TWI634210B - A peptide having inhibition of hsp expression and the composition comprising the same - Google Patents

Abstract

The present invention provides a composition for inhibiting the expression of heat shock protein (HSP). The composition includes: a peptide including an amino acid sequence of SEQ ID No. 1, including a sequence homology with an amino acid sequence of 80% or more The peptide of the amino acid sequence, or the HSP-binding peptide of the peptide fragment mentioned above, and provides a method of performing heat shock protein expression inhibition by using the composition. More specifically, due to the increased specificity of heat shock proteins in cancer cells, the present invention provides a method of inhibiting the expression of heat shock proteins by using peptides that specifically bind to heat shock proteins.

Description

Peptide and composition containing heat shock protein

One or more embodiments of the present invention relate to a peptide having heat shock protein expression inhibitory activity and a composition including the peptide, and more specifically, to a peptide derived from telomerase and having heat A peptide that exhibits inhibitory activity of a shock protein, a composition including the peptide, and a method of performing heat shock protein expression inhibition by using the composition.

Cancer cells express several types of heat shock proteins (HSP) at high levels to increase tumor aggressiveness and enable cancer cells to survive under lethal conditions such as apoptosis caused by cancer treatment. When HSP performance increases, it will inhibit programmed cell apoptosis and induce self-growth. In addition, it will gain resistance to cancer treatment so that cancer cells can grow.

When HSP was first reported in 1962, HSP was called a cell structural protein, which protects cells by acting as molecular chaperones under stress (such as heating, hypoxia, and radioactivity) (Ritossa F. Experimentia 1962; 18 : 571-3 .; and Lindquist S and others, Craig Annu Rev Genet 988; 22: 631-77). Recently, it is reported that HSP is involved in cell growth and apoptosis, and HSP is over-expressed in various tumor tissues, which indicates that HSP can play a series of roles in tumor development. HSP is important for cell survival under stress conditions such as hypoxia. Depending on its molecular weight, HSP can be classified into various groups (small HSP family, HSP 60 family, HSP 70 family, HSP 90 family).

Regarding tumors, in endometrial cancer, malignant osteosarcoma and kidney cancer, HSP 70 has enhanced performance, and it is believed that this enhanced performance is caused by HSP 70 inhibiting apoptosis. HSP 90 is equivalent to about 1% to about 2% of normal cell protein and has especially enhanced performance under adversity. HSP90 reacts with ATP to fold denatured proteins, has enhanced performance in lung cancer, leukemia, and Hodgkin's disease (Hodgkin's disease), and is also reported to be related to apoptosis; however, the exact role of HSP90 is not known.

As HSP participates in tumorigenesis becomes well known, in cancer treatment Has begun to work hard to use HSP performance inhibitors. Therefore, it was found that 17AAG (17-allylamino-17-demethoxy analog of geldanamycin; 17-allylamino-17-demethoxy analogue of geldanamycin) which is an HSP 90 inhibitor is a possible antitumor agent and the current In clinical trials. Quercetin is a flavonoid, a phytochemical with a polyphenol group, and has been found to inhibit cell proliferation in experiments with various cancer cell lines. Although the exact mechanism is not known, quercetin is known to inhibit the production of HSP70, therefore, quercetin is being studied as a drug used in combination with hyperthermia therapy.

HSP70 is a highly conserved protein chaperone associated with various intracellular mechanisms. HSP70 production and HSP70 inhibition can be induced by adversity in cells Apoptosis induced by adversity. In addition, it is known that HSP70 can immunomodulate and stimulate the production of anti-inflammatory cytokines (Van Eden, W. et al., Nat. Rev. Immunol. 5: 318-330 (2005)). In addition, HSP70 prevents inflammatory shock caused by tumor necrosis factors (TNF) and induces the activation of antigen presenting cells (APC).

HSP70 (structural performance Hsc70 and heat-induced HSP70-1 and HSP70-6) participates in the preparation phase, while HSP90 participates in the final phase. The functions of HSP70 and HSP90 require HSP70 regulators, HSP40, HSP110, and various cooperative companion proteins (such as BAG and HIP); HSP-organizing protein (HOP), which participates in the formation of intermediate molecule accompanying protein complexes. The client protein is transferred from HSP70 to HSP90, and other proteins (such as p23, cdc37), and the final or mature HSP90 complex immunoaffinity protein function. Identify HSP90 as a novel cancer target. HSP90 is a basic protein. The protein serves as a molecular companion protein to ensure the structural stability, form and function of the customer's protein, and is sufficiently universal (1-2% of the entire cell protein). Inhibiting HSP90's inherent ATPase blocks the interaction between HSP90 protein and client protein and induces the decomposition of HSP90 via the ubiquitin proteasome pathway. Imitators of HSP90 client proteins (such as the EGFR group including Raf, AKT, CDK4, and ErbB2) are oncogenic signaling molecules that are critically involved in cell growth, differentiation, and apoptosis of cells, and this process is relevant Extremely important.

Therefore, when a composition that suppresses the performance of HSP70 and HSP90 is developed, the ripple effect of the development is expected to be large.

【Prior Technical Literature】 【Patent case】

KR2012-0117954A

KR2006-0023576A

【Non-Patent Case】

Van Eden, W. et al., Nat. Rev. Immunol. 5: 318-330 (2005)

Ritossa F. Experimentia 1962; 18: 571-3.

Lindquist S and others, Craig Annu Rev Genet 988; 22: 631-77

【Invention Objective】

The inventors of the present invention developed a drug that inhibits the performance of HSP, and completed the present invention by specifically binding the drug to HSP.

The inventors of the present invention developed a peptide derived from telomerase, which specifically binds to HSP and specifically inhibits HSP performance.

One or more embodiments of the present invention include a peptide that binds to HSP to inhibit HSP performance and a method of inhibiting HSP performance by using the peptide.

Additional aspects will be partially explained in the description below and these aspects will be partially apparent from the description, or may be understood by the implementation of existing embodiments.

