Composition comprising pentapeptide as active ingredient

Abstract

The present invention relates to use of a composition comprising pentapeptides as an active ingredient for inhibiting excessive sweating, inhibiting body odor, or preventing or treating pain.

Description

Technical Field

[0001] The present invention relates to use of a composition comprising pentapeptides as an active ingredient for inhibiting excessive sweating, inhibiting body odor, or preventing or treating pain. Background Art

[0002] Hyperhidrosis is a skin disorder characterized by excessive and abnormally increased perspiration, or sweating, exceeding the amount needed for thermoregulation. Hyperhidrosis can be caused by genetics or physical or emotional stress and can be localized throughout the body or to specific areas. When excessive sweating occurs locally on the palms, soles, face, armpits, or scalp, it is called primary or localized hyperhidrosis. Generalized or secondary hyperhidrosis typically affects the entire body.

[0003] Currently, methods for treating hyperhidrosis include the use of antiperspirants, ion transfer methods that temporarily block sweat glands, and botulinum toxin therapy that temporarily blocks the nerves and sweat glands that stimulate sweating (see document WO2010-078242). Among these methods, in the case of methods using botulinum toxin to temporarily block nerves, the paralysis effect is reversible, averaging 6 months, so this treatment requires repeated injections of botulinum toxin. Because botulinum toxin has a size that can be recognized by the patient's immune system, it can induce an immune response to the drug. Therefore, it is necessary to develop such a molecule, which has a simpler and more stable molecular structure, does not induce an immune response, has a high activity in reducing sweat, and can be used for long-term non-invasive treatment with fewer side effects.

[0004] On the other hand, body odor is produced when microorganisms or bacteria break down sweat secreted by sweat glands. As microorganisms or bacteria break down the fat, protein, sebum, and dirt contained in sweat secreted by eccrine sweat glands distributed throughout the body and apocrine sweat glands distributed in areas with abundant body hair, such as the armpits, unpleasant body odors, such as underarm odor, foot odor, and head odor, are produced. Generally, methods such as deodorization, antibacterial treatment, and masking are used to control body odor. However, using the wrong fragrance to mask body odor can produce an unpleasant odor, which can have the opposite effect. Furthermore, when deodorant ingredients are applied to the skin, they only provide a temporary effect from the time of application until the next wash, leading to the inconvenience of reapplying the product after washing.

[0005] Therefore, the present inventors earnestly endeavored to develop a material that can suppress body odor by inhibiting excessive sweat production, which can be said to be the root cause of body odor. As a result, the present inventors confirmed that the pentapeptide newly synthesized by the present inventors has a stable molecular structure, penetrates through the dermis to reach the skin tissue, and exhibits high sweat-reducing activity, so that the pentapeptide can be effectively used to treat hyperhidrosis and control body odor, thereby completing the present invention.

[0006] On the other hand, various causes can cause pain, and if the pain lasts for a long time or the stimulation is too severe, it will interfere with daily life and sometimes cause anxiety and fear. In general, according to the occurrence site, pain is called headache, chest pain, abdominal pain, back pain, etc., according to the mechanism of occurrence, pain is divided into somatic pain, visceral pain, neuropathic pain, psychopathic pain, etc., and according to the time concept, pain is divided into acute pain and chronic pain. Chronic pain has clinical significance and refers to a disease in which pain lasts for 6 months or longer, although there is no clear cause. Most chronic pain manifests as the form of neuropathic pain, which is characterized by spontaneous pain, allodynia and hyperalgesia, and even slight temperature changes or slight contact with clothing can cause severe pain. Therefore, this causes serious interference to the patient's daily life and also responds poorly to existing various treatments.

[0007] Currently, invasive treatments such as nerve blockade and various types of drug treatments (opioids, drugs that regulate the activity of nervous system ion channels, anticonvulsants, etc.) are used to treat pain. However, these drugs are only effective when administered at high doses, and even if they are initially effective, tolerance develops with long-term use, and the effects often disappear. Furthermore, they can cause side effects such as gastrointestinal bleeding, gastrointestinal disorders, and liver damage.

[0008] Therefore, a method for using botulinum toxin A as a new therapy is being introduced, which can solve the side effects of existing treatments. Ranoux et al. reported that after intradermal injection of botulinum toxin in patients with post-herpetic pain and postoperative neuropathic pain with mechanical allodynia, pain and allodynia were alleviated (see document Ranoux et al., Annals of Neurology 64 (3): 274-283 (2008)), and Yuan et al. reported that pain relief and improvement of sleep disorders in patients with diabetic neuropathy were improved (see document Yuan et al., Neurology 72: 1473-1478 (2009)). However, in recent years, more and more reports have shown that the use of botulinum toxin A has a similar effect to that of administering a placebo, so more research results are needed.

[0009] On the other hand, because the effects of botulinum toxin are reversible and last an average of six months, repeated injections are required to treat pain. Furthermore, because botulinum toxin is sized to be recognized by the patient's immune system, it can induce an immune response to the drug. Therefore, there is a need to develop molecules with simpler and more stable molecular structures that do not induce immune responses, exhibit high pain-relieving activity, and enable long-term, non-invasive treatment with fewer side effects.

[0010] Therefore, the present inventors diligently worked to develop a new therapeutic agent for pain. As a result, the present inventors confirmed that the newly synthesized pentapeptide has a stable molecular structure, penetrates through the dermis to reach the skin tissue, and exhibits high pain-suppressing activity. Therefore, the pentapeptide can be effectively used for pain treatment, thereby completing the present invention. Summary of the Invention

[0011] [Technical Issues]

[0012] The object of the present invention is to provide a pharmaceutical composition for preventing or treating hyperhidrosis.

[0013] Another object of the present invention is to provide a method for preventing or treating hyperhidrosis.

[0014] Another object of the present invention is to provide a peptide for preventing or treating hyperhidrosis.

[0015] Another object of the present invention is to provide a composition for preventing or alleviating hyperhidrosis or inhibiting body odor.

[0016] Yet another object of the present invention is to provide a method for suppressing body odor.

[0017] Another object of the present invention is to provide a peptide for suppressing body odor.

[0018] Another object of the present invention is to provide a composition for relieving pain.

[0019] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating pain.

[0020] Another object of the present invention is to provide a method for preventing or treating pain.

[0021] Another object of the present invention is to provide a peptide for preventing or treating pain.

[0022] Another object of the present invention is to provide a quasi-drug composition for alleviating or improving pain.

[0023] Another object of the present invention is to provide a cosmetic composition for relieving or improving pain.

[0024] [Technical solution]

[0025] In order to solve the above problems, one aspect of the present invention provides a pharmaceutical composition for preventing or treating hyperhidrosis, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0026] Another aspect of the present invention provides a method for preventing or treating hyperhidrosis, comprising administering a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 to a subject.

[0027] Another aspect of the present invention provides a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1, for preventing or treating hyperhidrosis.

[0028] Another aspect of the present invention provides a composition for preventing or alleviating hyperhidrosis or inhibiting body odor, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0029] Another aspect of the present invention provides a method for inhibiting body odor, comprising administering a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 to a subject.

