KR101293777B1 - Peptides for treatment and prevention of allergic diseases - Google Patents

Abstract

The present invention relates to a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3. The present invention also relates to a polynucleotide encoding the peptide, a vector comprising the polynucleotide, and a host cell containing the vector. The present invention also relates to compositions and cosmetic compositions for the treatment and prevention of allergic diseases comprising the peptide as an active ingredient. The present invention also relates to a method for treating or preventing allergic diseases of an individual except a human, including a therapeutically effective amount of a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

Description

Peptides for treatment and prevention of allergic diseases

The present invention relates to a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3. The present invention also relates to a polynucleotide encoding the peptide, a vector comprising the polynucleotide, and a host cell containing the vector. The present invention also relates to compositions and cosmetic compositions for the treatment and prevention of allergic diseases comprising the peptide as an active ingredient. The present invention also relates to a method for treating or preventing allergic diseases of an individual except a human, including a therapeutically effective amount of a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

Allergic diseases are increasing in prevalence due to deterioration of environmental factors, and are known to affect about 20% of the population in many countries of the world (Wuthrich B., Int. Arch. Allergy Appl. Immunol., 90, pp 3-10, 1989). Allergic diseases have been reported in humans as well as animals, such as atopic dermatitis, asthma, and diarrhea and vomiting caused by food allergies.

Examples of representative allergic diseases include anaphylaxis, allergic asthma, allergic bronchitis, allergic rhinitis, atopic dermatitis, urticaria, urticaria Hey fever, insect allergy, food allergy, drug allergy, allergic gastritis, allergic diarrhea, allergic stomatitis, allergic conjunctivitis and allergic keratitis (allergic conjunctivitis) allergic keratitis).

Allergic diseases are most often caused by degranulation of mast cells in tissues activated by an antigen-antibody response. Mast cells are multifunctional effector cells that produce and release biologically active mediators (histamine, leukotriene, TNFa) that are involved in the pathogenesis of allergic diseases such as asthma. When the Fce-receptor, an IgE receptor present in the cell membrane of mast cells, is activated by a specific antigen / antibody reaction, it increases intracellular calcium ion (Ca2 +) influx (Weintraub W, et al., J Cell. Physiol. 1994; 160: : 389-399., Hide M., et al., J Biol Chem 266: P15221-P15229.) Ultimately mediators stored in intracellular granules (histamine, TNFa) and newly produced mediators (leukotriene, prostaglandin, platelet activity Factors such as platelet-activating factor) and various inflammatory cytokines (Plaut M, et al., Nature (Lond) 1989 339: 64-67., Kempuraj D, et al., Int. Arch. Allergy Immunol. 2003 132: 231-239., Kettner A, et al., J Exp Med 2004 199: 357-368). That is, when mast cells are activated by agonists or antigen / antibody reactions to increase calcium ion influx into the cytoplasm, various signaling mechanisms, especially the low-molecular G-protein family Rho, Rac1, Rac2, Ras and cdc42, are activated to degranulate mediators. Induced release of newly generated mediators and cytokines ultimately leads to allergic diseases (Hide I. Nippon Yakurigaku Zasshi 2003; 121 (3): 163-73.).

The substances released by mast cells mediate various biological effects such as vasodilation, intestinal and / or bronchial smooth muscle contraction, mucus secretion and local protein secretion. For example, in patients with allergic asthma, infiltration of inflammatory cells in bronchoalveolar fluids (BAL fluids) and production of IgE antibodies in serum may result in adhesions (VACM-1) and co-stimulatory molecules in lung tissue. molecules; CD40, CD40L) to increase expression (Ohkawara Y, et al., Am J Respir Cell Mol Biol 1995; 12: 4-12., Wills-Karp M, et al., Science 2004; 305: 1726-1729 .), Hyperproliferation of goblet cells (Chiappara G, et al., Curr Opin Allergy Clin Immunol 2001; 1: 85-93.) And structural modification of the airway wall (Vignola AM, et al., Am J.). Respir Crit Care Me. 1998; 158: 1945-1950.). Therefore, degranulation inhibition of mast cells causing early mediated responses of allergic reactions may be an important target of allergic disease treatment.