According to one or more embodiments of the present invention, a method for suppressing heat is provided A composition expressed by a shock protein (HSP), which includes: a peptide including the amino acid sequence of SEQ ID No. 1, and an amino acid sequence having 80% or more sequence homology with the amino acid sequence A peptide, or a fragment of a peptide mentioned above.

In the HSP binding peptide according to the embodiment of the present invention, the HSP may be HSP 70 or HSP 90.

In the HSP binding peptide according to the embodiment of the present invention, the peptide fragment includes three or more amino acids.

In the HSP binding peptide according to the embodiment of the present invention, the peptide includes 30 or fewer amino acids.

In the composition for inhibiting angiogenesis, the composition can be used to prevent or treat diseases or disorders related to non-regulated angiogenesis, such as tumor growth and metastasis, diabetic retinopathy, retinopathy of prematurity, corneal transplant rejection, neonatal Vascular glaucoma, skin redness, proliferative retinopathy, psoriasis, macular degeneration, hemophilic arthropathy, capillary proliferation in atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid Arthritis, chronic inflammation, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, cirrhosis, glomerulonephritis, Diabetic nephropathy, malignant renal sclerosis, thrombotic microangiopathy, organ transplant rejection, glomerular nephropathy, diabetes, inflammation, or neurodegenerative diseases.

In the composition for inhibiting blood vessel formation according to the embodiment of the present invention, the composition can be used for preventing or treating tumor growth and metastasis.

In the composition for inhibiting angiogenesis according to the embodiment of the present invention In this, the composition may be a pharmaceutical composition.

In the composition for inhibiting blood vessel formation according to the embodiment of the present invention, the composition may be a food composition.

According to another embodiment of the present invention, there is provided a method for preventing and treating diseases related to HSP performance, the method comprising administering a composition that inhibits HSP performance.

In conjunction with the accompanying drawings, these and / or other aspects will become apparent and easier to understand from the following description of the examples. In these drawings: Figure 1 illustrates the identification of proteins interacting with PEP 1 The data, etc. include the following: (A) Purified His tags including GFP, 11-mer-GFP (GFP fused to short PEP1) and PEP 1-GFP proteins were cultured with HEK293T cell lysate, and by using Each protein complex thus obtained was purified by Ni-NTA chromatography, followed by SDS-PAGE and silver staining. The protein specifically related to PEP 1-GFP was excised from the gel and subsequently identified by using a Maldi-TOF mass spectrometer. The results are shown in Figure 1 (A). * Refers to GFP, 11mer-GFP and PEP 1-GFP staining bands; (B) By using immunoblotting (IB) to identify proteins related to PEP 1 using antibodies, the results are shown in Section 1 (B) In the figure; (C) In order to identify the crossover effect between PEP 1 and HSP70 in different cells, the cell lysates of MCF7 and HepG2 cells were cultured with GST protein or PEP 1-GST protein. Figure 1 (C) illustrates the use of glutathione Resin pull-down, the result of the interaction between HSP70 and PEP 1 using IB and anti-HSP70 antibody; and (D) After separating the membrane portion from MCF7 cells, MCF7 cells were cultured with GFP or PEP 1-GFP. Figure 1 (D) shows the results obtained from the IB of HSP70 complex after Ni-NTA chromatography; Figures 2 and 3 show the inhibition of HSP70 and HSP90 due to PEP 1 treatment of cancer cells Low regulation results. Jurkat (Figure 2) and MCF7 (Figure 3) cells were treated in serum-free medium with increasing concentrations of PEP 1 or scrambled peptides for 2 hours. The amounts of HSP70 and HSP90 were analyzed by IB using antibodies to HSP70, HSP90 and GAPDH; Figures 4 to 5 show the experimental results of HSP production inhibition induced by hypoxia of PEP 1. MCF7 and HeLa cells were treated with PEP 1 (20 μM) or vehicle, and then cultured under hypoxia for a predetermined period of time. The cell lysates of MCF7 and HeLa cells thus obtained were subjected to IB to analyze the amount of HSP70 and HSP90; Figure 6 shows the results of treatment of Jurkat and MCF7 cells with vehicle, PEP 1, 17-AAG, and KNK437. Jurkat and MCF7 cells were treated in serum-free medium with vehicle, PEP 1 (5 μM for Jurkat and 20 μM for MCF7), 17-AAG (1 μM) and KNK437 (1 μM) for two hours. The cell lysates of Jurkat and MCF7 cells thus obtained were analyzed by IB using a method similar to that described in Figure 2; Figure 7 shows treatment with PEP 1 or PBS including MG132 (5 μM) or MG132 not included ( 5 μM) results of MCF7 cells. Stain intracellular HSP and surface HSP by surface intracellular staining and surface staining, and then by Use flow cytometry analysis. Red indicates DMSO, blue indicates PEP 1 plus DMSO, orange indicates PEP 1 plus MG132, and green indicates MG132; Figures 8 to 9 are graphs showing the inhibition of cancer cell proliferation by PEP 1 under hypoxia. MCF7 and HeLa cells were cultured under normoxia (left panel) or under hypoxia (right panel) with or without PEP 1. The number of cells was measured on the second day, fourth day and sixth day. The data represents the mean ± SD. * Indicates statistical significance when p <0.05. Perform a single factor t test; Figures 10 to 11 illustrate the drop in protein levels of HSP70 and HSP90 due to PEP 1 treatment. Using antibodies to HSP70 and HSP90, the protein levels of HSP70 and HSP90 samples were visualized by immunohistochemical staining (Figure 10), and these samples were quantified by using Leica Qwin software (Figure 11). Fields of view were randomly selected to quantify 10 fields of view from 6 slides in each processing group. The data represents the mean ± SD. *** When p <0.001, it indicates statistical significance, and a two-factor t test is performed. Figure 12 shows the decrease in protein levels of HSP70 and HSP90 in tumor samples treated with PEP 1 in an immuno-staining experiment using tumor lysate.

Figures 13 and 14 illustrate the effect of PEP 1 on the protein level of HSP70 secreted in the blood. Figure 13 shows the levels of HSP70 (left panel) and HSP90 (right panel); n = 20, via ELISA, using a mouse model treated with PEP 1 (50 μg / kg) or PBS (10 mice per group) Mouse). Figure 14 illustrates the analysis of the relationship between tumor weight (left panel) and tumor size (right panel). Perform a two-factor t test.