[0030] Another aspect of the present invention provides a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 for inhibiting body odor.

[0031] Another aspect of the present invention provides a composition for relieving pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0032] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0033] Another aspect of the present invention provides a method for preventing or treating pain, comprising administering a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 to a subject.

[0034] Another aspect of the present invention provides a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1, for use in preventing or treating pain.

[0035] Another aspect of the present invention provides a quasi-drug composition for preventing or ameliorating pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0036] Another aspect of the present invention provides a cosmetic composition for relieving pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0037] [Beneficial effects]

[0038] The pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention is absorbed through the skin into the muscle layer of the skin tissue, inhibiting the abnormal release of neurotransmitters from nerve endings and reducing sweating. Therefore, the pentapeptide of the present invention can be used alone to prevent, alleviate, improve or treat the symptoms of hyperhidrosis.

[0039] Furthermore, the pentapeptide of the present invention can preemptively block the cause of body odor by inhibiting excessive sweat production and provide a semi-permanent body odor suppressing effect.

[0040] The pentapeptide of the present invention consisting of the amino acid sequence of SEQ ID NO: 1 can inhibit the generation and transmission of pain by inhibiting the expression of proinflammatory cytokines in muscle cells and the release of pain transmitters in nerve endings. Therefore, the pentapeptide of the present invention can be used to alleviate, improve, prevent or treat pain.

[0041] Furthermore, since the pentapeptide of the present invention is absorbed through the skin to the muscle layer of the skin tissue, it can be used as a raw material for drugs, quasi-drugs, and cosmetics applied to the skin.

[0042] However, the effects of the present invention are not limited to the above-mentioned effects, and other effects that are not mentioned will be clearly understood by those skilled in the art from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1a and Figure 1b The results of the thermal stability evaluation of the KFLIK peptide are shown:

[0044] Figure 1a The results of the high temperature stability of the peptide confirmed during long-term storage at 45°C are shown, and Figure 1b The results of high temperature stability of the peptide confirmed at the highest heating temperature (121°C) are shown.

[0045] Figure 2a and Figure 2b The results show that KFLIK peptide treatment inhibits the formation of SNARE complexes in nerve cells. Figure 2a The results of confirming the formation of SNARE complex by Western blot are shown, and Figure 2b Shown are the results of confirming the degree of degradation of syntaxin 1a constituting the SNARE complex.

[0046] Figure 3 Shown is the inhibitory effect of KFLIK peptide treatment on neurotransmitter release in nerve cells.

[0047] Figure 4The relative sweat reduction effect of the palms of hyperhidrosis patients treated with the KFLIK peptide of the present invention is shown: A is an image of the iodine-starch test result, and B shows the result of intensity analysis using Image J software, i.e., the sweat reduction effect.

[0048] Figure 5 The relative sweat reduction effect of the soles of hyperhidrosis patients treated with the KFLIK peptide of the present invention is shown: A is an image of the iodine-starch test result, and B shows the result of intensity analysis using Image J software, i.e., the sweat reduction effect.

[0049] Figure 6 The relative sweat reduction effect of the axilla of a hyperhidrosis patient treated with the KFLIK peptide of the present invention is shown: A is an image of the iodine-starch test result, and B shows the result of intensity analysis using Image J software, i.e., the sweat reduction effect.

[0050] Figure 7 Fluorescence microscope images showing the penetration of KFLIK peptide into nerve cells by attaching the fluorescent substance rhodamine to the KFLIK peptide: A shows the coexistence of blue-stained neuronal nuclei and red rhodamine-pentapeptide, blue in B indicates DAPI-stained neuronal nuclei, and red in C indicates rhodamine-peptide.

[0051] Figure 8 Fluorescence microscope images, which were obtained by applying the rhodamine-KFLIK peptide to the backs of experimental animals and collecting skin tissue, show that the KFLIK peptide penetrates into the muscle layer: blue in A indicates DAPI-stained cell nuclei, green in B indicates TrkB neurofibrillary marker, red in C indicates rhodamine-pentapeptide, and yellow in D indicates that the pentapeptide of the present invention penetrates into the nerve cells of the muscle layer.

[0052] Figure 9 The body odor suppressing effect of the KFLIK peptide of the present invention is shown, and the absorbance measurement results using a spectrophotometer show the growth inhibitory effect on Corynebacterium. P / C is 1% triclosan as a positive control.

[0053] Figure 10a and Figure 10b The results show that KFLIK peptide treatment inhibits the expression of pro-inflammatory cytokines in myocytes: Figure 10a The results of RT-PCR confirmation of the mRNA expression levels of IL-1α, TNF-α, IL-1β, and COX-2 genes are shown, and Figure 10b The results of Western blotting confirm the expression levels of COX-2, IL-1β, and TNF-α proteins are shown.

[0054] Figure 11 This indicates that KFLIK peptide treatment inhibits the release of the pain transmitter substance P from nerve cells.

[0055] Figure 12a and Figure 12b The figure shows the effect of the KFLIK peptide of the present invention on relieving muscle pain through human efficacy evaluation: Figure 12a The results of calculating the mean value of pain intensity as measured by the Numerical Rating Scale (NRS), and Figure 12b The degree of pain relief by KFLIK peptide treatment is shown. DETAILED DESCRIPTION

[0056] Hereinafter, the present invention will be described in detail.

[0057] One aspect of the present invention is to provide a pharmaceutical composition for preventing or treating hyperhidrosis, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0058] Here, the term "peptide" refers to a linear or cyclic molecule formed by combining amino acid residues with each other via peptide bonds. The preparation of peptides can be accomplished by conventional biological or chemical synthesis methods known in the art, for example, by methods such as solid phase synthesis techniques.

[0059] Here, the term "pentapeptide" refers to a linear molecule consisting of five amino acid residues, and the pentapeptide of the present invention refers to a linear peptide molecule consisting of the amino acid sequence of SEQ ID NO: 1 (KFLIK).

[0060] "Peptides" and "pentapeptides" can be amino acid variants or fragments with different sequences obtained by deletion, insertion, substitution, or a 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 a peptide are known in the art. In some cases, amino acid exchanges can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc. Therefore, the present invention includes peptides having an amino acid sequence substantially identical to a pentapeptide composed of the amino acid sequence of KFLIK, as well as variants or active fragments of such peptides. Substantially identical proteins refer to amino acid sequences that share 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 with the amino acid sequence of KFLIK, but are not limited to these. If the amino acid sequence shares at least 75% sequence homology and possesses the same activity, the amino acid sequence is included within the scope of the present invention. Furthermore, the peptides of the present invention may also include targeting sequences, tags, labeled residues, or amino acid sequences specifically designed to increase the peptide's half-life or stability.

[0061] In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), modified specificity (e.g., a broad spectrum of biological activity) and reduced antigenicity, a protecting group can be bound to the N-terminus or C-terminus of the peptide of the present invention. For example, the protecting group can be an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG). However, when the protecting group is a component capable of modifying the peptide, in particular, improving the stability of the peptide, such a protecting group can be included without limitation. The "stability" used includes not only in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo, but also storage stability (e.g., storage stability at room temperature).

