KR102570768B1 - Composition comprising pentapeptide as an active ingredient for treatment of hyperhidrosis - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating hyperhidrosis or a composition for suppressing body odor, comprising a pentapeptide as an active ingredient, wherein the pentapeptide of the present invention inhibits the release of excessive neurotransmitters, thereby preventing excessive sweat production and body odor. It can be used as a raw material for pharmaceuticals or cosmetics for the purpose of preventing, alleviating, or treating the occurrence of

Description

Composition comprising pentapeptide as an active ingredient for treatment of hyperhidrosis}

The present invention relates to the use of a novel pentapeptide for suppressing excessive sweating or suppressing body odor.

Hyperhidrosis refers to a skin disorder characterized by abnormally increased sweating or secretion of sweat in excess of that required for body temperature regulation. Hyperhidrosis occurs due to genetic causes or physical and emotional stress, and may be localized throughout the body or in specific areas of the body. When excessive sweating appears locally on the palms, soles, face, armpits, scalp, etc., it is called primary or localized hyperhidrosis. Systemic or secondary hyperhidrosis usually involves the entire body.

Current methods of treating hyperhidrosis include the use of antiperspirants, iontophoresis to temporarily block sweat glands, botox therapy to temporarily block nerves that stimulate sweating (WO2010-078242), and endoscopic thoracic sympathectomy or sweat glands. There are surgical methods to remove it. Among them, in the case of the method of temporarily blocking nerves using botulinum toxin, the paralysis effect is reversible for an average of 6 months, so this treatment requires repeated injections of botulinum toxin, and botulinum toxin is a method that can be recognized by the patient's immune system. Because of its size, an immune response to the drug may be induced. Therefore, there is a need to develop a molecule that has a simple and stable molecular structure that does not induce an immune response, has high sweat reduction activity, and enables non-invasive treatment with long-lasting and low side effects.

On the other hand, body odor occurs in the process of decomposition of sweat secreted by the sweat glands by microorganisms or bacteria. In the process of decomposition of fat, protein, sebum, and contaminants contained in sweat secreted by microorganisms or bacteria, unpleasant body odors such as body odor, foot odor, and head odor are generated. In general, methods such as deodorization, antibacterial, and masking are used to control body odor, but if the wrong fragrance is used to cover body odor, it may rather emit a bad smell and cause adverse effects. In addition, when the deodorant component is used on the skin, only a temporary effect is provided until the next washing after treatment, and there is a hassle of reprocessing after washing.

Accordingly, the present inventors have made diligent efforts to develop a material capable of suppressing body odor by suppressing excessive sweating, which can be said to be the fundamental cause of body odor. As a result, the pentapeptide newly synthesized by the present inventors has a stable molecular structure and The present invention was completed by confirming that it can be usefully used for treatment of hyperhidrosis and control of body odor because it penetrates into skin tissue through the dermis and exhibits high sweat reduction activity.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating hyperhidrosis.

Another object of the present invention is to provide a cosmetic composition for preventing or alleviating hyperhidrosis or suppressing body odor.

To this end, one aspect of the present invention provides a pharmaceutical composition for preventing or treating hyperhidrosis, comprising as an active ingredient a pentapeptide composed of the amino acid sequence of KFLIK.

Another aspect of the present invention provides a cosmetic composition for preventing or alleviating hyperhidrosis, or suppressing body odor, comprising a pentapeptide composed of the amino acid sequence of KFLIK as an active ingredient.

According to the present invention, the pentapeptide composed of the amino acid sequence of KFLIK of the present invention is absorbed to the muscle layer of skin tissue through transdermal (skin), and suppresses abnormal release of neurotransmitters from nerve terminals to reduce sweating. Therefore, it can be used to prevent, alleviate, improve, or treat the symptoms of hyperhidrosis just by applying it.