Meanwhile, for the treatment and prevention of allergic diseases, compositions such as antihistamine, steroidal or nonsteroidal anti-inflammatory drugs, and leukotriene antagonists are generally used. Antihistamines, which are most commonly used as a popular therapy for allergic diseases, are used to suppress symptoms after the onset of allergy, but the most common side effects are tremor and hypnosis, and other excitement, irritability, insomnia, anxiety, dizziness, and nightmares. Central hallucinations, such as hallucinations, can also occur. In particular, paradoxical excitability has been reported in children.

While antihistamines are fast-acting by directly antagonizing histamine action, anti-allergic agents (Cromolyn Sodium (DSCG)) prevent allergic development by inhibiting the production and maintenance of histamine and leukotriene from cells involved in allergic reactions. Used as Therefore, acute symptoms cannot be suppressed.

In allergic diseases, steroid preparations have anti-inflammatory and immunosuppressive effects, making them very useful in lethal acute allergic reactions such as anaphylaxis, asthma and exfoliative dermatitis. However, long-term administration may cause side effects such as neurological sensitivity, insomnia, anxiety, obesity, and skin disorders, and risks requiring attention such as glaucoma, cataracts, gastritis, and osteoporosis.

As such, synthetic drugs that are currently used as treatments for allergic diseases have the disadvantage of causing serious side effects while improving the disease.

Against this background, the present inventors have found that the small synthetic peptides, which are modeled after the core complex of SNARE protein, reduce side effects of currently used drugs and competitively inhibit the formation of SNARE complexes in mast cells, thereby specifically preventing degranulation in mast cells. It confirmed that it suppresses effectively, and completed this invention.

An object of the present invention is to provide a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

Another object of the present invention is to provide a polynucleotide encoding the peptide.

Still another object of the present invention is to provide a vector comprising the polynucleotide and a host cell containing the vector.

Still another object of the present invention is to provide a composition and cosmetic composition for treating and preventing allergic diseases, including the peptide as an active ingredient.

Still another object of the present invention is to provide a method for treating or preventing allergic diseases of an individual except a human, including a therapeutically effective amount of a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

In order to achieve the above object, the present invention provides a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

The peptide of the present invention may comprise the amino acid sequence of SARHSEIQQLERTIREL (SEQ ID NO: 1 Syn4N), RRLQQTQAQVDEVVDIM (SEQ ID NO: Vp2N), and LQSEVEGVKNIMTQNVE (SEQ ID NO: 3: Vp8N) consisting of 17 amino acids. The peptide of the present invention is characterized by specifically binding to a soluble N- ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in vitro and in vivo. The peptide of the present invention has an effect of competitively inhibiting SNARE complex formation in mast cells to inhibit degranulation of mast cells, and may include any kind of peptides, proteins, peptide replicas, and compounds as long as such peptides are effective.

For degranulation of mast cells, the granule membrane should be fused with the cell membrane. At this time, it is the protein called SNARE (soluble N- ethylmaleimide-sensitive factor attachment protein receptor) that provides the fundamental force of membrane fusion. The t-SNARE present in the target membrane is a protein called syntaxin 4 and SNAP23, and granules are involved in membrane fusion, such as v-SNARE, VAMP2 and VAMP8.

The membrane fusion causes rearrangement of the lipid bilayer. Due to the characteristics of the biofilm, the membrane and the membrane are pushing each other and do not spontaneously fuse. Therefore, a strong force must be given from the outside, and SNARE protein provides the force to overcome this intermembrane repulsion. t-SNARE and v-SNARE are twisted together to form a SNARE complex, which acts as a force generator for membrane fusion. Therefore, it is possible to prevent the membrane fusion by inhibiting the formation of the SNARE complex.

Accordingly, the present inventors synthesized peptides that specifically bind to SNARE proteins to competitively inhibit SNARE protein complex formation, and conducted various experiments to confirm their function, and thus, the peptides of the present invention were mast cells in vivo. By inhibiting the formation of specific SNARE protein complexes, it was confirmed that they can be used for the treatment and prevention of allergic diseases by inhibiting mast cell degranulation.