[0062] The pentapeptide consisting of the amino acid sequence of KFLIK of the present invention can prevent or treat hyperhidrosis by inhibiting the formation of SNARE complexes and inhibiting the excessive release of neurotransmitters from nerve endings.

[0063] Furthermore, the pentapeptide consisting of the amino acid sequence of KFLIK of the present invention can prevent or alleviate hyperhidrosis by inhibiting the production of sweat in the skin area where hyperhidrosis occurs.

[0064] Furthermore, since the pentapeptide consisting of the amino acid sequence of KFLIK of the present invention is absorbed percutaneously and penetrates into the muscle layer of the skin tissue, the symptoms of hyperhidrosis can be improved or alleviated simply by applying it to the lesion.

[0065] Here, "hyperhidrosis" refers to a disease state characterized by excessive and uncontrolled sweating that exceeds the level required for cooling the body, and refers to a disease or symptom in which excessive sweat secretion occurs due to hyperactivity of sweat glands and disordered cholinergic stimulation. Here, hyperhidrosis includes localized hyperhidrosis (primary hyperhidrosis) and generalized hyperhidrosis (secondary hyperhidrosis), and may occur in any skin area selected from the group consisting of, for example, the face, forehead, neck, hands, feet, and armpits, but is not limited thereto.

[0066] In a specific embodiment, when a composition comprising the pentapeptide of the present invention is applied to one palm, sole, and armpit of a hyperhidrosis patient, a 45% to 90% reduction in sweating is confirmed compared to a control area on the opposite side to which the composition is not applied.

[0067] Here, the term "prevention" refers to any effect of inhibiting or delaying pain by administering a composition comprising a pentapeptide.

[0068] Herein, the term "treating" refers to any effect of "relieving" or "improving" or beneficially altering pain by administering a composition comprising a pentapeptide.

[0069] The pharmaceutical composition for preventing or treating hyperhidrosis according to the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injections according to conventional methods. The preparations may include suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.

[0070] Pharmaceutically acceptable carriers may include lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, etc., which are commonly used in formulations.

[0071] In addition to the above ingredients, the pharmaceutical composition may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like.

[0072] The pharmaceutical composition can be administered orally or parenterally (e.g., intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally or topically) according to the desired method. Dosage varies according to the patient's condition and body weight, the severity of the disease, the form of the drug, and the route of administration and time, and can be appropriately selected by those skilled in the art.

[0073] The pharmaceutical compositions of the present invention are administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to a sufficient amount to treat a disease at a reasonable benefit / risk ratio suitable for medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the disease, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, and concomitant medications, as well as other factors well known in the medical field. The pharmaceutical composition can be administered as a single therapeutic agent or in combination with other anti-obesity drugs. The pharmaceutical composition can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and the pharmaceutical composition can be administered alone or multiple times. It is important to administer an amount that achieves the maximum effect with the minimum dosage without side effects, taking all of the above factors into consideration, which can be readily determined by those skilled in the art.

[0074] The effective amount of the pharmaceutical composition may vary depending on the patient's age, sex, condition, body weight, absorption, inactivation rate, excretion rate of the active ingredient in the body, disease type, and drugs used in combination, and the effective amount of the pharmaceutical composition may be increased or decreased depending on the administration route, severity of obesity, sex, body weight, age, etc. For example, the pharmaceutical composition may be administered at about 0.0001 μg to 500 mg, preferably 0.01 μg to 100 mg, per kg of patient body weight per day.

[0075] In another aspect, the present invention provides a composition for preventing or alleviating hyperhidrosis or inhibiting body odor, comprising a pentapeptide consisting of the amino acid sequence of KFLIK as an active ingredient.

[0076] The pentapeptide consisting of the amino acid sequence of KFLIK of the present invention can prevent or treat hyperhidrosis by inhibiting the formation of SNARE complexes and excessive release of neurotransmitters from nerve endings, and can also inhibit body odor caused by excessive sweat production.

[0077] Furthermore, the pentapeptide consisting of the amino acid sequence of KFLIK of the present invention can prevent or alleviate hyperhidrosis by exhibiting an effect of inhibiting sweat production in skin areas where hyperhidrosis occurs.

[0078] Furthermore, the pentapeptide consisting of the amino acid sequence of KFLIK of the present invention can inhibit the growth of bacterial strains that cause body odor, thereby inhibiting the generation of body odor caused by the degradation of substances contained in sweat.

[0079] The specific details of peptides, pentapeptides and hyperhidrosis are as described above.

[0080] Here, "body odor" refers to the smell of degradation products produced when bacteria degrade sweat gland secretions (i.e., fat, protein, sebum and / or waste products secreted together with sweat), and specifically, "body odor" may be an odor produced in parts of the body where a large amount of sweat is produced, such as the armpits, feet, hands, forehead, neck, face or scalp.

[0081] Here, "body odor suppression" refers to preventing the odor produced by degradation products of sweat gland secretions by microorganisms, or suppressing sweat production, which is the cause of body odor. Specifically, body odor suppression can be preventing odor from the armpits, feet, hands, forehead, neck, face, or scalp, or suppressing sweat production in these areas, and more specifically, preventing underarm odor.

[0082] In yet another aspect, the present invention provides a cosmetic composition for preventing or alleviating hyperhidrosis or inhibiting body odor, comprising a pentapeptide consisting of the amino acid sequence of KFLIK as an active ingredient.

[0083] The cosmetic composition can be prepared in any formulation commonly prepared in the art, for example, it can be formulated in the form of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a cleanser containing a surfactant, an oil, a powder foundation, an emulsion foundation, a wax foundation and a spray, but is not limited thereto.

[0084] The cosmetic composition can be prepared in various forms such as solutions, sol-gels, emulsions, oils, waxes, aerosols, etc., for example, toners, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, facial masks, sprays, powders, deodorants, hair tonics, hair creams, shampoos, hair dyes, conditioners, hair sprays, hair aerosols, pomades, gels, etc., but are not limited thereto.

[0085] In addition to the pentapeptide consisting of the amino acid sequence of KFLIK, the cosmetic composition of the present invention may further comprise other additives such as excipients and carriers, and may apply and blend those commonly used ingredients incorporated into general cosmetics as needed.

[0086] When the cosmetic composition is formulated as a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone resin, bentonite, silicon dioxide, talc, zinc oxide and the like may be used as carrier components.

[0087] When the cosmetic composition is formulated as a powder or spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, when the formulation is a spray, the formulation may further contain a propellant such as, but not limited to, chlorofluorocarbons, propane / butane, or dimethyl ether.

[0088] 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, water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty esters, polyethylene glycol, or fatty acid esters of sorbitol may be used.

[0089] When the cosmetic composition is formulated as a suspension, liquid diluents such as water, ethanol and propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth gum can be used as carrier components.

[0090] When the cosmetic composition is prepared as a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glyceryl fatty acid esters can be used as carrier components.