In addition, the pentapeptide of the present invention can block the cause of body odor in advance by suppressing excessive sweat production and provide a semi-permanent effect of suppressing body odor.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Figures 1a and 1b show the thermal stability evaluation results of KFLIK peptides: Figure 1a shows the results of confirming the high-temperature stability of the peptide during long-term storage at 45 ℃, Figure 1b shows the results of the peptide at the maximum warming temperature (121 ℃) It shows the result of confirming high temperature stability.
Figures 2a and 2b show the effect of inhibiting the formation of SNARE complexes by KFLIK peptide treatment in neurons: Figure 2a shows the results of Western blotting to determine whether SNARE complexes are formed, and Figure 2b shows the formation of SNARE complexes It shows the result of confirming the degree of degradation of syntaxin 1a.
Figure 3 shows the effect of inhibiting the release of neurotransmitters by KFLIK peptide treatment in nerve cells.
Figure 4 shows the relative sweat reduction effect by the KFLIK peptide treatment of the present invention in the palm of the patient's hyperhidrosis, A is an image of the iodine-starch test result, B is the sweat reduction effect using Image J software It is shown as the result of intensity analysis.
Figure 5 shows the relative sweat reduction effect by the KFLIK peptide treatment of the present invention on the soles of patients with hyperhidrosis, A is an image of the iodine-starch test result, B is the sweat reduction effect using Image J software It is shown as the result of intensity analysis.
Figure 6 shows the relative sweat reduction effect by the KFLIK peptide treatment of the present invention in the axillary region of patients with hyperhidrosis, A is an image of the iodine-starch test result, and B is the sweat reduction effect using Image J software It is shown as the result of intensity analysis.
Figure 7 is a fluorescence micrograph confirming that KFLIK peptide penetrates into neurons by attaching rhodamine, a fluorescent substance, to KFLIK peptide: A shows the coexistence of red rhodamine-pentapeptide with blue-stained nuclei of neurons The blue color in B represents the nuclei of neurons stained with DAPI, and the red color in C represents the rhodamine-peptide.
8 is a fluorescence micrograph showing that the KFLIK peptide penetrates into the muscle layer by collecting skin tissue after applying the rhodamine-KFLIK peptide to the back of an experimental animal: Blue in A represents the nucleus of a cell stained with DAPI, and B is Green represents the TrkB neurofiber marker, C represents rhodamine-pentapeptide in red, and yellow in D represents penetration of the pentapeptide of the present invention into nerve cells in the muscle layer.
Figure 9 shows the body odor inhibitory effect of the KFLIK peptide of the present invention, and shows the growth inhibitory effect of Corynebacterium as a result of measuring absorbance using a spectrophotometer. P/C is 1% triclosan as a positive control.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating hyperhidrosis, comprising as an active ingredient a pentapeptide composed of the amino acid sequence of KFLIK.

In the present specification, the term 'peptide' refers to a linear or cyclic molecule formed by binding amino acid residues to each other by a peptide bond. Production of the peptide can be achieved by conventional biological or chemical synthesis methods known in the art, and for example, solid-phase synthesis techniques.

In the present specification, the term 'pentapeptide' refers to a linear molecule composed of five amino acid residues, and the pentapeptide of the present invention refers to a linear peptide molecule in which Lys, Phe, Leu, Ile, and Lys are linked in sequence.

The 'peptide' and 'pentapeptide' may be amino acid variants or fragments having different sequences due to deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function. Amino acid exchanges that do not entirely alter the activity of the peptide are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like. Accordingly, the present invention includes peptides having substantially the same amino acid sequence as the pentapeptide composed of the amino acid sequence of KFLIK, and variants thereof or active fragments thereof. The substantially identical protein is 75% or more, preferably 80% or more, such as 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence homology with the amino acid sequence of the KFLIK. It refers to an amino acid sequence, but is not limited thereto, and is included in the scope of the present invention if it has a homology of 75% or more of an amino acid sequence and has the same activity. In addition, the peptide of the present invention may additionally include a targeting sequence, a tag (tag), a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, uptake, potency, potency, etc.), altered specificity (eg broad biological activity spectrum), reduced antigenicity, N A protecting group may be bonded to the -terminus or C-terminus. For example, the protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but modification of the peptide, in particular, stability of the peptide may be improved. If there is a component, it can be included without limitation. The 'stability' is used to include not only in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo, but also storage stability (eg, storage stability at room temperature).