In addition, the peptide of the present invention is a small peptide of 17-mer has the advantage that it is easy to penetrate into the cell compared to the conventional polymer synthetic peptide. In addition, the material of the peptide of the present invention may be used by treating a reagent such as SLO (streptolysin o) or digitonin as a material to help the cell membrane permeation, but is not limited thereto as long as it can improve cell membrane permeability. According to one embodiment of the present invention, SLO was used to treat the peptide as a reagent to help the cell membrane permeation to the peptide of the present invention. Alternatively, cell membrane permeation auxiliary proteins such as PTD (protein transduction domain) can be further coupled to the peptide of the present invention to increase the efficiency of intracellular mass transfer. According to one embodiment of the invention, the cell membrane permeation auxiliary protein can be used to bind to the N terminal or the C terminal of the peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 of the present invention, preferably of the peptide It can be used in combination with the N terminal. The peptide and the cell membrane permeation auxiliary protein may be covalently linked directly or via a linker. When linked through a linker, the linker may be used without limitation as long as the linker is flexible by connecting the peptide and the cell membrane permeation auxiliary protein, but may be linked using 1 to 5 amino acids selected from the group consisting of glycine, serine, and alanine. And preferred amino acids that can be used include glycine. In one embodiment of the present invention was used in connection with the PTD sequence through a linker linking three glycine to the N-terminal of the peptide of the present invention.

In addition, since the peptide of the present invention specifically inhibits the allergic reaction by specifically binding to the SNARE protein of the mast cells, compared to the conventional polymer synthetic peptide, there is a feature that can minimize the side effects due to the non-specific reaction.

More specifically, in one embodiment of the present invention, by screening a 17-mer synthetic peptide modeled after the SNARE protein core complex domain of mast cells, Syn4N (SEQ ID NO: 1), Vp2N has an excellent effect in suppressing allergic diseases Peptides of (SEQ ID NO: 2) to Vp8N (SEQ ID NO: 3) were selected and the inhibitory effect of degranulation of mast cells was confirmed on the selected peptides to inhibit β-hexosaminidase and histamine release, which are high in allergic diseases. It was confirmed that the effect (Fig. 3b and 3c). In addition, the cytotoxicity test results of the synthetic peptides confirmed that there is no intracellular toxicity (Fig. 3a) confirmed that it can be used as a pharmaceutical composition for the prevention and treatment, artificial atopic dermatitis to examine the in vivo effect It was confirmed that the effect of reducing the amount of serum IgE and degranulated adipocytes by the synthetic peptide in mice induced by (Figs. 4 and 5).

Peptides of the present invention can be prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press) , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).

In addition, the peptide of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using automated DNA synthesizers (eg, sold by Biosearch or Applied iosystems). The constructed DNA sequence is inserted into a vector comprising one or more expression control sequences (eg, promoters, enhancers, etc.) operably linked to and regulate expression of the DNA sequence, and recombinant expression formed therefrom. Transform the host cell with the vector. The resulting transformants are cultured under media and conditions appropriate to allow the DNA sequence to be expressed to recover substantially pure peptides encoded by the DNA sequences from the culture. The recovery can be carried out using methods known in the art (for example, chromatography). As used herein, "substantially pure peptide" means that the peptide according to the invention is substantially free of any other protein derived from the host. For genetic engineering methods for peptide synthesis of the present invention, reference may be made to Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080,1990.

The present invention also provides a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 3, a vector comprising the polynucleotide and a host cell which is a transformant transformed with the vector.

The polynucleotide comprises an equivalent nucleic acid sequence, that is, a codon degeneracy sequence that encodes the peptides of the invention that differ in the DNA sequence but are identical. Compositions of the various forms of the present invention include a polynucleotide or equivalent nucleic acid sequence consisting of a DNA sequence encoding the peptide, that is, a vector comprising a codon degeneracy sequence encoding a peptide having a different sequence but the same DNA sequence. In addition, the gene delivery method of the nucleotides is also included in the technical scope of the present invention. The nucleotides may be delivered intracellularly in a known form, for example, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, which have been used as conventional gene carriers. Viral vectors, including liposomes, polylysine, polyethylenimine (PEI), protamine, histones, polyesteramines and their respective variants Non-viral vectors such as micelles, emulsions, nanoparticles, and the like may be used, as well as sex polymers, and in addition to the vector system, they may be efficiently delivered into cells using known intracellular substance transfer peptides.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Vectors of the present invention may be conventional cloning vectors or expression vectors, which may be used for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers (promoter). It includes a signal sequence or leader sequence and can be prepared in various ways depending on the purpose. The vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable vector, the origin of replication.