[0091] When the cosmetic composition is formulated as a shampoo, basic ingredients used in shampoo compositions, such as thickeners, surfactants, viscosity modifiers, humectants, pH modifiers, preservatives, and essential oils, can be mixed with the Trolox-peptide conjugate of the present invention. CDE can be used as a thickener, the anionic surfactant LES and the amphoteric surfactant cocobetaine 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 modifiers. Grapefruit extract and the like can be used as preservatives, and essential oils such as fir, mint, and rosemary, silk amino acids, amyl alcohol, or vitamin E can also be added.

[0092] In addition to the Trolox-peptide conjugate of the present invention as an active ingredient and a carrier component, 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, but are not limited thereto.

[0093] In still another aspect, the present invention provides a method for preventing or treating hyperhidrosis, comprising administering a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 to a subject.

[0094] In another aspect, the present invention provides a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 for use in preventing or treating hyperhidrosis.

[0095] In still another aspect, the present invention provides a method for inhibiting body odor, comprising administering a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 to a subject.

[0096] In yet another aspect, the present invention provides a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 for use in inhibiting body odor.

[0097] "Subject" may include humans. In addition, the term "subject" may be an object to which the pentapeptide of the present invention is to be administered, and the subject in need of administration may include individuals who have been diagnosed with hyperhidrosis or odorosis, individuals who have developed symptoms associated with hyperhidrosis or odorosis, and individuals who wish to be administered to prevent the development of a disease or symptom or to improve health.

[0098] "Administering" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pentapeptide of the present invention can be oral administration or parenteral administration by any general route, as long as the target tissue can be reached. In addition, the pentapeptide can be administered by any device capable of delivering the active agent to the target cells.

[0099] As described above, the pentapeptides of the present invention inhibit the formation of SNARE complexes in nerve cells, thereby suppressing the excessive release of neurotransmitters. Furthermore, simply by applying the pentapeptides of the present invention to areas where excessive sweat production occurs, they penetrate into the nerve cells of the muscle layer, thereby exhibiting an excellent effect of suppressing sweat production. Therefore, the pentapeptides of the present invention can be used as a raw material for pharmaceutical or cosmetic compositions for preventing, ameliorating, alleviating, or treating hyperhidrosis or suppressing body odor.

[0100] Another aspect of the present invention provides a composition for relieving pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0101] Here, the term "peptide" refers to a linear or cyclic molecule formed by combining amino acid residues with each other via peptide bonds. The preparation of peptides can be accomplished by conventional biological or chemical synthesis methods known in the art, for example, by methods such as solid phase synthesis techniques.

[0102] Here, the term "pentapeptide" refers to a linear molecule consisting of five amino acid residues, and the pentapeptide of the present invention refers to a linear peptide molecule consisting of the amino acid sequence of SEQ ID NO: 1 (KFLIK).

[0103] "Peptides" and "pentapeptides" can be amino acid variants or fragments with different sequences obtained by deletion, insertion, substitution, or a 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 a peptide are known in the art. In some cases, amino acid exchanges can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc. Therefore, the present invention includes peptides having an amino acid sequence substantially identical to a pentapeptide composed of the amino acid sequence of KFLIK, as well as variants or active fragments of such peptides. Substantially identical proteins refer to amino acid sequences that share 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 with the amino acid sequence of KFLIK, but are not limited to these. If the amino acid sequence shares at least 75% sequence homology and possesses the same activity, the amino acid sequence is included within the scope of the present invention. Furthermore, the peptides of the present invention may also include targeting sequences, tags, labeled residues, or amino acid sequences specifically designed to increase the peptide's half-life or stability.

[0104] In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), modified specificity (e.g., a broad spectrum of biological activity) and reduced antigenicity, a protecting group can be bound to the N-terminus or C-terminus of the peptide of the present invention. For example, the protecting group can be an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG). However, when the protecting group is a component capable of modifying the peptide, in particular, improving the stability of the peptide, such a protecting group can be included without limitation. The "stability" used includes not only in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo, but also storage stability (e.g., storage stability at room temperature).

[0105] The pentapeptide composed of the amino acid sequence of KFLIK of the present invention inhibits the expression of pro-inflammatory cytokines in muscle cells and suppresses inflammatory response, and thus can prevent or treat pain caused by microlacerations.

[0106] In addition, the pentapeptide consisting of the amino acid sequence of KFLIK of the present invention can prevent the transmission of pain by inhibiting the release of pain transmitter substance P secreted by synaptic neurons, thereby preventing or treating pain.

[0107] Furthermore, the pentapeptide consisting of the amino acid sequence of KFLIK of the present invention is absorbed percutaneously and penetrates into the muscle layer of the skin tissue, and thus pain can be prevented, alleviated or treated by applying the pentapeptide of the present invention to the lesion.

[0108] Here, "pain" refers to an unpleasant sensation caused by stimulation of specific nerves having pain receptors, and includes all symptoms of such sensations that can be alleviated, prevented or treated by the pentapeptides of the present invention. Pain according to the present invention can be acute pain or chronic pain.

[0109] Here, the pain may be at least one selected from the group consisting of: inflammatory pain, neuropathic pain, nociceptive pain and psychiatric pain. For example, the pain may be at least one selected from the group consisting of cancer pain or postoperative pain, wherein nociceptive pain, inflammatory pain, etc. act in combination; trigeminal neuralgia, idiopathic pain and diabetic neuropathic pain belonging to neuropathic pain; and migraine and menstrual pain belonging to visceral pain, but not limited thereto.

[0110] In addition, the composition comprising the pentapeptide of the present invention can be applied to pain occurring in the skin or musculoskeletal system. For example, the pain can be associated pain selected from at least one of the following: diabetic neuropathy, diabetic peripheral neuropathy, pain caused by viral infection, nociceptive pain, facial neuralgia, postherpetic neuralgia, back pain, intervertebral disc, radiculopathy, neuropathy, hyperalgesia, central sensitization-related pain, allodynia, cancer pain, myofascial pain syndrome, carpal tunnel syndrome, cubital tunnel syndrome (ulnar neuropathy), de Quervain (de Quervain), ulnar neuropathy, neuropathy, neuropathy, hyperalgesia, central sensitization-related pain, allodynia, cancer pain, myofascial pain syndrome, carpal tunnel syndrome, cubital tunnel syndrome (ulnar neuropathy), de Quervain (de Quervain), ulnar neuropathy, ... Quervain's syndrome, rotator cuff syndrome, osteoarthritis, tendinitis, synovitis, lateral epicondylitis, medial epicondylitis, tendon injury, bursitis, ligament inflammation, tennis elbow, golfer's elbow, knee pain, patellar tendinitis, meniscus injury, finger joint pain, wrist tendinitis, wrist sprain, ankle sprain, sprain, fibromyalgia, plantar fasciitis, inflammatory arthritis, infectious arthritis, rheumatoid arthritis, degenerative joint pain, frozen shoulder, gout, postpartum joint pain, osteomyelitis, ankylosing spondylitis, degenerative spinal disease, lumbar stenosis, cruralgia, sprains, bruises, joint pain, and sciatica.