The pentapeptide composed of the amino acid sequence of KFLIK of the present invention inhibits the formation of the SNARE complex to inhibit excessive release of neurotransmitters from nerve terminals, thereby preventing or treating hyperhidrosis.

In addition, the pentapeptide composed of the amino acid sequence of KFLIK of the present invention can prevent or alleviate hyperhidrosis by exhibiting the effect of suppressing the generation of sweat in the skin area where hyperhidrosis appears.

In addition, since the pentapeptide composed of the amino acid sequence of KFLIK of the present invention penetrates into the muscle layer of skin tissue through transdermal absorption, the symptoms of hyperhidrosis can be improved or alleviated only by applying to the lesion site. 'Hyperhidrosis' in the present invention means a disease state characterized by excessive and uncontrolled sweating beyond what is necessary to cool the body, and means a disease or condition in which excessive sweat is secreted due to hyperfunction of the sweat glands and disturbance of their cholinergic stimulation. In the present invention, hyperhidrosis includes both local hyperhidrosis and systemic hyperhidrosis, and may occur in any one skin area selected from the group consisting of, for example, the face, forehead, neck, hands, feet, and armpits, but is not limited thereto. don't

In a specific example, when the composition containing the pentapeptide of the present invention is applied to the palm, sole and armpit of a patient with hyperhidrosis, sweat production is increased by 45% to 90% compared to the control area in the opposite direction to which the above composition is not applied. % reduction was confirmed.

The pharmaceutical composition for preventing or treating hyperhidrosis according to the present invention is an oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injection solution according to conventional methods, respectively. It may be formulated and used in a form, and suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions may be included for formulation.

The pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl, which are commonly used in formulation. pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

The pharmaceutical composition may be administered orally or parenterally (for example, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or locally applied) depending on the desired method, and the dosage depends on the patient's condition and It varies depending on body weight, severity of disease, drug type, administration route and time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other anti-obesity therapeutic agents, may be administered simultaneously with, separately from, or sequentially with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The effective amount of the pharmaceutical composition may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivity rate, excretion rate, disease type, concomitant drug of the active ingredient in the body, route of administration, severity of obesity, It may increase or decrease depending on gender, weight, age, etc., and for example, about 0.0001 μg to 500 mg, preferably 0.01 μg to 100 mg per 1 kg of the patient's body weight per day may be administered.

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

The pentapeptide composed of the amino acid sequence of KFLIK of the present invention can prevent or treat hyperhidrosis by inhibiting the formation of the SNARE complex to inhibit excessive release of neurotransmitters from nerve terminals, and can also prevent or treat body odor caused by excessive sweat production. can suppress

In addition, the pentapeptide composed of the amino acid sequence of KFLIK of the present invention exhibits the effect of suppressing the generation of sweat in the skin area where hyperhidrosis appears, thereby preventing or alleviating hyperhidrosis and suppressing body odor caused by excessive sweat production. can

In addition, the pentapeptide composed of the amino acid sequence of KFLIK of the present invention inhibits the growth of odor-causing strains, thereby suppressing the decomposition of substances contained in sweat to prevent body odor from occurring.

Details of the peptide, pentapeptide and hyperhidrosis are as described above.

In the present specification, the 'body odor' refers to the odor of decomposition products generated when sweat gland secretions, that is, fat, protein, sebum, and/or waste products discharged with sweat are decomposed by bacteria, and specifically, sweat in the body. It may be an odor generated in areas of the body where odor is frequently generated, such as armpits, feet, hands, forehead, neck, face, or scalp.

