control group, the expression levels of collagen, fibronectin and elastin were significantly increased when treated with Trolox-peptide conjugate.

[0149] As a result, it can be seen that Trolox-peptide conjugates can prevent or improve skin wrinkles by increasing the expression levels of proteins that make up the dermis. <110> Kelgen Ltd. <120> Peptides with botulinum toxin-like activity and their uses <130> 2021-OPA-5320 <150> KR 10-2020-0071661 <151> 2020-06-12 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 1 Lys Phe Leu Ile Lys 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Col1a1 <400> 2 caccctcaag agcctgagtc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 reverse primer <400> 3 agacggctga gtagggaaca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for fibronectin <400> 4 ccaggaaccg agtacaccat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for fibronectin <400> 5 atacccaggt tgggtgatga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for elastin <400> 6 ggacccctga ctcgcgacct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for elastin <400> 7 ggggaggtgg gactgcccaa 20 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primers for GAPDH <400> 8 ggtgtgaacg gatttggccg tattg 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 9 ccgttgaatt tgccgtgagt ggagt 25

Claims

1. A Trolox-peptide conjugate, wherein a peptide consisting of the amino acid sequence of SEQ ID NO: 1 is conjugated with Trolox. The carboxyl group of Trolox is bound to the N-terminus of the peptide.

2. The Trolox-peptide conjugate according to claim 1, wherein the Trolox-peptide conjugate relieves muscle contraction by inhibiting the release of a neurotransmitter, and wherein the neurotransmitter is acetylcholine.

3. A composition for muscle relaxation comprising the Trolox-peptide conjugate according to claim 1.

4. The composition for muscle relaxation according to claim 3, wherein the composition relieves muscle contraction by inhibiting the release of a neurotransmitter, and wherein the neurotransmitter is acetylcholine.

5. A pharmaceutical composition for the prevention or treatment of neuromuscular disorders, comprising the Trolox-peptide conjugate of claim 1 as an active ingredient. The neuromuscular disorders mentioned therein are selected from the group consisting of: strabismus, temporomandibular joint disorder, overactive bladder, and hyperhidrosis.

6. The pharmaceutical composition of claim 5, wherein the overactive bladder syndrome includes urge incontinence.

7. The pharmaceutical composition according to claim 5, wherein the temporomandibular joint disorder comprises at least one selected from the group consisting of myoclonus and myalgia.

8. A composition for improving skin condition, comprising the Trolox-peptide conjugate of claim 1 as an active ingredient. The improvement in skin condition mentioned herein is selected from the group consisting of: reducing the appearance of fine lines, reducing the appearance of wrinkles, making the eyes appear larger, lifting the corners of the mouth, reducing muscle mass, reducing facial asymmetry, and flattening the line extending from the upper lip.

9. The composition for improving skin condition according to claim 8, wherein the composition relieves facial muscle contraction by inhibiting the release of a neurotransmitter, and wherein the neurotransmitter is acetylcholine.

10. The composition for improving skin condition according to claim 8, wherein the composition improves wrinkles by i) increasing the expression of ColIa1 (Iα1 type collagen), ii) increasing the expression of fibronectin gene, or iii) increasing the expression of elastin gene.

11. The composition for improving skin condition according to claim 8, wherein the composition is a cosmetic composition.

12. The composition for improving skin condition according to claim 8, wherein the composition is a quasi-pharmaceutical composition.

13. Use of the Trolox-peptide conjugate according to claim 1 in the manufacture of a medicament for the prevention or treatment of neuromuscular disorders. The neuromuscular disorders mentioned therein are selected from the group consisting of: strabismus, temporomandibular joint disorder, overactive bladder, and hyperhidrosis.

14. The use according to claim 13, wherein the overactive bladder syndrome includes urge incontinence.

15. The use according to claim 13, wherein the temporomandibular joint disorder comprises at least one selected from the group consisting of myoclonus and myalgia.

16. Use of the Trolox-peptide conjugate according to claim 1 in the manufacture of a medicament for improving skin condition. The improvement in skin condition mentioned herein is selected from the group consisting of: reducing the appearance of fine lines, reducing the appearance of wrinkles, making the eyes appear larger, lifting the corners of the mouth, reducing muscle mass, reducing facial asymmetry, and flattening the line extending from the upper lip.

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