[0111] Specifically, the pentapeptides according to the present invention can reach the muscle layer simply by applying them to the skin and be absorbed through the skin, thereby showing an excellent analgesic effect. In particular, the pentapeptides of the present invention can effectively act on pain around the skin or tissues close to the skin, especially areas with thin subcutaneous fat. For example, the pentapeptides of the present invention can effectively prevent or treat pain associated with sprains, abrasions, joint pain, tendinitis, ligamentitis, frozen shoulder, rotator cuff disease, tennis elbow, golfer's elbow, and de Quervain syndrome in areas such as the knees, elbows, fingers, shoulders, ankles, and feet.

[0112] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0113] Here, the term "prevention" refers to any effect of inhibiting or delaying pain by administering a composition comprising a pentapeptide.

[0114] Herein, the term "treating" refers to any effect of "relieving" or "improving" or beneficially altering pain by administering a composition comprising a pentapeptide.

[0115] The pharmaceutical composition for preventing or treating pain according to the present invention can be prepared and used in the form of oral preparations, external preparations, suppositories and sterile injections such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols according to conventional methods. For the preparation, suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions can be included.

[0116] Pharmaceutically acceptable carriers may include lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., which are commonly used in formulations.

[0117] In addition to the above ingredients, the pharmaceutical composition may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like.

[0118] The pharmaceutical composition can be administered orally or parenterally (e.g., intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally or topically) according to the desired method. Dosage varies according to the patient's condition and body weight, the severity of the disease, the form of the drug, and the route of administration and time, and can be appropriately selected by those skilled in the art.

[0119] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to a sufficient amount for treating a disease at a reasonable benefit / risk ratio suitable for medical treatment, and effective dosage levels can be determined based on factors including disease type and severity, drug activity, drug sensitivity, administration time, route of administration and discharge rate, duration of treatment, and concurrently used drugs, as well as other elements well known in the medical field. The pharmaceutical composition can be administered as a separate therapeutic agent or can be administered in combination with other analgesics, the pharmaceutical composition can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and the pharmaceutical composition can be administered alone or multiple times. Importantly, the amount that can achieve the maximum effect without side effects with the minimum dosage, taking all of the above factors into consideration, can be easily determined by those skilled in the art.

[0120] The effective amount of the pharmaceutical composition may vary depending on the patient's age, sex, condition, body weight, absorption, inactivation rate, excretion rate of the active ingredient in the body, disease type, and drugs used in combination, and the effective amount of the pharmaceutical composition may be increased or decreased depending on the administration route, severity of obesity, sex, body weight, age, etc. For example, the pharmaceutical composition may be administered at about 0.0001 μg to 500 mg, preferably 0.01 μg to 100 mg, per kg of patient body weight per day.

[0121] Another aspect of the present invention provides a quasi-drug composition for preventing or ameliorating pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0122] The specific details of peptides, pentapeptides and pain are as described above.

[0123] The term "quasi-drug" refers to products intended for the diagnosis, treatment, amelioration, alleviation, cure, or prevention of human or animal diseases that have a milder effect than drugs. For example, under the Pharmaceutical Affairs Act, quasi-drugs exclude products intended for pharmaceutical purposes and include products intended for the treatment or prevention of human or animal diseases, as well as products with minimal or no direct effect on the human body.

[0124] The quasi-drug composition of the present invention can be prepared using at least one selected from the group consisting of, but not limited to, body cleansers, foams, soaps, facial masks, ointments, creams, lotions, serums, and sprays. Furthermore, the composition can be prepared in the form of, but not limited to, strips or sanitary napkins.

[0125] When the pentapeptide of the present invention is used as a quasi-drug additive, the composition can be added as it is or used together with other quasi-drugs or quasi-drug components, and can be appropriately used according to conventional methods.

[0126] In yet another aspect, the present invention provides a cosmetic composition for relieving pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0127] The specific details of peptides, pentapeptides and pain are as described above.

[0128] The cosmetic composition can be prepared in any formulation commonly prepared in the art, for example, it can be formulated in the form of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a cleanser containing a surfactant, an oil, a powder foundation, an emulsion foundation, a wax foundation and a spray, but is not limited thereto.

[0129] The cosmetic composition can be prepared into various forms such as solutions, sol-gels, emulsions, oils, waxes, aerosols, etc., for example, toners, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, facial masks, sprays, powders, hair tonics, hair creams, shampoos, hair dyes, conditioners, hair sprays, hair aerosols, pomades, gels, etc., but are not limited thereto.

[0130] In addition to the pentapeptide consisting of the amino acid sequence of KFLIK, the cosmetic composition of the present invention may further comprise other additives such as excipients and carriers, and may apply and blend those commonly used ingredients incorporated into general cosmetics as needed.

[0131] When the cosmetic composition is formulated as a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone resin, bentonite, silicon dioxide, talc, zinc oxide and the like may be used as carrier components.

[0132] When the cosmetic composition is formulated as a powder or spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, when the formulation is a spray, the formulation may further contain a propellant such as, but not limited to, chlorofluorocarbons, propane / butane, or dimethyl ether.

[0133] 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, water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty esters, polyethylene glycol, or fatty acid esters of sorbitol may be used.

[0134] When the cosmetic composition is formulated as a suspension, liquid diluents such as water, ethanol and propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth gum can be used as carrier components.

[0135] When the cosmetic composition is prepared as a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glyceryl fatty acid esters can be used as carrier components.

[0136] When the cosmetic composition is formulated as a shampoo, basic ingredients used in shampoo compositions, such as thickeners, surfactants, viscosity modifiers, humectants, pH regulators, preservatives, and essential oils, can be mixed with the pentapeptide conjugates of the present invention. CDE can be used as a thickener, anionic surfactants LES and amphoteric surfactants coconut oil betaine can be used as surfactants, polyquaternium salts can be used as viscosity modifiers, glycerol can be used as a humectant, and citric acid and sodium hydroxide can be used as pH regulators. Grapefruit extract and the like can be used as preservatives, and essential oils such as fir, mint, and rosemary, silk amino acids, amyl alcohol, or vitamin E can also be added.

[0137] In addition to the pentapeptide conjugate of the present invention and the carrier component as an active ingredient, 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, but are not limited thereto.

[0138] In still another aspect, the present invention provides a method for preventing or treating pain, comprising administering a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 to a subject.

[0139] In another aspect, the present invention provides a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 for use in preventing or treating pain.

[0140] In yet another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0141] In yet another aspect, the present invention provides a cosmetic composition for relieving pain, comprising a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0142] "Subject" may include humans. In addition, the term "subject" may be an object to which the pentapeptide of the present invention is to be administered, and the subject in need of administration may include individuals who have been diagnosed with pain, individuals who have developed pain, and individuals who wish to be administered to prevent the development of a disease or symptom or to improve health.

[0143] "Administering" means providing a predetermined substance to a patient by any suitable method, and the administration route of the composition of the present invention can be oral administration or parenteral administration by any general route, as long as the target tissue can be reached. In addition, the composition can be administered by any device capable of delivering the active agent to the target cells.

[0144] As described above, the pentapeptides of the present invention suppress pain caused by inflammatory reactions by inhibiting the expression of proinflammatory cytokines in muscle cells, prevent the transmission of pain by inhibiting the release of substance P in nerve endings, and effectively suppress pain by penetrating into nerve cells in the muscle layer simply by applying it to the skin. Therefore, the pentapeptides of the present invention can be effectively used as raw materials for pharmaceutical, quasi-drug, or cosmetic compositions to relieve, improve, prevent, or treat pain.