In the present specification, the 'body odor suppression' means to prevent odors caused by decomposition products of sweat gland secretions by microorganisms or to suppress the generation of sweat, which is the cause of body odor. Specifically, the suppression of body odor may be to prevent odor from the armpits, feet, hands, forehead, neck, face, or scalp, or to suppress sweat production in the area, and more specifically, to prevent odor. there is.

In another aspect, the present invention provides a cosmetic composition for preventing or alleviating hyperhidrosis or suppressing body odor, comprising as an active ingredient a pentapeptide composed of the amino acid sequence of KFLIK.

The cosmetic composition may be prepared in any formulation commonly prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto.

The cosmetic composition includes flexible lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, deodorant, hair tonic, hair cream, hair lotion, hair shampoo, It may be prepared in various forms such as hair rinse, hair conditioner, hair spray, hair aerosol, pomade, solution such as gel, sol gel, emulsion, oil, wax, aerosol, etc., but is not limited thereto.

The cosmetic composition of the present invention may include other additives such as excipients and carriers in addition to the pentapeptide composed of the amino acid sequence of KFLIK, and it is possible to apply and blend ordinary ingredients formulated in general skin cosmetics as needed.

When the formulation of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or oxide as a carrier component Zinc and the like may be used.

When the form of the formulation of the cosmetic composition is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, especially in the case of a spray, additionally chlorofluorohydrocarbon , propellants such as, but not limited to, propane/butane or dimethyl ether.

When the form of the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl Benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, or sorbitan fatty acid esters may be used.

When the form of the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethyl sorbitol ester and polyoxyethylene sorbitan ester , microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the formulation of the cosmetic composition is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid Amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

When the formulation of the cosmetic composition is a hair shampoo, base components for forming the shampoo, such as a thickener, a surfactant, a viscosity modifier, a moisturizer, a pH modifier, a preservative, and an essential oil, may be mixed with the Trolox-peptide conjugate of the present invention. there is. CDE may be used as the thickener, LES, an anionic surfactant, and coco betaine, an amphoteric surfactant, may be used as the surfactant, and polyquater may be used as the viscosity modifier. Glycerin may be used, and citric acid and sodium hydroxide may be used as the pH adjusting agent. Grapefruit extract and the like may be used as the preservative, and in addition, essential oils such as cedarwood, peppermint, and rosemary, and silk amino acids, pentaol, or vitamin E may be added.

Ingredients included in the cosmetic composition are components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and Conventional adjuvants such as spices may be further included, but are not limited thereto.

As described above, the pentapeptide of the present invention inhibits the excessive release of neurotransmitters by inhibiting the formation of the SNARE complex in nerve cells, and penetrates into the nerve cells of the muscle layer just by applying to areas where sweat is excessively produced. Since it is excellent in suppressing sweat production, it can be used as a raw material for pharmaceutical or cosmetic compositions for the purpose of preventing, improving, alleviating or treating hyperhidrosis or suppressing body odor.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following Examples and Experimental Examples are only for exemplifying the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

[Example 1]

Synthesis of novel pentapeptide and confirmation of physical properties

1-1. Synthesis of pentapeptides

A novel peptide sequence 'KFLIK' composed of the amino acid sequence shown in SEQ ID NO: 1 was prepared using a known method. As a result of measuring the molecular weight of the peptide including the amino acid sequence of SEQ ID NO: 1 using a molecular weight measuring instrument, it was confirmed that the molecular weight corresponds to 647.4 Da.

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

The peptide of the present invention composed 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 HPLC analysis was performed.

As a result, as shown in Figure 1a, it was confirmed that the peptide of the present invention maintains stability for 75 days, which is the maximum observation day, at 45 °C.

1-3. High temperature stability evaluation

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 HPLC analysis was performed.