This disclosure can be modified and embodied in various ways. In the following, will The present disclosure is described in more detail via exemplary embodiments. However, the following examples are not intended to limit this disclosure. Specifically, this disclosure can make various changes based on the scope of the accompanying patent application. It should be understood that this disclosure includes any changes, equivalents, or substitutions that fall within the technical concepts and scope of this disclosure. In the description, details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the presented embodiments.

Telomeres are known as repetitive sequences of genetic material found at the ends of chromosomes and are used to prevent chromosomes from being damaged or merging onto other chromosomes. The length of telomeres is shortened each time a cell divides, and after several cell divisions, the length of telomeres is extremely shortened to the point where the cells stop dividing and die. On the other hand, it is known to extend telomeres to extend the lifespan of cells. For example, cancer cells secrete an enzyme called telomerase, which prevents telomere shortening, which leads to cancer cell proliferation. The inventors of the present disclosure have identified that peptides derived from telomerase are effective in treating and preventing ischemic reperfusion injury and have completed the present disclosure.

Benzoquinone ansamycin antibiotics (such as geldanamycin and herbimycin) and radicicol are known in the art as compounds that bind to HSP90 family proteins. The report states that all of these compounds bind to HSP90 family proteins and inhibit the function of HSP90 family proteins to exhibit pharmacological activity (such as anti-tumorigenic activity). Therefore, it is known that compounds bound to HSP90 family proteins are useful as therapeutic agents for diseases associated with HSP90 family proteins or proteins bound to HSP90 family proteins (ie, HSP90 client proteins).

In an exemplary embodiment of the present disclosure, the polynucleotide is directed to a peptide comprising the amino acid sequence of SEQ ID No. 1, including an amino acid sequence having 80% or more sequence homology with the amino acid sequence Of peptides or as mentioned above Encoding of peptide fragments of peptides. Polynucleotides can be used to produce peptides on a large scale. For example, a vector including the polynucleotide encoding the peptide can be inserted into the host cell, and the host cell can be cultured to produce the peptide on a large scale.

The peptides disclosed herein may include peptides comprising amino acid sequences, and these peptides have at least 80%, at least 85%, at least 90%, and fragments of the peptide of sequence number 1 or the peptide of sequence number 1. At least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence homology. In addition, the peptides disclosed in the present invention may include at least one amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least A peptide having a difference among 5 converted amino acids, at least 6 converted amino acids, or at least 7 amino acids.

In one embodiment of the invention, the changes in the amino acid include modifications in the physical and chemical properties of the peptide. For example, amino acid modification can be performed in order to improve the thermal stability of the peptide, change the substrate specificity, and change the optimal pH.

In the exemplary embodiment of the present disclosure, the peptide of SEQ ID NO: 1, the peptide of the fragment of the peptide of SEQ ID NO: 1, or a peptide having 80% or more sequence homology with the peptide includes peptides derived from The peptide of telomerase (specifically, human (Homo sapiens) telomerase).

The term "amino acid" herein includes not only the 22 standard amino acids naturally integrated into the peptide, but also D-isomers and modified amino acids. Therefore, in specific embodiments of the present invention, the peptides herein include peptides with D-amino acids. On the other hand, peptides may include non-standard amino acids, such as those amino acids that have been modified after translation. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including acetylation, nutmeg acylation, palm Acylation), alkylation, carboxylation, hydroxylation, saccharification, biotinylation, ubiquitination, modification of chemical properties (for example, β-removal deamidation, deamidation) and Structural modification (eg, formation of disulfide bonds). Also, changes in amino acids include changes in amino acids, such as changes in amino groups, carboxyl groups, or side chains, due to chemical reactions during the combined process of forming a peptide conjugate with a cross-linking agent.

The peptide disclosed herein may be a wild-type peptide that has been identified and isolated from natural sources. On the other hand, when compared with the peptide of sequence number 1 or a fragment thereof, the peptide disclosed herein can be an artificial variant, which includes one or more amino acids substituted, deleted and / Or insert. Amino acid changes in wild-type polypeptides (not only in artificial variants) include protein folding and / or conservative substitutions of amino acids that do not significantly affect activity. Examples of conservative substitutions can be found in basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamic acid and Aspartic acid), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small Within the group of amino acids (glycine, alanine, serine and threonine). It is known in the art that amino acid substitutions do not substantially change the specific activity. The most common changes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly and the reverse changes of the above. Other examples of conservative substitutions are shown in Table 1 below:

Substantial modification of the biological characteristics of peptides is performed by selecting distinctly different substitutions according to the following functions: (a) Maintain the efficacy of the structure of the multiple peptide backbone in the substituted region (such as a sheet or spiral three-dimensional structure), (b ) The effect of maintaining the charge or hydrophobicity of the molecules in the target area, or (c) the effect of maintaining the size of the side chain. The natural residues are divided into the following groups by general side chain characteristics: (1) hydrophobicity: n-leucine, met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr (3) Acidity: asp, glu; (4) Basicity: asn, gln, his, lys, arg; (5) Residues affecting chain orientation: gly, pro; and (6) Aromaticity: trp, tyr, phe.

Non-conservative substitutions can be performed by swapping members of the above categories with members of different categories. Generally, serine can be used to replace any cysteine residues that are not relevant for maintaining the proper three-dimensional structure of the peptide, thereby increasing the oxidative stability of the molecule and preventing inappropriate cross-linking. Conversely, stability can be improved by adding cysteine bonds to the peptide.

Another type of peptide amino acid variant system has their peptides with a changing pattern of peptide glycosylation. The term "alteration" herein means deleting at least one carbohydrate residue found in the peptide and / or adding at least one glycosylation residue that is not present in the peptide.