[0145] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0146] However, the following embodiments and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following embodiments and experimental examples.

[0147] [Example 1]

[0148] Synthesis and confirmation of physical properties of new pentapeptides

[0149] 1-1. Synthesis of pentapeptides

[0150] A novel peptide sequence "KFLIK" consisting of the amino acid sequence of SEQ ID NO: 1 was prepared using a known method. The molecular weight of the peptide comprising the amino acid sequence of SEQ ID NO: 1 was measured using a molecular weight meter and confirmed to be 647.4 Da.

[0151] 1-2. Evaluation of high temperature stability during long-term storage

[0152] 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, stored at 45° C. for 7 days, 14 days, 28 days, 60 days and 75 days, and then subjected to HPLC analysis.

[0153] As a result, Figure 1a As shown, it was confirmed that the peptide of the present invention maintained stability at 45°C for a maximum observation period of 75 days.

[0154] 1-3. Evaluation of high temperature stability

[0155] 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 subjected to HPLC analysis.

[0156] As a result, Figure 1b As shown, it was confirmed that the peptide of the present invention maintained stability at 121°C for a maximum heating time of 30 minutes.

[0157] [Experimental Example 1]

[0158] Confirmation of the inhibitory effect on the formation of SNARE complexes in nerve cells

[0159] 1-1. Confirmation of the inhibitory effect on SNARE complex formation

[0160] In order to confirm the effect of the pentapeptide of the present invention in inhibiting the release of neurotransmitters in nerve cells, a neuroblastoma-derived cell line (SH-SY5Y) was used to determine whether the formation of the SNARE complex was inhibited.

[0161] Specifically, SH-SY5Y cells were cultured at 3 × 10 5Cells were seeded at a density of 10 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours, after which the medium was replaced with serum-free medium. Subsequently, the pentapeptide was added to the 6-well plate at concentrations of 10 μM, 50 μM, and 100 μM. As a positive control, 0.2 μM botulinum toxin type C light chain (BoNT / C LC) was added and cultured. After 24 hours, cells were harvested, lysates were obtained, and Western blotting was performed against a syntaxin 1a antibody (synaptic systems, Germany).

[0162] As a result, Figure 2a As shown, when treated with the pentapeptide of the present invention, the formation of the SNARE complex was inhibited, which was similar to the positive control treated with botulinum toxin.

[0163] It can be seen from this that the pentapeptide of the present invention inhibits the formation of the SNARE complex and prevents the fusion of synaptic vesicles with the presynaptic membrane, thereby inhibiting the release of neurotransmitters.

[0164] 1-2. Confirmation of the effect on syntaxin 1a protein degradation

[0165] In order to confirm the effect of the pentapeptide of the present invention in inhibiting neurotransmitter release in nerve cells, it was confirmed whether the pentapeptide of the present invention has the ability to degrade the syntaxin 1a protein constituting t-SNARE.

[0166] Specifically, in the experimental group, 1 μg of recombinant syntaxin 1a protein (Novus Biologicals, USA) and 20 μM, 100 μM, and 200 μM of the pentapeptide of the present invention were added to the reaction buffer (50 mM HEPES, 40 mM 2-ME, 20 μM ZnCl2, pH 7.4). In the negative control group, only recombinant syntaxin 1a protein was added. After reacting at 37°C for 4 hours, Western blotting was performed using a syntaxin 1a antibody (Synaptic Systems, Germany).

[0167] As a result, Figure 2b As shown, it was confirmed that the band of the recombinant syntaxin 1a protein was attenuated in a concentration-dependent manner when treated with the pentapeptide of the present invention.

[0168] It can be seen from this that the pentapeptide of the present invention has the effect of inhibiting neurotransmitter release by degrading syntaxin 1a protein and preventing the formation of SNARE complex.

[0169] [Experimental Example 2]

[0170] Confirmation of the inhibitory effect on the release of neurotransmitters in nerve cells

[0171] In order to confirm the effect of the pentapeptide of the present invention in inhibiting the release of neurotransmitters in nerve cells, the secretion of the neurotransmitter acetylcholine was measured using a neuroblastoma-derived cell line (SH-SY5Y).

[0172] Specifically, SH-SY5Y cells were injected into a culture dish and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was then replaced with a serum-free medium and cultured for 48 hours. Subsequently, the pentapeptide was added to the culture dish at concentrations of 1 μM, 10 μM, and 50 μM. As a positive control group, 50 nM tetanus toxin was added thereto and cultured. After a period of time, the release of acetylcholine was induced by nicotine (NIC) and potassium chloride (KCl). The amount of acetylcholine secreted in the culture medium was then measured using an acetylcholine assay kit.

[0173] As a result, Figure 3 As shown, compared with the control group not treated with any agent, the amount of acetylcholine release increased in the group treated with only nicotine (NIC) and potassium chloride (KCl), but was significantly inhibited in the group treated with the pentapeptide of the present invention.

[0174] [Experimental Example 3]

[0175] Human efficacy evaluation confirms the inhibitory effect of pentapeptide on hyperhidrosis

[0176] In order to confirm that the pentapeptide according to the present invention is actually effective in inhibiting sweat secretion in the palms, soles and armpits, images of sweat secretion levels were taken using starch and iodine, and intensity analysis was performed using Image J software.

[0177] Specifically, the sweat secretion degree of 10 subjects suffering from local hyperhidrosis (palms, soles, armpits) was evaluated for human efficacy. A solution of 2000ppm of the pentapeptide of the present invention was applied to the palms, soles or armpits of the subject's side using a microneedle roller. At this time, the palms, soles and armpits of the opposite side were set as a control group, and in the case of the control group, PBS was applied with a microneedle roller to replace the solution containing the pentapeptide. After sufficient absorption and natural drying, iodine solution was applied to the same area with a sterile gauze pad and allowed to dry naturally. When the iodine solution was completely dry, corn starch was evenly applied to the iodine-coated area with cotton to form a starch layer. When sweat secretion occurs, iodine and starch dissolve, and the color becomes bluish purple. Therefore, the change of color over time is observed in the palms, soles or armpits of the subject. After collecting the image, intensity analysis was performed using Image J software.

[0178] As a result, Figure 4 As shown in FIG, it was confirmed that the amount of sweat secreted by the hands of the subjects who applied the care solution containing the pentapeptide of the present invention was reduced by about 74%. Figure 5 As shown in FIG, it was confirmed that the amount of sweat secreted from the soles of the feet of the subjects who applied the care solution containing the pentapeptide of the present invention was reduced by about 58%, and as shown in FIG. Figure 6 As shown, it was confirmed that the amount of sweat secreted from the armpits of the subjects who applied the care solution containing the pentapeptide of the present invention was reduced by about 88%.

[0179] It can be seen from this that simply by applying the pentapeptide of the present invention to the skin of a hyperhidrosis patient, a therapeutic effect can be provided in terms of reducing sweating and preventing excessive sweating, thereby providing an effect of fundamentally blocking the occurrence of body odor.