The transformant may be transformed by the vector. Preferably the transformant is a method known in the art, including, but not limited to, a vector comprising a polynucleotide encoding a peptide of the present invention, such as transient transfection, microinjection, transduction transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextranmediated transfection, polybrene-mediated transfection ), Electroporation, gene gun, and other known methods for introducing nucleic acids into cells, which can be obtained by introducing them into host cells, and the transformants can be obtained from Escherichia coli. , Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, star Lokomotiv kusu (Staphylococcus), may be an Agrobacterium tumefaciens Tome Pacific Enschede (Agrobacterium tumefaciens), but is not limited thereto.

As another aspect, the present invention relates to a composition for treating and preventing allergic diseases, comprising the peptide as an active ingredient.

The synthetic peptide has a prophylactic and therapeutic activity for allergic diseases in the nature of the present invention, the allergic diseases include anaphylaxis, allergic rhinitis, asthma, urticaria, atopic dermatitis atopic dermatitis, contact dermatitis and allergic dermatitis, but are not limited thereto.

The composition may further include a cell membrane permeation aid material to improve cell membrane permeability, and may include, but are not limited to, a cell membrane permeation aid protein such as a protein transduction domain (PTD) as an example of the cell membrane permeation aid material. Do not. According to one embodiment of the present invention, a cell membrane permeation auxiliary protein such as PTD may be included in the composition at the N or C terminus of a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 of the present invention. The peptide and the cell membrane permeation auxiliary protein may be directly or covalently linked through a linker, and when linked through a linker, the linker may be used without limitation as long as the linker is flexible by connecting the peptide and the cell membrane permeation auxiliary protein. One to five amino acids selected from the group consisting of glycine, serine, and alanine may be linked, and preferably, glycine may be linked.

The composition may be a pharmaceutical composition, in which case the pharmaceutically acceptable non-toxic carrier according to the method of administration is prepared in various forms by mixing the peptide of the present invention to prevent allergic diseases disclosed above And as a therapeutic agent.

Pharmaceutically acceptable carriers according to various formulations include all forms of diluents or solvents, fillers, scattering agents, binders, dispersants, disintegrants, surfactants, lubricants, excipients and wetting agents. If necessary, commercially available dissolution aids, buffers, preservatives, colorants, flavors, sweeteners and the like can also be added to the drug.

In addition, the composition may further include one or more selected from the group consisting of antihistamines, leukotriene antagonists, steroidal anti-inflammatory agents, and nonsteroidal anti-inflammatory agents that are known to be effective in preventing and treating allergic diseases.

The dosage unit form of the composition according to the present invention is not specifically limited and may be widely selected according to various therapeutic purposes, for example, tablets, capsules, granules, pills, syrups, emulsions, solutions, emulsions, suspensions Oral dosages such as preparations, injections (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.) and parenteral administration such as suppositories. Among these formulations, oral dosage formulations are preferred. In addition, the composition may be prepared in the form of aerosols, external preparations, ointments or patches.

Further, intracellular delivery methods of peptides known in the art, such as microinjection, electroporation using electroshock, delivery using cations or liposomes, and protein transduction domain (PTD), can be used.

The various types of medicaments disclosed above can be prepared by conventional methods. For example, in preparing oral dosage preparations such as tablets, capsules, granules and pills, these include excipients such as white sugar, lactose, glucose, starch, mannitol; Binders such as syrup, gum arabic, sorbitol, trigacanth rubber, methylcellulose, polyvinylpyrrolidone and the like; Disintegrants such as starch, carboxymethyl cellulose and its calcium salts, microcrystalline cellulose, polyethylene glycol and the like; Lubricants such as talc, magnesium stearate, calcium stearate, silica and the like; It may be prepared by conventional methods using wetting agents such as sodium laurate, glycerol and the like.