Glycosylation in peptides is usually N-linked or O-linked. The term "N-linked" as used herein refers to the attachment of carbohydrate residues to the side chain of asparagine residues. As a tripeptide sequence, asparagine-X-serine and aspartate-X-threonine (where X is any amino acid except proline) are carbohydrate residues The recognition sequence is enzymatically attached to the side chain of asparagine. Therefore, when one of the three peptide sequences in the multiple peptides is present, a potential glycosylation site is created. "O-linked glycosylation" means that one of the sugar N-acetylgalactosamine, galactose, or xylose is attached to the hydroxyl amino acid. Hydroxyamino acids are most typically serine or threonine, but 5-hydroxyproline or 5-hydroxylamine can be used.

The addition of glycosylation sites to the peptide (for N-linked glycosylation sites) is conveniently performed by changing the amino acid sequence to contain the three peptide sequences discussed above. These changes can be made by adding at least one serine or threonine residue to the first antibody sequence or by substitution with their residues (for O-linked glycosylation sites).

According to an embodiment of the invention, the polynucleotide is a nuclear molecule, which may be a natural or artificial DNA or RNA molecule and may be single-stranded or double-stranded. There may be one or more nuclear molecules, where the nuclear molecules may be of the same type (eg, with the same nucleotide sequence) or of different types. Polynucleotides include one or more of DNA, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomers, plastids, and modified nucleic acids described above.

According to an aspect of the present invention, there is provided a method of performing HSP expression inhibition, the method comprising the steps of: administering to a subject in need of HSP expression inhibition a peptide comprising an amino acid sequence of SEQ ID NO: 1, including an A peptide of an amino acid sequence with an acid sequence of 80% or more homology or a peptide fragment mentioned above.

According to another embodiment of the present invention, there is provided a peptide comprising the amino acid sequence of SEQ ID No. 1, a peptide comprising an amino acid sequence having 80% or more sequence homology with the amino acid sequence or above The use of the peptide fragment of the peptide in question is to produce a composition that inhibits the expression of HSP.

As used herein, sequence number 1 (hereinafter "PEP 1") is a telomerase-derived peptide composed of 16 amino acids.

Serial number 1 EARPALLTSRLRFIPK

The peptide described in SEQ ID No. 1 is the same as in Table 2 below. The "name" in Table 2 below is used to distinguish peptides. In one aspect, the peptide of sequence number 2 is the entire peptide of human telomerase. In different specific embodiments of the invention, the peptide having the sequence of SEQ ID No. 1, the peptide of the fragment of the peptide having the sequence of SEQ ID No. 1, or the peptide having the peptide according to the present disclosure Peptides with 80% or more sequence homology include "synthetic peptides" synthesized by selecting and synthesizing peptides corresponding to related positions within telomerase. Sequence number 2 is the amino acid sequence of the entire telomerase.

Furthermore, in a specific embodiment of the present invention, a peptide comprising the amino acid sequence of SEQ ID No. 1, a fragment of the peptide comprising the amino acid sequence of SEQ ID No. 1 or a peptide having an amino acid sequence of 80% The peptides of the amino acid sequence of the above sequence homology have high in vivo biological stability due to low intracellular toxicity.

According to an embodiment of the present invention, a group for inhibiting HSP performance is provided The product comprises: a peptide comprising the amino acid sequence of SEQ ID No. 1, the above-mentioned fragment peptide, or a peptide comprising an amino acid sequence having 80% or more sequence homology with the amino acid sequence Peptide, where the composition is used to deliver one or more active ingredients.

According to an embodiment of the present invention, a pharmaceutical composition or food is provided A composition that effectively transports an active ingredient to a cell. The pharmaceutical composition or food composition includes: a peptide containing an amino acid sequence of SEQ ID NO. 1, or a fragment peptide as described above, including an amino acid group The amino acid sequence HSP of 80% or more homology of the acid sequence binds the peptide and the conjugate of the active ingredient.

In one embodiment of the present invention, the composition may contain 0.1 μg / mg To 1 mg / mg, specifically 1 μg / mg to 0.5 mg / mg, more specifically 10 μg / mg to 0.1 mg / mg peptides containing the amino acid sequence of at least one of sequence number 1, including and above The peptide of the amino acid sequence with at least 80% sequence homology of the sequence mentioned in this article or the fragment of the peptide mentioned above. When a peptide within the range mentioned above is contained, both the safety and stability of the composition can be satisfied and these ranges are suitable in terms of cost efficiency.

In one embodiment of the present invention, the composition can be applied to all animals including humans, dogs, chickens, pigs, cows, sheep, guinea pigs and monkeys.

In an embodiment of the present invention, oral, rectal, The pharmaceutical composition is administered percutaneously, intravenously, intramuscularly, intraperitoneally, intramedullaryly, epidurally or subcutaneously.

The form of oral administration can be (but not limited to) lozenges, pills, soft Capsules or hard capsules, granules, powders, solutions or emulsions. Non-oral administration can be (but not limited to) injection, drip, lotion, ointment, gel, cream, suspension, emulsion, suppository, patch or spray.

In one embodiment of the present invention, if necessary, the pharmaceutical composition may Contains additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, fragrances or sweeteners. In one embodiment of the invention, the pharmaceutical composition may be manufactured by conventional industrial methods in the art.

In one embodiment of the invention, the active ingredient of the pharmaceutical composition The dosage may vary according to the patient's age, sex, weight, pathology and status, administration route, or diagnosis by the prescribing physician. The dosage based on these factors may be determined to be within the level of those skilled in the art, and the daily dosage (for example) may be (but not limited to) 0.1 μg / kg / day to 1 g / kg / day, specifically 1 μg / kg / day to 10 mg / kg / day, more specifically 10 μg / kg / day to 1 mg / kg / day, more specifically 50 μg / kg / day to 100 μg / kg / day. In one embodiment of the present invention, the pharmaceutical composition may be administered 1 to 3 times a day, but it is not limited thereto.

In one embodiment of the present invention, the food composition is not limited to Form, but for example, can be in the form of tablets, granules, powders, liquids and solids. In addition to active ingredients, various forms can be formed together with ingredients commonly used in the industry appropriately selected by those skilled in the art, and each form combined with other ingredients can produce a synergistic effect.