[0180] [Experimental Example 4]

[0181] Confirmation of intracellular or skin tissue penetration patterns

[0182] 4-1. Preparation of rhodamine-pentapeptide conjugate

[0183] In order to confirm whether the peptide of the present invention can penetrate cells or tissues, a rhodamine-pentapeptide conjugate conjugated to a fluorescent material was prepared. First, a 10 mg / ml pentapeptide solution was prepared using 100 mM sodium bicarbonate (pH 9.0), and a 1 mg / ml NHS-rhodamine (Thermo Scientific, 46406) solution was prepared using dimethylformamide. The prepared solutions were mixed so that the pentapeptide: NHS-rhodamine = 1:10 (molar ratio). After shading, the solution was inverted and reacted at room temperature for 1 hour, the reactant was dialyzed, and then conjugation was confirmed by LC / MS.

[0184] 4-2. Confirmation of intracellular penetration

[0185] In order to confirm that the pentapeptide of the present invention penetrates into the nerve cells, SH-SY5Y cells were plated at 3×10 5 The cells were seeded at a density of 10 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours, after which the culture medium was replaced with a serum-free medium. After treatment with various concentrations of rhodamine-peptide conjugates for 4 hours, the cells were fixed by treatment with 4% paraformaldehyde and the cell nuclei were stained using a DAPI staining kit (Invitrogen, USA). Fluorescence microscopy was used to observe the intracellular peptide penetration. Blue represents the cell nuclei stained with DAPI, and red represents the "rhodamine-pentapeptide" conjugate.

[0186] As a result, Figure 7 As shown, the coexistence of blue and red confirmed that the pentapeptide of the present invention permeated into nerve cells.

[0187] 4-3. Penetration into tissues middle Confirmation

[0188] After depilation of the backs of 7-week-old SD rats, rhodamine-pentapeptide was applied thereto, and 1 hour later, the rats were killed for analysis. Skin tissue was collected from the application site and formalin fixed for one day. After making paraffin blocks using fixed tissue and sectioning them, immunohistochemical staining was performed using neuronal cell marker TrkB Ab (Cell signaling, USA). Then, nuclear staining was performed using a DAPI staining kit (Invitrogen, USA), and images of peptide penetration into the tissue were observed using a fluorescence microscope. Blue represents cell nuclei stained with DAPI, red represents rhodamine-pentapeptide conjugates, and green represents TrkB (nerve fiber marker).

[0189] As a result, Figure 8 As shown, it was confirmed that the pentapeptide of the present invention penetrated into the muscle layer of the skin tissue and co-localized with the nerve cell marker.

[0190] From this, it can be seen that since the pentapeptide of the present invention can penetrate into nerve cells, it is absorbed percutaneously and penetrates into the muscle layer of the skin tissue and coexists with nerve cell markers in the muscle layer. Therefore, the pentapeptide can show the effect of inhibiting sweat secretion and blocking the production of body odor caused by sweat secretion.

[0191] It can be seen from this that since the pentapeptide of the present invention can penetrate into nerve cells, it is absorbed percutaneously into the muscle layer of the skin tissue and coexists with nerve cell markers in the muscle layer. Therefore, the pentapeptide can inhibit the expression of pro-inflammatory cytokines in muscle cells and the release of pain transmitters in nerve endings, thereby showing the effect of inhibiting the generation and transmission of pain.

[0192] [Experimental Example 5]

[0193] Confirmation of the body odor-inhibiting effect of pentapeptides using strains that cause body odor

[0194] The antibacterial effect of the pentapeptide of the present invention was confirmed using Corynebacterium, which is a representative odor-causing bacterium known to produce odor by decomposing substances contained in sweat.

[0195] Specifically, dried Corynebacterium (C. xerosis, KCTC9105) purchased from the Korean Collection of Type Cultures was used as the strain causing body odor. This strain was inoculated into brain heart infusion (Difco, USA) medium and cultured in a 37°C incubator for 2 days. The cultured strain was diluted to 10% by adding the same medium to the cultured strain. 6A diluted solution with a concentration of 100 CFU / ml was used as the test bacterial solution. 100 μL of the test bacterial solution was aliquoted into a 96-well plate and the pentapeptide was added according to the concentration. In the positive control, 1% triclosan was added instead of the pentapeptide. After 48 hours of incubation, the absorbance at 600 nm was measured using a spectrophotometer to confirm the effectiveness of inhibiting the growth of odor-causing bacteria.

[0196] As a result, Figure 9 As shown, it was confirmed that the proliferation of the strains that cause body odor was significantly inhibited when treated with the pentapeptide of the present invention. In particular, it was confirmed that the antibacterial effect was excellent in the groups treated with 1 mg and 2 mg of the pentapeptide, and was at the same level as the antibacterial effect of the positive control group.

[0197] It can be seen from this that the pentapeptides of the present invention can effectively inhibit the unique body odor produced during sweating by inhibiting the proliferation of strains that cause body odor.

[0198] [Experimental Example 6]

[0199] Confirmation of the inhibitory effect of pentapeptide treatment on the release of proinflammatory cytokines in myocytes 6-1. Proinflammatory cytokines Confirmation of subgene expression levels

[0200] To confirm the pain-alleviating effects of the pentapeptides of the present invention, the expression patterns of inflammation-related cytokines were examined using mouse myoblasts. Proinflammatory cytokines are involved in pain in neuropathy. When proinflammatory cytokines such as TNF-α are expressed after nerve damage, an inflammatory response is triggered, leading to pain.

[0201] Specifically, mouse myoblast C2C12 cells were cultured at a density of 2 × 10 5 The density of each cell / well was seeded in a 6-well plate and then cultured in DMEM (10% PBS) culture medium for 3 days. When the cells grew to about 70% to 80% of the plate, the culture medium was replaced with differentiation medium (DMEM, 5% horse serum) and cultured for 3 days. Subsequently, the culture medium was replaced with differentiation medium (DMEM, 5% horse serum) and the cells were cultured for 4 days to induce myoblast differentiation to myotubes. The culture medium was then replaced with serum-free medium and incubated for 2 hours, and pretreated with 1 μM, 10 μM and 100 μM pentapeptides for 1 hour. Then, 20 nM TNF-α was applied to it and the cells were cultured for 24 hours to induce an inflammatory response.

[0202] Then, reclaim cells, isolate RNA, and use cDNA synthesis kit (Intron, Korea) to synthesize cDNA.Use synthetic cDNA, PCR premix (Intron, Korea) and IL-1α, TNF-α, IL-1β and COX-2 primers to carry out polymerase chain reaction, and the amplified PCR product is electrophoresed on agarose gel, and DNA bands are examined to compare the mRNA expression level of the gene.The primer sequences used now are as shown in [Table 1].

[0203] [Table 1]

[0204]

[0205]

[0206] As a result, Figure 10a As shown in the results, it was confirmed that the mRNA expression levels of IL-1α, IL-1β, COX-2, and TNF-α (i.e., proinflammatory cytokines) were significantly increased when myotube inflammation was induced by treatment with TNF-α compared to the untreated group (control). However, it was confirmed that the magnitude of the increase in proinflammatory cytokines was reduced in the treatment groups using 1 μM and 10 μM of the pentapeptide of the present invention.