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

[Experimental Example 1]

Confirmation of inhibitory effect on SNARE complex formation in nerve cells

1-1. Confirmation of inhibitory effect on SNARE complex formation

In order to confirm the effect of the pentapeptide of the present invention on inhibiting neurotransmitter release in nerve cells, a neuroblastoma-derived cell line (SH-SY5Y) was used to determine whether or not SNARE complex formation was inhibited.

Specifically, SH-SY5Y cells were seeded in a 6-well plate at a density of 3X10 ⁵ cells/well and cultured in a 37°C, 5% CO ₂ incubator for 24 hours, and then the medium was replaced with a serum-free medium. Thereafter, pentapeptides at concentrations of 10 μM, 50 μM, and 100 μM were treated in a 6-well plate, and as a positive control, 0.2 μM of botulinum toxin type C light chain (BoNT/C LC) was treated and cultured. After 24 hours, cells were collected, lysates were obtained, and western blotting was performed for synaptic 1a antibodies (synaptic systems, Germany).

As a result, as shown in [FIG. 2a], treatment with the pentapeptide of the present invention suppressed the formation of SNARE complexes, similar to the positive control group treated with botulinum toxin.

From this, it can be seen that the pentapeptide of the present invention has an effect of inhibiting neurotransmitter release by inhibiting the formation of a SNARE complex and preventing synaptic vesicles from fusing with the presynaptic membrane.

1-2. Syntaxin 1a proteolytic effect confirmed

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

Specifically, in the experimental group, 1 μg of recombinant syntaxin 1a protein (Novus biologicals, USA) and the pentapeptide of the present invention at concentrations of 20 μM, 100 μM, and 200 μM were mixed in a reaction buffer (50 mM HEPES, 40 mM 2-ME, 20 μM ZnCl ₂ , pH 7.4). and, as a negative control, only recombinant syntaxin 1a protein was treated. After reacting for 4 hours at 37° C., Western blotting was performed using Syntaxin 1a antibody (synaptic systems, Germany).

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

Through this, it can be seen that the pentapeptide of the present invention has the effect of inhibiting the release of neurotransmitters by degrading the syntaxin 1a protein and preventing the formation of the SNARE complex.

[Experimental Example 2]

Confirmation of neurotransmitter release inhibitory effect in nerve cells

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

Specifically, the SH-SY5Y cells were injected into the dish and cultured in a 37°C, 5% CO ₂ incubator for 24 hours, and then the medium was replaced with a serum-free medium and cultured for 48 hours. Thereafter, the dishes were treated with pentapeptides at concentrations of 1 μM, 10 μM, and 50 μM, and as a positive control, 50 nM tetanus was treated with the dishes and cultured. After a certain period of time, the release of acetylcholine was induced by treatment with nicotine (NIC) and potassium chloride (KCl). Then, the amount of acetylcholine secreted in the medium was measured using an acetylcholine assay kit.

As a result, as shown in [Figure 3], in the group treated with only nicotine (NIC) and potassium chloride (KCl), the amount of acetylcholine released increased compared to the control group treated with nothing, but the group treated with the pentapeptide of the present invention In all cases, the release of acetylcholine was significantly suppressed.

[Experimental Example 3]

Confirmation of hyperhidrosis inhibitory effect of pentapeptide through human efficacy evaluation

In order to confirm that the pentapeptide according to the present invention actually has an effect of inhibiting sweat secretion in the palms, soles and armpits, the degree of sweat secretion was taken as an image using starch and iodine, and intensity analysis using Image J software proceeded.

Specifically, 10 subjects with local hyperhidrosis (palms, soles, armpits) were evaluated for human efficacy on the degree of sweat secretion. A solution containing 2000 ppm of the pentapeptide of the present invention was applied to the palm, sole, or armpit of the experimenter in one direction using a meso roller. At this time, the palms, soles, and armpits in the opposite direction were set as the control group, and in the case of the control group, PBS was applied with a mesoroller instead of the solution containing the pentapeptide. After sufficient absorption and air drying, an iodine solution was applied to the same area with a sterile gauze pad and air dried. When the iodine solution was completely dry, corn starch was applied evenly over the iodine-coated area with cotton to form a starch layer. When sweat is secreted, iodine and starch dissolve and change color to blue-purple, so the color change over time was observed in the subject's palms, soles, or armpits. After image capture, intensity analysis was performed using Image J software.