For addressing the above dose of the active ingredient is determined by those skilled in the art within the level, and the daily dose (for example) may be 1 μ g / kg / day to 10mg / kg / day, more particularly in terms of 10 μ g / kg / day to 1mg / kg / day, more particularly 50 μ g / kg / day to 100 μ g / kg / day, but not limited to these figures and according to the age, health, other complications and Various factors vary.

The terminology used herein is intended to describe the embodiments, but not to limit Making the invention. Terms without numbers in front do not limit the number, but show that there can be more than one of the terms used. The terms "include", "have", "include" and "include" (that is, "including but not limited to") should be interpreted openly.

Use references to numerical ranges, rather than to state individual numbers within the range Words, so unless expressly stated, this range should be considered as a separate description of all numbers within the range. The end values of all ranges are included in the range and can be combined independently.

Unless otherwise stated or clearly contradicted in context, all methods discussed herein can be performed in an appropriate order. Unless included in the scope of the patent application, the use of any embodiment and all embodiments or exemplary language (eg, "such as", "similar") is used to more clearly describe the invention, not to limit the invention category. Any language outside the scope of the patent application herein should not be interpreted as necessary for the invention. Unless otherwise defined, the technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this invention belongs.

The preferred embodiments of the invention include the best mode known to the inventors for carrying out the invention. Changes in the preferred embodiment can be familiar after reading the above statement Those who learn this technique become clear. The inventor hopes that those skilled in the art can make full use of the changes and can implement the invention in other ways than those listed here. Therefore, as permitted by the patent law, the present invention includes equivalents, modifications, and changes to the key points of the present invention stated in the scope of the accompanying patent application. In addition, unless expressly stated otherwise or contradicted in context, all possible variations in any combination of the components discussed above are included in the present invention. Although the present invention has been described and illustrated by exemplary embodiments, those skilled in the art will better understand that in the form and details without departing from the spirit and scope of the present invention defined by the scope of the following patent There can be various changes in.

Hereinafter, the present disclosure will be described in detail through examples and test examples. However, the following examples and test examples are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited to these examples and test examples.

Examples Example 1: Synthesis of peptides

The peptide of sequence number 1 was synthesized according to the conventional method of solid phase peptide synthesis. More specifically, peptides are synthesized by coupling various amino acids from the C-terminus via Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon ROK). Use those peptides with the first amino acid attached to the resin at the C-terminus as follows: NH ₂ -Lys (Boc) -2-chloro-trityl resin

NH ₂ -Ala-2-chloro-trityl resin

NH ₂ -Arg (Pbf) -2-chloro-trityl resin

Protect all amine groups of the peptide to be synthesized by Fmoc at the N-terminus Acid, and by Trt, Boc, t-Bu (t-butylester; third butyl ester) soluble in acid, Pbf (2,2,4,6,7-pentamethyldihydro-benzofuran- 5-sulfonyl) protects amino acid residues. Examples include the following: Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe- OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Met -OH, Fmoc-Asn (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ahx-OH, Trt-mercaptoacetic acid.

HBTU [2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethylammonium hexafluorophosphate] / HOBt [N-hydroxybenzotriazole] / NMM [4 -Methylmorpholine] is used as a coupling agent. Fmoc was removed using pyridine in 20% DMF. To remove protection from residues or to separate peptides from synthetic resins, use decomposition mixture [TFA (trifluoroacetic acid; trifluoroacetic acid) / TIS (triisopropylsilane; triisopropylsilane) / EDT (ethanedithiol; ethanedithiol) /H2O=92.5/2.5/2.5/2.5].

Peptide synthesis is performed by using a solid-phase scaffold and repeating the following process: starting with amino acid protection, each amino acid reacts separately, washed with solvent and deprotected. By using the solid-phase scaffold combined with the starting amino acid and amino acid protection, the corresponding amino acids are reacted separately, washed and deprotected with a solvent, and repeating these processes to synthesize each peptide. After being released from the resin, the synthesized peptide was purified by HPLC, confirmed by mass spectrometry, and lyophilized, and verified by MS for synthesis, and then lyophilized.

The purity of the peptide prepared by high performance liquid chromatography was found to be 95% or higher.

The specific synthesis process of PEP 1 can be as follows:

1) Coupling of amino acid (8 equivalents) protected with NH ₂ -Lys (Boc) -2-chloro-trityl resin and the coupling agent HBTU (8 equivalents) / HOBt (8 equivalents) melted in DMF ) / NMM (16 equivalents) were mixed together and incubated at room temperature (RT) for 2 hours. After incubation, the reaction mixture was successively washed with DMF, MeOH and DMF.

2) Fmoc deprotection of pyridine in 20% DMF was added and incubated at RT for 5 minutes, 2 times, and then washed successively with DMF, MeOH and DMF.

3) By repeating the reactions 1) and 2) mentioned above, the basic framework for making peptides NH2-E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR ( Pbf) -FIPK (Boc) -2-chloro-trityl resin.

4) Decomposition: Add the decomposition mixture to the fully synthesized peptide, thereby separating the synthesized peptide from the resin.

5) Add pre-cooled diethyl ether to the obtained mixture, and then use centrifugation to precipitate the aggregated peptides.

6) After purification by preparative HPLC, the molecular weight was confirmed by LC / MS and produced in powder form after lyophilization.

Experiments of coupling with HSP and HSP inhibition were performed using PEP 1 prepared by the method described in Example 1.

Example 2: Method for culturing and analyzing cell lines Cultured cell line

Store human breast cancer cell lines (MCF7), human T lymphocyte cell lines (Jurkat) and murine colon adenocarcinoma cell lines (MC38) in RPMI1460 medium containing 10% fetal bovine serum, 100 U / ml penicillin and streptomycin . The human cervical adenocarcinoma cell line (HeLa) was stored in Dulbecco's modified Eagle's medium (DMEM) including 10% fetal bovine serum, 100 U / ml penicillin and streptomycin.