The dosage of the composition for the prevention and treatment of allergic diseases comprising a therapeutically effective amount of the peptide according to the present invention is appropriate depending on the method of administration, the type of preparation, the age of the patient, the weight of the patient, the sensitivity of the patient, and the condition of the disease. May be selected and is not specifically limited.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be envisaged, for example, by oral, subcutaneous, intravenous, intramuscular, nasal, intraperitoneal, rectal, intrauterine dural or intracerebroventricular injection. Since the peptide of the present invention has almost no toxicity and side effects, it can be used safely even when taken for long periods of time.

As another aspect, the present invention provides a cosmetic composition for improving or preventing allergy symptoms comprising a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 disclosed in the present invention as an active ingredient.

The cosmetic composition that can be prepared by including the peptide of the present invention is not particularly limited in the formulation, for example, may have a formulation such as lotion, nutrition lotion, nutrition cream, massage cream, essence, pack. In addition, other components may be arbitrarily selected and blended according to the formulation or purpose of use of the cosmetic.

The components to be incorporated into the cosmetic composition of the present invention include oils and fats, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, antioxidants, pH adjusters, alcohols, pigments, perfumes and the like. The fat or oil component may be an ester fat, a hydrocarbon fat, a silicone fat, a fluorine fat, an animal fat, a vegetable fat or the like. The moisturizing agent may be a water-soluble low molecular moisturizer, a fat-soluble low molecular moisturizer, a water-soluble polymer moisturizer, a fat-soluble polymer moisturizer or the like. Examples of the emollient agent include long-chain acyl glutamic acid cholesteryl esters, hydroxystearate choleteryl esters, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like. As the surfactant, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and the like can be used. The alcohol may be cetearyl alcohol or the like. The cosmetic composition may be usefully used as a cosmetic composition for the prevention or improvement of allergic diseases.

The present invention also provides a method for treating or preventing an allergic disease in a subject other than a human, including a therapeutically effective amount of a peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 disclosed herein.

As used herein, the term "individual" means any non-human primate and allergic diseases, including dogs, wheat, cats, sheep, pigs, cattle, etc., that require treatment, including livestock, breeding, pet or sport animals, such as humans. Refers to the mammal.

As used herein, the term “treatment” refers to therapeutic treatment and prophylactic or prophylactic means. Thus, an individual in need of treatment may include invasive or non-invasive medical acts on all mammals, except humans, for the purpose of preventing allergic diseases, as well as those already performed on individuals with allergic diseases described above. Can be.

As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease, and an effective dose level may be defined as the severity of the disease; The age, body weight, health, sex of the individual; Sensitivity to the subject's drugs; Administration time, route of administration and rate of release; Treatment period; It may be determined according to factors including drugs which are used in combination or coincidental with the composition of the present invention and other factors well known in the medical field.

Diseases applicable to the method for treating or preventing allergic diseases using the peptides according to the present invention include anaphylaxis, allergic rhinitis, asthma, urticaria, atopic dermatitis dermatitis, contact dermatitis and allergic dermatitis, and the application of the method disclosed in the present invention is not limited by the above examples.

The peptide of the present invention and a composition for preventing or treating allergic diseases including the same have an excellent effect of treating and preventing allergic diseases by inhibiting degranulation of mast cells by using small-molecule peptides that are mast cell specific and have fewer side effects.

Figure 1 shows the amino acid sequence of the 17-mer synthetic peptides modeled after the core complex of the SNARE protein tested to inhibit the formation of the SNARE complex of the present invention.
2 shows the results of membrane fusion assay inhibition to search for inhibitors of SNARE complex formation of the synthetic peptides of the present invention. Each peptide concentration used in (a) and (b) is 200 uM, and the black circle represents the path through which the control reaction proceeds. (c) shows a bar graph of the results of (a) and (b) based on the black control group. 2 (c), "SN23 Stx4 Vp8" and "SN23 Stx Vp2" at the top are two systems used when performing a membrane fusion assay. V-SNARE, which is typically present in mast cells, is VAMP2 and VAMP8. For this reason, the fusion assay was performed using two VAMPs for t-SNARE.
Figure 3 confirms whether the synthetic peptide of the present invention has a toxic effect in RBL-2H3 cells (a), β-hexosaminidase (b) and histamine release (degranulation phenomenon) (c) inhibits whether It is shown as a graph.
Figure 4 illustrates the effect of reducing the amount of serum IgE by synthetic peptides in mice artificially induced atopic dermatitis. In FIG. 4, peptide 1 is linked to Syn4N, peptide 2 to Vp2N, and peptide 3 to Vp8N, respectively.
Figure 5 shows the number of fat cells (mast cell) in the skin tissue of the lower ear of the mouse induced atopic dermatitis and stained it.
Figure 6 is stained with Hematoxylin & Eosin the skin tissue was observed 200 times with an optical microscope.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are intended to illustrate the present invention in detail, but the scope of the present invention is not limited by these examples.