As a result, through [Figure 4], it was confirmed that the amount of sweat secreted from the hands of the test subjects who applied the lotion containing the pentapeptide of the present invention was reduced by about 74%. In addition, through [Figure 5], it was confirmed that the amount of sweat secreted from the soles of the test subjects who applied the lotion containing the pentapeptide of the present invention decreased by about 58%. It was confirmed that the amount of sweat secreted from the armpit of the test subject who applied the lotion containing the peptide was reduced by about 88%.

Through this, the pentapeptide of the present invention can provide a therapeutic effect in reducing sweating and preventing excessive sweat production just by applying it to the skin of patients with hyperhidrosis, thereby fundamentally blocking the occurrence of body odor It can be seen that it can provide.

[Experimental Example 4]

Intracellular or skin tissue penetration pattern confirmation

4-1. Preparation of rhodamine-pentapeptide conjugates

In order to confirm whether or not the peptide of the present invention penetrates cells or tissues, a rhodamine-pentapeptide conjugate conjugated with 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 solution was mixed so that pentapeptide: NHS-rhodamine = 1:10 (molar ratio). After shading, reacting at room temperature for 1 hour while inverting, and after dialysis of the reactants, conjugation was confirmed by LC/MS.

4-2. Confirmation of intracellular penetration

In order to confirm whether the pentapeptide in the present invention penetrates into the nerve cells, SH-SY5Y cells were seeded in a 6-well plate at a density of 3X10 ⁵ cells/well. After culturing for 24 hours in a 37°C, 5% CO ₂ incubator, the medium was replaced with a serum-free medium. After treatment with the rhodamine-peptide conjugate for 4 hours at each concentration, cells were fixed by treatment with 4% paraformaldehyde, and nuclei were stained using a DAPI staining kit (Invitrogen, USA). Intracellular peptide penetration was observed through a fluorescence microscope. Blue represents the nuclei of cells stained with DAPI, and red represents the 'rhodamine-pentapeptide' conjugate.

As a result, as shown in [Fig. 7], it was confirmed that the pentapeptide of the present invention penetrated into the nerve cells from the coexistence of blue and red colors.

4-3. Confirmation of tissue penetration

After hair removal on the back of 7-week-old SD rats, rhodamine-pentapeptide was applied, and the rats were sacrificed for analysis after 1 hour. The skin tissue of the application site was collected and formalin fixation was performed for one day. Paraffin blocks were made using the fixed tissue, and after sectioning, immunohistochemical staining was performed using the nerve cell marker TrkB Ab (Cell signaling, USA). Thereafter, nuclear staining was performed using a DAPI staining kit (Invitrogen, USA). Intra-tissue penetration of peptides was observed using a fluorescence microscope. Blue represents the nuclei of cells stained with DAPI, red represents the rhodamine-pentapeptide conjugate, and green represents TrkB (a nerve fiber marker).

As a result, as shown in [Fig. 8], 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.

Through this, the pentapeptide of the present invention can penetrate into nerve cells, penetrates into the muscle layer of skin tissue through percutaneous absorption, and coexists with nerve cell markers in the muscle layer, so it is involved in the formation of the SNARE complex and excessive release of neurotransmitters It can be seen that it can exhibit the effect of suppressing the secretion of sweat caused by and blocking the source of body odor caused by secretion of sweat.

[Experimental Example 5]

Confirmation of body odor suppression effect of pentapeptide using body odor inducing strain

The antibacterial effect of the pentapeptide of the present invention was confirmed using Corynebacterium, which is known as a representative deodorizing bacterium that causes odor by decomposing substances contained in sweat.