[0207] 6-2. Confirmation of pro-inflammatory cytokine protein expression levels

[0208] On the other hand, in order to confirm the expression level of inflammatory cytokine proteins, for some cells recovered in 6-1 (WB), the proteins were quantified after separation of the cell lysate and mixed with sample buffer to prepare samples. The samples were developed on SDS-PAGE and nitrocellulose membranes and blocked with 5% skim milk. COX-2 antibody (Cell Signaling Technology, USA), IL-1β antibody (Cell Signaling Technology, USA) and TNF-α antibody (Cell Signaling Technology, USA) were first bound to the sample and then bound to the secondary antibody. After treatment with ECL solution, the expression level of the target protein was observed.

[0209] As a result, Figure 10b As shown in the results, it was confirmed that when TNF-α treatment induced inflammation in myotubes, the protein expression levels of COX-2, IL-1β, and TNF-α (i.e., the amount of proinflammatory cytokine protein) were significantly increased compared to the untreated group (control). However, it was confirmed that in the case of the pentapeptide-treated group of the present invention, the magnitude of the increase in the expression of proinflammatory cytokines was reduced in a concentration-dependent manner.

[0210] From this, it can be seen that the pentapeptide of the present invention exhibits the effect of reducing the expression of IL-1α, IL-1β, COX-2 and TNF-α in myotubes (i.e., the expression of proinflammatory cytokines) at both the mRNA level and the protein level, and therefore can be effectively used as a substance for preventing or treating pain.

[0211] [Experimental Example 7]

[0212] Confirmation of the inhibitory effect on neurotransmitter release in nerve cells

[0213] To confirm the pain-relieving effect of the pentapeptide of the present invention, the release of substance P at synapses was determined using mouse nerve cells. Substance P transmits pain from the periphery to the central nervous system, so inhibiting the release of substance P can produce a pain-relieving effect.

[0214] Specifically, dorsal root ganglia (DRG) were isolated from the spinal cord of 6-week-old rats and washed, and then nerve cells were isolated. After the isolated nerve cells were equally divided in a 24-well plate and cultured for one day, mitosis inhibitors were mixed with the culture medium for culturing nerve cells, and only pure nerve cells were cultured for 6 days. Then, in order to add nutrients, the culture medium was replaced with B-27 culture medium (serum-free, 1% penicillin), and the cells were cultured for 24 hours, followed by pretreatment with 1 μM and 10 μM pentapeptides for 1 hour. Then, the release of substance P was induced by 100 μM capsaicin for 30 minutes, and the amount of substance P released was analyzed by ELISA.

[0215] As a result, Figure 11 As shown, it was confirmed that the release of substance P increased when treated with capsaicin compared to the untreated group (control), but the magnitude of the increase in substance P release was significantly reduced in the group treated with the pentapeptide of the present invention.

[0216] From this, it can be seen that the pentapeptide of the present invention can be effectively used as a substance for preventing or treating pain by inhibiting the release of substance P from nerve cells and preventing the transmission of pain signals.

[0217] [Experimental Example 8]

[0218] Human efficacy evaluation confirms the pain relief effect of pentapeptides

[0219] In order to confirm whether the pentapeptide according to the present invention actually has the effect of inhibiting pain, after inducing delayed onset muscle soreness in 20 subjects, a care solution containing the pentapeptide of the present invention was applied to the painful area to evaluate the efficacy of relieving human pain.

[0220] Specifically, in order to produce delayed onset muscle soreness in the subjects, a total of 3 sets of exercises were performed, in which the subjects were required to stand with their feet hip-width apart, with their backs straight, and to raise and lower their heels as much as possible for 2 minutes, followed by a 3-minute rest. After 24 hours, a solution containing 2000ppm of the pentapeptide of the present invention was applied to the painful area. Then, after 24 hours, 48 hours, and 72 hours, the subjects were self-assessed on a numerical rating scale (NRS) (0 for no pain, 10 for the worst pain imaginable) felt while walking. The average value of pain intensity (average PID score) was calculated using the following equation, and after the 72-hour test, the degree of pain relief was confirmed by a subject questionnaire.

[0221] [Equation]

[0222] Pain intensity difference (PID) = pain intensity after administration of the pentapeptide of the present invention - pain intensity before administration of the pentapeptide of the present invention

[0223] As a result, Figure 12a and Figure 12b As shown, it was confirmed that in the experimental group to which the pentapeptide of the present invention was applied, the pain level induced 24 hours after exercise was significantly improved 72 hours after exercise (48 hours after application of the care solution).

[0224] It can be seen from this that simply by applying the pentapeptide of the present invention to the skin of a patient suffering from muscle pain, a therapeutic effect of reducing pain can be provided. <110> Kelgen GmbH <120> Composition comprising pentapeptide as active ingredient <130> 2021-OPA-5102 <150> KR 10-2020-0071659 <151> 2020-06-12 <150> KR 10-2020-0071660 <151> 2020-06-12 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Pentapeptide <400> 1 Lys Phe Leu Ile Lys 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1α forward primer <400> 2 gaagtcaggc agaagtgctc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1α reverse primer <400> 3 gtgcacccga ctttgttctt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-α forward primer <400> 4 cgtcagccga tttgctatct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-α reverse primer <400> 5 cggactccgc aaagtctaag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1β forward primer <400> 6 caaggagaac caagcaacga 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1β reverse primer <400> 7 ttggccgagg actaaggagt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 forward primer <400> 8 tgagtggtag ccagcaaagc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 reverse primer <400> 9 ctgcagtcca ggttcaatgg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 10 accacagtcc atgccatcac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 11 tccaccaccc tgttgctgta 20

Claims

1. Use of a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of a medicament for preventing or treating hyperhidrosis, in, The hyperhidrosis includes localized hyperhidrosis and generalized hyperhidrosis. Wherein, the hyperhidrosis occurs in any one skin area selected from the group consisting of face, forehead, neck, hands and feet.

2. The use according to claim 1, wherein the amount of sweating is reduced by 45% to 90% compared with a control group not treated with the drug.

3. Use of a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of a composition for preventing or alleviating hyperhidrosis or inhibiting body odor, in, The hyperhidrosis includes localized hyperhidrosis and generalized hyperhidrosis. Wherein, the hyperhidrosis occurs in any one skin area selected from the group consisting of face, forehead, neck, hands and feet.

4. The use according to claim 3, wherein the body odor is an unpleasant odor caused by decomposition products produced by bacteria decomposing sweat gland secretions.

5. The use according to claim 4, wherein the body odor is an unpleasant odor from the armpits, feet, hands, forehead, neck, face or scalp.

6. Use of a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 in the preparation of a cosmetic for alleviating hyperhidrosis or inhibiting body odor, in, The hyperhidrosis includes localized hyperhidrosis and generalized hyperhidrosis. Wherein, the hyperhidrosis occurs in any one skin area selected from the group consisting of face, forehead, neck, hands and feet.

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