Example 1: Synthetic Peptide Preparation

In the present invention, 17-mer synthetic peptides modeled after SNARE complex domain were consigned to Peptron. The PTD sequence is a 14mer peptide (SEQ ID NO: 19) that enhances cell permeability, and a PTD-peptide (SEQ ID NO: 16 to SEQ ID NO: 18) was synthesized by adding this sequence to the front of the peptide obtained from the SNARE motif. Hereinafter, the 31mer sequences in which the 17mer peptide and the PTD sequence were combined were compared with each other in the experiment. (FIG. 1).

Example 2: Membrane Fusion Inhibitory Effect

Membrane fusion inhibitory effects were tested on the synthetic peptides shown in FIG. 1 to search for membrane fusion inhibitors.

To produce SNARE proteins, SNARE proteins called SNAP23, Syntaxin 4, VAMP2 and VAMP8, respectively, were purified from E. coli transformed with biological techniques. First, POPC (62mol%), DOPS (35mol%), NBD-PS (1.5mol%), and Rodamin-PE (1.5mol%) were mixed to make a liposome, a microencapsulated particle labeled with fluorescent material. Lymphosomes (v-vesicles) were made, and 50 mM liposomes (t-vesicles) were prepared by mixing DOPS and POPC at a molar concentration of 35:65 to make up liposomes that were not labeled with fluorescent material. To make the binary complex of purified SNAP23 and Syntaxin 4, the two proteins were mixed at a molar concentration of 1: 1 and allowed to react at room temperature for 1 hour, followed by mixing at a molar ratio of 50: 1 with a liposome that was not labeled with fluorescent material. And, VAMP2 and VAMP8 were mixed to 50: 1 to the phosphor-labeled liposomes, respectively. The two lyposomes were then dialyzed and mixed at a ratio of 1: 9 (v-vesicle: t-vesicle) to measure fluorescence intensity using a fluorescence intensity measuring device (Model name: Spectramax M2, manufacturer: Molecular Device). The results are shown in Fig.

The black circle is the control reaction and progresses when DMSO is added instead of the synthetic peptide. In FIG. 2, the increase in fluorescence intensity means a membrane fusion phenomenon due to SNARE protein, and thus, low fluorescence intensity indicates that membrane fusion was inhibited by that amount. Most of the synthetic peptides follow a reaction pathway similar to the control, while Syn4N, Vp2N, and Vp8N peptides have been shown to inhibit membrane fusion. In particular, the Syn4N peptide showed a significant effect in the combination of both VAMP2 and VAMP8, whereas Vp2N showed an inhibitory effect in membrane fusion with VAMP2, and in the case of Vp8N in membrane fusion with VAMP8.

The bar graph below summarizes the membrane fusion inhibitory effects of various synthetic peptides. Based on the response of the control group, the synthetic peptides showing a significant effect by statistical processing are Syn4N, Vp2N, Vp8N. Vp2S and Vp8S were used as a positive control and compared with t-SNARE to control membrane fusion.