Identification of protein expression and cell growth under hypoxia

In order to test the effect of PEP 1 on HSP levels under hypoxia, MCF7 and HeLa cell lines were treated with 20 μMPEP 1 and these cell lines were subsequently cultured under hypoxia or normoxia. By using BBL GasPak (Becton Dickinson) to induce a catalytic reaction within 90 minutes to induce hypoxia, the oxygen level is reduced to a point where oxygen cannot be detected. The cultivation time is about 2 hours to about 24 hours. After extracting cells as described above, immunotransfection is performed using α-HSP70, α-HSP90, α-HIF-1α or α-GAPDH antibodies. The amount of HSP70 / 90 was normalized to the amount of GAPDH using α-GAPDH for protein quantification.

To investigate the effect of PEP 1 on the growth of cancer cells under hypoxia, MCF7 and HeLa cells were inoculated so that 1 × 10 ⁴ cells were in each well of a 96-well dish and then in complete medium including 10% FBS at 37 ° C and 5% Incubate under CO ₂ conditions. After 2 hours of serum starvation treatment, MCF7 and HeLa cells were cultured in complete medium with or without PEP 1 (20 μM). As described above, MCF7 and HeLa cells were cultured for 1 to 6 days under hypoxia or normoxia. The number of viable cells is measured daily using trypan blue staining. Repeat all calculation experiments.

Method of analyzing protein levels of HSP70 and HSP90 by using immunostaining

Jurkat and MCF7 cells (5 × 10 ⁵ cells / well) were seeded and subsequently cultured for 12 hours. OPTI-MEM medium was added to the cells to treat the cells in starvation for 2 hours, and the cells were treated with different concentrations of PEP 1, scrambled peptide and 17-AAG (1 μM) or KNK437 (1 μM). After culturing the cells for 2 hours, the cells were recovered and then lysed by using cell lysis buffer (Thermo Scientific, IL, USA). The protein concentration was quantified using the Bradford protein assay (Bio-Rad, USA), and the samples thus obtained were subjected to SDS-PAGE and by using α-HSP70 (sc-32239 and sc-66048, Santa Cruz, CA, USA), α -HSP90 (ab1429, abcam, USA), α-GRP78 (sc-13968, Santa Cruz, CA, USA), α-HIF-1a (sc-10790, Santa Cruz, CA, USA) or α-GAPDH (sc- 25778, Santa Cruz, CA, United States) antibody immunization. The immunoreactive ribbons were visualized by using an enhanced chemiluminescence kit (iNtRoN Biotechnology, INC, Korea) and then the ribbons were quantified by using ImageQuant ™ LAS-4000 (GE Healthcare Life Science, NJ, USA).

Method for analyzing protein levels of HSP70 and HSP90 by using flow cytometry

MCF7 cells were treated with PEP 1 or as a control group. To perform the proteasome inhibition test, MCF7 cells were treated with 5 μM proteasome inhibitor MG132 (Calboicam) while culturing. MCF7 cells were isolated using trypsin, and MCF7 cells were washed with cold phosphate buffered saline (PBS) and FACS buffer (PBS containing 1% BSA and 0.1% NaN ₃ ). MCF7 cells were treated with osmosis buffer (eBioscience, CA, USA) according to the protocol described by the manufacturer for intracellular staining. MCF7 cells were reacted with α-HSP70-FITC (ab61907, Abcam) or α-HSP90-PE (ab65171, Abcam) at a temperature of 4 ° C for 30 minutes. Flow cytometry was performed using FACScan flow cytometer (Becton Dickinson Co., CA, USA). The data thus obtained was analyzed by using FlowjoTM software (version 10.0.5, Tree Star, Inc., OR, USA).

Method for evaluating the effect of PEP 1 on tumor growth in vivo

Seven-week-old BALB / c athymic (Nu / Nu) mice (10 mice per group; n = 20, female, Orient Bio Co. Gyunggido, South Korea) were injected subcutaneously with murine colon carcinoma MC38 (each Site 200 μl 5 × 10 ⁵ cells / ml) cells in PBS, and then the mice were randomly divided into two groups. Mice were injected intraperitoneally with PEP 1 (50 μg / kg PEP 1 in 100 μl 0.9% NaCl solution) or PBS once every two days. When the tumor size reached 10 mm, PEP 1 or PBS was injected into the tumor. Measure the tumor size every two days and use the following formula: volume (mm ³ ) = ((area ² × length) / 2) to calculate the tumor volume. On the 14th day of the experiment, mice were sacrificed and tumor weight was measured. All experiments were approved by the Laboratory Animal Institute of the Seoul National University School of Medicine in Seoul, South Korea.

Evaluation of proliferating cells and apoptosis in tumor slices

To assess tumor cell apoptosis, DNA breaks were analyzed by using the Tunnel test, which used formalin-fixed and paraffin-embedded tumor sections. Tumor sections were stained by using the ApopTag peroxidase in situ apoptosis detection kit (Millipore) according to the protocol described by the manufacturer. By using proliferating cell nuclear antigens (PNCAs) Measure the proliferating cells in the tumor. To detect antigens, tumor sections were dewaxed in 10 μM citrate buffer at pH 6 for 40 minutes. The resulting tissue sections are subsequently hydrated and heated. The resulting tumor sections were stained by using anti-mouse PCNA monoclonal antibody (ab29, Abcam). After secondary antibody treatment and color development, the tissue sections were counterstained using H & E staining. Thereafter, the field of view was randomly selected from 6 slides of each processing group and analyzed by Leica Qwin software for quantification.

Immunotransfection analysis of HSP manifestation in tumor

Immunohistochemical staining was performed in a similar manner to PCNA staining to evaluate the performance of HSP70 and HSP90 proteins in tumors. Antibodies to heat shock proteins (HSP70; sc-7298 and HSP90; ab1429) were used as primary antibodies. Using tumor lysate, the performance of HSP70 and HSP90 proteins caused by tumors was evaluated by immunostaining. After freezing the tumor with liquid oxygen, the tumor was ground by using a mortar, and by using extraction buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 0.05% Triton X-100, 1 mM DTT, 0.5 mM sodium orthovanadate, 1 mM EDTA, 0.5 mM PMSF, 10 μg / ml aprotinin, 5 μg / ml leupeptin and 2 μg / ml gastrin)) homogenized the thus obtained ground tumor. After repeated centrifugation, the thus obtained supernatant was subjected to SDS-PAGE and immunostaining as described above.