Specifically, Corynebacterium xerosis (C.xerosis, KCTC 9105) was purchased from the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology as a strain causing body odor. The strain was inoculated into brain heart infusion (Difco, USA) medium and cultured for 2 days in an incubator at 37°C. A diluted solution diluted to a concentration of 10 ⁶ CFU/ml by adding the same medium to the cultured strain was used as a test strain. After dispensing 100 μL of the bacterial solution for the test in a 96-well plate, the pentapeptide was treated by concentration. In the positive control group, 1% triclosan was added instead of pentapeptide. After culturing for 48 hours, the absorbance at 600 nm was measured with a spectrophotometer to confirm the growth inhibitory effect of the extracting bacteria.

As a result, as shown in [FIG. 9], it was confirmed that the growth of odor-inducing strains was clearly inhibited when the pentapeptide of the present invention was treated. In particular, in the group treated with 1 mg and 2 mg of pentapeptide, it was confirmed that the antibacterial effect was excellent at the same level as that of the positive control group.

From this, it can be seen that the pentapeptide of the present invention can effectively suppress the unique body odor generated by sweating by suppressing the growth of body odor-inducing strains.

<110> CAREGEN CO., LTD. <120> Composition comprising pentapeptide as an active ingredient for treatment of hyperhidrosis <130> 2021-DPA-4154 <150> KR 10-2020-0071660 <151> 2020-06-12 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> artificial sequence <220> <223> Pentapeptides <400> 1 Lys Phe Leu Ile Lys 1 5

Claims (15)

A pharmaceutical composition for preventing or treating hyperhidrosis, comprising as an active ingredient a pentapeptide composed of the amino acid sequence of KFLIK. The method of claim 1,
Wherein the hyperhidrosis includes local hyperhidrosis and systemic hyperhidrosis, the pharmaceutical composition. The method of claim 1,
The hyperhidrosis is a pharmaceutical composition that occurs in any one skin area selected from the group consisting of the face, forehead, neck, hands, feet and armpits. The method of claim 1,
Sweat production compared to the control group not treated with the composition is reduced by 45% to 90%, the pharmaceutical composition. The method of claim 1,
A pharmaceutical composition that further comprises a pharmaceutically acceptable carrier, excipient or diluent. The method of claim 1,
The pharmaceutical composition is formulated in the form of oral preparations, injection preparations or external preparations, a pharmaceutical composition. The method of claim 6,
The pharmaceutical composition for external use is at least one selected from the group consisting of creams, gels, ointments, emulsions, suspensions, sprays, and transdermal delivery patches. A composition for preventing or alleviating hyperhidrosis or suppressing body odor, comprising as an active ingredient a pentapeptide composed of the amino acid sequence of KFLIK. The method of claim 8,
The body odor is an odor caused by decomposition products generated by decomposition of sweat gland secretions by bacteria. In claim 9,
The body odor is an odor generated in the armpits, feet, hands, forehead, neck, face, or scalp, composition. The method of claim 8,
Wherein the hyperhidrosis includes local hyperhidrosis and systemic hyperhidrosis. The method of claim 8,
The hyperhidrosis is a composition that occurs in any one skin area selected from the group consisting of the face, forehead, neck, hands, feet and armpits. A cosmetic composition for preventing or alleviating hyperhidrosis or suppressing body odor, comprising as an active ingredient a pentapeptide composed of the amino acid sequence of KFLIK. The method of claim 13,
The cosmetic composition further comprises a cosmetically acceptable carrier, excipient or diluent, the cosmetic composition. The method of claim 13,
The cosmetic composition is skin, lotion, cream, essence, emulsion, gel, hand cream, lipstick, cleansing foam, cleansing cream, cleansing water, spray, shampoo, rinse, treatment, body cleanser, soap, pack, massage agent, face A cosmetic composition prepared in one or more formulations selected from the group consisting of powder, compact, foundation, two-way cake, and makeup base.