Example 3: Inhibition of Degranulation in RBL-2H3 Cells

3-1. RBL-2H3 Cell Culture

RBL-2H3 cells were purchased from ATCC and cultured in DMEM medium containing 10% fetal calf serum and antibiotics. Subculture was treated with 0.25% Trypsin-EDTA to separate the cells from the plate and centrifuged at 1,000 xg for 5 minutes to collect the cells, add new medium and disperse the cell pellet by pipetting into a new culture plate at 37 ℃, The cells were cultured in an incubator fed with 5% CO ₂ gas. RBL-2H3 cells were detached from the plate wall, and then the number of cells was measured by a hematocytometer and dispersed and inoculated in fresh medium at a concentration of 2 × 10 ⁵ cells / well. In order to label IgE on the outer wall of mast cells, DNP-IgE was treated with 10ng / ml in starved media and incubated for 3 hours. After washing three times with PBS, SLO (Streptolysin o) was added to synthetic peptide (10μM) and synthetic peptide treatment buffer (Tyrodes's buffer, 10mM Hepes, 130mM NaCl, 5mM KCl, 1.4mM CaCl ₂ , 1mM MgCl ₂ to increase cell permeability). , 5.6mM glucose, 0.1% BSA, pH7.4) was added together and reacted for 30min in a carbon dioxide incubator. At this time, PTD-peptides (31mer) did not process SLO. Thereafter, 30 min of DNP-BSA (0.2 / ml) was incubated for cross-linking IgE, and the plate was placed on ice for 10 minutes to stop the reaction, and the supernatant was collected to measure degranulation.

3-2. MTT assay

Prior to measuring the degree of release, MTT assay was performed to measure the intracellular toxicity of each synthetic peptide. After 10μM of the synthetic peptide was treated to the cells, the cells were cultured in the same manner as the above experiment, and the supernatant was discarded and the cells were treated with MTT solution. After reacting for 3 ~ 4 hours, DMSO was treated to completely dissolve the formazam at the bottom, shaken for about 20 minutes, and OD was measured at 570 nm. The degree of survival of the cells when the synthetic peptide was treated by comparing the control group without the synthetic peptide (control) and the OD value was shown as a graph. When 10 μM of the synthetic peptide was treated, the cell viability was similar to that of the non-peptide-treated control group, indicating no intracellular toxicity.

3-3. β- hexosaminidase  Emission measurement

β-hexosaminidase release was carried out by mixing the same amount of the supernatant 50 and the substrate solution containing 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide in Citrate buffer (0.1 mM citrate, pH 4.5) for 3 hours. After the reaction was completed, 100 stop buffer (0.1M Na ₂ CO ₃ / NaHCO ₃ pH 10.0) was added to stop the reaction, and the absorbance was measured at 405 nm. 17mer synthetic peptide was used to treat SLO for intracellular permeation, and PTD-peptide was not treated separately because it has intracellular permeability. As a result, Syn4N and Vp8N peptides showed an inhibitory effect on both peptides.

3-4. Histamine Release Measurement

Histamine release was measured by the ELASA reaction with the supernatant using a Histamine Enzyme immunoassay kit (Cayman, SPI-BIO).

Using the above method, β-hexosaminidase and histamine were used as markers to confirm whether synthetic peptides inhibited degranulation in mast cells, and the results are shown in FIG. 3. Peptides treated with SLO and PTD-peptide had similar effects (FIG. 3A). As a result, it was observed that the Syn4N peptide inhibited both β-hexosaminidase and histamine release, whereas Vp2N inhibited histamine release and Vp8N inhibited hexosaminidase release.

Example  4: Efficacy in treating atopic dermatitis

4-1. Experimental animal

All animal experiments used in this evaluation were conducted after approval of the Animal Experiment Ethics Committee, which is operated by Gyeonggi Bio Center.

Nc / Nga female mice at 7 weeks of age were purchased from a central laboratory animal (Seoul, Korea). All mice were reared in a 12-hour light and dark cycle in a laboratory maintained at an optimal environmental condition with a temperature of 24 ± 50% humidity. The experiment was started at 8 weeks after the mouse was quarantined and purified for 1 week before the experiment.

4-2. Induced atopic dermatitis

In order to induce atopic dermatitis, the hair from the lower part of the ear to the upper part of the tail of the mouse was epilated using a depilatory cream, and then 200 μl of DNCB solution was applied one day later. Dermatitis was induced by first application of 1% DNCB solution (acetone: olive oil = 4: 1) three times per week and twice with 0.2% DNCB solution three times for three weeks.