Enzyme-linked immunosorbent assay (ELISA) analysis

Identify the VEGF secretion of cancer cells by using ELISA analysis. PEP 1 or vehicle was added to MCF7 and HeLa cells, and then MCF7 and HeLa cells were cultured under hypoxia or normoxia for 24 hours. According to the manufacturer's description It was determined that the amount of VEGF in the clear solution on the cells was identified by using the human VEGF immunoassay kit (R & D Systems, USA). To analyze the concentration of HSP70 and HSP90 in the blood, blood was drawn from a mouse model with tumors. After preparing the serum, the HSP70 in the blood was confirmed by using the immunoassay kit purchased from R & D systems (USA) for HSP70 and the immunoassay kit purchased from Cusabio Biotech co., Ltd (DE, USA) for HSP90 And the concentration of HSP90.

Confocal microscope analysis

The cut tumor sections were fixed by using 4% paraformaldehyde at room temperature for 15 minutes. Subsequently, the cut tumor sections were washed twice, stored in PBS including 0.25% Triton X-100 for 10 minutes, and then washed three more times with PBS. The tissue of the cut tumor section was blocked by using 1% BSA-PBST for 30 minutes. Subsequently, the tissue was incubated with a mixture of mouse anti-Tie2 (557039, BD Pharmigen) and rat anti-CD11b antibody (ab8878, abcam) in a humidified chamber at a temperature of 4 ° C. After washing, the tissue was incubated with a mixture of AlexaFlour 488 goat anti-mouse IgG and AelxaFlour633 goat anti-rat IgG. To visualize the nuclei, the tissues were treated with DAPI (Sigma Aldrich) for one minute and then analyzed with a confocal microscope.

Statistical analysis

The statistical comparison between the control group and the experimental group was analyzed by using the Student's t test, and when the P value was P <0.05 (*) or 0.01 (**), the results obtained from this were considered statistically significant.

Example 3: PEP 1 binding protein screening using pull-down assay

By using His tags including PEP 1-GFP (sequence number: 3 And sequence number: 4) Perform a pull-down test to identify proteins that interact with PEP 1. PEP 1-GFP was cultured with total cell lysate of HEK293T cells (Korean Cell Line Bank) and then treated with Ni-NTA affinity chromatography. The GFP protein and GFP fused PEP 1 peptide was used as a control group. The dissociated protein was separated by 12% SDS-PAGE and then stained with silver (product number # _PROT-SIL1, Sigma Aldrich, MO, USA) for observation. Protein ribbons showing specific interactions were cut from the gel and then analyzed by using MALDI-TOF (Proteomix, Seoul, South Korea) to distinguish protein ribbons.

In more detail, a protein that fused GFP to the N-terminus of PEP 1 was selected and subsequently purified from E. coli. PEP 1-GFP, GFP-PEP 1 or GFP was cultured with total cell lysate of HEK293T cells. Each protein complex was purified and subsequently subjected to SDS-PAGE and silver staining. Several proteins are especially found in PEP 1-GFP protein complexes. However, no protein was found in the GFP or GFP-PEP 1 complex.

Thereafter, the proteins were identified as HSP90, HSP70, enolase 1, and creatine kinase B by using mass spectrometry (Figure 1A). The interaction between the protein identified above and PEP 1 was also observed in the immunostaining experiment by using antibodies (Figure 1B).

PEP 1-GST can successfully pull down HSP70 from cell lysates of MCF7 cells and HepG2 cells (Figure 1C). Thereafter, fragments of the MCF7 cell membrane were purified and subsequently cultured with GFP or GFP-PEP 1. As shown in Figure 1D, HSP70 is associated with GFP-PEP 1 and submerged with GFP-PEP 1. The results show the localization of HSP70 and the interaction between PEP 1 and HSP70.

Statistical analysis

By using the Student's t test to analyze the statistical comparison between the control group and the experimental group, and when the P value is P <0.05 (*) or 0.01 (**), the data obtained from this is considered statistically significant.

Example 4: Proof of inhibition of HSP70 and HSP90 performance due to PEP 1 treatment

Confirm whether PEP 1 may or may not affect the protein levels of HSP70 and HSP90. As shown in Figures 2 and 3, Jurkat T cell lymphoma cells and MCF7 breast cancer cells were treated with PEP 1 for 2 hours, and Jurkat T lymphoma cells and MCF7 breast cancer cells showed a significant decrease in protein levels of HSP70 and HSP90. In experiments using Jurkat cells, 5 μM PEP 1 reduced the protein levels of HSP70 and HSP90 by 50% or more.

In MCF7 cells, the group treated with 5 μM PEP 1 showed a decrease in protein levels of HSP90 by up to 20% compared to the control group. In the group treated with 20 μM PEP 1, the protein level of HSP70 decreased by approximately 50% compared to the control group. However, in the group treated with scrambled peptides having a sequence similar to PEP 1 but different from PEP 1, the protein levels of HSP70 and HSP90 were not substantially affected (refer to Figure 2 and Figure 3).

In addition, an experiment to recognize the effect of PEP 1 on HSP70 and HSP90 according to time was performed under hypoxia. Figures 4 and 5 illustrate the results thus obtained. As shown in Figure 4 and Figure 5, the performance of HSP70 and HSP90 in the simulated treatment control group was not affected by time, but both MCF7 and HeLa cells treated with PEP 1 showed the performance of HSP70 and HSP90 according to time Substantial decline. This clearly shows that the PEP 1 treatment triggers the decomposition of HSP and can be controlled thereafter HSP client protein (Figure 4 and Figure 5). The results indicate that PEP 1 can affect various cellular responses related to hypoxia by reducing the protein level of HSP.