4-3. Test Material Manufacturing and Processing

Peptides 1, 2, and 3 (SEQ ID NOs: 16 to 18, respectively) in which PTDs were bound to the N-terminus of these peptides were respectively mixed into Syn4N, Vp2N, and Vp8N as test substances, respectively, by mixing EtOH: H ₂ O: butyleneglycol = 1: 4: 5 Dissolved in 0.1% of the solvent was applied for 20 days 100μl once a day. Negative control was applied to the solvent and dexamethasone dissolved in 0.1% of the solvent in the same manner as the test material.

4-4. serum IgE  Measure

In order to measure serum IgE, blood was collected from mouse eyes using microtubules at the beginning and end of sample material treatment. After collecting blood for 1 hour and stored at room temperature for 1 hour, the serum was separated by centrifugation at 1000g, 4 ℃ conditions, and stored in -70 ℃ freezer until use. Serum IgE levels were measured using the BD OptEIA ^™ ELISA set provided by BD Bioscienc according to the method presented.

After 4 weeks of application, the amount of IgE in serum was significantly decreased in the group treated with dexametasone, peptide 3, peptide 2 + peptide 3 at 0.1% compared to vehicle group. The results show that the amount of IgE increased by DNCB treatment is reduced by treatment with Vp8N and Vp2N + Vp8N, which can be expected to improve atopic dermatitis (FIG. 4).

4-5. Histopathological examination

In order to observe the skin tissue, the skin tissue of the lower part of the ear of the mouse was extracted at the end of the test material treatment. The extracted tissue was fixed in 10% formalin solution, and then paraffin embedding, tissue sections and staining were commissioned to the central laboratory animals. Tissue sections stained with Hematoxylin & Eosin were visually observed 200 times by optical microscope. In addition, mast cells were stained by toluidine blue staining and observed 100 times with an optical microscope, and 5 sites were randomly selected to measure the number of mast cells.

Tissues of NC / Nga mice were biopsied, stained with Toluidin Blue, and the number of mast cells was measured. (FIG. 5) After randomly selecting five places on each slide, the cells were measured and statistically treated as the average value, and the dexametasone 0.1% treatment group and the Vp8N 0.1% treatment group showed a significant decrease compared to the vehicle group. The rest of the peptide treatment group also showed a reduction effect.

As a result of H & E staining, the vehicle treatment group showed that the epidermis was thickened and inflammatory cells were infiltrated by DNCB. Dexametasone treated group and peptide treated group were found to be normal due to the markedly reduced epidermal thickening.

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Claims (15)

delete delete delete delete delete delete Comprised of the amino acid sequence of SEQ ID NO: 3, comprising a peptide that inhibits the formation of soluble N- ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex in mast cells and inhibits degranulation as an active ingredient, treatment or prevention of allergic diseases Pharmaceutical composition for.
The method according to claim 7, wherein the allergic disease is anaphylaxis, allergic rhinitis, asthma, urticaria, atopic dermatitis, contact dermatitis and allergic dermatitis. allergic dermatitis).
8. The composition of claim 7, wherein the cell membrane permeation aid is further mixed with Streptolysin o (SLO).
The composition of claim 7, wherein the peptide has a protein transduction domain (PTD), which is a cell membrane permeation auxiliary protein, attached to its N- or C-terminus.
8. The composition of claim 7, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.
8. The composition of claim 7, further comprising at least one selected from the group consisting of antihistamines, leukotriene antagonists, steroidal anti-inflammatory agents, and nonsteroidal anti-inflammatory agents.
Consisting of the amino acid sequence of SEQ ID NO: 3, comprising a peptide that inhibits the formation of SNARE complexes in mast cells and inhibits degranulation as an active ingredient, cosmetic composition for improving or preventing allergy symptoms.
A method for treating or preventing an allergic disease in an individual except a human using a peptide consisting of the amino acid sequence of SEQ ID 3, which inhibits SNARE complex formation and inhibits degranulation in mast cells.
15. The method of claim 14, wherein the allergic disease is anaphylaxis, allergic rhinitis, asthma, urticaria, atopic dermatitis, contact dermatitis, and allergic dermatitis. allergic dermatitis).

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