Thereafter, the HSP inhibitory activity of PEP 1 was compared with the HSP inhibitory activity of 17-AAG and KNK437, which are well known as inhibitors of HSP90 and HSP70, respectively. 17-AAG inhibits the ATPase activity of HSP90 to directly inhibit HSP90 activity [Uehara Y, Current cancer drug targets, 3: 325-30, 2003]; and KNK437 inhibits the synthesis of HSP induced by adversity. The experimental results show that in Jurkat and MCF7 cells, only PEP 1 shows a decrease in protein levels of both HSP90 and HSP70 (refer to Figure 6).

In Jurkat cells, only PEP 1 reduced the protein levels of HSP70 and HSP90, and although 17-AAG and KNK437 reduced the protein levels of HSP90, 17-AAG and KNK437 could not reduce the protein levels of HSP70. In MCF7 cells, PEP 1 and KNK437 reduced the protein levels of both HSP90 and HSP70, while 17-AAG had a minimal effect on the protein levels of HSP90 and HSP70.

The decrease in protein levels of HSP90 and HSP70 caused by PEP 1 can be more clearly confirmed by flow cytometry analysis. The surface staining and intracellular staining of HSP90 and HSP70 showed that the effect of PEP 1 treatment on cell surface HSP90 and HSP70 may be less than that on cytoplasmic HSP90 and HSP70, but the protein levels of intracellular and cytoplasmic HSP90 and HSP70 may be reduced (Figure 7). When PEP 1 and proteasome inhibitor MG132 were treated together, there was no action of PEP 1, indicating that PEP 1 could induce HSP90 and HSP70 Proteasome-dependent decomposition (refer to Figure 7).

Example 5: Identification of tumor cell growth under hypoxia and normoxia

The effect of PEP 1 on tumor cell growth was investigated under hypoxia and normoxia. PEP 1 exhibits a weak inhibitory effect on MCF7 and HeLa cells under normoxia, but exhibits a substantially increased inhibitory effect under hypoxia (refer to FIGS. 8 and 9).

Example 6: Due to PEP 1, the protein levels of HSP70 and HSP90 in the tumor decreased

To see if PEP 1 inhibits HSP70 and HSP90 in vivo, immunohistochemical staining was performed by using α-HSP70 or α-HSP90 antibodies. According to the data obtained from the cancer cell lines, the tumor samples taken from the PEP 1 treatment group showed a weaker staining pattern than the other samples of the PBS treatment control group (refer to Figure 10). The positively stained portions in the tumor samples treated with PEP 1 were substantially smaller than those of the control group (refer to Figure 11).

The protein levels of HSP70 and HSP90 in tumor samples treated with PEP 1 can be confirmed by immunotransfection experiments using tumor lysate. Decreased protein levels of HSP70 and HSP90 were observed in all three PEP 1 treated tumor samples (see Figure 12). In detail, HSP90 was not found in PEP 1 treated tumor samples. In the PEP 1 treated samples, the protein level of GRP78, which is regarded as another family member of HSP, also decreased. In summary, the results indicate that PEP 1 reduces HSP levels in vivo and can inhibit tumor growth.

Example 7: Effect of PEP 1 on the protein level of HSP70 secreted in the blood

Both HSP70 and HSP90 can be secreted from tumor cells, and recent research Research indicates that HSP70 and HSP90 play some roles in tumor production and anti-tumor response. Regarding the secretion of HSP90 and HSP70, the concentrations of HSP70 and HSP90 were measured from the blood of mice with tumors in order to describe the role of PEP 1 in more detail. The measurement results indicate that PEP 1 treatment is particularly effective for reducing the protein level of HSP70 in PEP 1 treated mice (see Figure 13).

Furthermore, the results of analyzing HSP70 concentration, tumor weight, and tumor size showed that the correlation between tumor weight and HSP70 concentration had a statistical significance of P = 0.037, and the correlation between tumor size and HSP70 concentration had a statistical value of P = 0.039. Significance (R ² = 1). These results indicate that the low protein level of HSP70 is related to the size and weight of the tumor (refer to Figure 14).

Preset sequence table

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As described above, according to one or more of the above-described embodiments of the present invention, HSP performance can be effectively suppressed by using a peptide that specifically binds to HSP. More specifically, due to the increased expression of HSP in cancer cells, a method for effectively inhibiting tumor growth by using a peptide that specifically binds to HSP is provided.

It should be understood that the exemplary embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should generally be considered as available for other similar features or aspects in other embodiments.

Although one or more embodiments of the present invention have been described with reference to the drawings, those of ordinary skill in the art should understand that the forms and forms can be carried out without departing from the spirit and scope of the present invention defined by the scope of the following patent applications In detail

Various changes.

<110> Korean businessmen. Kyle Jim Vickers Co., Ltd.

<120> Peptides that inhibit the expression of heat shock proteins and compositions containing them

<130> OF14P081 / TW

<150> 10-2013-0049983

<151> 2013-05-03

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<170> PatentIn version 3.2

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Claims (9)

A peptide is used for manufacturing a composition for inhibiting heat shock protein expression, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 1. The use according to claim 1, wherein the heat shock protein is HSP 70 or HSP 90. The use according to claim 1, wherein the heat shock protein is HSP 70. The use according to claim 1, wherein the peptide contains 30 or fewer amino acids. The use according to claim 1, wherein the composition is used to prevent or treat a disease or disorder, wherein the disease or disorder is selected from the group consisting of tumor growth and metastasis, endometrial cancer , Malignant osteosarcoma, kidney cancer, leukemia, Hodgkin's disease, skin redness, psoriasis, macular degeneration, hemophilic arthropathy, wound granulation, vascular adhesion, rheumatoid arthritis, chronic inflammation, osteoarthritis, Autoimmune diseases, Crohn's disease, vascular restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, cirrhotic nephropathy, glomerulonephritis, diabetic nephropathy, malignant renal sclerosis, organs Transplant rejection, glomerulonephropathy, diabetes and inflammation. The use according to claim 5, wherein the tumor can develop in the stomach, liver, kidney, pancreas, intestine, uterus, ovary, bladder, breast, prostate, thyroid, lymph, spine, brain, bone, or esophagus. The use according to claim 1, wherein the composition is used for growth inhibition of a tumor. The use according to claim 1, wherein the composition is a pharmaceutical composition. The use according to claim 1, wherein the composition is a food composition.